RU2786441C2 - Peptide vaccines against interleukin-31 - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: vaccine composition for immunization and/or protection of a mammal – a dog, a cat, or a horse – from a disease mediated by IL-31 is proposed, where the specified composition contains a combination of a polypeptide carrier and at least one mimotope of peptide IL-31, and an adjuvant. Mimotope of peptide IL-31 has a length from about 5 to about 40 amino acids, and it is selected from a group consisting of mimotope of dog IL-31, mimotope of cat IL-31, mimotope of horse IL-31, and mimotope of human IL-31. A method for protection of a mammal from a disease mediated by IL-31 is presented. The method includes administration to a mammal of the presented vaccine composition.

EFFECT: invention allows for obtaining a vaccine for reduction in skin itch and skin damage in dogs, cats, horses with atopic dermatitis.

20 cl, 39 dwg, 2 ex

Description

FIELD OF THE INVENTION

This invention relates to the field of peptide vaccines and from applications in clinical and scientific procedures, including diagnostic procedures. The peptide vaccines of this invention may find use in immunizing and/or protecting a mammal, such as a cat, dog, horse, or human, against an IL-31 mediated disease.

PRIOR ART

According to the American College of Veterinary Dermatology Working Group, atopic dermatitis is "an inflammatory, pruritic, allergic skin disease with characteristic clinical features and a genetic predisposition" (Olivry, et al. Veterinary Immunology and Immunopathology 2001; 81: 143-146). The Working Group also recognized that in dogs the disease was associated with allergen-specific IgE (Olivry, et al. 2001, supra; Marsella & Olivry Clinics in Dermatology 2003; 21: 122-133). Severe pruritis along with secondary alopecia and erythema are the most noticeable and disturbing symptoms for pet owners.

The possible factors involved in allergic dermatitis are numerous and poorly understood. Atopic dermatitis can be triggered 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, etc. Genetic factors also play an important role. Although there is no evidence of breed predisposition, it is believed that there is some influence of heredity in increasing the predisposition to atopic dermatitis (Sousa & Marsella Veterinary Immunology and Immunopathology 2001; 81: 153-157; Schwartzman, et al. Clin. Exp. Immunol. 1971;9:549-569).

The estimated prevalence of atopic dermatitis in the general dog population is 10% (Marsella & Olivry 2003 supra; Scott, et al. Canadian Veterinary Journal 2002; 43: 601-603; Hillier Veterinary Immunology and Immunopathology 2001; 81: 147-151 ). Overall, approximately 4.5 million dogs are affected by this chronic and lifelong condition. Apparently, the incidence rate is increasing. There was thought to be a predisposition in relation to breed and sex, but there could be strong differences depending on the geographic region (Hillier, 2001, see above; Picco, et al. 2008, see above).

In cats, allergic dermatitis is an inflammatory, itchy skin disorder thought to be caused by an abnormal reaction of the immune system to substances that do not cause a reaction in healthy cats. The most characteristic sign of allergic dermatitis in cats is chronic recurrent itching. Typical clinical manifestations of allergic dermatitis in cats include self-induced alopecia, miliary dermatitis, eosinophilic granuloma complex (including plaques, granulomas, and indolent ulcer), and focused head and neck pruritus characterized by excoriations, erosions, and/or ulcers. Breed and sex predisposition has not been demonstrated, and younger cats seem to be more prone to 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).

Current treatments for cats diagnosed with atopic dermatitis depend on the severity of clinical signs, duration of illness, and owner preference, 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 clinical improvement may not be seen for 6-9 months (Buckley, supra). Immunosuppressants such as glucocorticoids and cyclosporins are generally effective, but their long-term use often leads to unwanted side effects.

Atopic dermatitis in horses is considered a potential cause of pruritus. The role of environmental allergens in the development of equine atopic dermatitis is increasingly recognized. The disease may be seasonal or non-seasonal, depending on the allergen(s). No predisposition was reported for age, breed, or sex. In preliminary work at the UC Davis School of Veterinary Medicine (SVM-UCD), the median age of onset was 6.5 years, Thoroughbreds were the most common breed, accounting for 25% of horses, and males (usually geldings) outnumbered mares by almost twice; however, these data were for only 24 horses and have not yet been compared with the general clinic population. Itching, often affecting the muzzle, hind legs, or trunk, is the most common clinical sign of equine AD. Alopecia, erythema, urticaria, and papules may be present. Urticaria can be quite severe, but not accompanied by itching. The horse may have a family history of urticaria. Horses may have secondary pyoderma, which presents as excessive scaling, small epidermal collars, or crusted papules (“miliary dermatitis”). The diagnosis of atopic dermatitis is based on clinical signs and the exclusion of other diagnoses, especially insect (lice) hypersensitivity (White Clin Tech Equine Pract 2005; 4: 311-313; Fadok Vet Clin Equine 2013; 29 541-550). Currently, both symptomatic treatment of atopic dermatitis in horses is carried out, suppressing inflammation and itching caused by an allergic reaction, and etiotropic (i.e., identifying the allergens responsible for the disease and creating allergen-specific vaccines). Symptomatic management is usually needed in the short term to keep the patient comfortable and minimize self-injury. This approach is based on the use of a combination of local and systemic therapies, including antihistamines, essential fatty acids, pentoxifylline, and glucocorticoids. The primary approach to environmental allergy control involves identifying the allergens causing the hypersensitivity reaction. Dermatologists believe that allergen-specific immunotherapy can help horses with atopy. However, in general, most horses 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 immunosuppressants in horses can lead to unwanted side 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; US Patent No. 8,790,651 to Bammert et al.; Gonzalez et al. Vet Dermatl 2013; 24(1): 48-53. IL-31 binds to a co-receptor consisting of the IL-31 A receptor (IL-31 RA) 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 receptor leads to STAT phosphorylation via the JAK receptor(s). Coreceptor expression was found in macrophages, keratinocytes, and dorsal root ganglia.

Recently, IL-31 has been found to play a role in dermatitis, pruritus, allergies, and airway hypersensitivity. Cytopoint®, an anti-canine 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 alternative approaches to the prevention and treatment of IL-31-mediated diseases in mammals. It would be particularly desirable to provide vaccines to reduce pruritus and skin lesions in dogs, cats, horses and humans with atopic dermatitis.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a vaccine composition for immunizing and/or protecting a mammal against an IL-31 mediated disorder, wherein the composition comprises: a combination of a carrier polypeptide and at least one mimotope selected from feline IL-31 mimotope, IL-31 mimotope, 31 dogs, equine IL-31 mimotope or human IL-31 mimotope, and adjuvant.

In one embodiment of the vaccine composition, the dog IL-31 mimotope is and/or contains as part of the amino acid sequence SVPADTFECKSF (SEQ ID NO: 186), SVPADTFERKSF (SEQ ID NO: 187), NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ ID NO: 192), APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO: 200), or variants thereof that retain binding to an anti-1L-31 agent.

In another embodiment of the vaccine composition, the feline IL-31 mimotope is and/or contains as part of the amino acid sequence SMPADNFERKNF (SEQ ID NO: 188), NG SAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ ID NO: 193), APAHRLQPSDIRKIILELRPM SKG (SEQ ID NO: 197) , IGLPES (SEQ ID NO: 201) or variants thereof that retain binding to an anti-1L-31 agent.

In another embodiment of the vaccine composition, the equine IL-31 mimotope is and/or contains as part of the amino acid sequence SMPTDNFERKRF (SEQ ID NO: 189), NS SAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ ID NO: 194), GPIYQLQPKEIQAIIVELQNLS KK (SEQ ID NO: 198) , KGVQKF (SEQ ID NO: 202) or variants thereof that retain binding to an anti-1L-31 agent.

In one embodiment of the vaccine composition, the human IL-31 mimotope is and/or contains as part of the amino acid sequence SVPTDTHECKRF (SEQ ID NO: 190), SVPTDTHERKRF (SEQ ID NO: 191), HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195), LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203), or variants thereof that retain binding to an anti-1L-31 agent.

In one embodiment, the mimotope contained in the vaccine composition binds to an anti-IL-31 antibody, or an antigen-binding portion thereof, that specifically binds to a region of the mammalian IL-31 protein involved in the interaction of the IL-31 protein with its co-receptor. In one embodiment, binding of said antibody to said region is affected by mutations in the 15H05 epitope binding region selected from the group consisting of:

a) the region between amino acid residues 124 and 135 of the feline IL-31 sequence shown in SEQ ID NO: 157 (wild-type feline IL-31);

b) the region between amino acid residues 124 and 135 of the canine IL-31 sequence represented by SEQ ID NO: 155 (canine IL-31); and

c) the region between amino acid residues 118 and 129 of the horse IL-31 sequence shown in SEQ ID NO: 165 (equine IL-31).

In a specific embodiment, the mimotope binds to an anti-IL-31 antibody, or antigen-binding portion thereof, comprising at least one of the following combinations of complementarity determining region (CDR) sequences:

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

2) ZIL1 antibody: Heavy chain variable region (VH) CDR1 with the sequence SYGMS (SEQ ID NO: 13), VH-CDR2 with the sequence HINSGGSSTYYADAVKG (SEQ ID NO: 14), VH-CDR3 with the sequence VYTTLAAFWTDNFDY (SEQ ID NO: 15 ), light chain variable region (VL) of the sequence SGSTNNIGIL A AT (SEQ ID NO: 16), VL-CDR2 with the sequence of SDGNRPS (SEQ ID NO: 17) and VL-CDR3 with the sequence of QSFDTTLDAYV (SEQ ID NO: 18);

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

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

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

6) ZIL69 antibody: VH-CDR1 with SYAMK sequence (SEQ ID NO: 37), VH-CDR2 with TINNDGTRTGYADAVRG sequence (SEQ ID NO: 38), VH-CDR3 with GNAESGCTGDHCPPY sequence (SEQ ID NO: 39), VL-CDR1 with the sequence SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 with the sequence KDTERPS (SEQ ID NO: 41), VL-CDR3 with the sequence ESAVSSETNV (SEQ ID NO: 42); 7) ZIL94 antibody: VH-CDR1 with TYFMS sequence (SEQ ID NO: 43), VH-CDR2 with LISSDGSGTYYADAVKG sequence (SEQ ID NO: 44), VH-CDR3 with FWRAFND sequence (SEQ ID NO: 45), VL-CDR1 with the sequence GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 with the sequence DTGSRPS (SEQ ID NO: 47), VL-CDR3 with the sequence SLYTDSDILV (SEQ ID NO: 48);

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

9) ZIL159 antibody: VH-CDR1 with SYVMT sequence (SEQ ID NO: 55), VH-CDR2 with GFNSEGSRTAYADAVKG sequence (SEQ ID NO: 56), VH-CDR3 with GDIVATGTSY sequence (SEQ ID NO: 57), VL-CDR1 sequences SGETLNRF YTQ (SEQ ID NO: 58), VL-CDR2 with the sequence KDTERPS (SEQ ID NO: 59), VL-CDR3 with the sequence KSAVSIDVGV (SEQ ID NO: 60);

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

11) variant (1) - (10), which differs from the corresponding parental antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 or ZIL171 by the addition, deletion and / or replacement of one or more amino acid residues at at least one of CDR1, CDR2 or CDR3 VH or VL.

In some embodiments, the mimotope used in the vaccine compositions of the present invention binds to an anti-IL-31 antibody, or an antigen-binding portion thereof, that binds to feline IL-31, wherein the antibody comprises a VL chain containing frame region 2 (FW2) changes selected of 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 refer to the numbering of SEQ ID NO: 127 (FEL_15H05_VL1).

In one embodiment of the vaccine compositions described above, the mimotope is a mimotope with limited conformational freedom. In a specific embodiment, the mimotope with limited conformational freedom is a chemically linked cyclic peptide.

In some embodiments of the vaccine compositions described above, the mimotope is chemically conjugated to a carrier polypeptide. In other embodiments, the carrier polypeptide and the mimotope are part of a recombinant fusion protein.

In one embodiment of the vaccine compositions described above, the carrier polypeptide that is combined with mimotope comprises a bacterial toxoid or derivative thereof, keyhole hemocyanin (KLH) or a virus-like particle. In one embodiment, mimotope is combined with a bacterial toxoid or a derivative selected from tetanus toxoid, diphtheria toxoid, tetanus toxoid, N. meningitidis group B outer membrane protein complex, Pseudomonas exotoxin, or a non-toxic diphtheria toxin mutant (CRM197). In another embodiment, mimotope is combined with a virus-like particle selected from HBsAg, HBcAg, E. coli bacteriophage Qbeta, Norwalk virus, canine distemper virus (CDV), or influenza virus hemagglutinin (HA). In a specific embodiment, the mimotope is combined with a carrier polypeptide that contains or consists of CRM197.

In one embodiment, the adjuvant contained in the vaccine compositions of the present invention described above is selected from an oil in water adjuvant, a polymer and water adjuvant, a water in oil adjuvant, an aluminum hydroxide adjuvant, a vitamin E adjuvant, and combinations thereof.

In one embodiment, the adjuvant is a formulation containing a saponin, a sterol, a quaternary ammonium compound, and a polymer. In a specific embodiment, the saponin is Quil A or a purified fraction thereof, the sterol is cholesterol, the quaternary ammonium compound is dimethyldioctadecylammonium bromide (DDA), and the polymer is polyacrylic acid.

In another embodiment, the adjuvant comprises a combination of one or more isolated immunostimulatory oligonucleotides, a sterol and a saponin. In a specific embodiment, the one or more isolated immunostimulatory oligonucleotides comprise CpG, the sterol is cholesterol, and the saponin is Quil A or a purified fraction thereof.

The present invention also provides a method for protecting a mammal from an IL-31 mediated disease. Such a method includes administering to a mammal a vaccine composition according to the present invention. In one embodiment, the mammal to which the vaccine of the present invention is administered is selected from a dog, cat, horse, or human. In a specific embodiment, the vaccine composition comprises an IL-31 peptide mimotope that is administered to a mammal at a dose of about 10 μg to about 100 μg, or an appropriate dose, to elicit an equivalent immune response. In one embodiment, the vaccine composition comprises IL-31 mimotope, which is administered to a mammal, such as a cat, at a dose of approximately 10 μg.

In one embodiment, the IL-31 mediated disease 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 pruritus or allergic condition is an allergic condition selected from allergic dermatitis, summer eczema, urticaria, emphysema, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, and autoimmunity-induced inflammation. In other embodiments, the IL-31 mediated disorder is tumor progression. In some embodiments, the IL-31 mediated disorder is eosinophilic disease or mastocytomas.

Also provided herein is a method for qualitative or quantitative detection of anti-IL-31 antibody in a sample. Such a method comprises incubating a sample containing an anti-IL-31 antibody with at least one mimotope selected from feline IL-31 mimotope, canine IL-31 mimotope, equine IL-31 mimotope, and human IL-31 mimotope and qualitative or quantitative detection in the sample of anti-IL-31 agent.

In one embodiment, a canine IL-31 mimotope used in a method for qualitatively or quantitatively detecting an anti-IL-31 antibody in a sample is and/or contains as part of the amino acid sequence SVPADTFECKSF (SEQ ID NO: 186), SVPADTFERKSF (SEQ ID NO: 187), NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ ID NO: 192), APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO: 200), or variants thereof that retain binding to an anti-IL-31 agent.

In another embodiment, the feline IL-31 mimotope used in such a method is and/or contains as part of the amino acid sequence SMPADNFERKNF (SEQ ID NO: 188), NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ ID NO: 193), APAHRLQPSDIRKIILELRPM SKG (SEQ ID NO: : 197), IGLPES (SEQ ID NO: 201) or variants thereof that retain binding to an anti-IL-31 agent.

In a further embodiment, the equine IL-31 mimotope used in such a method is and/or contains as part of the amino acid sequence SMPTDNFERKRF (SEQ ID NO: 189), NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ ID NO: 194), GPIYQLQPKEIQAIIVELQNLS KK (SEQ ID NO: 189). : 198), KGVQKF (SEQ ID NO: 202), or variants thereof that retain binding to an anti-IL-31 agent.

In addition, the human IL-31 mimotope used in such a method is and/or contains as part of the amino acid sequence SVPTDTHECKRF (SEQ ID NO: 190), SVPTDTHERKRF (SEQ ID NO: 191), HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195), LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203), or variants thereof that retain binding of the anti-IL-31 agent.

In one embodiment of the diagnostic method described above, the mimotope is a capture reagent bound to a solid surface. In one embodiment, the sample is added to the mimotope, which is a capture reagent, and then secondary detection reagents are added to determine the amount of antibody in the sample.

The present invention also provides a method for determining the amount of IL-31 in a sample from a mammal. Such a method comprises incubating a sample from a mammal containing IL-31 with a labeled anti-IL-31 antibody: IL-31 mimotope complex bound to a solid surface, wherein the mimotope in the complex is selected from the group consisting of feline IL-31 mimotope, canine mimotope IL-31, mimotope equine IL-31 and mimotope human IL-31; and determining the level of IL-31 in the sample, wherein the labeled anti-IL-31 antibody in the complex has a lower affinity for mimotope in the complex compared to its affinity for IL-31 in the sample. In one embodiment of this method, the detection step includes measuring the signal from the labeled antibody that is released from the solid surface when IL-31 in the sample binds to the labeled antibody against the IL-31 complex, with the level of IL-31 in the sample being inversely proportional to the signal.

In one embodiment, the canine IL-31 mimotope used in the method for quantifying IL-31 in a sample is and/or contains as part of the amino acid sequence SVPADTFECKSF (SEQ ID NO: 186), SVPADTFERKSF (SEQ ID NO: 187), NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ ID NO: 192), APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO: 200) or variants thereof that retain binding to an anti-IL-31 agent.

In another embodiment, the feline IL-31 mimotope used in such a method is and/or contains as part of the amino acid sequence SMPADNFERKNF (SEQ ID NO: 188), NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ ID NO: 193), APAHRLQPSDIRKIILELRPM SKG (SEQ ID NO: : 197), IGLPES (SEQ ID NO: 201) or variants thereof that retain binding to an anti-IL-31 agent.

In yet another embodiment, the equine IL-31 mimotope used in such a method is and/or contains as part of the amino acid sequence SMPTDNFERKRF (SEQ ID NO: 189), NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ ID NO: 194), GPIYQLQPKEIQAIIVELQNLS KK (SEQ ID NO: 198), KGVQKF (SEQ ID NO: 202), or variants thereof that retain binding to an anti-IL-31 agent.

In a further embodiment, the human IL-31 mimotope used in such a method is and/or contains as part of the amino acid sequence SVPTDTHECKRF (SEQ ID NO: 190), SVPTDTHERKRF (SEQ ID NO: 191), HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195), LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203), or variants thereof that retain binding to an anti-IL-31 agent.

In some embodiments of the diagnostic methods of the invention described above, the mimotope binds to an anti-IL-31 antibody, or an antigen-binding portion thereof, that specifically binds to a region of the mammalian IL-31 protein involved in the interaction of the IL-31 protein with its co-receptor. In one embodiment of the diagnostic methods of the invention, binding of said antibody to said region is affected by mutations in the region of the 15H05 binding epitope selected from the group consisting of:

a) the region between amino acid residues 124 and 135 of the feline IL-31 sequence shown in SEQ ID NO: 157 (wild-type feline IL-31);

b) the region between amino acid residues 124 and 135 of the canine IL-31 sequence represented by SEQ ID NO: 155 (canine IL-31); and

c) the region between amino acid residues 118 and 129 of the horse IL-31 sequence shown in SEQ ID NO: 165 (equine IL-31).

In one specific embodiment of any of the diagnostic methods of the invention, mimotope binds to an anti-IL-31 antibody, or antigen-binding portion thereof, comprising at least one of the following combinations of complementarity determining region (CDR) sequences:

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

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

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

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

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

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

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

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

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

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

11) variant (1) (10) that differs from the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 or ZIL171 by the addition, deletion and/or substitution of one or more amino acid residues at least at least one of CDR1, CDR2 or CDR3 VH or VL.

In some embodiments, the mimotope used in the diagnostic methods of the present invention binds to an anti-IL-31 antibody, or an antigen-binding portion thereof, that binds to feline IL-31, wherein the antibody comprises a VL chain containing frame region 2 (FW2) changes selected of 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 refer to the numbering of SEQ ID NO: 127 (FEL_15H05_VL1).

BRIEF DESCRIPTION OF GRAPHICS

Fig. 1 is an alignment showing amino acid sequence conservation between IL-31 from different species. Specifically, 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 (IL-31 person). Also indicated are the percentage amino acid sequence identity of canine, feline, equine and human IL-31.

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

Fig. 3 is a table showing the potency (IC50 (µg/mL)) of mouse-derived CDR candidate antibodies in dog and cat 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 for canine-derived CDR candidate monoclonal antibodies to various proteins using indirect ELISA and Biacore methods. For indirect ELISA, binding (ELISA OD) to wild-type feline IL-31 and 15H05 feline IL-31 mutant IL-31, which had mutations in the region of the epitope recognized by the 15H05 monoclonal antibody, was evaluated. To confirm binding, a biacore assay was performed using canine, feline, equine, human IL-31 proteins, 15H05 and 11E12 mutant feline IL-31 proteins as surfaces, and a single antibody concentration tested. The 11E12 mutant of feline IL-31 had mutations in the epitope region recognized by the 11E12 monoclonal antibody.

On FIG. 5 - Fig. 5A shows a VL sequence alignment of the murine antibody 11E12 (SEQ ID NO: 73) comparing previously disclosed caninized 11E12 sequences designated as Can_11E12_VL_cUn_1 (SEQ ID NO: 182) and CAN_llE12_VL_cUn_FW2 (SEQ ID NO: 184) with felinized versions designated as FEL_V_11H12 SEQ ID NO: 111) and FEL_11E12_VL1_FW2 (SEQ ID NO: 117). Dots are marked below the alignment in FIG. 5A showing the positions of the corresponding 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, herein designated MU_15H05_VL (SEQ ID NO: 69), with the VL sequences of felinized 15H05, herein designated FE1_15H05_VL1 (SEQ ID NO: 127) and FE1_15H05_VL_FW2 (SEQ ID NO: 135). ). The dots under alignment in Figure 5B indicate the necessary changes in the VL of felinized 15H05 (Fe1_15H05_VL1) that were required 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.

Figure 6 - Figure 6A shows the 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 at which the substitutions occur to alanine. FIG. 6B shows a homology model of feline IL-31, highlighting the positions of two amino acids involved in antibody binding 11E12 (site 1) and 15H05 (site 2). 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), when wild-type protein and these mutants were used as immobilized antigens.

Fig. 7 is a graph showing competitive binding scores of mAb 15H05 and 11E12 using Biacore. On FIG. 7A shows competitive binding data for mouse 15H05 and 11E12 antibodies to canine IL-31. On FIG. 7B shows competitive binding data for antibodies 15H05 and 11E12 to surface-immobilized feline IL-31.

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 IL-31 mutant proteins 15H05 (SEQ ID NO: 163) and 11E12 (SEQ ID NO: 161) when the wild-type protein and these mutants were used as immobilized antigens.

Fig. 9 is a graph showing preliminary efficacy data for the mouse:feline 11E12 chimera, mouse:feline 15H05 chimera, and feline 11E12 (feline 11E12 1.1) chimera in an IL-31 induced itch model in cats.

Fig. 10 is a graph showing the in vivo efficacy evaluation of a felinized anti-IL-31 antibody 15H05, designated ZTS-361, in a feline pruritus model. On FIG. 10A shows the baseline behavior before pruritus in the placebo group T01 and the ZTS-361 antibody group T02 from day 7 to day 28, where day 0 was the day the antibody was administered to the T02 group. On 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 induction of IL-31 by compared with placebo-treated controls.

Fig. 11 - Fig. 11A is a graph showing plasma levels of IL-31 in client-owned dogs with atopic and allergic dermatitis compared to normal laboratory animals. 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 US geographic areas. Fig. 11C is a graph showing the pharmacokinetic profile of canine IL-31 in dogs following subcutaneous administration of canine IL-31 at a dose of 1.75 μg/kg.

On FIG. 12 is a table showing the results of scanning mutagenesis of canine IL-31 with complete substitution of the amino acids indicated in FIG. 12. Each depicted position in the full-length canine IL-31 protein (SEQ ID NO: 155) was separately replaced with one of the other possible 19 amino acids and binding of the 15H05 antibody was assessed by indirect ELISA. For comparison, the respective regions of feline (SEQ ID NO: 157), equine (SEQ ID NO: 165) and human IL-31 (SEQ ID NO: 181) are shown.

Fig. 13 - Fig. 13A is a table showing the sequences and chemical linkers of various conformationally constrained peptides. The ZTS-561 peptide contains the amino acid sequence N-TEISVPADTFERKSFILT-C, which corresponds to positions 121-138 of SEQ ID NO: 155 with the replacement of arginine (R) by cysteine (C) at position number 132. The ZTS-562 peptide contains the amino acid sequence N- EISVPADTFERKSF-C, which corresponds to positions 122-135 of SEQ ID NO: 155 with the substitution of arginine (R) for cysteine (C) at position number 132. The ZTS-563 peptide contains the amino acid sequence N-AKVSMPADNFERKNFILT-C, which corresponds to positions 121- 138 of SEQ ID NO: 157 with a threonine (T) replaced by alanine (A) at position number 138. The ZTS-564 peptide contains the amino acid sequence N-TEISVPADTFERKSFILT-C, which corresponds to positions 121-138 of SEQ ID NO: 155. Each of the peptides ZTS-561, ZTS-562, ZTS-563 and ZTS-564 also includes N and C terminal cysteines as shown to facilitate conjugation using free thiol groups. On FIG. 13B shows the affinity results of each of the ZTS-561, ZTS-562, ZTS-563, and ZTS-564 peptides that were independently conjugated to a carrier polypeptide (CRM-197). For affinity assessment, each peptide was independently immobilized on the surface of the biacore chip and KD was determined for the felinized anti-IL-31 mAb 15H05 (ZTS-927).

On FIG. 14 depicts a schematic of an immunogenicity assay conducted to evaluate the ability of CRM-197-conjugated IL-31 mimotopes to elicit an epitope-specific immune response directed to the appropriate region of the IL-31 protein where the 15H05 antibody and other anti-IL antibodies described herein bind. -31.

On FIG. 15 is a graph showing serum titers obtained after vaccination of dogs with canine and feline mimotopes IL-31 15H05 and feline full-length IL-31 protein, presented by treatment group, showing the response on each day the serum was taken. On FIG. 15A shows mean canine antibody titers to full-length feline IL-31 protein (SEQ ID NO: 159). On FIG. 15B shows mean canine antibody titers to full-length feline IL-31 mutant 15H05 (SEQ ID NO: 163). On FIG. 15C shows mean canine antibody titers to full-length canine IL-31 (SEQ ID NO: 155). On FIG. 15D shows mean canine antibody titers to full-length equine IL-31 (SEQ ID NO: 165). On FIG. 15E shows mean canine antibody titers to full-length human IL-31 (SEQ ID NO: 181).

On FIG. 16 - Fig. 16A depicts a design of an immunogenicity study conducted to evaluate the ability of CRM-197-conjugated full-length canine IL-31 protein or mimotopes to elicit an immune response in laboratory beagle dogs. Each mimotope described herein is designed to elicit an epitope-specific immune response directed to the appropriate region of the IL-31 protein where the 15H05 antibody and other anti-IL-31 antibodies described herein bind. The sequences and chemical linkers of the peptides of various mimotopes are shown as groups T02-T04. The ZTS-420 peptide contains the amino acid sequence N-TEISVPADTFERKSFILT-C, which corresponds to positions 121-138 of SEQ ID NO: 155 with the replacement of arginine (R) with cysteine (C) at position number 132. The ZTS-421 peptide contains the amino acid sequence N- TNISVPTDTHECKRFILT-C, which corresponds to positions 122-139 of SEQ ID NO: 181. The ZTS-766 peptide contains the amino acid sequence N-NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF-C, which corresponds to positions 83-115 of SEQ ID NO: 155. Each of the ZTS-420 peptides, ZTS-421 and ZTS-766 also include N and C terminal cysteines as shown to facilitate conjugation using free thiol groups. ZTS-766 also contains an additional three amino acid spacer sequence (GSG) next to the N-terminal cysteine. On FIG. 16B shows homologous sequences to compare the BC helix mimotope of canine IL-31 (ZTS-766) with the corresponding sequence of feline, equine, and human IL-31 and includes the reference sequence number and amino acid position for each.

On FIG. 17 is a graph showing serum titers obtained after vaccination of dogs with 15H05 canine and human IL-31 mimotope, canine IL-31 helix mimotope, and feline full-length IL-31 protein, presented by treatment group, showing the response on each sampling day. serum. Dogs were administered on days 0, 28 and 56, indicated by arrows. Figure 17A shows mean canine antibody titers to full length canine IL-31 protein (SEQ ID NO: 155). On FIG. 17B shows mean canine antibody titers to full-length human IL-31 (SEQ ID NO: 181) on days 0, 42 and 84 in group T03 only. Dogs in the T03 group (human 15H05 mimotope) lacked CRAR for canine IL-31 (data not shown).

On FIG. 18 - Fig. 18A depicts a design of an immunogenicity study conducted to evaluate the ability of CRM-197-conjugated full-length feline IL-31 protein or mimotopes to elicit an immune response in laboratory cats. All exposure groups received a mixture of adjuvants including the Bay R1005 glycolipid adjuvant (N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-ocgadecyl dodecanoylamide hydroacetate) as well as CpG oligonucleotides.

Each mimotope described herein is designed to elicit an epitope-specific immune response directed to the appropriate region of the IL-31 protein where the 15H05 antibody and other anti-IL-31 antibodies described herein bind. The sequences and chemical linkers of the peptides of various mimotopes are shown as groups T02-T05. The ZTS-563 peptide contains the amino acid sequence N-AKVSMPADNFERKNFILT-C, which corresponds to positions 121-138 of SEQ ID NO: 157 with a threonine (T) replaced by alanine (A) at position number 138. The ZTS-418 peptide contains the amino acid sequence N- TEVSMPTDNFERKRFILT-C, which corresponds to positions 115-132 of SEQ ID NO: 165. The ZTS-423 peptide contains the amino acid sequence N-NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF-C, which corresponds to positions 83-115 of SEQ ID NO: 157. The ZTS-422 peptide contains the amino acid sequence N-AKVSMPADNFERKNFILT-C, which corresponds to positions 121-138 of SEQ ID NO: 157 with threonine (T) replaced by alanine (A) at position number 138. Each of the peptides ZTS-563, ZTS-418, ZTS-423 and ZTS -422 also includes N and C terminal cysteines as shown to facilitate conjugation using free thiol groups. ZTS-422 also contains an additional aminohexano linker voic acid (Ahx) between two N-terminal cysteines. ZTS-423 also contains an additional three amino acid spacer sequence (GSG) next to the N-terminal cysteine. On FIG. 18B shows mean feline antibody titers to full-length feline IL-31 (SEQ ID NO: 157) in all treatment groups except T03. Cats in group T03 (equine mimotope 15H05) lacked CRAR for feline IL-31 (data not shown).

On FIG. 19A shows the amino acid sequence of the minimum epitope associated with the M14 anti-canine IL-31 antibody according to WO 2018/156367 (Kindred Biosciences, Inc.). A comparison of sequences from different biological species is given, their identification numbers and relative positions of amino acids are indicated. Figure 19B shows this minimum amino acid sequence of canine IL-31 highlighted in black box. In this Fig. the alignment of the sequences in the region surrounding the protein is also shown, and the relative positions of the corresponding amino acids in the reference sequence are indicated.

On FIG. 20 shows a fragment of the IL-31 protein with a loop formed by the approximation of helix A with the random helix sequence behind it, which has a similar position and structure to the 15H05 loop. Comparison of amino acid sequences of different species is shown and sequence identification numbers and amino acid positions are indicated.

On FIG. 21A shows the amino acid sequences of three peptide mimotopes of equine IL-31 representing different key epitope regions on the protein. The 15H05 mimotope contains the amino acid sequence N-TEVSMPTDNFERKRFILT-C, which corresponds to positions 115-132 of SEQ ID NO: 165. The BC helix mimotope contains the amino acid sequence N-NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF-C, which corresponds to positions 77-109 of SEQ ID NO: 165. Mimotope helix A contains the amino acid sequence N-GPIYQLQPKEIQAIIVELQNLSKK-C, which corresponds to positions 20-43 of SEQ ID NO: 165. Mimotope 15H05 also includes N and C terminal cysteines as shown to facilitate conjugation using free thiol groups. All three mimotopes also contain an additional three amino acid spacer sequence (GSG) next to the N-terminal biotin group, shown in bold and underlined sequences. The respective positions of each amino acid residue in SEQ ID NO: 165 are shown. FIG. 21B shows the results of a binding assay using biolayer interferometry. These mimotopes were immobilized on streptavidin biosensors and used to study various dilutions of mouse serum. Serum from mice vaccinated with equine IL-31 protein (SEQ ID NO: 165) or control sera from mice vaccinated with foreign protein was used.

Definitions

Before a detailed description of the present invention will be given definitions of several terms used in the context of this invention. In addition to these terms, other terms are defined in the specification as necessary. Unless otherwise indicated, the terms of the prior art used in this description will have the meaning generally accepted in this field.

As used in the specification and claims, the singular forms include references to the plural unless the context clearly provides otherwise. For example, reference to "antibody" includes many such antibodies. As another example, "mimotope", "IL-31 mimotope" and the like, including a plurality of such mimotopes, can be cited.

As used herein, the term "comprising" is intended to mean that the compositions and methods include the listed elements, but do not exclude other elements.

As used herein, the term "vaccine composition" includes at least one antigen or immunogen in a pharmaceutically acceptable carrier suitable for inducing an immune response in a host. Vaccine compositions may be administered at doses and by methods well known to those skilled in the art of medicine or veterinary medicine, taking into account factors such as the age, sex, weight, species and condition of the mammalian recipient, as well as the route of administration. The route of administration may be transdermal, transmucosal (eg, oral, nasal, anal, vaginal) or parenteral (intradermal, transdermal, intramuscular, subcutaneous, intravenous, or intraperitoneal). Vaccine compositions may be administered alone or may be administered concomitantly or sequentially with other treatments or therapies. Dosage forms may include suspensions, syrups, or elixirs, as well as preparations for parenteral, subcutaneous, intradermal, intramuscular, or intravenous (eg, injection) administration, such as sterile suspensions or emulsions. Vaccine compositions can be administered as a spray, or mixed with food and/or water, or delivered in admixture with a suitable carrier, diluent, or excipient such as sterile water, saline, glucose, or the like. The compositions may contain adjuvants such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity increasing agents, preservatives, flavoring agents, coloring agents, and the like, depending on the route of administration and the desired formulation. For the preparation of suitable formulations without undue experimentation, reference may be made to standard pharmaceutical texts such as "Remington's Pharmaceutical Sciences" (1990).

The term "immune response" as used herein refers to a response elicited in an animal or human. The immune response may refer to cellular immunity (CMI), humoral immunity, or may include both. The present invention also contemplates a response limited to part of the immune system. Typically, an "immune response" includes one or more of the following: production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or gamma-delta T cells, specifically directed to the antigen or antigens included in the composition or vaccine of interest, without limitation. Preferably, the host exhibits either a therapeutic or protective immune response such that resistance to the disease or disorder is enhanced and/or the clinical severity of the disease is reduced. Such protection will be demonstrated by either a reduction or absence of the symptoms normally shown by the affected host, a faster recovery time, and/or a reduced antigen (eg, IL-31) titer in the affected host.

The term "protective" as used herein means providing a therapeutic immune response to a mammalian host such that resistance to a disease or disorder is increased in the mammalian host and/or the clinical severity of the disease is reduced.

As used herein, the term "immunogenicity" means the ability to elicit an immune response against an antigen or antigens in a mammalian host. This immune response forms the basis of the protective immunity elicited by a vaccine against a specific antigen.

In this context, the terms "immunize", "immunization" and the like means the process by which a mammal acquires immunity or becomes resistant to a disease, usually by administration of a vaccine. Vaccines stimulate a mammal's own immune system to protect it from subsequent disease.

Used in this document, the term "adjuvant" means a composition consisting of one or more substances that enhance the immune response to the antigen(s). The mechanism of action of the adjuvant is not fully known. Some adjuvants are believed to enhance the immune response by slow release of the antigen, while other adjuvants are themselves highly immunogenic and are believed to act synergistically.

An epitope, as used herein, 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 an antibody can recognize and bind to. Unless otherwise indicated, the term "epitope" as used herein refers to the region of IL-31 with which the anti-IL-31 agent reacts.

An "antigen" is a molecule or region of a molecule to which an antibody can bind, which in addition is able to recognize and to which the antibody can bind (the corresponding binding region of an antibody may be called a paratope). In general, epitopes are composed of reactive surface groupings of molecules, such as amino acid or sugar side chains, and have certain spatial structural characteristics as well as certain charge characteristics. Epitopes are antigenic determinants of a protein that are recognized by the immune system. The epitope-recognizing components of the immune system are antibodies, T cells, and B cells. T cell epitopes are displayed on the surface of antigen presenting cells (APC) and are typically 8-11 (MHC class I) or more than 15 (MHC class II) amino acids in length. Recognition of the exposed MHC-peptide complex by T cells is critical for their activation. These mechanisms make it possible to adequately recognize self and "foreign" proteins, such as bacterial and viral ones. Independent amino acid residues, which are not necessarily contiguous, facilitate 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 that are recognized by soluble antibodies and cell surface-associated B-cell receptors vary greatly in length and degree of continuity (Sivalingam and Shepherd, Immunol. 2012 Jul;51(3-4):304-309 9). Again, even linear epitopes or epitopes found in a contiguous stretch of a protein sequence will often have non-contiguous amino acids that represent key points of contact with antibody paratopes or the B cell receptor. Epitopes recognized by antibodies and B cells may be conformational and contain amino acids that form a common area of contact on the protein in three-dimensional space, and depend on the tertiary and quaternary structural features of the protein. These residues are often located in spatially distinct regions of the primary amino acid sequence.

As used herein, the term "mimotope" means a linear peptide or a peptide with limited conformational freedom that mimics an epitope of an antigen. The mimotope may have a primary amino acid sequence capable of eliciting an effector T cell response and/or a three-dimensional structure required for B cell binding resulting in the maturation of an acquired immune response in the animal. An antibody to a given antigen epitope recognizes a mimotope that mimics that epitope. Alternatively, an IL-31 mimotope may be referred to herein as an IL-31 peptide mimotope. In some embodiments, a mimotope (linear or conformationalally constrained) for use in the compositions and/or methods of the present invention is and/or includes as part of a peptide that is from about 5 amino acid residues to about 40 amino acid residues in length.

The term "specifically" in relation to binding to an antibody refers to the highly avid and/or high affinity binding of an antibody to a particular antigen, i.e. polypeptide or epitope. In many embodiments, the specific antigen is the antigen (or antigen fragment or subfraction) used to immunize the host animal from which the antibody-producing cells have been 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 have the ability to bind other polypeptides at a low, yet detectable level (eg, 10% or less of the binding shown for the polypeptide of interest). Such weak binding or background binding is easily distinguished from specific binding of the antibody to the polypeptide of interest, for example by using appropriate controls. In general, specific antibodies bind to an antigen with a KD binding affinity of 10 ^-7 M or less, e.g., 10 ^-8 M or less (e.g., 10 ^-9 M or less, 10 ^-10 or less, 10 ^-11 or less , 10 ^-12 or less, or 10 ^-13 , etc.).

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, a fully equine antibody, or a fully human antibody. . The term "antibody" preferably refers to monoclonal antibodies and fragments thereof (for example, including, but not limited to, antigen-binding portions of an antibody) and immunological binding equivalents thereof, which can bind to the IL-31 protein and fragments or modified fragments thereof. Such fragments and modified fragments of IL-31 may include mimotopes of the IL-31 peptide used in various embodiments of this invention. For example, an antibody to a given epitope on IL-31 will recognize a mimotope of an IL-31 peptide that mimics that epitope. The term "antibody" is used to refer to both a homogeneous molecular substance and a mixture, such as a whey product, consisting of many different molecular substances.

"Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins with a molecular weight of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a single covalent disulfide bond, while the number of disulfide bonds in the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has well-placed intrachain disulfide bridges. 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. It is believed that certain amino acid residues form a contact surface between the variable domains of the light and heavy chains.

The term "antibody fragment" refers to a structure smaller than an intact antibody, including, without limitation, an isolated single chain antibody, Fv construct, Fab construct, Fc construct, light chain variable region or complementarity determining region (CDR) sequence, etc. . For example, an antibody fragment may include an antigen-binding portion of an antibody.

The term "variable" region includes the framework and CDRs (also known as hypervariable regions) and refers to the fact that certain portions of the variable domains differ significantly in sequence among antibodies and are used in binding each particular antibody to its particular antigen and determine the specificity of that antibody. However, variability is not evenly distributed across all antibody variable domains. It is concentrated in three segments of the variable domains of both the light chain and the heavy chain called hypervariable regions. The more highly conserved regions of variable domains are called the framework region (FR). Each of the native heavy and light chain variable domains contains numerous FRs, in most cases adopting a β-sheet configuration, connected by three hypervariable regions that form loops connecting and in some cases forming part of the α-sheet structure. The hypervariable regions in each chain are held together in close proximity to each other by FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antibody antigen-binding site (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 antibody-antigen binding, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. A hypervariable region contains amino acid residues from a "complementarity determining region" or "CDR" (Kabat, et al. (1991), supra) and/or those residues from a "hypervariable loop" (Chothia and Lesk J. Mol. Biol. 196 :901-917 (1987) "Framework" or "FR" residues are those variable domain residues that differ from the hypervariable region residues defined above.

Cleavage of antibodies with papain produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to easily crystallize. Pepsin treatment produces an F(ab')2 fragment that has two antigen binding sites and is still capable of crosslinking between antigens.

"Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in close non-covalent association. 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. Collectively, six hypervariable regions confer antigen-binding specificity to an antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific hypervariable regions) has the ability to recognize and bind an antigen, albeit at a lower 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 in that they have several residues added at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH as used herein is the designation for Fab' in which the cysteine residue(s) of the constant domains bear(ut) a free thiol group. F(ab') ₂ antibody fragments are initially prepared as pairs of Fab' fragments that contain hinged cysteines between them. In addition, other methods of chemical coupling of antibody fragments are known.

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

Depending on the amino acid sequence of the constant domain 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), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 (mouse designations and person). The heavy chain constant domains that correspond 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 activities associated with these constant domains are species-specific and must be determined experimentally.

"Monoclonal antibody", as defined herein, is an antibody produced by cells of a single clone (specifically, hybridoma cells of a single clone), and therefore refers to one 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 cells of one 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 therefore a single pure homogeneous type of antibody. Antibodies from single B cells of immunized mammals, such as dogs, are generated as recombinant IgG proteins after their variable domain sequences have been identified. The grafting of these variable domains onto constant domains of canine origin (heavy chain and light chain kappa or lambda constant domains) 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 cells of one 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 therefore a single pure homogeneous type of antibody. Antibodies from single B cells of immunized mammals, such as dogs, are generated as recombinant IgG proteins after their variable domain sequences have been identified. The transplantation of these variable domains into constant domains of feline origin (heavy chain and light chain kappa or lambda constant domains) results in the formation of 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 cells of one clone, one cell and the progeny of that cell.

"Full equine antibody", as defined herein, is a monoclonal antibody produced by a clone of cells (usually a CHO cell line) and, therefore, a single pure homogeneous type of antibody. Antibodies from single B cells of immunized mammals, such as dogs, are generated as recombinant IgG proteins after their variable domain sequences have been identified. The grafting of these variable domains onto horse constant domains (heavy chain and light chain kappa or lambda constant domains) 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 cells of one clone, one cell and the progeny of that cell.

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

Monoclonal antibodies as used herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species, while the remainder of the chain(s) ) is identical or homologous to the corresponding sequences in antibodies derived from another species, as well as fragments of such antibodies, if they exhibit the desired biological activity. Typically, chimeric antibodies are antibodies whose light and heavy chain genes are constructed, usually by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segment genes of a mouse monoclonal antibody can be fused to the constant segment genes of a dog antibody. In one embodiment of the chimeric mouse:canine IgG, the antigen binding site is from a mouse while the Fc portion is canine.

"Caninized" forms of non-canine (eg, murine) antibodies are genetically engineered antibodies that contain a minimal sequence derived from a non-canine immunoglobulin. Caninized antibodies are canine immunoglobulin (recipient antibody) sequences 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 binding ability. In some cases, framework region (FR) residues of canine immunoglobulin sequences are replaced with corresponding non-canine residues. In addition, caninized antibodies may include residues that are not present in the recipient antibody or in the donor antibody. Such modifications are made to further improve the performance of the antibodies. In general, a caninized 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 those of a non-canine immunoglobulin sequence, and all or substantially all FRs refer to a canine immunoglobulin sequence. The caninized antibody will optionally also contain the entire immunoglobulin constant region (Fc) or at least a portion thereof, typically that of a canine immunoglobulin sequence. In one embodiment of modifying antibodies to increase their similarity to natural antibodies, or caninization of mouse IgG, mouse CDRs are grafted onto canine-derived frameworks.

"Felinized" forms of non-feline (eg, murine) antibodies are genetically engineered antibodies that contain 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 with hypervariable region residues of a non-feline species (donor antibody), such as a mouse, having the desired specificity, affinity, and binding ability. In some cases, framework region (FR) residues of feline immunoglobulin sequences are replaced with corresponding non-feline residues. In addition, felinized antibodies may include residues that are not present in the recipient antibody or in the donor antibody. Such modifications are made to further improve the performance of the antibodies. In general, a felinized 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 those of a non-feline immunoglobulin sequence, and all or substantially all FRs refer to the feline immunoglobulin sequence. The felinized antibody will optionally also comprise the entire immunoglobulin constant region (Fc) or at least a portion thereof, typically that of a feline immunoglobulin sequence.

"Equinized" forms of non-equine (eg, murine) antibodies are genetically engineered antibodies that contain a minimal sequence derived from a non-equine immunoglobulin. Equinized antibodies are equine immunoglobulin sequences (recipient antibody) 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 binding ability. In some cases, framework region (FR) residues of equine immunoglobulin sequences are replaced with corresponding non-equine residues. In addition, equinized antibodies may include residues that are not present in the recipient antibody or in the donor antibody. Such modifications are made to further improve the performance of the antibodies. In general, an equinized antibody will include substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of the non-equine immunoglobulin sequence, and all or substantially all of the FRs. refer to the equine immunoglobulin sequence. The equinized antibody will optionally also comprise the entire immunoglobulin constant region (Fc) or at least a portion thereof, typically that of an equine immunoglobulin sequence.

"Humanized" forms of non-human (eg, murine) antibodies are genetically engineered antibodies that contain a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies are human immunoglobulin sequences (recipient antibody) in which the hypervariable region residues of the recipient are replaced with hypervariable region residues from a non-human species (donor antibody) such as a mouse having the desired specificity, affinity and binding ability. In some cases, framework region (FR) residues of human immunoglobulin sequences are replaced with corresponding non-human residues. In addition, humanized antibodies may include residues that are not present in the recipient antibody or in the donor antibody. Such modifications are made to further improve the performance of the antibodies. In general, a humanized antibody will include substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin sequence, and all or substantially all FRs refer to the human immunoglobulin sequence. The humanized antibody will optionally also comprise the entire immunoglobulin constant region (Fc) or at least a portion thereof, typically that of a human immunoglobulin sequence.

"All-canine" antibodies are genetically engineered antibodies that do not contain a sequence derived from a non-canine immunoglobulin. 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 binding capacity. In some cases, framework region (FR) residues of canine immunoglobulin sequences are replaced with corresponding non-canine residues. In addition, fully canine antibodies may include residues that are not present in the recipient antibody or in the donor antibody, for example, including, but not limited to, changes in the CDR to modify affinity. Such modifications are made to further improve the performance of the antibodies. In general, a fully canine antibody will include substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable regions correspond to those of the canine immunoglobulin sequence and all or substantially all of the FRs are from the sequence canine immunoglobulin. A fully canine antibody will also contain the entire immunoglobulin constant region (Fc) or at least a portion thereof, typically that of a canine immunoglobulin sequence.

"Full feline" antibodies are genetically engineered antibodies that do not contain a sequence derived from a non-feline immunoglobulin. 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 binding capacity. In some cases, framework region (FR) residues of feline immunoglobulin sequences are replaced with corresponding non-feline residues. In addition, fully feline antibodies may include residues that are not absent in the recipient antibody or in the donor antibody, for example, including but not limited to changes in the CDR to modify affinity. Such modifications are made to further improve the performance of the antibodies. In general, a fully feline antibody will include substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable regions correspond to those of the feline immunoglobulin sequence, and all or substantially all of the FRs are from the immunoglobulin sequence. cats. A fully feline antibody will also optionally comprise the entire immunoglobulin constant region (Fc) or at least a portion thereof, typically that of a feline immunoglobulin sequence.

"Full equine" antibodies are genetically engineered antibodies that do not contain a sequence derived from a non-equine immunoglobulin. 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 binding capacity. In some cases, framework region (FR) residues of equine immunoglobulin sequences are replaced with corresponding non-equine residues. In addition, fully equine antibodies may include residues that are not present in the recipient antibody or in the donor antibody, for example, including but not limited to changes in the CDR to modify affinity. Such modifications are made to further improve the performance of the antibodies. In general, a fully equine antibody will include substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable regions correspond to those of the equine immunoglobulin sequence, and all or substantially all of the FRs are from the sequence horse immunoglobulin. A fully equine antibody will also optionally comprise the entire immunoglobulin constant region (Fc) or at least a portion thereof, typically that of an equine immunoglobulin sequence.

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

The term "heterochimeric", as defined herein, refers to an antibody in which one of the antibody chains (heavy or light) is, for example, caninized, felinized, equinized, or humanized, 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, both the heavy chain variable regions and the light chain variable regions are fused to a constant region of feline origin.

As used herein, the term "variant" refers to a peptide, polypeptide, or nucleic acid sequence encoding a peptide or polypeptide that has one or more conservative amino acid variations or other minor modifications such that the corresponding peptide or polypeptide has substantially equivalent function when compared with a wild-type peptide or polypeptide. Typically, peptide mimotope variants for use in the present invention will have at least 30% identity with the parent mimotope, more preferably at least 50%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85% more preferably at least 90% and most preferably at least 95% sequence identity with the parent mimotope.

A "variant" of an anti-IL-31 antibody, as used herein, refers to a molecule that differs in amino acid sequence from the amino acid sequence of the "parent" anti-IL-31 antibody due to the addition, deletion, and/or substitution of one or more amino acid residues in the sequence of the parent antibody. and retains 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 signaling in cell assays. 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 having an amino acid sequence identity with the heavy or light chain variable domain sequences of the parent antibody of at least 50%, more preferably at least 65%, more preferably at least 75%, more preferably at least at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity. Identity or homology to that sequence is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to those of the parent antibody, after sequence alignment and gaps, if necessary, to achieve the maximum percent sequence identity. None of the N-terminal, C-terminal or internal extensions, deletions or insertions in the sequence of an antibody should be considered as affecting sequence identity or homology. The variant retains the ability to bind IL-31 and preferably has the desired activity that is superior to that of the parent antibody. For example, a variant may have stronger binding affinity, increased ability to reduce, inhibit, or neutralize IL-31 activity in animals, and/or increased ability to inhibit IL-31 mediated pSTAT signaling in cell assays.

A "variant" nucleic acid, as used herein, refers to a molecule that differs in sequence from the "parent" nucleic acid. Variations in polynucleotide sequences may result from mutational changes such as deletions, substitutions, or additions of one or more nucleotides. Each of these changes in a given sequence can occur individually or in combination, one or more times.

A "parent" antibody, as used herein, is an antibody that is encoded by the amino acid sequence used to generate the variant. In one embodiment, the parent antibody has a canine framework region and canine antibody constant region(s), if present. 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. In another example, the parent antibody may be a humanized or human antibody. In yet another example, the parent antibody is a mouse monoclonal antibody.

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

The term "isolated" means that a substance (eg, antibody or nucleic acid) is separated from components of its natural environment and/or isolated. Contaminant components of its natural environment are substances that may 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 the nucleic acid, the isolated nucleic acid may include one that is separated from the 5'-3' sequences to which it is normally associated on the 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 isolated material is obtained using at least one purification step.

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

The terms "nucleic acid", "polynucleotide", "nucleic acid molecule" and the like can be used interchangeably herein and refer to a number 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. The term includes both 5'-3' (sense) and 3'-5' (antisense) polynucleotides.

"Subject" or "patient" refers to a mammal in need of treatment that may be affected by the molecules of the invention. Mammals that can be treated in accordance with the invention include vertebrates, among which particularly preferred examples are mammals such as dogs, cats, horses and humans.

A "therapeutically effective amount" (or "effective amount") refers to an amount of an active ingredient, such as a substance of the invention, sufficient to achieve beneficial or desired results when administered to a subject or patient. An effective amount may be administered in one or more administrations, applications, or dosages. A therapeutically effective amount of the composition according to the invention can be easily determined by a person skilled in the art. In the context of this invention, a "therapeutically effective amount" is an amount that causes an objectively measurable change in one or more parameters associated with the treatment of an IL-31 mediated disorder, such as an pruritic condition, an allergic condition, or tumor progression, including clinical improvement in symptoms. Of course, the therapeutically effective amount will vary depending on the particular subject and the condition being treated, the weight and age of the subject, the severity of the condition, the particular compound chosen, the administration schedule, the time of administration, the route of administration, and the like, all of which can be readily determined by those skilled in the art. in this area.

As used herein, the term "therapeutic" encompasses the full spectrum of treatment for a disease or disorder. The "therapeutic" agent of the present invention may act in a prophylactic or preventive manner, including those that include procedures designed to affect animals that may be at risk (pharmacogenetics); or in a manner that is ameliorative or curative in nature; or may slow the rate or extent of progression of at least one symptom of the disease or disorder being treated.

"Treatment", "treat", etc. refer to both therapeutic effects and prophylactic or preventive measures. Animals in need of treatment include those that already have the disease as well as those in which the disease is to be prevented. The terms "treating" a disease or disorder or "treating" a disease or disorder include preventing or protecting against the disease or disorder (ie, preventing the development of clinical symptoms); inhibiting the disease or disorder (ie stopping or suppressing the development of clinical symptoms) and/or alleviating the disease or disorder (ie reversing the clinical symptoms). Obviously, it is not always possible to distinguish between "preventing" and "suppressing" a disease or disorder, since the resulting event or events may be unknown or latent. Accordingly, "prevention" is to be understood as a type of "treatment" which includes both "prevention" and "suppression". Thus, the term "treatment" includes "prophylaxis".

The term "allergic condition" is defined herein as a disorder or disease caused by an interaction between the immune system and a substance foreign to the body. This foreign substance is called an "allergen". Common allergens include aeroallergens such as pollen, dust, mold, dust mite proteins, saliva from insect bites, etc. Examples of allergic conditions include the following: allergic dermatitis, summer eczema, urticaria, emphysema, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease, and inflammatory conditions resulting from autoimmune diseases such as irritable bowel syndrome ( SRK), but are not limited to them.

The term "pruritic condition" is defined herein as a disease or disorder characterized by severe itching that causes a desire to rub or scratch the skin for relief. Examples of pruritic conditions include, but are not limited to, atopic dermatitis, allergic dermatitis, eczema, psoriasis, scleroderma, and pruritus.

In the context of the present description, the terms "cell", "cell line" and "cell culture" can be used interchangeably. All these terms also include their offspring, i.e. any possible subsequent generations. It is clear that all offspring cannot be identical due to intentional or accidental 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) in vitro or in vivo. For example, the host cells may be in a transgenic animal. A host cell may be used as a recipient for vectors and may include any transformable organism that is capable of replicating the vector and/or expressing the heterologous nucleic acid encoded by the vector.

"Composition" is intended to refer to a combination of an active substance and another compound or composition, which may be inert (eg, a label) or active, such as an adjuvant.

As used herein, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable carrier medium" are used interchangeably and refer to a liquid carrier medium containing vaccine antigens that can be administered to a host without side effects. Pharmaceutically acceptable carriers suitable for use in the invention are well known to those skilled in the art. Such carriers include water, saline, buffered saline, phosphate buffer, alcohol/aqueous solutions, emulsions or suspensions, without limitation. It is fashionable to add other commonly used diluents, adjuvants and excipients, in accordance with standard practice. Such carriers may include ethanol, polyols and suitable mixtures thereof, vegetable oils and injectable organic esters. Buffers and pH adjusting agents can also be used. Buffers include salts derived from organic acids or bases, without limitation. Typical buffers include salts of organic acids such as salts of citric acid, for example citrates, ascorbic acid, gluconic acid, histidine-HC1, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid, Tris, trimethylamine hydrochloride or phosphate buffers, without restrictions. Carriers for parenteral administration may include sodium chloride solution, Ringer's solution with dextrose, dextrose, trehalose, sucrose and sodium chloride, Ringer's lactate, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers such as Ringer's dextrose solutions, and the like. Preservatives and other additives, such as, for example, antimicrobials, antioxidants, chelating agents (eg, EDTA), inert gases, and the like, may also be included in pharmaceutical carriers. The present invention is not limited to the choice of carrier. The preparation of these pharmaceutically acceptable compositions from the components described above, having appropriate pH, isotonicity, stability and other usual characteristics, is within the skill of the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott Williams & Wilkins, publ., 2000, and The Handbook of Pharmaceutical Excipients, 4th edition, revised, ed. 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 so chemically similar to this residue that does not significantly reduce the function of the polypeptide (eg, enzymatic activity). Conservative amino acid substitutions are well known in the art and examples are described in, for example, US Pat.

• 1. Small aliphatic, essentially 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, which are given as pairs of native residues (conservative substitutions): Ala (Ser); Arg(lys); Asn (Gin; 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), a polynucleotide may contain a conservative(s) codon substitution(s). A codon substitution is considered conservative if, when expressed, it results in a conservative amino acid substitution as described above, degenerate codon substitution that does not result in an amino acid substitution can also be used in the polynucleotides of the present invention. Thus, for example, a polynucleotide encoding a selected polypeptide that can be used in an embodiment of the present invention can be mutated by changing a degenerate codon to approximate the codon usage displayed by the expression host cell being transformed by it, or to improve expression in another way.

INFORMATION CONFIRMING THE POSSIBILITY OF IMPLEMENTING THE INVENTION

It should be understood that the invention is not limited to the specific methods, protocols and reagents and the like described in this document, and therefore may vary. It should also be understood that the terminology used herein is for the sole purpose of describing particular embodiments and is not intended to limit the scope of the invention, which is solely defined by the claims.

Unless otherwise indicated, all scientific and technical terms used herein in relation to the vaccine compositions and antibodies described herein have the usual meaning known to those skilled in the art. In addition, unless the context requires otherwise, terms in the singular include terms in the plural, and terms in the plural include terms in the singular. The commonly used terms and methods of cell and tissue culture, molecular biology as well as protein and oligo- or polynucleotide chemistry and hybridization described herein are well known and widely used in the art.

Standard methods are used for recombinant DNA, oligonucleotide synthesis, tissue culture and transfection (eg electroporation, lipofection). Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's specifications or in accordance with generally accepted practice in this field, or as described in this document. The above methods and procedures are usually performed in accordance with standard methods well known in the art and described in various general and more specific documents that are cited and discussed in the present description, 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. In this invention, the terms and laboratory procedures and methods related to analytical chemistry, organic synthesis chemistry, medicinal and pharmaceutical chemistry described in this document are well known and are widely used in this field of technology. Standard methods are used for chemical synthesis, chemical analysis, pharmaceutical formulation and delivery, and patient treatment.

Except for working examples or where not indicated otherwise, it is to be understood that all numbers expressing amounts of ingredients or reaction conditions used in this specification are in all cases adjusted using the term "about".

All patents and other publications found are expressly incorporated herein by reference for the purpose of setting forth and disclosing, for example, the methodologies described in such publications that may be used in connection with the present invention. These publications are given solely in view of their disclosure prior to the filing date of this application.

Compositions

The present invention provides mimotopes (peptides) of IL-31 and variants thereof, as well as their use in clinical and scientific techniques, including diagnostic techniques. As used herein, such an IL-31 mimotope is a linear or conformationally restricted peptide that mimics an antigen epitope. An anti-IL-31 antibody to a given epitope of the IL-31 antigen will recognize an IL-31 mimotope that mimics that epitope.

IL-31 mimotopes (peptides) are used in the vaccine compositions of the present invention. Such vaccine compositions may be used to protect a mammal from an IL-31 mediated disorder such as an pruritic or allergic condition. In some embodiments, the IL-31 mediated pruritic or allergic condition is an pruritic condition selected from atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. In other embodiments, the IL-31 mediated pruritic or allergic condition is an allergic condition selected from allergic dermatitis, summer eczema, urticaria, emphysema, inflammatory airway disease, recurrent airway obstruction, airway hypersensitivity, chronic obstructive pulmonary disease and inflammatory processes caused by autoimmunity. In other embodiments, the IL-31 mediated disorder is tumor progression. In some embodiments, the IL-31 mediated disorder is eosinophilic disease or mastocytomas.

In one embodiment, the vaccine composition of the present invention comprises a combination of a carrier polypeptide and at least one mimotope selected from feline IL-31 mimotope, canine IL-31 mimotope, equine IL-31 mimotope, and human IL-31 mimotope and an adjuvant. In some embodiments, the vaccine compositions of the present invention may include more than one IL-31 mimotope of a given species, or even a combination of IL-31 mimotopes from different species. In some embodiments, a mimotope (linear or conformationalally constrained) for use in the compositions and/or methods of the present invention is and/or includes as part of a peptide that is from about 5 amino acid residues to about 40 amino acid residues in length.

In one embodiment, at least one mimotope used in the compositions and methods of the present invention is selected from IL-31 15H05 mimotope, IL-31 helix region mimotope IL-31, IL-31 helix region mimotope A, IL-31 AB loop mimotope, or any their combinations.

In one embodiment, at least one mimotope for use in the compositions and methods of the present invention induces the production of antibodies that neutralize the bioactivity of IL-31.

In another embodiment, the vaccine compositions of the present invention are capable of eliciting a targeted immune response to generate antibodies in a mammal directed against at least one neutralizing epitope on IL-31, but not against non-neutralizing epitopes on IL-31.

In one embodiment, the vaccine composition comprises a canine IL-31 mimotope which is and/or contains as part of the amino acid sequence SVPADTFECKSF (SEQ ID NO: 186), SVPADTFERKSF (SEQ ID NO: 187), NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ ID NO: 192 ), APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO: 200), or variants thereof that retain binding to an anti-IL-31 agent.

In another embodiment, the vaccine composition comprises a feline IL-31 mimotope that is and/or contains as part of the amino acid sequence SMPADNFERKNF (SEQ ID NO: 188), NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ ID NO: 193), APAHRLQPSDIRKIILELRPM SKG (SEQ ID NO: 197), IGLPES (SEQ ID NO: 201) or variants thereof that retain binding to an anti-IL-31 agent.

In another embodiment, the vaccine composition comprises an equine IL-31 mimotope which is and/or contains as part of the amino acid sequence SMPTDNFERKRF (SEQ ID NO: 189), NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ ID NO: 194), GPIYQLQPKEIQAIIVELQNLS KK (SEQ ID NO: 189). : 198), KGVQKF (SEQ ID NO: 202) or variants thereof that retain binding to an anti-IL-31 agent.

In a further embodiment, the vaccine composition comprises a human IL-31 mimotope which is and/or contains as part of the amino acid sequence SVPTDTHECKRF (SEQ ID NO: 190), SVPTDTHERKRF (SEQ ID NO: 191), HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195 ), LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203), or variants thereof that retain binding to an anti-IL-31 agent.

In one embodiment, the mimotope used in the vaccine compositions of this invention is a mimotope with limited conformational freedom. In one embodiment, such mimotope with limited conformational freedom is a chemically linked cyclic peptide.

Linear IL-31 mimotopes can be chemically synthesized or produced by recombinant methods. Mimotopes of IL-31 with limited conformational freedom, such as a chemically linked cyclic peptide, can be synthesized chemically or can be obtained using a combination of chemical synthesis and recombinant technology.

In some embodiments, the IL-31 mimotope used in the vaccine composition is chemically conjugated to a carrier polypeptide. In other embodiments, the carrier polypeptide and mimotope are part of a recombinant fusion protein.

The carrier polypeptide that is combined with the IL-31 mimotope may be or may include as part of it a bacterial toxoid or derivative thereof, keyhole hemocyanin (KLH), or a virus-like particle. As non-limiting examples, the bacterial toxoid or derivative can be tetanus toxoid, diphtheria toxoid, tetanus toxoid, outer membrane protein complex from N. meningitidis group B, Pseudomonas exotoxin, or a non-toxic diphtheria toxin mutant (CRM197). As other non-limiting examples, the virus-like particle can be HBsAg, HBcAg, E. coli Qbeta bacteriophage, Norwalk virus, canine distemper virus (CDV), or influenza virus hemagglutinin (HA). In one preferred embodiment, the IL-31 mimotope is in combination with a carrier polypeptide that includes or consists of CRM197.

Vaccine compositions of the present invention include at least one adjuvant or adjuvant formulation, as will be described in more detail below.

The vaccines of the present invention may be formulated in accordance with accepted convention to include pharmaceutically acceptable carriers for animals, including humans (if applicable), such as standard buffers, stabilizers, diluents, preservatives and/or diluents, and may also be formulated to provide extended release. Diluents include water, saline, dextrose, ethanol, glycerin, and the like. Isotonicity additives include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include, inter alia, albumin. Other suitable carrier vehicles and vaccine additives, including those particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, the contents of which are incorporated herein by reference.

The vaccines of the present invention may further include one or more additional immunomodulatory components such as, for example, an adjuvant or a cytokine. Suitable adjuvants for use in the compositions of the present invention include the following: oil-in-water adjuvant, polymer and water adjuvant, water-in-oil adjuvant, aluminum hydroxide adjuvant, vitamin E adjuvant, and combinations thereof. Some specific examples of adjuvants include complete Freund's adjuvant, incomplete Freund's adjuvant, Corynebacterium parvum, Bacillus Calmette-Guerin, aluminum hydroxide gel, glucan, dextran sulfate, iron oxide, sodium alginate, bacto adjuvant, certain synthetic polymers such as polyamino acids and amino acid copolymers. , block copolymer (CytRx, Atlanta, GA), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville CA), AMPHIGEN® Adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A and adjuvant avridine lipid-amine (N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine), REGRESSIONS (Vetrepharm, Athens, GA), paraffin oil, RIBI adjuvant system (Ribi Inc. ., Hamilton, Mont.), muramyl dipeptide, and the like, without limitation.

Non-limiting examples of oil-in-water emulsions that can be used in the vaccine of the invention include modified formulations of SEAM62 and SEAM 1/2. Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) ./v) of TWEEN® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 µg/ml Quil A, 100 µg/ml cholesterol and 0.5% (v/v) ) lecithin. Modified SEAM 1/2 is an oil-in-water emulsion containing 5% (v/v) squalene, 1% (v/v) SPAN® 85 detergent, 0.7% (v/v) ) Tween 80 detergent, 2.5% (v/v) ethanol, 100 µg/ml Quil A and 50 µg/ml cholesterol.

Another example of an adjuvant that can be used in the composition of the invention is SP oil. Used in the description and the claims, the term "SP oil" means an oil emulsion containing a block copolymer of polyoxyethylene and polyoxypropylene, squalane, polyoxyethylene sorbitan monooleate and buffered saline. Block copolymers of polyoxyethylene and polyoxypropylene are surfactants that aid in the suspension of solid and liquid components. These surfactants are commercially available as polymers under the trade name Pluronic®. A preferred surfactant is poloxamer 401 which is commercially available under the trade name Pluronic® L-121. Typically, the SP oil emulsion is an immunostimulatory adjuvant mixture that will include from about 1 to 3% v/v. block copolymer, from about 2 to 6% vol./about. squalane, more specifically from about 3 to 6% squalane and about 0.1-0.5% v/v. polyoxyethylene sorbitan monooleate, and the remainder is buffered saline.

"Immunomodulators" that may be included in a vaccine include, for example, immunostimulatory oligonucleotides, one or more interleukins, interferons, or other known cytokines. In one embodiment, the adjuvant may be a cyclodextrin derivative or a polyanionic polymer such as those described in US Pat. Nos. 6,165,995 and 6,610,310, respectively.

In one embodiment, the adjuvant is a formulation containing a saponin, a sterol, a quaternary ammonium compound, and a polymer. In a specific embodiment, the saponin is Quil A or a purified fraction thereof, the sterol is cholesterol, the quaternary ammonium compound is dimethyldioctadecylammonium bromide (DDA), and the polymer is polyacrylic acid.

In another embodiment, the adjuvant comprises a combination of one or more isolated immunostimulatory oligonucleotides, a sterol and a saponin. In a specific embodiment, the one or more isolated immunostimulatory oligonucleotides comprise CpG, the sterol is cholesterol, and the saponin is Quil A or a purified fraction thereof. In this context, the ZA-01 adjuvant mentioned in the examples section includes Quil A (saponin), cholesterol, CpG and diluent.

In another embodiment, a useful adjuvant for use in the compositions of the present invention comprises CpG-containing immunostimulatory oligonucleotides. CpG-containing oligonucleotides are described, for example, in US patent 8580280. In one specific embodiment, the adjuvant for use in the present invention is a mixture comprising at least one glycolipid adjuvant and CpG-containing oligonucleotides. A specific example of a suitable adjuvant is a mixture that includes the Bay R1005 glycolipid adjuvant (N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyl dodecanoylamide hydroacetate) as well as CpG oligonucleotides.

In one embodiment, the adjuvant or mixture of adjuvants is added in an amount of from about 100 μg to about 10 mg per dose. In another embodiment, the adjuvant/adjuvant mixture is added in an amount of from about 200 μg to about 5 mg per dose. In yet another embodiment, the adjuvant/adjuvant mixture is added in an amount of from about 300 μg to about 1 mg/dose.

With the advent of molecular biology techniques and recombinant technology, it has become possible to obtain the aforementioned peptides and polypeptides by recombinant methods and thus generate gene sequences that encode specific amino acid sequences found in the structure of a peptide or polypeptide. In one embodiment, the peptide is an IL-31 mimotope, or at least part of an IL-31 mimotope. In another embodiment, the polypeptide is a carrier polypeptide that is present in combination with an IL-31 mimotope. In yet another embodiment, the polypeptide is an antibody, such as an antibody that binds to an IL-31 mimotope used in a vaccine composition or diagnostic methods of the present invention. Such antibodies can be obtained either by cloning the sequences of genes encoding the polypeptide chains of these antibodies, or by direct synthesis of these polypeptide chains with the assembly of the synthesized chains with the formation of active tetrameric (H ₂ L ₂ ) structures with affinity for specific epitopes and antigenic determinants. This made it possible to obtain ready-made antibodies with sequences characteristic of neutralizing antibodies from different species and sources.

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

The "antigen-binding region" or "antigen-binding portion" of an antibody refers to that portion of an antibody molecule that contains amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. The binding region of an antibody includes "framework" amino acid residues necessary to maintain the correct conformation of the antigen-binding residues. The antigen-binding portion of an antibody referred to in the specification and claims may be referred to herein, for example, as an IL-31-specific peptide or polypeptide, or as an anti-IL-31 peptide or polypeptide.

In 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 with different specificities. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDR" regions. These CDR regions determine the basic specificity of the antibody for a particular antigenic determinant structure.

The CDRs are non-contiguous stretches of amino acids in the variable regions, but the positions of these critical amino acid sequences have been found to be similar in the amino acid sequences of the heavy and light chain variable regions, regardless of species. Each heavy and light chain variable region of all antibodies has three CDR regions, each of which is not adjacent to the others.

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

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

An "antigen" is a molecule or portion of a molecule capable of binding to 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. The aforementioned specific reaction indicates that the antigen will react in a highly selective manner with its respective antibody, and not with the multitude of other antibodies that may be elicited by other antigens.

Antibodies referred to herein are intended to include both intact immunoglobulin molecules and portions, fragments, peptides and derivatives thereof, 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 an antigen or epitope. An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule, thereby binding the molecule to the antibody.

Antibodies referred to herein also include chimeric antibodies, heterochimeric antibodies, caninized antibodies, felinized antibodies, equinized antibodies, humanized antibodies, fully canine antibodies, fully feline antibodies, fully equine antibodies, fully human antibodies, as well as fragments, parts, regions, peptides or derivatives obtained by any known method such as enzymatic cleavage, peptide synthesis or recombinant methods, without limitation. Such antibodies mentioned herein are capable of specifically binding to at least one of canine IL-31, feline IL-31, equine IL-31, or human IL-31. Fragments or portions of antibodies may not have the Fc fragment of an intact antibody, are more rapidly cleared from the circulation, and may be less non-specifically bound to tissue compared to an intact antibody. Exemplary antibody fragments can be prepared from intact antibodies using methods well known in the art, such as proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(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 an appropriate expression vector. For example, the CDR region(s) of a recombinant antibody can be isolated and subcloned into an appropriate expression vector. See, for example, US patent No. 6680053.

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

In some embodiments of the present invention, IL-31 mimotopes are provided that bind to novel monoclonal antibodies that bind to at least one of canine IL-31, feline IL-31, or equine IL-31. Such monoclonal antibodies can be used in the diagnostic methods of the present invention together with IL-31 mimotope. In one embodiment, the monoclonal antibody mentioned in the specification and claims binds to canine IL-31, feline IL-31, or equine IL-31 and prevents it from binding to and activating its co-receptor complex containing the IL-31 receptor A (IL-31Ra ) and an oncostatin M-specific receptor (OsmR or IL-31Rb). Examples of such monoclonal antibodies are referred to herein as "15H05", "ZIL1", "ZIL8", "ZIL9", "ZIL11", "ZIL69", "ZIL94", "ZIL154", "ZIL159" and "ZIL171", in according to the numbers assigned to their clones. In this document, "15H05", "ZIL1", "ZIL8", "ZIL9", "ZIL11", "ZIL69", "ZIL94", "ZIL154", "ZIL159", and "ZIL171" also refer to the monoclonal antibody portion, the paratope or CDRs that specifically bind to an IL-31 epitope designated as 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 and ZIL171 due to its ability to bind antibodies 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 and ZIL171, respectively. Several 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, and ZIL171 described herein may be referred to by the same name.

In one embodiment, the vaccine composition of the invention comprises a mimotope that binds to an anti-IL-31 antibody, or an antigen-binding portion thereof, that specifically binds to a region of the mammalian IL-31 protein involved in the interaction of the IL-31 protein with its co-receptor. In one embodiment, binding of said antibody to said region is affected by mutations in the 15H05 binding epitope region selected from: a) the region between amino acid residues 124 and 135 of the feline IL-31 sequence represented by SEQ ID NO: 157 (feline_IL-31_wild type) ; b) the region between amino acid residues 124 and 135 of the canine IL-31 sequence represented by SEQ ID NO: 155 (canine_IL-31) and c) the region between amino acid residues 118 and 129 of the equine IL-31 sequence represented by SEQ ID NO: 165 (equine_IL -31). In one embodiment, a mimotope for use in the compositions of this invention binds to an anti-IL-31 antibody, or antigen-binding portion thereof, that specifically binds to the aforementioned epitope region recognized by 15H05.

In one specific embodiment of the vaccine composition of the invention, the mimotope binds to an anti-IL-31 antibody, or antigen-binding portion thereof, comprising at least one of the following sequence combinations of complementarity determining regions (CDRs):

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

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

3) ZIL8 antibody: VH-CDR1 with DYAMS sequence (SEQ ID NO: 19), VH-CDR2 with GIDSVGS GTS YADA VKG sequence (SEQ ID NO: 20), VH-CDR3 with GFPGSFEH sequence (SEQ ID NO: 21), VL-CDR1 with the sequence TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 with the sequence YNSDRPS (SEQ ID NO: 23), VL-CDR3 with the sequence SVYDRTFNAV (SEQ ID NO: 24);

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

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

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

7) ZIL94 antibody: VH-CDR1 with TYFMS sequence (SEQ ID NO: 43), VH-CDR2 with LIS SDGSGTY YADA VKG sequence (SEQ ID NO: 44), VH-CDR3 with FWRAFND sequence (SEQ ID NO: 45), VL-CDR1 with the sequence GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 with the sequence DTGSRPS (SEQ ID NO: 47), VL-CDR3 with the sequence SLYTDSDILV (SEQ ID NO: 48);

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

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

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

11) variant (1)-(10) that differs from the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 or ZIL171 by the addition, deletion and/or substitution of one or more amino acid residues at at least one of CDR1, CDR2 or CDR3 VH or VL.

In one embodiment, the IL-31 mimotope used in the vaccine composition binds to an antibody that specifically binds to feline IL-31, wherein the antibody binds to the region between amino acid residues 125 and 134 of the feline IL-31 sequence represented by SEQ ID NO: 157 ( feline_IL-31_wild type). In some embodiments, such an antibody comprises a VL chain containing frame region 2 (FW2) changes selected from the following: asparagine for lysine at position 42, isoleucine for valine at position 43, valine for leucine at position 46, asparagine for lysine at position 49, and combinations thereof, where the provisions refer to the numbering of SEQ ID NO: 127 (FEL_15H05_VL1).

In some embodiments, mimotope binds to an antibody characterized in that:

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

a) light chain variable region containing CAN-ZIL8_VL:

b) heavy chain variable region containing CAN-ZIL8_VH:

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

a) light chain variable region containing CAN-ZIL169_VL:

b) heavy chain variable region containing CAN-ZIL69_VH:

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

a) light chain variable region containing CAN-ZIL171_VL:

b) heavy chain variable region containing CAN-ZIL171_VH:

The host cell can be used to produce the antibody described above. Such antibodies can be used in the diagnostic procedures described as part of this invention, although the diagnostic procedures are not limited to these particular antibodies.

Nucleotide sequences encoding the variable regions of the light and heavy chains of an anti-IL-31 antibody can be used to generate the anti-IL-31 antibodies described herein. Such nucleotide sequences include, but are not limited to, any nucleotide sequence that codes for the amino acid sequence of antibodies 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, or ZIL171 or IL-31-specific polypeptides or peptides thereof. In addition, in one embodiment, nucleotide sequences encoding IL-31 mimotopes can be used to recombinantly produce mimotopes alone or as part of a fusion protein along with a carrier polypeptide. Alternatively, or in addition, mimotopes can be chemically synthesized.

In some embodiments, an isolated nucleic acid can be used to generate a useful antibody (such as an antibody used in one of the diagnostic methods described herein), wherein the nucleic acid sequence encodes at least one of the following combinations of complementarity determining region (CDR) sequences: heavy chain variable region:

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

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

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

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

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

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

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

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

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

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

11) variant (1)-(10) differing from the CDR of the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 or ZIL171 by the addition, deletion and/or substitution of one or more amino acid residues in at least one of CDR1, CDR2 or CDR3 VH.

In another embodiment, the isolated nucleic acid comprises a nucleic acid sequence encoding at least one of the following combinations of light chain variable region complementarity determining region (CDR) sequences:

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

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

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

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

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

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

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

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

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

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

11) variant (1)-(10) differing from the CDR of the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 or ZIL171 by the addition, deletion and/or substitution of one or more amino acid residues in at least one of CDR1, CDR2 or CDR3 VL.

In yet another embodiment, the isolated nucleic acid used in the manufacture of the antibody described herein comprises a nucleic acid sequence encoding the above-described complementarity-determining regions (CDR) light chain variable region sequences as well as the above-described heavy chain variable region CDR sequences of the corresponding parental 15H05 antibody. , ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, or ZIL171, or variants thereof.

A vector comprising at least one of the nucleic acids described above may be used in the production of these antibodies. As will be described in more detail below, a nucleic acid sequence encoding at least one of the combinations of complementarity determining regions (CDR) sequences of the heavy chain variable region described above may be contained in the same vector along with a nucleic acid sequence encoding at least one of combinations of light chain variable region CDR sequences described above. Alternatively, a nucleic acid sequence encoding at least one of the above-described combinations of heavy chain variable region 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 contained 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 identified, each of which is capable of encoding an amino acid. The likelihood that a particular oligonucleotide will in fact be the actual sequence encoding XXX can be estimated by considering the abnormal base-pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic or prokaryotic cells expressing an antibody against IL-31 or a portion specific for IL-31. Such "codon usage rules" are set forth by Lathe, et al., 183 J. Molec. Biol. 1-12 (1985). Using Lathe's "codon usage rules", a single nucleotide sequence or set of nucleotide sequences can be identified that contains a theoretical "most likely" nucleotide sequence capable of encoding anti-IL-31 sequences. It is also contemplated that antibody coding regions can also be obtained by altering existing antibody genes using standard molecular biology techniques that result in the antibody variants (agonists) and peptides described herein. Such variants include deletions, additions and substitutions in the amino acid sequence of antibodies against IL-31 or IL-31-specific polypeptides or peptides (such as parts or fragments of antibodies), without limitation. In addition, variants of the peptide mimotopes described herein can be obtained by changing the nucleotide sequence encoding the parental peptide mimotope.

For example, one class of substitutions are conservative amino acid substitutions. Such substitutions are substitutions in which a given amino acid in an anti-IL-31 antibody, IL-31-specific polypeptide or peptide is replaced with another amino acid with similar characteristics. Likewise, IL-31 mimotopes that bind to such an antibody, or antigen-binding portion thereof, may include conservative substitutions or other types of amino acid substitutions. Typically, conservative substitutions are substitutions of the aliphatic amino acids Ala, Val, Leu, and Ile; mutual substitutions of Ser and Thr hydroxyl residues, substitution of negatively charged Asp and Glu residues, substitution between Asn and Gin amide residues, substitution of positively charged Lys and Arg residues, substitutions between aromatic Phe, Tyr residues, and the like. Guidance as to which amino acid changes are likely to be phenotypically inconspicuous can be found in Bowie et al., 247 Science 1306-10 (1990).

Anti-IL-31 variant or agonist antibodies or IL-31-specific polypeptides or peptides may be fully functional or may lack one or more activities. Similarly, IL-31 variant or agonist mimotopes may be fully functional or may lack one or more activities. Fully functional variants usually contain only conservative variations or variations in non-key residues or regions. Functional variants may also contain similar amino acid substitutions that result in little or no change in function. Alternatively, such substitutions may positively or negatively affect function 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 at a key residue or key region.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The last procedure introduces single mutations of each residue in the molecule with a replacement for alanine. The resulting mutant molecules are then examined for biological activity such as epitope binding or ADCC activity in vitro. Sites that are key for ligand-receptor binding can also be determined using 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).

In addition, polypeptides often contain amino acids other than the twenty "naturally occurring" amino acids. Moreover, 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 methods well known in the art. Known modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent attachment, heme covalent attachment, nucleotide or nucleotide derivative covalent attachment, lipid or lipid derivative covalent attachment, phosphotidylinositol covalent attachment, crosslinking, cyclization, disulfide bonding, demethylation , formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, formation of the GPI-anchor (glycosylphosphatidylinositol anchor), hydroxylation, iodination, methylation, myristiolation (acetylation with a myristic acid residue), oxidation, proteolysis, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA mediated addition of amino acids to proteins such as, but not limited to, 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 glycosylation, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation, are described in most basic texts such as Proteins-Structure and Molecular Properties (2nd ed., TE Creighton WH Freeman & Co., NY, 1993). There are many detailed reviews on this subject, 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 IL-31-specific antibodies, polypeptides and peptides described herein, as well as IL-31 peptide mimotopes described herein, also encompass derivatives or analogs in which the substituted amino acid residue is not encoded by the genetic code.

Similarly, additions and substitutions in amino acid sequence, as well as variations and modifications described, may be equally applicable to the amino acid sequence of an IL-31 antigen and/or its epitope or peptides, and thus are covered by the present invention.

Derivatives of antibodies and mimotopes

The scope of this invention includes derivatives of antibodies and mimotopes. A "derivative" of an antibody or mimotope contains additional chemical moieties, usually not part of the protein or peptide. Covalent modifications of a protein or peptide are within the scope of this invention. Such modifications may be introduced into the molecule by reacting the target amino acid residues of the antibody or mimotope with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents well known in the art can be used to form crosslinks between an antibody or fragment or mimotope and a water insoluble carrier matrix or other macromolecular carriers.

Derivatives also include radiolabeled monoclonal antibodies or mimotopes. For example, with radioactive iodine ( ¹²⁵ I, ¹³¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), indium ( ¹¹¹ In), tritium ( ³ H), etc.; conjugates of monoclonal antibodies with biotin or avidin, with enzymes such as horseradish peroxidase, alkaline phosphatase, bepha-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose-6-phosphate dehydrogenase; as well as conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemiluminescent agents (such as acridine esters) or fluorescent agents (such as phycobiliproteins). Likewise, mimotopes may be labeled in some embodiments.

Another derivative of a bifunctional antibody is a bispecific antibody obtained by combining parts of two separate antibodies that recognize two different antigenic groups. This can be achieved by crosslinking or recombinant techniques.

In addition, moieties may be added to an antibody or portion thereof, or mimotopes of IL-31 described herein, to increase in vivo half-life (eg, by increasing clearance time). Such methods include, for example, the addition of PEG moieties (also referred to as PEGylation) and are well known in the art. See US Patent Application No. 20030031671.

Recombinant expression of antibodies, mimotopes and carrier polypeptides

In some embodiments, nucleic acids encoding the monoclonal antibody or fusion protein of interest, comprising both the mimotope and the carrier polypeptide, are introduced directly into a host cell and the cell is incubated under conditions sufficient to induce expression of the encoded antibody or fusion protein. After the nucleic acids in question have been introduced into the cell, the cell is typically incubated, usually at 37° C., sometimes under selective conditions, for about 1-24 hours to allow expression of the antibody or fusion protein bearing the peptide mimotope and the carrier polypeptide. . In one embodiment, the antibody or fusion protein is secreted into the supernatant of the medium in which the cells are grown.

Traditionally, monoclonal antibodies have been produced as native molecules in mouse hybridoma lines. In addition to this technology, the present invention provides for the expression of recombinant DNA monoclonal antibodies. This allows the production of canine, felinized, equinized, humanized, fully canine, fully feline, fully equine, and fully human antibodies, as well as a spectrum of antibody derivatives and fusion proteins, in selected host species.

A nucleic acid sequence encoding at least one anti-IL-31 antibody, a portion thereof, or an IL-31-specific polypeptide, or a nucleic acid sequence encoding as its portion at least one mimotope of an IL-31 peptide that binds to such the antibody, or portion thereof, can be recombined with the vector DNA according to conventional techniques, including blunt ends or sticky ends for ligation, restriction enzyme digestion to obtain suitable ends, filling in sticky ends if necessary, treatment with alkaline phosphatase to avoid undesirable joining, and ligation with appropriate ligases. The technique for such manipulations is described, for example, by 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 that encode a monoclonal antibody molecule or antigen-binding region or IL-31 peptide mimotope.

A nucleic acid molecule, such as DNA, is considered "capable of expressing" a polypeptide if it contains nucleotide sequences that contain information for regulating transcription and translation, and such sequences are "operably linked" to the nucleotide sequences encoding the polypeptide. A functional linkage is a linkage in which DNA regulatory sequences and a DNA sequence to be expressed are linked in such a way as to allow expression of genes as anti-IL-31 peptides or antibody moieties, or as fusion proteins bearing IL-31 mimotopes. in allocated quantities. The precise nature of the regulatory regions required for gene expression may vary from organism to organism, as is well known in the art. See, for example, Sambrook et al., 2001, supra, Ausubel et al., 1993, supra.

The present invention accordingly encompasses the expression of an antibody against IL-31 or an IL-31-specific polypeptide or peptide or fusion protein comprising an IL-31 mimotope in prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including bacteria, yeast, insects, fungi, in vivo or in situ avian and mammalian cells, or mammalian, insect, avian or yeast derived host cells. The mammalian cell or tissue may be from a human, primate, hamster, rabbit, mouse, cow, pig, sheep, horse, goat, dog, or cat, but any other mammalian cell may be used.

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

Examples of prokaryotic vectors known in the art include plasmids, such as plasmids capable of replication in E. coli (eg, such as pBR322, ColE1, pSC101, pACYC 184, .pi. VX). Such plasmids are described, for example, Maniatis et al., 1989, supra; Ausubel et al, 1993, supra. Bacillus plasmids include pC194, pC221, pT127 and others. Such plasmids are disclosed by 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) Review of Pseudomonas plasmids by John et al., 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 expressing cDNA encoding anti-IL-31 antibodies or peptides or fusion proteins described herein include (a) viral transcription promoters and enhancer elements thereof, 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 sites and polyadenylation sites such as those derived from the late transcribed region of SV40 (Okayama et al., MCB, 3:280 (1983) and (c) polyadenylation sites such as in SV40 (Okayama et al. , 1983, supra), but are not limited to them.

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

For immunoglobulin genes consisting of part cDNA, part genomic DNA (Whittle et al., 1 Protein Engin. 499-505 (1987)), the transcription promoter can be human cytomegalovirus, promoter enhancers can be cytomegalovirus and mouse/human immunoglobulin enhancers, and the mRNA splicing-regulating regions and the polyadenylation site may be native immunoglobulin chromosomal sequences.

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

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

In one embodiment, the fusion genes encoding the 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 fused genes encoding the chimeric H and L chains can be assembled in the same expression vector.

For transfection of expression vectors and production of a chimeric antibody, the recipient cell line may be a myeloma cell. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by transfected immunoglobulin genes and have an immunoglobulin glycosylation mechanism. Myeloma cells can be grown in culture or in the mouse abdomen, where secreted immunoglobulin can be obtained from ascitic fluid. Other suitable recipient cells include lymphoid cells such as B-lymphocytes with or without human origin, hybridoma cells with or without human origin, or cells of interspecies heterohybridomas.

An expression vector carrying a nucleotide sequence encoding chimeric, canine, felinized, equinized, humanized, fully canine, fully feline, fully equine, or fully human sequences of an anti-IL-31 antibody construct or an IL-31-specific polypeptide or peptide (e.g., an antigen-binding portion antibodies described herein), or an expression vector carrying a nucleotide sequence encoding a fusion protein described herein, can be introduced into a suitable host cell by any of a variety of suitable methods, including biochemical means such as transformation, transfection, conjugation , protoplast fusion, calcium phosphate precipitation using polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means 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 immunoglobulin. Yeasts carry out post-translational modifications of peptides, including glycosylation. There are currently a number of recombinant DNA strategies 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 bearing 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, antibodies, and assembled murine and chimeric, heterochimeric, caninized, felinized, equinized, humanized, fully canine, fully feline, fully equine, or fully human. antibodies, their fragments and regions. Any of a series of yeast gene expression systems can be used, including promoter and terminator elements of actively expressed genes encoding glycolytic enzymes produced in large quantities when yeast is grown in a glucose-rich medium. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, phosphoglycerate kinase (PGK) gene promoter and terminator signals can be used. A number of approaches can be used to evaluate optimal expression 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 the production of antibody molecules or peptides or fusion proteins described in this invention. With such bacterial hosts, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used. The vector carries a replication site, as well as specific genes that are capable of providing the phenotypic selection of transformed cells. To evaluate expression plasmids to produce murine, chimeric, heterochimeric, caninized, felinized, equinized, humanized, fully canine, fully feline, fully equine, or fully human antibodies, antibody fragments and regions or chains encoded by cloned immunoglobulin cDNAs in bacteria, a range of approaches (see Glover, 1985, see above; Ausubel, 1993, see above; Sambrook, 2001, see above; 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 the leader peptide, folding and assembly of H and L chains, glycosylation of antibody molecules, and secretion of functional antibody protein.

Mammalian cells that can be used as hosts for the production of antibody proteins, in addition to cells of lymphoid origin described above, include cells of regional origin fibers 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 peptide H and L chain genes in mammalian cells (see Glover, 1985, supra). Various approaches can be used to obtain complete H ₂ L ₂ antibodies. It is possible to co-express the H and L chains in the same cells to achieve intracellular association and binding of the H and L chains into full tetrameric H ₂ L ₂ antibodies and/or anti-IL-31 peptides. Co-expression can occur 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, thereby directly selecting cells expressing both chains. Alternatively, cells can first be transfected with a single chain coding plasmid, such as 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 route can be transfected with plasmids encoding additional copies of the peptides, H, L, or H+L chains in combination with additional selectable markers to generate cell lines with improved properties such as higher 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 engineered that stably express an antibody molecule. Instead of using expression vectors that contain viral origins of replication, host cells can be transformed with expression cassettes encoding an immunoglobulin and a selectable marker. After the introduction of foreign DNA engineered cells can be allowed to grow for 1-2 days in enriched medium, and then transferred to 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 into colonies, which in turn can be cloned and propagated in cell lines. Such engineered cell lines can be particularly useful in screening and evaluating compounds/components that interact directly or indirectly with an antibody molecule.

Once an antibody has been obtained, it can be purified by any method known in the art for purifying an immunoglobulin molecule, such as chromatography (e.g., ion exchange, affinity, especially based on affinity for a specific antigen after protein A chromatography, and size exclusion column chromatography), centrifugation, method based on differential solubility or any other standard protein purification method. In many embodiments, the antibodies are secreted from the cell into the culture medium and are harvested from the culture medium.

Practical use

The vaccine compositions of the present invention can be used, for example, to treat and/or protect against IL-31 mediated diseases such as pruritic and/or allergic conditions in mammals such as dogs, cats, horses and humans. The pharmaceutical compositions of the present invention are suitable for parenteral administration, for example subcutaneously, intramuscularly or intravenously. Other suitable routes of administration are described herein.

The vaccines of the present invention may be administered either as therapeutic agents on their own or in combination with other therapeutic agents. They can be administered alone, but are usually administered with a pharmaceutical carrier selected based on the chosen route of administration and standard pharmaceutical practice.

Administration of the vaccine compositions described herein may be by any suitable route, including parenteral injection (eg, intraperitoneal, subcutaneous, or intramuscular injection), orally, or by topical administration of vaccines to the surface of the respiratory tract. Topical administration to the surface of the respiratory tract can be by intranasal administration (eg, using a pipette, swab or inhaler). Topical administration of vaccines to the surface of the respiratory tract can also be done by inhalation, for example, by creating respirable particles of a pharmaceutical composition (including solid and liquid particles) containing vaccines in the form of an aerosol suspension, and then inhaling the respirable particles by the subject. Methods and devices for administering inhalable particles of pharmaceutical formulations are well known and any conventional method may be used. Oral administration may, for example, be in the form of a liquid or solid oral preparation.

In some preferred embodiments, the vaccines are administered by parenteral injection. For parenteral administration, vaccines may be formulated as a solution, suspension, emulsion, or lyophilized powder in combination with a pharmaceutically acceptable parenteral carrier. For example, the carrier may be a solution of a combination of mimotope and a carrier polypeptide (e.g. mimotope conjugate) or a cocktail thereof dissolved in a suitable carrier such as an aqueous vehicle, such carrier media are water, saline, Ringer's solution, dextrose, trehalose, or sucrose solution. or 5% serum albumin, 0.4% saline, 0.3% glycine, and the like. Liposomes and non-aqueous carriers such as fatty oils can also be used. These solutions are sterile and usually free of particulate matter. These compositions can be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable excipients necessary to approach physiological conditions such as pH adjusters and buffering agents, agents for adjusting tonicity and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. . Also, as described herein, the vaccine compositions of the present invention include an adjuvant or adjuvant formulation. The mimotope conjugate concentration in these vaccine compositions can vary widely, such as from less than about 0.5%, typically from at least about 1% to 15% or 20% by weight, and will be selected primarily based on volumes of liquid, viscosity, etc. in accordance with the selected specific route of administration. The carrier or lyophilized powder may contain additives that maintain isotonicity (eg sodium chloride, mannitol) and chemical stability (eg buffers and preservatives). The composition is sterilized using conventional methods.

The actual methods for preparing compositions for parenteral administration 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 vaccines of this invention can be lyophilized for storage and reconstituted in a suitable vehicle prior to use. Any suitable lyophilization and reconstitution methods may be used. Those skilled in the art will appreciate that lyophilization and reconstitution can result in varying degrees of loss of activity and that their levels used may need to be adjusted to compensate for the loss.

Compositions containing true IL-31 mimotopes (eg, IL-31 mimotope conjugates) or a mixture thereof can be administered to prevent recurrence and/or treat an existing disease. Suitable pharmaceutical carriers are described in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES, the standard reference text 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 or alleviate the disease and its complications. An amount sufficient to achieve this is defined as a "therapeutically effective dose" or "therapeutically effective amount". Effective amounts for such use will depend on the severity of the disease and the general state of the subject's own immune system. A therapeutically effective amount of a vaccine composition according to the invention can be easily determined by a person skilled in the art.

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

As a non-limiting example, treatment of IL-31 related pathologies in dogs, cats, horses, or humans can be by administering the vaccines of the present invention every other week or once a month in the dose range described above.

Single or multiple administration of vaccine compositions can be carried out in accordance with dose levels and schedule selected by the attending physician or veterinarian. In any case, the pharmaceutical compositions should provide an amount of the vaccine compositions of the present invention sufficient to effectively treat the subject.

Diagnostic Application

The present invention also provides IL-31 mimotopes and anti-IL-31 antibodies for use in diagnostic assays for the detection of IL-31 or anti-IL-31 antibodies in mammalian specimens, including specimens from mammals known or suspected to have associated pruritus. and/or an allergic condition.

For example, the present invention provides a method for qualitatively or quantitatively detecting an anti-IL-31 antibody in a sample. Such a method includes incubating a sample containing an anti-IL-31 antibody with at least one mimotope, such as feline IL-31 mimotope, dog IL-31 mimotope, equine IL-31 mimotope, and human IL-31 mimotope, and qualitatively and /or quantify the anti-IL-31 agent in the sample.

In one embodiment, the canine IL-31 mimotope used in the method for qualitative and/or quantitative determination of IL-31 in a sample is and/or contains as part of the amino acid sequence SVPADTFECKSF (SEQ ID NO: 186), SVPADTFERKSF (SEQ ID NO : 187), NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ ID NO: 192), APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO: 200), or variants thereof that retain binding to an anti-IL-31 agent.

In another embodiment, the feline IL-31 mimotope used in such a method is and/or contains as part of the amino acid sequence SMPADNFERKNF (SEQ ID NO: 188), NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ ID NO: 193), APAHRLQPSDIRKIILELRPMSKG (SEQ ID NO: 197) , IGLPES (SEQ ID NO: 201) or variants thereof that retain binding to an anti-IL-31 agent.

In a further embodiment, the equine IL-31 mimotope used in such a method is and/or includes as part of the amino acid sequence SMPTDNFERKRF (SEQ ID NO: 189), NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ ID NO: 194), GPIYQLQPKEIQAIIVELQNLSKK (SEQ ID NO: 198), KGVQKF (SEQ ID NO: 202) or variants thereof that retain binding to an anti-IL-31 agent.

In addition, the human IL-31 mimotope used in such a method is and/or contains as part of the amino acid sequence SVPTDTHECKRF (SEQ ID NO: 190), SVPTDTHERKRF (SEQ ID NO: 191), HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195) , LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203), or variants thereof that retain binding of the anti-IL-31 agent.

In one embodiment of the diagnostic method described above, the mimotope is a capture reagent bound to a solid surface. In one embodiment, the sample is added to the mimotope, which is a capture reagent, and then secondary detection reagents are added to determine the amount of antibody in the sample.

The present invention also provides a method for determining the amount of IL-31 in a sample from a mammal. Such a method would be useful for determining IL-31 from many species. Such a method comprises incubating a sample from a mammal containing IL-31 with a labeled anti-IL-31 antibody: IL-31 mimotope complex bound to a solid surface, wherein the mimotope in the complex is selected from the group consisting of feline IL-31 mimotope, canine mimotope IL-31, mimotope equine IL-31 and mimotope human IL-31; and determining the level of IL-31 in the sample, wherein the labeled anti-IL-31 antibody in the complex has a lower affinity for mimotope in the complex compared to its affinity for IL-31 in the sample. In one embodiment of this method, the detection step includes measuring the signal from the labeled antibody that is released from the solid surface when IL-31 in the sample binds to the labeled antibody against the IL-31 complex, with the level of IL-31 in the sample being inversely proportional to the signal.

In one embodiment, the canine IL-31 mimotope used in the method for quantifying IL-31 in a sample is and/or contains as part of the amino acid sequence SVPADTFECKSF (SEQ ID NO: 186), SVPADTFERKSF (SEQ ID NO: 187), NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ ID NO: 192), APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO: 200) or variants thereof that retain binding to an anti-IL-31 agent.

In another embodiment, the feline IL-31 mimotope used in such a method is and/or contains as part of the amino acid sequence SMPADNFERKNF (SEQ ID NO: 188), NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ ID NO: 193), APAHRLQPSDIRKIILELRPMSKG (SEQ ID NO: 197) , IGLPES (SEQ ID NO: 201) or variants thereof that retain binding to an anti-IL-31 agent.

In another embodiment, the equine IL-31 mimotope used in such a method is and/or contains as part of the amino acid sequence SMPTDNFERKRF (SEQ ID NO: 189), NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ ID NO: 194), GPIYQLQPKEIQAIIVELQNLSKK (SEQ ID NO: 194). : 198), KGVQKF (SEQ ID NO: 202), or variants thereof that retain binding to an anti-IL-31 agent.

In a further embodiment, the human IL-31 mimotope used in such a method is and/or contains as part of the amino acid sequence SVPTDTHECKRF (SEQ ID NO: 190), SVPTDTHERKRF (SEQ ID NO: 191), HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195), LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203), or variants thereof that retain binding of the anti-IL-31 agent.

In some embodiments of any of the diagnostic methods of the invention described above, the mimotope binds to an anti-IL-31 antibody, or an antigen-binding portion thereof, that specifically binds to a region of the mammalian IL-31 protein involved in the interaction of the IL-31 protein with its co-receptor. In one embodiment of the diagnostic methods of the invention, binding of said antibody to said region is affected by mutations in the 15H05 binding epitope region selected from the group consisting of:

a) the region between amino acid residues 124 and 135 of the feline IL-31 sequence shown in SEQ ID NO: 157 (wild-type feline IL-31);

b) the region between amino acid residues 124 and 135 of the canine IL-31 sequence represented by SEQ ID NO: 155 (canine IL-31); and

c) the region between amino acid residues 118 and 129 of the equine IL-31 sequence shown in SEQ ID NO: 165 (equine IL-31).

In one embodiment of the diagnostic methods of the present invention, the mimotope binds to an anti-IL-31 antibody, or antigen-binding portion thereof, that specifically binds to the aforementioned region of the 15H05 epitope. In one specific embodiment of any of the diagnostic methods of the present invention, mimotope binds to an anti-IL-31 antibody, or antigen-binding portion thereof, comprising at least one of the following combinations of complementarity determining region (CDR) sequences:

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

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

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

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

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

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

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

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

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

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

11) variant (1)-(10) differing from the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 or ZIL171 by the addition, deletion and/or substitution of one or more amino acid residues at at least one of CDR1, CDR2 or CDR3 VH or VL.

In some embodiments, the mimotope used in the diagnostic methods of the present invention binds to an anti-IL-31 antibody, or an antigen-binding portion thereof, that binds to feline IL-31, wherein the antibody comprises a VL chain containing frame region 2 (FW2) changes selected of 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 refer to the numbering of SEQ ID NO: 127 (FEL_15H05_VL1).

Anti-IL-31 antibodies, polypeptides and/or peptides of the present invention and IL-31 peptide mimotopes are useful for immunoassays that detect or quantify IL-31 or anti-IL-31 antibodies in a sample. An IL-31 immunoassay typically involves incubating a clinical or biological specimen in the presence of a detectable, labeled, high affinity (high avid) anti-IL-31 antibody, polypeptide, or peptide of the present invention capable of selectively binding to IL-31, and detecting the labeled polypeptide, peptide, or antibody. that are linked in the sample. In a preferred embodiment, the IL-31 mimotope binds to a solid surface and is used to capture the labeled anti-IL-31 antibody such that the labeled anti-IL-31 antibody: IL-31 mimotope complex becomes fixed to the solid surface. The labeled anti-IL-31 antibody in the complex has a lower affinity for mimotope in the complex compared to its affinity for IL-31 in the sample. Thus, the level of IL-31 in a sample can be determined by measuring the signal from the labeled antibody that is released from the solid surface when IL-31 in the sample binds to the labeled anti-IL-31 antibody of the anti-IL-31 antibody: mimotope IL- 31. In this case, the level of IL-31 in the sample is inversely proportional to the signal. Various clinical research methods 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 biopsies, blood, serum, and faecal or fluid samples taken from animals 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 determined in a liquid phase assay.

In other embodiments, an IL-31 peptide mimotope or an anti-IL-31 antibody, polypeptide, or peptide can, 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 appropriate buffers followed by treatment with a detectable labeled IL-31 specific polypeptide, peptide or antibody. The solid phase support can then be washed with buffer a second time to remove unbound polypeptide, peptide or antibody. The amount of bound label on the solid support can then be determined using known method steps.

"Solid phase 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, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. By its nature, the carrier may be either soluble to some extent, or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration, so long as the bound molecule is capable of binding to an IL-31 peptide mimotope, IL-31, or an anti-IL-31 antibody. It is contemplated that the support-bound IL-31 mimotope may itself be conjugated to a carrier polypeptide if desired. Thus, the substrate configuration can be spherical, like a bead, or cylindrical, like on the inner surface of a test tube, or on the outer surface of a rod. Alternatively, the surface may be flat, such as a sheet, culture dish, test strip, etc. For example, the substrates may include polystyrene beads. Many other suitable carriers for binding an antibody, polypeptide, peptide, or antigen are known to those skilled in the art, or can be selected by routine experimentation.

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

Detectable labeling of an IL-31-specific polypeptide, peptide, and/or antibody, as well as labeling of a mimotope of an IL-31 peptide (or conjugate thereof) can be accomplished in several different ways, including binding to an enzyme for use in an enzyme-linked immunosorbent assay (EIA) or enzyme-linked immunosorbent assay. (ELISA). The bound enzyme reacts with the exposed substrate to form a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric, or visual means. Enzymes that can be used to detectably label IL-31-specific antibodies or mimotopes described herein include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase , but not limited to asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

By radiolabeling IL-31-specific antibodies or mimotopes, IL-31 can be detected by 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 means such as using a gamma or scintillation counter or autoradiography. Isotopes that are particularly effective 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 IL-31-specific antibodies or mimotopes 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 most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phtaldehyde, and fluorescamine.

IL-31 specific antibodies or mimotopes can also be labeled with a detectable label using fluorescent metals such as ¹²⁵ Eu or others from the lanthanide series. These metals can be attached to IL-31-specific antibodies or mimotopes using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Antibodies specific for IL-31 can also be labeled with a detectable label by binding to a chemiluminescent compound. The presence of a chemiluminescently labeled antibody or mimotope is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, acridinium theromatic ester, imidazole, acridinium salts, and oxalic acid ester.

Similarly, a bioluminescent compound can be used to label a mimotope or an IL-31 specific antibody, portion, fragment, polypeptide, or derivative thereof. Bioluminescence is a type of chemiluminescence found in biological systems in which a protein that has catalytic activity increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase and aequorin.

Detection of a mimotope, IL-31 specific antibody, moiety, fragment, polypeptide, or derivative can be performed using a scintillation counter, for example, if the label to be detected is a gamma radiation source, or using a fluorometer, for example, if the label is a fluorescent substance. In the case of an enzyme label, detection can be performed by colorimetric methods that use a substrate for the enzyme. Detection can also be carried out by visual comparison of the intensity of the enzymatic reaction with the substrate and similarly prepared standards.

For the purposes of the present invention, IL-31, which is determined using the above studies, 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 studies using only these samples, however, a person of ordinary skill in the art, having read this description, can establish suitable conditions that allow the use of other samples.

In situ detection can be performed by taking a histological sample from a subject animal and adding a labeled antibody (alone or in combination with the IL-31 mimotope described herein) to such a sample. It is also contemplated that an antibody in a complex may include only a portion of the antibody. An antibody (or part thereof) can be presented as a labeled antibody (or part thereof) applied or layered on a biological sample. Using this procedure, it is possible to determine not only the presence of IL-31, but also the distribution of IL-31 in the tissue under study. Using the present invention, those of ordinary skill in the art will readily appreciate that any of a wide variety of histological methods (such as staining procedures) can be modified for such in situ determination.

The mimotope, antibody, fragment or derivative of the present invention can be adapted for use in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, an amount of unlabeled antibody (or antibody fragment) is bound to a solid support that is insoluble in the test fluid and a certain amount of detectable, labeled, soluble antibody is added to allow detection and/or quantification of the ternary complex formed between the bound solid phase antibody, antigen, and labeled antibody.

Antibodies or antibody: mimotope complexes can be used to quantify or qualitatively detect IL-31 in a sample, or to detect the presence of cells expressing IL-31. This can be achieved by immunofluorescent techniques using a fluorescently labeled antibody or antibody: mimotope complex (see below) in combination with fluorescence microscopy, flow cytometry, or fluorometric detection. For diagnostic purposes, antibodies can be either labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that react with the antibody, such as antibodies specific for canine or feline immunoglobulin constant regions. Alternatively, antibodies can be labeled directly. A wide variety of labels can be used, such as radionuclides, fluorescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially haptens), etc. Numerous types of immunoassays are available, such as those discussed previously, and are well known to those skilled in the art.

In one embodiment, the diagnostic method for determining IL-31 is an immunochromatographic assay. It is also known as immunochromatographic assay, rapid immuno-migration (RIM™) or strip test. An immunochromatographic assay is essentially an immunoassay adapted to function along a single axis to fit the format of the test strip. Several variations of this technology have been developed into commercial products, but they all operate on the same basic principle. A typical test strip consists of the following components: (1) sample pad - an absorbent pad onto which the test sample is applied; (2) a conjugate or reagent pad - this contains antibodies specific to the target analyte conjugated to colored particles (usually colloidal gold particles or latex microspheres); (3) reactive membrane - usually a hydrophobic nitrocellulose or cellulose acetate membrane on which antibodies against the target analyte are immobilized as a line crossing the membrane to form a capture zone or test line (a control zone may also be present containing antibodies specific for the conjugated antibodies ); and (4) an absorbent zone or waste reservoir - an additional absorbent platform designed to move the sample through the reaction membrane due to capillary forces and collect it. The components of the strip are usually attached to an inert backing material and can be presented as a simple strip or having a plastic case with a sample port and a reaction window where the capture zone and the control zone are visible. In microbiological research, two main types of immunochromatographic assays are used: sandwich tests with two antibodies and competitive tests. In dual antibody sandwich assays, the sample migrates from the sample pad through the conjugate pad, where any target analyte present binds to the conjugate. The sample then continues to migrate across the membrane until it reaches the capture zone, where the target/conjugate complex will bind to the immobilized antibodies, producing a visible line on the membrane. The sample is then moved further down the strip until it reaches the control zone, where the excess conjugate binds and a second visible line appears on the membrane. This control line indicates that the sample passed through the membrane as expected. Two clear lines on the membrane - a positive result. One line in the control zone is a negative result. Competitive assays differ from dual antibody sandwich assays in that the conjugate pad 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. As the sample travels along the membrane and reaches the capture zone, excess unlabeled analyte binds to the immobilized antibody and blocks the capture of the conjugate so that no visible lines appear. The unbound conjugate will then bind to the antibodies in the control zone with the appearance of a visible control line. The only control line on the membrane is a positive result. Two visible lines in the zone of capture and control - a negative result. However, if there is no excess of the unlabeled target analyte, a faint line may appear in the capture zone, indicating a questionable result. There are a number of varieties of immunochromatographic assay technology. The capture zone on the membrane may contain not antibodies, but immobilized antigens or enzymes, depending on the target analyte. Multiple capture zones can be used to create a multiplex test. For example, commercial test strips have been developed that are capable of detecting Shiga toxins ST1 and ST2 of enterohemorrhagic strains of E. coli separately in a single sample.

It is important to note that the mimotopes and antibodies described herein can be used in the diagnosis of pruritus and/or allergies in dogs, cats or horses. More specifically, an antibody in an antibody:mimotope complex can bind to IL-31 in a sample and help detect overexpression of IL-31 in mammals, including domestic animals. Thus, the antibodies described herein, which can be used in combination with mimotope, can serve as an important tool in immunohistochemistry. In one embodiment, this document provides an assay scheme whereby an IL-31 mimotope (peptide) is used to capture an antibody of the present invention labeled for assay detection. This captured antibody will have a lower affinity for the attached mimotope than for native IL-31 circulating in the host species. In this embodiment, a fluid obtained from a host species is incubated with a labeled antibody: mimotope complex bound to a solid surface. IL-31 present in the test fluid obtained from the host species will have a higher affinity for the antibody, thus the labeled antibody will be released from the solid surface and can be removed during the washing steps. Thus, the level of IL-31 in the test fluid may be inversely proportional to the signal generated at the surface to which the mimotop is bound. It is contemplated that such an assay could be used as a diagnostic test in research or in the clinic to measure IL-31.

The antibodies and mimotopes described herein can be used in chips that are very useful for measuring gene expression profiling.

Sets

Also included within the scope of the present invention are kits for the practice of the therapeutic methods and diagnostic methods in question. In one embodiment, the kit according to the present invention at least includes a vaccine composition according to the present invention. In one embodiment, the vaccine of the present invention may be presented in a container, usually in lyophilized form. In another embodiment, the kit according to the present invention may include components necessary for the implementation of diagnostic methods according to the present invention. For example, a kit of the present invention may include as one of its components an IL-31 mimotope as described herein, such as a feline IL-31 mimotope, a canine IL-31 mimotope, an equine IL-31 mimotope, or a human IL-31 mimotope. . Such a mimotope may already be bound to a solid surface. The kit according to the present invention may also include antibodies. Antibodies, which can be labeled or toxin-conjugated or unconjugated, are typically kitted with buffers such as tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, such as serum albumin, and the like. Usually these materials 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 the concentration of antibodies. It is often 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% by weight of the total composition. If the assay uses a second antibody that can bind to the primary antibody, it is usually in a separate vial. The second antibody is usually conjugated to a label and formulated with the antibody preparations described above in a similar manner. Kits usually also include a set of instructions for use.

In one embodiment, the kit of the present invention is a test strip kit (immunochromatographic assay kit) that can be used to detect IL-31, such as canine, feline, equine or human IL-31 protein in a sample. Such a test strip typically includes a sample pad onto which the test sample is applied; a conjugate or reagent pad comprising an antibody or antibody:mimotope specific for canine, feline, equine, or human IL-31, wherein the antibody or antibody:mimotope complex is conjugated to colored particles (usually colloidal gold particles); a reactive membrane on which anti-IL-31 antibodies or an antibody:mimotope complex is immobilized as a line crossing the membrane, forming a capture zone or test line (a control zone may also be present containing antibodies or an antibody:mimotope complex for which the conjugated antibodies are specific) ; and another absorbent pad designed to move the sample through the reaction membrane due to capillary forces and collect it. The test strip kit usually also includes instructions for use.

Further, the invention will be described below by examples, not limiting the scope of the invention. In the Examples section below and in the graphics, any data presented for antibodies labeled "11E12" is provided for comparison with the antibodies of the present invention.

Examples

1. Example 1.

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

The amino acid sequence conservation of the interleukin 31 protein differs among homologous species (Fig. 1), but it is thought to share a common structural architecture with 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). The topology of the up-down bundles is important for the way these cytokines share the receptor recognition (Dillon et al. supra, Cornelissen et al. 2012 Eur J Cell Biol. Jun-Jul; 91(6-7):552- 66). Due to differences in IL-31 protein sequences between species, it is not possible to predict whether antibodies raised against one species will cross-react with others, given the different frequencies of amino acids in the epitopes and the local amino acid composition. As a consequence, many forms of the IL-31 protein were considered for this work, representing many species and expression systems. Canine IL-31 protein (cIL-31) was prepared for use as an immunogen and reagent for testing the affinity and potency of antibody variants. Recombinant cIL-31 was generated in CHO cells using the CHROMOS ACE system (Artificial Chromosome Expression) (Chromos Molecular Systems, Inc., Burnaby, British Columbia) to generate a secreted canine IL-31 protein having the sequence (SEQ ID NO: 155; Canine_IL -31), the corresponding nucleotide sequence for which is (SEQ ID NO: 156; Canine_IL-31). Conditioned medium from 400 ml of cell culture (CHO cell line) was prepared and dialyzed against 10 volumes of QA buffer (20 mM Tris pH 8.0, 20 mM NaCl) for 4.5 hours. The dialyzed medium was filtered through a 0.2 µm filter and loaded at 1 ml/min onto a SOURCE™ Q column (GE Healthcare, Uppsala, Sweden) pre-equilibrated with QA buffer. The protein was eluted using a multi-stage linear gradient. Most cIL-31 remained in the flow fraction (FT), a small amount of cIL-31 was eluted at the beginning of the gradient. The authenticity of the protein was previously confirmed by Western blotting and mass spectrometry (MS) analysis of the tryptic digest. The protein in the FT fraction was concentrated 4-5 times and dialyzed overnight against phosphate buffered saline (PBS) at 4°C. After dialysis in PBS, the stability of the protein was checked. No precipitation was observed and no proteolysis was observed after a few days at 4°C. Deglycosylation using N-glycosidase F gave the protein migrating on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as a single band corresponding to approximately 15 kDa. Protein concentration was determined using a bicinchonin assay (BCA assay) with bovine serum albumin (BSA) as standard (ThermoFisher Scientific, Inc., Rockford, IL). The protein solution was divided into aliquots, quickly frozen (liquid N ₂ ) and stored at -80°C.

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

To aid in the identification of antibodies with the ability to bind appropriate epitopes, wild-type and mutant feline IL-31 proteins were expressed in a mammalian expression system for production, purification, and affinity studies in cell assays. The binding site of the antibody 11E12 on IL-31 has been described previously (patent US 8790651, Bammert, et al.). This document characterizes a new binding site on IL-31 recognized by the 15H05 antibody. Wild type refers to the full-length feline IL-31 protein without changes in native amino acid residues. Mutant proteins were named according to their antibody names (11E12 and 15H05), indicating amino acid mutations in the IL-31 protein that (when altered) affect binding to each respective antibody. Identification of the relevant mutations required for the 15H05 feline IL-31 protein is described below in Section 1.10. The challenge was to change the amino acids in the IL-31 epitope and observe the loss of binding to each respective antibody. A comparison can then be made during screening to see if the new candidate antibodies bind to the wild-type protein but not to the mutant. New antibody variants can then be sorted by their binding to the same epitope as the 11E12 or 15H05 antibody or a similar epitope.

Expression constructs were optimized for codon usage and synthesized for expression in Chinese Hamster Ovary (CHO) cells. The synthesized genes were cloned into pD2529 (ATUM vector) for use in transient expression. The wild-type feline IL-31 protein is shown (SEQ ID NO: 157; Feline_IL-31_wildtype) and corresponds to the nucleotide sequence (SEQ ID NO: 158; Feline_IL-31_wildtype). The 11E12 feline IL-31 mutant protein is shown (SEQ ID NO: 161; Feline_IL-31_11E12_mutant), the corresponding nucleotide sequence for it is (SEQ ID NO: 162; Feline_IL-31_11E12_mutant). The 15H05 feline IL-31 mutant protein is shown (SEQ ID NO: 163; Feline_IL-31_15H05_mutant), the corresponding nucleotide sequence for it is (SEQ ID NO: 164; Feline_IL-31_15H05_mutant). Recombinant feline IL-31 proteins were expressed in ExpiCHO-S™ cells (ThermoFisher Scientific, Inc., Rockford, IL) following the manufacturer's Max Titer protocol for transient expression in CHO cells. Twelve days after transfection, cells were centrifuged and filtered to capture the secreted protein from the 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) equilibrated with 5 mM imidazole, 20 mM sodium phosphate, 300 mM NaCl, pH 7.4. Each sample and resin was left to stir at 4° C. overnight. Resins were collected (and separated from the unbound fraction) by passing through BioRad Econcolumn columns (Bio-Rad, Hercules, CA). The resins were washed with 5×5 ml of buffer (as above) and then eluted with 5×5 ml of 500 mM imidazole in the same buffer. Fractions were evaluated using SDS-PAGE. Protein concentration was measured by BCA analysis according to standard methods.

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

Recombinant feline IL-31 protein was generated in an E. coli expression host for use as a reagent in the assay and in vivo experiments to elicit an itch response in cats. A gene representing feline IL-31 was synthesized for optimal expression in E. coli. Expression constructs were created with the full-length feline IL-31 gene containing an N-terminal hexahistidine tag for detection and purification. This feline IL-31 protein is (SEQ ID NO: 159; Feline_IL-31_E_coli), the corresponding nucleotide sequence for which is (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.

E. coli cell mass (262.3 g) was lysed as follows. The cell mass was resuspended in 500 ml of 50 mM Tris pH 8, filtered through a stainless mesh filter to remove particles, and then lysed by passing through a microfluidizer twice at 8.963 MPa (1300 psi). The lysate (volume approximately 1200 ml) was divided into four vials, centrifuged at 12000 g for 20 minutes at 10°C. The supernatant was decanted and discarded. Each pellet was washed by dissolving in 300 ml 5 mM EDTA, 0.5% Triton X-100, pH 9.0 and then centrifuged at 12,000 g for 50 minutes at 10°C. The supernatant was decanted and discarded. The washed precipitate was 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 decanted again. 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 centrifugation again at 10,000 g for 20 minutes at 4°C. This time the supernatant was decanted and left for further processing. The pellet (resuspended in water to original volume) was left for SDS-PAGE only. To improve purity, primary immobilized metal ion affinity chromatography (IMAC) was performed before folding. In this case, 15 ml Ni-NTA Superflow (Qiagen Inc, Germantown, MD, cat. no. 30450, pre-equilibrated with 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 (same as solubilization buffer). The resin was eluted (first with 7.5 ml and then several times with 15 ml, protein elution monitored by Bradford assay) with 200 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 6 M guanidine-HCl, pH 7.4. The eluted fractions containing protein (according to Bradford) were pooled (125 ml) for further processing.

Folding of the IL-31 protein was performed as follows. IL-31 was reduced by adding dithiothreitol to a final concentration of 10 mm and left to mix at room temperature for 2 hours. The diluted sample was then diluted dropwise in 2500 ml (20X volume) PBS + 1 M NaCl with rapid stirring. The theoretical concentration of urea at this point should be about 0.4 M. The remaining urea was slowly removed by dialysis in PBS, making 3 changes (each time in a volume of 4 l) at 4°C during the night. After dialysis, the sample was filtered through a 0.2 µm filter to remove any unfolded/precipitated protein.

The sample was then purified in a second round of IMAC, this time with linear gradient elution. 15 ml of Ni-NTA Superflow resin was added to the sample and left to bind in batches by stirring (with a magnetic stirrer) overnight at 4°C. Again, the unbound fraction was decanted and left. Ni-NTA Superflow resin was loaded onto an XK16 column (GE Healthcare Lifesciences, Marlborough, MA) and connected to an AKTA brand chromatography system (GE Healthcare Lifesciences, Marlborough, MA). The column was then washed with 50 mM Tris, 300 mM NaCl, pH 8.2 and eluted with 150 ml linear gradient from 0 to 500 mM imidazole in wash buffer. Fractions were analyzed by SDS-PAGE. Fractions having IL-31 of sufficient purity were pooled and the buffer was changed again by dialysis in PBS, making 3 changes (each time 2 L) at 4° C. overnight. Finally, the folded and purified sample was collected after dialysis, filter sterilized, 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 to IL-31 using surface plasmon resonance

The affinity with which mAb candidates bind canine and feline IL-31 was determined using surface plasmon resonance (SPR) in the Biacore system (Biocore Life Sciences (GE Healthcare), Uppsala, Sweden). To avoid differences in affinity associated with different surface preparation, which can occur when antibodies are immobilized on surfaces, a strategy was used in which IL-31 was directly conjugated to the surface. Immobilization was performed by amino coupling with 5 μg/ml IL-31 using N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The chips were blocked with ethanolamine and the affinity with which all mAb candidates bound to immobilized IL-31 was assessed. A 1:1 model was used to fit all curves. Affinity constants (KD) less than 1×10 ^-11 M (1E-11 M) less than the lower limit of quantitative determination of the device. The results of affinity measurements are described in this document.

1.5. Method for Evaluating the Efficacy of Anti-IL-31 Antibodies in Inhibiting PSTAT3 Signaling Induced by Canine and Feline IL-3L in Canine and Cat Macrophage Cells

To identify candidates with inhibitory activity, the ability of antibodies to influence IL-31 mediated STAT3 phosphorylation in a dog or cat cell assay was evaluated. STAT3 phosphorylation was determined in canine DH-82 macrophage-like cells (ATCC® CRL-10389™) or feline FCWF4 (ATCC CRL-2787). DH82 and FCWF4 cells were primed with canine interferon gamma (R&D Systems, Minneapolis, MN) at 10 ng/mL for 24 hours or feline interferon gamma (R&D Systems, Minneapolis, MN) at 125 ng/mL for 96 hours, respectively, to increase receptor expression. Both types of cells were kept in the absence of serum for 2 hours before exposure to IL-31 and mAb. Using two independent methods, the ability of all mAb candidates to inhibit STAT3 phosphorylation induced by 1 μg/ml canine or 0.2 μg/ml feline IL-31 was assessed. Studies were also conducted to demonstrate the cross-reactivity of canine and feline cytokines and the cross-functional ability of antibodies to inhibit signaling in both species. To ensure complex formation, mAb and IL-31 cytokine were co-incubated for one hour prior to cell stimulation. Stimulation of IL-31 cells was carried out for five minutes. Phosphorylation of STAT3 was measured using AlphaLISA SureFire ULTRA™ technology (Perkin Elmer, Waltham, MA). In the case where antibody concentration and purity are unknown, the ability of hybridoma supernatants to inhibit STAT3 phosphorylation after 1 hour co-incubation with 1 mg/ml canine or 0.2 mg/ml feline IL-31 was quantified. The efficacy of individual monoclonal antibodies, as determined by their ability to inhibit IL-31 mediated STAT3 phosphorylation in these studies, was considered a key selection criterion for further refinement of selected antibodies. The term "potency" refers to the IC50 value calculated from these studies and is the antibody concentration at which IL-31-induced signaling is reduced to half of the maximum value. The increased efficiency described in this document correlates with a lower IC50 value.

1.6. Finding mouse and canine monoclonal antibodies recognizing canine and feline interleukin 31

Mice and dogs were immunized with recombinant canine IL-31 (SEQ ID No. 155) to detect antibodies. Serum antibody titers from immunized animals were determined using enzyme-linked immunosorbent assay (ELISA). Canine or feline IL-31 (50 ng/well) was immobilized on polystyrene microtiter plates 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 determined using a suitable secondary antibody labeled with HRP. After addition of chromogenic substrate (SureBlue Reserve TMB, 1-component microplate peroxidase substrate, KPL, Inc., Gaithersburg, MD) and a ten minute incubation at room temperature (RT), the reaction was stopped by adding 100 μl of 0.1 N HCl. Optical density (OD) was determined in each well at a wavelength of 450 nm. Antibodies were selected for their ability to bind canine and feline IL-31 using ELISA. In some cases, additional characterization was performed during screening using an ELISA with a mutated form of feline IL-31 protein as the capture antigen. Cells producing antibodies with desired binding and inhibition properties were selected for sequence analysis of RNA transcripts representing the IgG heavy (VH) and light (VL) chain variable regions.

For murine 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 protein by ELISA. This allowed the identification of a single mouse Mu-15H05 antibody having sub-nanomolar affinity for both IL-31 species (FIG. 2A). The mouse anti-IL-31 antibody 15H05 was further subcloned to obtain a homogeneous antibody producing hybridoma and to sequence the heavy and light chain variable regions. The sequences of the murine anti-IL-31 antibody variable regions determined for the 15H05 antibody are as follows: 15H05 heavy chain variable region (SEQ ID NO: 67; MU-15H05-VH), the corresponding nucleotide sequence for which is (SEQ ID NO: 68; MU-15H05-VH), the 15H05 light chain variable region (SEQ ID NO: 69; MU-15H05-VL) whose corresponding nucleotide sequence is (SEQ ID NO: 70; MU-15H05-VL).

In addition to the mouse antibody 15H05, additional attention has been paid to the mouse antibody 11E12, which was previously described in US patent 8790651 issued by Bammert, et al. This document provides 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 feline IL-31 made this antibody a suitable candidate for felinization and potential therapeutic use in cats. The sequences of the mouse anti-IL-31 antibody variable regions previously determined for the 11E12 antibody are as follows: 11E12 heavy chain variable region (SEQ ID NO: 71; MU-11E12-VH), the corresponding nucleotide sequence for which is (SEQ ID NO: 72 ; MU-11E12-VH), the 11E12 light chain variable region (SEQ ID NO: 73; MU-11E12-VL), the corresponding nucleotide sequence for which is (SEQ ID NO: 74; MU-11E12-VL).

Dogs having elevated anti-IL-31 antibody titers 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 separated into individual wells and analyzed for the presence of secreted IgG capable of binding to wild-type canine IL-31 and 11E12 and 15H05 mutants (AbCellera, Vancouver, BC) using the methods described in US 2012/0009671 A1, US 2016/0252495 A1, US 9188593, WO 2015/176162 A9 and WO 2016/123692 A1.

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

The above nine monoclonal antibodies that were selected for further characterization may be referred to elsewhere in the description, in the drawings or in the 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 antibodies are responsible for antigen binding. It is expected that transplantation of the entire variable domain into the appropriate constant region will have little or no effect on the ability of the antibody to bind the IL-31 immunogen. To simultaneously confirm that the correct heavy and light chain variable region sequence was identified and to obtain a homogeneous material, expression vectors were constructed to produce recombinant chimeric or fully canine antibodies in mammalian expression systems. The chimeric antibodies described herein consist of the variable sequence (both CDR and framework region) of an antibody from a host species grafted onto the respective heavy and light chain constant regions of a cat or dog IgG molecule (e.g.: mouse-derived variable region: constant region canine origin are referred to as mouse:canine chimeric antibody). The fully canine antibodies described herein consist of the variable region sequence (both CDR and framework region) of an antibody from a host species (dog) grafted onto the respective heavy and light chain constant regions of a canine IgG molecule. Synthetic DNA sequences were designed for the heavy chain variable region (VH) and light chain variable region (VL) sequences of selected antibodies. These sequences contain unique endonuclease restriction sites, a Kozak consensus sequence, and an N-terminal secretion leader 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 cell expression plasmid containing constant regions of either the cat IgG heavy chain (SEQ ID NO: 173; Feline_HC_AlleleA_1) corresponding to the nucleotide sequence for which is (SEQ ID NO: 174 ; Feline_HC_AlleleA_1), or a light chain (SEQ ID NO: 175; Feline_LC_Kappa_G_minus) whose corresponding nucleotide sequence is (SEQ ID NO: 176; Feline_LC_G_K_minus). For mouse:canine chimeric antibodies or fully canine antibodies, each respective variable region was cloned into a mammalian cell expression plasmid containing constant regions of either canine IgG heavy chain (SEQ ID NO: 177; Canine_HC_65_1) corresponding to the nucleotide sequence for which is (SEQ ID NO: 178; Canine_HC_65_1) or a light chain (SEQ ID NO: 179; Canine_LC_Kappa) whose corresponding nucleotide sequence is (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 Flealthcare, Uppsala, Sweden) according to standard protein purification methods. a Nanosep Omega device with a nominal molecular weight cutoff of 10,000 (Pall Corp., Port Washington, NY) was dialyzed overnight at 4°C in 1×PBS, pH 7.2 and stored at 4°C for later use. The affinity and potency of selected recombinant antibodies in cell assays are described below.

On FIG. 2 shows in detail the affinity of antibodies to mouse-derived CDRs assayed using biacore. On FIG. 2a shows the surface affinity of mouse anti-IL-31 antibodies 11E12 and 15H05 and the corresponding feline and canine chimeric forms for surface with feline and canine IL-31. These observations confirm the correct sequence of both mouse antibodies and indicate that conversion to the chimeric form results in antibodies with equivalent or higher affinity than the parental mouse antibody, with the exception of the mouse:feline chimera 15H05, which has lost some affinity for IL- 31 of both species as a result of transformation into a chimeric form.

Also studied the activity of fully murine and chimeric forms of antibodies 11E12 and 15H05 in relation to cells of dogs and cats, as described in section 1.5. On FIG. 3 shows the results of these analyses. The activity of mouse 11E12 and 15H05 antibodies against canine and feline cells was studied using both canine and feline IL-31 to stimulate signaling. The efficacy of both mouse antibodies against both canine and feline cells using the feline cytokine was comparable, with the exception of 15H05 against feline IL-31 in feline FCWF4 cells, which showed a slight increase in IC50. Mouse 15H05 was able to block canine IL-31 signaling in both feline and canine cells with slightly better efficacy in canine cells. These results indicate that the respective epitopes recognized by these antibodies exist on both canine and feline IL-31, and binding of these antibodies is able to neutralize receptor-mediated cell signaling in the respective cell lines of both species.

On FIG. 3 also shows the performance of selected chimeras in assays on both cell types. Conversion of mouse antibodies to feline and canine chimeras had minimal effect on potency against feline IL-31 in the feline cell assay (IC50 range 1.15-3.45 µg/mL). Similar results were observed when these chimeras were tested for feline IL-31 signaling in the canine DH82 cell line, with a slight increase in activity for the chimeric mouse:canine antibody 15H05 (IC50=0.71 μg/mL). Overall, there was an increase in IC50 values for canine IL-31 in both dog and cat cells. The mouse:feline 15H05 chimera was slightly less efficient in this format compared to the mouse:dog form (IC50 28.61 vs 12.49 µg/mL). Conversion to canine and feline chimeric forms resulted in minimal changes in potency, consistent with mouse antibody data.

The antibodies described above, produced by single B cells of immunized dogs, were constructed as recombinant IgG proteins after identification of their variable domain sequences. The grafting of these variable domains onto a canine Fc heavy chain (isotype 65_1) resulted in recombinant all-canine antibodies. It was of interest to identify additional canine antibodies that bind wild-type feline IL-31 and have reduced binding to 15H05 mutant feline IL-31 (ie directed against the 15H05 epitope). Such antibodies derived from this alternative source (canine versus murine) provide additional paratopes (the part of the antibody that recognizes the IL-31 protein includes CDRs) that recognize the 15H05 epitope and thus increase the selection of antibodies with different physical properties.

On FIG. 4 shows the binding results of these recombinant canine antibodies to various proteins using ELISA and Biacore methods. Antibody binding to wild-type feline IL-31 and mutant 15H05 protein was assessed by indirect ELISA. 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 binding was affected by mutations in the epitope region recognized by mAb 15H05, confirming the correct binding phenotype established during the initial screening used to find them. By comparison, the 11E12 antibody bound to wild-type feline IL-31 and its binding was not affected by mutations in the epitope region recognized by 15H05, as shown in FIG. 4. To confirm binding, a biacore assay was performed using canine, feline, equine, human IL-31 proteins, mutant feline IL-31 protein, and mutant feline 11E12 protein immobilized on the surface and the only antibody concentration tested. All antibodies tested bound to wild-type feline IL-31, which was consistent with data obtained using ELISA. As described above in this section, mouse 11E12 and 15H05 antibodies bound surface-immobilized canine and feline IL-31. Three more antibodies showed this double binding property: ZIL69 (partial binding to canine), ZIL94 and ZIL159. Of this group of nine all-canine antibodies, only ZIL1 and ZIL9 cross-reacted with equine IL-31.

Of note, the 15H05 antibody was the only antibody analyzed here that bound to canine, feline, and equine IL-31, indicating some degree of epitope conservation across the three species. In contrast, none of the antibodies described herein bound to human IL-31. Additional biacore experiments were performed to validate ELISA data showing different antibody binding to wild-type feline IL-31 and two proteins with mutations in epitopes recognized by 15H05 (15H05 mutant) or 11E12 (11E12 mutant). As expected, the control mouse 11E12 antibody bound to the 15H05 IL-31 mutant and did not bind to the 11E12 IL-31 mutant due to mutations in the epitope. Similarly, mouse 15H05 did not bind to the 15H05 mutant and retained binding to the 11E12 IL-31 mutant, further distinguishing the individual binding epitopes recognized by the two antibodies. The 15H05 mutation affected all fully canine antibodies except for ZIL94, ZIL154 and ZIL171 (partial effect), which was consistent with the ELISA results. Differences in results can be explained by differences in the two methods of analysis. In addition, the 11E12 mutation has been shown to affect the binding of three antibodies: ZIL1 (partial effect), 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 support the characterization of nine canine B cell-derived antibodies binding to a region of the feline IL-31 protein that is recognized by the 15H05 antibody.

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

The formation of anti-drug antibodies (ADA) can be associated with the loss of efficacy of any biotherapeutic protein, including monoclonal antibodies. Careful review of the literature has shown that modifying monoclonal antibodies to increase their similarity to natural antibodies of a given species can reduce the propensity of mAbs to be immunogenic, although examples of immunogenic fully human mAbs and non-immunogenic chimeric mAbs can be found. To help reduce the risks associated with the formation of ADA to the anti-IL-31 monoclonal antibodies proposed herein, a felinization strategy was used. This felinization strategy is based on finding the most appropriate feline germline antibody sequence for CDR transplantation. After extensive examination of all available feline germline sequences for both the heavy and light chain variable regions, candidate germlines were selected based on their homology to mouse mAbs, and CDRs from mouse mAb progenitors were used to replace native feline CDRs. The challenge was to maintain high affinity and activity against cells using feline antibody framework regions to minimize possible in vivo immunogenicity. The felinized mAbs were expressed and characterized for their affinity for feline IL-31 and their potency in cell assays. In the event that a felinized antibody loses its ability to bind IL-31, a systemic analysis was performed to identify: 1) the chain responsible for the loss of function, 2) the framework region responsible for the loss of function, and 3) the amino acid(s) responsible for the loss of function. ) for loss of function.

Created synthetic nucleotide constructs representing the felinized variable regions of the heavy and light chains of mAb 11E12 and 15H05. After subcloning each variable chain region into plasmids containing the corresponding feline heavy chain or kappa constant region, the plasmids were co-transfected into HEK 293 cells for antibody expression. Initial attempts at felinization of the 11E12 antibody focused on using a single feline VH framework region (SEQ ID NO: 111; FEL_11E12_VH1), whose corresponding nucleotide sequence is (SEQ ID NO: 112; FEL_11E12_VH1), independently paired with VL framework regions (SEQ ID NO: : 113; FEL_11E12_VL1), whose corresponding nucleotide sequence is (SEQ ID NO: 114; FEL_11E12_VL1), and (SEQ ID NO: 115; FEL_11E12_VL2), whose corresponding nucleotide sequence is (SEQ ID NO: 116; FEL_11E12_VL2), to form Feline 11E12 1.1 and Feline 11E12 1.2, respectively. This attempt to modify monoclonal antibodies to increase their similarity to natural feline antibodies resulted in a loss of affinity of mAb Feline 11E12 1.1 for both feline and canine IL-31 proteins and a complete loss of binding of mAb Feline 11E12 1.2 compared to the mouse form of the antibody ( Fig. 2b). The efficacy of these antibodies, modified to increase their similarity to natural feline antibodies, was tested in canine DH82 cells and feline FCWF4 cells using the feline cytokine IL-31. The potency of feline 11E12 1.1 against feline IL-31 in the feline FCWF assay was approximately two-fold lower compared to the murine version of the antibody. This antibody showed a complete loss of potency in the cell assay, consistent with the loss of affinity of felinized 11E12 1.2 (FIG. 3). In an attempt to recover the loss of affinity upon felinization, a strategy similar to that used in the previous caninization experiment on the mAb 11E12 ortholog (US Patent 8,790,651, Bammert, et al.) was adopted. To create the Feline 11E12 VL1 FW2 antibody, felineized framework region 2 (FW2) of the Feline 11E12 VL1 antibody was replaced with a mouse FW2 from (SEQ ID NO: 73; Mu_11E12_VL), the corresponding nucleotide sequence of which is (SEQ ID NO: 74; Mu_11E12_VL). In addition, a single substitution was made at position 46 with feline VL (K46Q) to obtain (SEQ ID NO: 119; FEL_11E12_VL1_K46Q), the corresponding nucleotide sequence of which is (SEQ ID NO: 120; FEL_11E12_VL1_K46Q). Pairing the above VLs 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 and KD protein equivalent to that of the mouse and chimeric forms (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 nature of the ability of the 11E12 antibody to bind this epitope on feline and canine cytokines. A single amino acid substitution in Feline 11E12 1.1 K46Q failed to affect the affinity of this antibody. Increasing the affinity of the 11E12 1.1 FW2 antibody for feline IL-31 protein resulted in increased potency for the feline cytokine in the canine DH82 cell assay (FIG. 3).

The felinization effort of the mouse 15H05 antibody focused on combining three feline VH frameworks with three feline VL frameworks to form a total of 9 felinized mAbs. FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1) whose corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1) was combined with (SEQ ID NO: 127; FEL_15H05_VL1) whose corresponding nucleotide sequence is (SEQ ID NO: 128; FEL_15H05_VL1), (SEQ ID NO: 129; FEL_15H05_VL2) whose corresponding nucleotide sequence is (SEQ ID NO: 130; FEL_15H05_VL2), and (SEQ ID NO: 131; FEL_15H05_VL3) whose corresponding nucleotide sequence is (SEQ ID NO: 132 ; FEL_15H05_VL3), with the formation of Feline 15H05 1.1, Feline 15H05 1.2 and Feline 15H05 1.3, respectively. FEL_15H05_VH2 (SEQ ID NO: 123; FEL_15H05_VH2) whose corresponding nucleotide sequence is (SEQ ID NO: 124; FEL_15H05_VH2) was combined with (SEQ ID NO: 127; FEL_15H05_VL1) whose corresponding nucleotide sequence is (SEQ ID NO: 128; FEL_15H05_VL1), (SEQ ID NO: 129; FEL_15H05_VL2) whose corresponding nucleotide sequence is (SEQ ID NO: 130; FEL_15H05_VL2), and (SEQ ID NO: 131; FEL_15H05_VL3) whose corresponding nucleotide sequence is (SEQ ID NO: 132 ; FEL_15H05_VL3), with the formation of Feline 15H05 2.1, Feline 15H05 2.2, and Feline 15H05 2.3, respectively. FEL_15H05_VH3 (SEQ ID NO: 125; FEL_15H05_VH3) whose corresponding nucleotide sequence is (SEQ ID NO: 126; FEL_15H05_VH3) was co-binned with (SEQ ID NO: 127; FEL_15H05_VL1) whose corresponding nucleotide sequence is (SEQ ID NO: 128; FEL_15H05_VL1), (SEQ ID NO: 129; FEL_l5H05_VL2) whose corresponding nucleotide sequence is (SEQ ID NO: 130; FEL_15H05_VL2), and (SEQ ID NO: 131; FEL_15H05_VL3) whose corresponding nucleotide sequence is (SEQ ID NO: 132 ; FEL_15H05_VL3), with the formation of Feline 15H05 3.1, Feline 15H05 3.2 and Feline 15H05 3.3, respectively. Similar to the results with the 11E12 antibody, the first attempt at felinization of the 15H05 antibody resulted in a loss of affinity for the feline IL-31 protein compared to mouse 15H05 and a neutral effect compared to the chimeric mouse:feline 15H05 antibody (FIGS. 2A and 2C). Similar to the results of binding of feline antibody 11E12 to canine IL-31, some combinations of the VH and VL framework regions of feline 15H05 had a neutral or positive effect on affinity for canine IL-31 (see Fig. 2C Feline 15H05 1.1, 2.2 and 3.2).

In an attempt to restore the affinity of the felinized 15H05 antibody, each felinized 15H05 VH was paired with mouse 15H05 VL to form heterochimeric antibodies. FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1) whose corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1) was combined with MU_15H05_VL (SEQ ID NO: 69; MU_15H05_VL) whose corresponding nucleotide sequence is (SEQ ID NO: 70 ; MU_15H05_VL), with the formation of Feline 15H05 VH1 mouse VL. FEL_15H05_VH2 (SEQ ID NO: 123; FEL_15H05_VH2) whose corresponding nucleotide sequence is (SEQ ID NO: 124; FEL_15H05_VH2) was combined with MU_15H05_VL (SEQ ID NO: 69; MU_15H05_VL) whose corresponding nucleotide sequence is (SEQ ID NO: 70 ; MU_15H05_VL), with the formation of Feline 15H05 VH2 mouse VL. FEL_15H05_VH3 (SEQ ID NO: 125; FEL_15H05_VH3) whose corresponding nucleotide sequence is (SEQ ID NO: 126; FEL_15H05_VH3) was combined with MU_15H05_VL (SEQ ID NO: 69; MU_15H05_VL) whose corresponding nucleotide sequence is (SEQ ID NO: 70 ; MU_15H05_VL), with the formation of Feline 15H05 VH3 mouse VL. The affinity of these heterochimeric antibodies with felinized VH and mouse VL for canine and feline IL-31 was analyzed. Pairing of 15H05 felinized VH1 and VH3 with mouse 15H05 VL restored affinity for feline IL-31 to equivalent or greater than that of the murine and chimeric forms. This upward trend in affinity was also observed for canine IL-31 protein (FIGS. 2A and 2C).

To further analyze positions in the 15H05 framework regions responsible for the loss of affinity, one felinized VH from 15H05 (FEL_15H05_VH1) was paired with a mouse VL from 15H05 with separate substitutions in the framework. FEL_15H05_VH1 (SEQ ID NO: 122; FEL_15H05_VH1) whose corresponding nucleotide sequence is (SEQ ID NO: 123; FEL_15H05_VH1) was independently combined with FEL_15H05_VL1_FW1 (SEQ ID NO: 133; FEL_15H05_VL1_FW1) whose corresponding nucleotide sequence is: 134; FEL_15H05_VL1_FW1), FEL_15H05_VL1_FW2 (SEQ ID NO: 135; FEL_15H05_VL1_FW2) соответствующей нуклеотидной последовательностью которой является (SEQ ID NO: 136; FEL_15H05_VL1_FW2), и FEL_15H05_VL1_FW3 (SEQ ID NO: 137; FEL_15H05_VL1_FW3), соответствующей нуклеотидной последовательностью которой является (SEQ ID NO: 138; FEL_15H05_VL1_FW3) to form Feline 15H05 1.1 FW1, Feline 15H05 1.1 FW2, and Feline 15H05 1.1 FW3, respectively. Replacing mouse FW1 15H05 with Feline 15H05 1.1 adversely affected affinity for both feline and canine IL-31, however, when mouse FW2 or FW3 was substituted with Feline 15H05 1.1, excellent affinity for both feline and feline IL-31 was achieved while FW2 showed superior properties for both species (FIG. 2C). To determine the degree of change in affinity using this approach, additional pairwise replacements of the framework regions were carried out. FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1) whose corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1) was independently combined with FEL_15H05_VL1_FW1_2 (SEQ ID NO: 139; FEL_15H05_VL1_FW1_FW2) whose corresponding nucleotide sequence is (SEQ ID: SEQ 140; FEL_15H05_VL1_FW1_FW2), FEL_15H05_VL1_FW2_3 (SEQ ID NO: 143; FEL_15H05_VL1_FW2_FW3), соответствующей нуклеотидной последовательностью которой является (SEQ ID NO: 144; FEL_15H05_VL1_FW2_FW3), и FEL_15H05_VL1_FW1_3 (SEQ ID NO: 141; FEL_15H05_VL1_FW1_FW3), соответствующей нуклеотидной последовательностью которой является (SEQ ID NO: 142; FEL_15H05_VL1_FW1_FW3), to form Feline 15H05 1.1 FW1_2, Feline 15H05 1.1 FW2_3 and Feline 15H05 1.1 FW1_3, respectively. Interestingly, substitution of mouse FW1 alone had a negative effect on affinity, while combination 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 backmutations in feline scaffolds, starting with the most promising combinations of feline VH and VL sequences. To this end, FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1) whose corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1) was independently combined with FEL_15H05_VL1_FW2_K42N (SEQ ID NO: 145; FEL_15H05_VL1_FW2_K42Q), whose corresponding nucleotide sequence is NO: 146; FEL_15H05_VL1_FW2_K42N), FEL_15H05_VL1_FW2_V43I (SEQ ID NO: 147; FEL_15H05_VL1_FW2_V43I), соответствующей нуклеотидной последовательностью которой является (SEQ ID NO: 148; FEL_15H05_VL1_FW2_V43I), FEL_15H05_VL1_FW2_L46V (SEQ ID NO: 149; FEL_15Н05_VL1_FW2_L46V), соответствующей нуклеотидной последовательностью которой является ( SEQ ID NO: 150; FEL_15Н05_VL1_FW2_L46V), FEL_15H05_VL1_FW2_Y49N (SEQ ID NO: 151; FEL_15H05_VL1_FW2_Y49N), соответствующей нуклеотидной последовательностью которой является (SEQ ID NO: 152; FEL_15H05_VL1_FW2_Y49N) и FEL_15H05_VL1_FW2_K42N_V43I (SEQ ID NO: 153; FEL_15H05_VL1_FW2_K42N_V43I), соответствующей нуклеотидной последовательностью which is (SEQ ID NO: 154; FEL_15H05_VL1_FW2_K42N_V43I), with the formation of 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 of the entire murine FW2 framework with that of Felinized 15H05 VL1 resulted in an antibody with excellent affinity for canine and feline IL-31 (Figure 2C, Feline 15H05 1.1 FW2), individual backmutations of FW2 amino acid residues were neutral or negative. an effect indicating that all 4 substitutions are required to maintain the optimal tertiary structure for CDR positioning on the IL-31 epitope. Due to the increased affinity of felinized 15H05 1.1 FW2 for feline and canine IL-31, this antibody was selected for further work.

On FIG. 5A shows the alignment of the VL sequence of the murine 11E12 antibody, where the previously indicated caninized 11E12 sequence is compared to the felinized versions. Under the alignment dots indicate the positions of the corresponding changes in Fel 11E12 VL1, which were necessary to restore the affinity of this antibody to the IL-31 protein. Similarly, in FIG. 5B shows the necessary changes to felinized VL 15H05 (Fel_15H05_VL1) that were required 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.

1.9. Generation of cell lines expressing felinized antibodies against IL-31 from plasmids encoding glutamine synthetase (GS)

Felinized 15H05 1.1 FW2 was chosen as a candidate for establishing stable cell lines that would produce homogeneous antibodies for further characterization. Genes encoding felinized heavy and light chains for cell line production were cloned into GS plasmids pEE 6.4 and pEE 12.4, respectively (Lonza, Basel, Switzerland). The resulting plasmids were digested according to the manufacturer's protocol and ligated together to form a single plasmid for expression in mammalian cells. For ZTS-927, a heavy chain (SEQ ID NO: 121; FEL_15H05_VH1) whose corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1) was combined with a cat IgG heavy chain constant region (SEQ ID NO: 171; Feline_HC_AlleleA_wt), the corresponding nucleotide sequence for which is (SEQ ID NO: 172; Feline_HC_AlleleA_wt). For ZTS-927, the heavy chain (SEQ ID NO: 135; FEL-15H05-VL1_FW2) whose corresponding nucleotide sequence is (SEQ ID NO: 136; FEL-15H05-VL1_FW2) was combined with the cat IgG light chain constant region (SEQ ID NO: 175; Feline LC Kappa G minus), the corresponding nucleotide sequence for which is (SEQ ID NO: 176; Feline_LC_Kappa_G_minus). For ZTS-927, a heavy chain (SEQ ID NO: 121; FEL_15H05_VH1) whose corresponding nucleotide sequence is (SEQ ID NO: 122; FEL_15H05_VH1) was combined with a cat IgG heavy chain constant region (SEQ ID NO: 173; Feline_HC_AlleleA_1), the corresponding nucleotide sequence for which is (SEQ ID NO: 174; Feline_HC_AlleleA_1). For ZTS-927, the heavy chain (SEQ ID NO: 135; FEL-15H05-VL1_FW2) whose corresponding nucleotide sequence is (SEQ ID NO: 136; FEL-15H05-VL1_FW2) was combined with the cat IgG light chain constant region (SEQ ID NO: 175; Feline_LC_Kappa_G_minus), the corresponding nucleotide sequence for which is (SEQ ID NO: 176; Feline_LC_Kappa_G_minus).

To demonstrate transient antibody production, each plasmid was used to transfect HEK 293 cells and expressed in cultures of various scales. Protein was isolated from conditioned HEK medium using protein A affinity chromatography according to standard protein purification methods. The medium was applied to the chromatographic resin and eluted by changing the pH. The eluted protein was adjusted to pH before use, dialyzed and filter sterilized. ZTS-361 was subsequently used in a pruritus cat model to evaluate in vivo efficacy. The affinity and potency of antibodies derived from a single GS plasmid, ZTS-927 and ZTS-361, was studied. 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 the parental murine mAb 15H05. The efficacy of these two antibodies against canine and feline IL-31 was determined in tests using both canine and feline cells (FIG. 3). Consistent with previous results, IC50 values were proportionally higher when using the canine form of IL-31 with both cell types. The IC50 values for ZTS-927 and ZTS-361 for feline IL-31 were also highly consistent with those obtained for the chimeric and murine forms of the antibody, indicating that the final felinized version of mAb 15H05 produced from a single GS plasmid was suitable for the creation of a cell line.

To create a stable cell line producing candidate antibodies, the GS plasmid was linearized prior to transfection with the restriction enzyme Pvul, which cuts one site in the backbone of the plasmid. GS-CHOK1SV cells (clone 144E12) were transfected with linearized plasmid DNA by electroporation. After transfection, cells were placed in 48-well plates (48WP) to create stable pools. When pools in 48WP reached at least 50% confluence, IgG expression in 100 μl of supernatant was analyzed using ForteBio Octet and protein A biosensors (Pall ForteBio, Fremont, CA). Clones with the best expression were placed in 6-well plates (6 WP) and then in shake flasks (SF) with a volume of 125 ml. After the cells had adapted to suspension culture in 125 ml flasks, 2 flasks of each cell line pool were placed in a liquid nitrogen storage jar. Because the producing cell lines must be from a single clone, the 3 best expressing pools were subcloned by limiting dilution in 96-well culture plates. To confirm single-clone descent and avoid a second round of dilution, 96-well plates were imaged with Molecular Devices Clone-Select Imager (CSI) (Molecular Devices LLC, San Jose, CA), which captures images of individual cells and their subsequent growth. . Clones were selected based on CSI images, growth and productivity in 96WP.

To evaluate cell culture growth and productivity, maximal expression pools were further evaluated in 14 cultures fed with 125 ml SF. Cells were seeded in Life Technologies CD CHO medium supplemented with 4 amino acids as an in-house CDF v6.2 supplement and 10% glucose. After 14 days of fed-batch culture, the pools were centrifuged and mAB produced by CD CHO were isolated by filtering the supernatant through a 0.20 μm polyethersulfone (PES) membrane followed by purification.

A typical purge involves loading two liters of conditioned medium (from a CHO cell culture filtered through a 0.2 µm filter) onto a 235 ml MabSelect column (GE healthcare, catalog number 17-5199-02). The column was pre-equilibrated with PBS. The retention time of the loaded sample was more than 2.5 minutes. After loading, the column was washed again with PBS and then with 25 mM sodium acetate at neutral pH. The column was eluted with 25 mM acetic acid, pH 3.6, and then desorbed with 250 mM acetic acid, 250 mM sodium chloride, pH approximately 2.2. Fractions (50 ml) were collected during the elution and stripping steps. At all stages, UV absorption was determined at A280. The peak fractions were pooled, the pH was adjusted to about 5.5 with 20 mM sodium acetate, and then dialyzed by three buffer changes. The dialysate was collected, filter sterilized and stored at 4°C.

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

Studying the IL-31 epitope that is recognized by an antibody is critical to understanding the mechanism by which it neutralizes cytokine binding to the IL-31 Ra:OSMR co-receptor. In addition, epitope studies allow optimization of antibody binding affinity and generation of peptide epitope mimetics (mimotopes) that can be widely used as capture assays and as subunit vaccines to induce an appropriate targeted immune response (but not limited to this). A multi-step process using CLIPS technology (Chemical Attachment of Peptides to Scaffolds) (Timmerman et al. J Mol Recognit. 2007; 20(5): 283-299) was used to identify and optimize a peptide capable of binding to the paratope of mAb 15H05 (Pepscan, Lelystad 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 suitable for these purposes. To identify peptides capable of binding mAb 15H05, a peptide library representative of canine IL-31 protein was created and used in an indirect ELISA. After identifying peptides whose primary amino acid sequences represent the 15H05 mAb binding region on IL-31, a targeted analysis was performed with complete amino acid substitution in peptides representing the IL-31 segment, replacing each of the 12 amino acids in this 15H05 mAb binding region with 19 others. possible amino acid residues. This analysis was necessary to identify key IL-31 amino acid residues involved in mAb 15H05 binding and also showed where substitutions in the canine primary sequence resulted in increased antibody binding.

The amino acids of the canine IL-31 protein that are recognized by the 11E12 antibody have been previously described (US Patent 8,790,651 to Bammert, et al.). This document describes a mutational analysis of canine IL-31 protein showing positions on canine IL-31 protein at which alanine substitutions affect mAb 11E12 binding. Based on the full replacement assay described above for mAb 15H05 and the available 11E12 binding epitope knowledge, feline IL-31 protein mutants were generated by alanine substitution for two key residues in the epitope recognized by each antibody (mutants described in section 1.2 above). Mutations of each epitope were designated by the name of the antibody that recognizes the site of the mutation (the 11E12 and 15H05 mutant as opposed to the native wild-type protein sequence).

On FIG. 6A shows the 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 positions where substitutions to alanine were made. IL-31 belongs to the IL-6 family of cytokines with four antiparallel helix bundles having an up and down architecture (CATH database, Dawson et al., 2017 Nucleic Acids Res. 2017 Jan 4; 45 (Database issue): D289 -D295). Based on homology with the 1P9M structure of human IL-6, a model was created (Boulanger et al. 2003 Science. Jun 27; 300(5628):2101-4) using MOE (Chemical Computing Group, Montreal, QC, Canada) software. On FIG. 6B shows a homology model of feline IL-31 highlighting amino acid positions involved in binding of antibodies 11E12 (site 1) and 15H05 (site 2). The binding sites for each antibody appear to be located at distinct positions on the IL-31 protein.

To determine the effect of the ability of mAbs 11E12 and 15H05 to bind these feline IL-31 mutants, an indirect ELISA was performed using mutants directly coated on the 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 the 11E12 mutant was used for capture, 11E12 mAb binding was severely attenuated and 15H05 mAb binding was partially attenuated. Previous analysis of the 11E12 epitope on canine IL-31 (described in U.S. Patent 8,790,651 to Bammert et al.) showed that 4 amino acid residues affect mAb binding when mutated to alanine, so in this case a 2 residue mutation may not be sufficient to completely eliminate high affinity binding of mAb 11E12 in this ELISA format. The slight weakening of mAb 15H05 binding to the 11E12 mutant is likely due to the translational effects of the mutations due to the movement of the two forward helices affecting the 15H05 binding site. Mutations designed to affect the binding of mAb 15H05 (15H05 mutant) show a complete loss of the ability of mAb 1505 to bind this IL-31 mutant in ELISA. In contrast to the 11E12 mutant, changes in the random helix recognized by the 15H05 mAb (15H05 mutant) did not affect the binding of the 11E12 mAb, further confirming the difference between the two epitopes (FIG. 6C).

1.11. Assessment of mAbs 15H05 and 11E12 by competitive binding using biacore

To further characterize IL-31 epitopes associated with mAbs 15H05 and 11E12, blocking experiments were performed using biacore by immobilizing IL-31 protein on the chip surface and then sequentially passing antibodies. On FIG. 7 shows the relative binding of each antibody to IL-31 after 11E12 or 15H05 capture. Bars labeled HBS-EP (assay buffer) indicate the maximum signal obtained when each antibody binds to surface-immobilized IL-31 in the absence of competition. On FIG. 7A shows the results of competitive 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 on the protein. The sensorgrams shown in Fig. 7A show that both antibodies dissociate very slowly from this newly formed surface of the biacore chip, so no additional binding sites can be occupied when the same antibody is added (data not shown).

On FIG. 7B shows the results of competitive binding of 15H05 and 11E12 antibodies to surface-immobilized feline IL-31, again demonstrating that the epitopes they recognize do not overlap. The binding of additional antibodies in the presence of the same antibody is the result of an increase in the rate of dissociation due to the lower quality of the surface used. An increase in dissociation rate was observed, see KD values on newly formed surfaces with immobilized feline IL-31 shown in FIG. 2.

These results further support the epitope mapping data in section 1.10 indicating that the CDRs contained in the MU-15H05 antibody recognize a different epitope than MU-11E12. The epitope recognized by the 15H05 antibody differs from that recognized by the 11E12 antibody described in US Pat. These data highlight the different spatial arrangement of binding sites described by the homology-based model of feline IL-31 (FIG. 6B) and support the hypothesis that this side of the cytokine is key to interacting with the IL-31 Ra:OSMR receptor complex.

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

The heteromeric human IL-31 receptor complex, consisting of IL31Ra and OSMR subunits, has been shown to be required for IL-31 mediated cell-to-cell 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). It was later described that the human IL-31 Ra subunit mediates the initial binding that occurs when IL-31 is in contact with cell surface receptors, and this event is a necessary condition for attracting OSMR with subsequent formation of a high-affinity co-receptor complex (Le Saux et al ., 2010 J Biol Chem Jan 29;285(5):3470-7). In this document, the inventors provide evidence that the feline IL-31 protein is able to independently bind to both OSMR and Ra IL-31. These data are new and important for understanding how the IL-31 protein interacts with the IL-31Ra:OSMR co-receptor, as well as for the biological role of IL-31, since it independently interacts 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 IL-31 receptor subunits, IL-31Ra (SEQ ID NO: 169; Feline_IL31Ra_HIgG1_Fc_X1_Fn3), whose corresponding nucleotide sequence is (SEQ ID NO: 170; Feline_IL31Ra_HIgG1_Fc_X1_Fn3) and the OSMR subunit (SEQ ID NO: 167; Feline_OSMRF_hIgG1), corresponding to the nucleotide the sequence of which is (SEQ ID NO: 168; Feline_OSMR_hIgG1_Fc) were constructed in human IgG1 Fc fusion form. By homology with human homologues, a cytokine-binding domain, a type III fibronectin-like domain, and an Ig-like domain were identified. To evaluate individual receptor subunits, the extracellular domains of OSMR and IL-31Ra (with its expected N-terminal proximal type III fibronectin-like domain) were generated in a human IgG1 Fc fusion form, both 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 receptor forms to bind wild-type IL-31 protein and mutant IL-31 proteins, an indirect ELISA was performed by applying 100 µl of each respective protein to an immulon 2HB plate (1 µg/ml) in carbonate/bicarbonate buffer (sigma C3041-100CAP ) and incubated at 4°C overnight. The ELISA plates were then blocked with 5% NFDM blocking buffer in PBST for 1 hour at room temperature, followed by binding of 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 by incubation with mouse anti-human IgG1 (Lifetech A10684, 1:500 dilution) for 1 hour at room temperature. The wells were washed again with PBST and developed with sureblue 3,3',5,5'-tetramethylbenzidine (TMB) KPL microwell substrate. On FIG. 8 shows the results of this indirect ELISA using wild-type and mutant forms to capture feline IL-31 protein. These data demonstrate the ability of wild-type feline IL-31 to independently bind to IL-31Ra and OSMR receptor subunits. These data contrast with previous reports showing that the IL-31 protein initially binds to the IL-31Ra subunit and then recruits to this OSMR site. Because the biological role of IL-31 has not yet been determined, it is important to understand the dynamics of IL-31 binding and the possible consequences of impaired function in diseases such as atopic dermatitis. For this reason, these data were also taken into account when characterizing the binding of antibodies to epitopes that can interfere with the ability of IL-31 to recognize IL-31Ra and OSMR.

Section 1.2 describes the weakened binding of antibodies 11E12 and 15H05 to mutants in which key amino acids in their binding sites are converted to 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 subunits of the IL-31Ra receptor and OSMR. On FIG. 8 shows that a mutation in the 11E12 or 15H05 binding site completely disrupts the ability to bind IL-31Ra and OSMR, indicating that both antibodies bind epitopes that are necessary for IL-31 to interact with both receptor subunits. The lack of binding could also be due to conformational changes in IL-31 as a result of the mutation, however, these mutants are still able to bind to the antibody, suggesting that this is not the case. This key finding confirms the ability of both 11E12 and 15H05 antibodies (and their derivatives) to recognize epitopes on IL-31 that neutralize the ability of cytokines to signal through its co-receptor and further block cytokine binding to any cellular receptor during this process. These data confirm that antibodies have been found that are able to remove IL-31 from the bloodstream and render it unable to bind to cell-surface or soluble forms of receptors.

1.13. Evaluation of Chimeric Antibodies in an In Vivo Pruritus Provocation Model with Feline IL-31

The ability of an antibody to effectively neutralize its target can be assessed in vitro by assaying binding to the appropriate epitope on the target protein with appropriate affinity and potency in cell assays that can be extrapolated to in vivo potency. Described above are the steps taken to characterize two series of antibodies derived from mouse mAb precursors 11E12 and 15H05. Section 1.7 describes the generation of chimeric mouse:feline mAbs 11E12 and 15H05 that have comparable affinity for canine and feline IL-31 to the parent mouse monoclonal antibody (FIG. 2A). Chimeric mouse:feline forms of 11E12 and 15H05 also had comparable IC50 values indicating inhibition of feline IL-31-induced pSTAT3 signaling in canine and feline macrophage cells (FIG. 3). During the felinization process described in Section 1.8, the mouse 11E12 mAb was converted to a felinized version (Feline 11E12 1.1) resulting in loss of affinity for canine and feline IL-31 (Figure 3) and loss of efficacy against feline IL-31 signaling in cells of dogs and cats (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 pruritic activity of feline IL-31 in a cat model. Of interest was the pharmacodynamic effect of these various antibodies on neutralizing pruritus and understanding any correlation with affinity, potency in cell assays, or epitope recognition that might affect efficacy. In the future, further optimization can be made based on the range of efficacy in in vitro cell assays correlated with in vivo efficacy in the pruritus provocation model.

To test the preliminary efficacy of mouse:feline chimera 11E12, mouse:feline chimera 15H05, and feline 11E12 (Feline 11E12 1.1), a model of IL-31-induced itch in cats was developed. After intravenous administration of feline IL-31 at a dose of 0.5 μg/kg (SEQ ID NO: 159; Feline_IL-31_E_coli), the corresponding nucleotide sequence for which is (SEQ ID NO: 160; Feline_IL-31_E_coli), transiently observed behavior associated with itching, including licking, chewing, scratching and shaking the head or body, without limitation. Cage rubbing was not considered an pruritic activity. Pruritus observations were made by a trained investigator 30 minutes prior to IL-31 protein administration and 1 hour after. In this study, an initial challenge with feline IL-31 was performed 1 month prior to antibody administration. On day 0, the antibody at 0.5 mg/kg was combined with 0.5 μg/kg feline IL-31 at room temperature 60 minutes prior to administration of the pre-bound complex to each animal. A “no mAb” control was included as a control. At this dose of mAb, the antibody is in molar excess to the cytokine. Pruritic activity was monitored as described on days 0, 7 and 21. The results in FIG. 9 shows a significant improvement (p < 0.05) in pruritus score with the 15H05 chimeric mouse:feline mAb on days 0, 7 and 21 compared to control placebo. Although the mouse:feline 11E12 chimeric antibody initially showed a trend toward efficacy at day 0, there was no significant reduction in itching at any time point compared to placebo. Feline 11E12 1.1 did not improve pruritus on day 0 and did not show a trend towards efficacy compared to placebo, so no further IL-31 challenges were performed on days 7 and 21.

Taken together, these results demonstrate a clear difference between the activities of these antibodies, with no efficacy of feline 11E12 1.1 in preventing IL-31-induced pruritic behavior in cats. The loss of affinity and potency of feline 11E12 1.1 likely resulted in a lack of in vivo potency. When comparing the efficacy results of the mouse:feline 11E12 chimeric form and the mouse:feline 15H05 chimeric form, the difference was less pronounced. The chimeric forms of both mAbs had KD values comparable to their murine progenitor, while the affinity of the murine:feline form of 11E12 for both feline and canine IL-31 was somewhat higher (FIG. 2A). However, this higher affinity did not directly translate into increased potency, as the IC50 for feline IL-31-induced pSTAT3 signaling in feline FCWF4 cells in the chimeric mouse:feline form of 15H05 was approximately twice that of the chimeric mouse: feline form 11E12 (Fig. 3). These data suggest that the way the 15H05 CDR recognizes feline IL-31 allows for better neutralization of cytokine signaling through its co-receptor, which in turn makes it more effective in blocking itch in cats. The differences in IC50 observed in these cell-based assays appear promising for predicting in vivo efficacy and for detecting minor differences in epitope recognition between antibodies of the same and different lots.

1.14. Evaluation of the Efficacy of the Felinized 15H05 Anti-IL-31 Antibody in an In Vivo Cat Pruritus Model

Based on the positive results obtained with regard to the efficacy of the chimeric mouse:feline form of 15H05 described above, additional experiments were performed to increase the affinity and potency of felinized 15H05 (described above in section 1.8). Systematic substitution of the light chain variable region frameworks in the feline 15H05 1.1 antibody revealed Feline 15H05 1.1 FW2, which has increased affinity for both feline and canine IL-31 compared to murine 15H05 (FIG. 2). Combining the heavy and light chains of Feline 15H05 1.1 FW2 into a single plasmid resulted in the formation of ZTS-927 and ZTS-361 antibodies produced in the HEK and CHO expression systems. The affinity and potency of both antibodies expressed from the same plasmid are also described in FIG. 2 and 3, respectively.

The efficacy of the fully feline anti-feline IL-31 mAb ZTS-361 was evaluated by its ability to neutralize IL-31-induced pruritus in an in vivo feline model. On FIG. 10A shows the baseline behavior before pruritus in the placebo group T01 and the ZTS-361 antibody group T02 from day -7 to day 28, where day 0 was the day the antibody was administered to the T02 group. As shown in this graph, the pruritic behaviors assessed in the T01 and T02 groups prior to IL-31 challenge did not vary significantly, with the number of pruritic events during the 30-minute follow-up period ranging from 0 to 10. What makes this study different from the preliminary feline itch provocation model described in section 1.13 above was that on day 0, cats were administered 4 mg/kg ZTS-361 subcutaneously without pre-complexed feline IL-31. This is a more accurate measure of efficacy because the ZTS-361 antibody will be in circulation for seven days prior to the first IL-31 challenge requiring that the amount of antibody exposed to the body be sufficient to bind and neutralize circulating IL-31.

In this study, pruritic behavior was assessed on days 7, 21 and 28 for 1 hour after challenge with 0.5 μg/kg IL-31 protein intravenously. On FIG. 10B shows the efficacy of the ZTS-361 antibody showing a significant reduction in pruritus observed on day 7 (p less than 0.0001), day 21 (p less than 0.0027), and day 28 (p less than 0.0238) after IL-31 challenge. compared with a placebo-treated control. The results obtained in this challenge model confirm previous observations demonstrating the efficacy of the chimeric mouse:feline form of 15H05 and confirm the efficacy and importance of the epitope on feline IL-31 recognized by the 15H05 CDR in cell assays. These data also support the ability of the ZTS-361 antibody to neutralize feline IL-31 pruritus in vivo and suggest that this antibody may serve as a therapeutic agent in the treatment of feline IL-31 mediated diseases, including atopic dermatitis.

Recent data on plasma levels of IL-31 in client animals show an increased amount of cytokines in the circulation in 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) in several different geographic regions of the United States. On FIG. 11B shows the results of this assessment showing that, as in dogs with atopic and allergic dermatitis, the mean circulating IL-31 level in 73 screened cats with this putative diagnosis was 8799 fg/mL as opposed to 205 fg/mL in 17 control cats. comparable age. To understand levels of canine IL-31 in a previous model building study, the pharmacokinetic profile of canine IL-31 was analyzed in dogs following a subcutaneous dose of 1.75 μg/kg. On FIG. 11C shows peak plasma levels during the first hour, peaking at about 30 ng/mL and remaining at about 400 pg/mL after three hours. Based on these results, it is reasonable to believe that with the intravenous administration of 0.5 μg/kg of feline IL-31 used in this feline model, the amount in the circulation will be much higher than the amount observed in the natural course of the disease in dogs and cats.

2. Example 2 Characterization and use of IL-31 mimotopes in vaccines and diagnostics

2.1. Canine IL-31 amino acid residues involved in binding to 15H05 antibody

As described in section 1.10 of this application, scanning mutagenesis of canine IL-31 protein was performed with a complete replacement of the amino acids indicated in FIG. 12. Each position in this region of IL-31 was individually replaced in the full-length protein with one of the other possible 19 amino acids and the binding of the 15H05 antibody was assessed by indirect ELISA. For those substitutions that did not affect binding, the ELISA signal was equivalent to (or higher than) that of the IL-31 protein, while for substitutions that affected antibody binding, the signal in the assay was lower (or absent). As shown in detail in FIG. 12, certain positions of canine IL-31 were tolerant of certain substitutions of the indicated amino acids (SEQ ID NO: 155; positions 124, 125, 129, and 132-135), while others were not (SEQ ID NO: 155; positions 126, 127, 128, 130 and 131). For comparison, the corresponding region of feline (SEQ ID NO: 157), equine (SEQ ID NO: 165) and human IL-31 (SEQ ID NO: 181) is shown. The results of mutations in canine IL-31 can be extrapolated to other species to generate homologous peptides. This precise mapping of positions in the epitope region on canine IL-31 allowed the construction of both linear and conformationally constrained peptides that mimic the binding site on the IL-31 protein recognized by the 15H05 antibody. These results, in combination with the feline IL-31 model described in Section 1.10 (FIG. 6B), show that the epitope recognized by mAb 15H05 is a region of consecutive amino acids that are contiguous in a random helix forming the cytokine's fourth helical domain. Exposure of this epitope allows mAb 15H05 to bind to both linear and conformationally constrained peptides, with greater affinity for the conformationally constrained form. The mutation results described above (section 1.12) further highlight the importance of the location of this epitope (and the 11E12 epitope previously described in US 8,790,651 to Bammert et al.) on the surface of the IL-31 protein for binding to the coreceptor complex.

2.2. Characterization of peptide mimetics. representing an epitope on IL-31 recognized by the 15H05 antibody

As mentioned earlier, the construction of peptide epitope mimetics (mimotopes) may find wide application as capture assays and as subunit vaccines to induce an appropriate targeted immune response to produce antibodies in animals directed to neutralize the epitope to IL-31. To achieve this goal, canine and feline peptides were constructed and their affinity for the ZTS-927 antibody and their ability to block the ability of the ZTS-361 antibody to suppress IL-31 mediated receptor signaling on feline FCFW4 cells was characterized. The construction of the ZTS-927 and ZTS-361 antibodies (both containing the mouse antibody 15H05 CDRs) is described in section 1.9 above. The ZTS-561 peptide contains the amino acid sequence N-TEISVPADTFERKSFILT-C, which corresponds to positions 121-138 of SEQ ID NO: 155 with the arginine (R) replaced by cysteine (C) at position number 132. The ZTS-561 peptide also includes N and C terminal cysteines to facilitate conjugation using free thiol groups. The ZTS-562 peptide contains the amino acid sequence N-EISVPADTFERKSF-C, which corresponds to positions 122-135 of SEQ ID NO: 155 with the substitution of arginine (R) for cysteine (C) at position number 132. The ZTS-562 peptide also includes N and C terminal cysteines to facilitate conjugation using free thiol groups. The ZTS-563 peptide contains the amino acid sequence N-AKVSMPADNFERKNFILT-C, which corresponds to positions 121-138 of SEQ ID NO: 157 with a threonine (T) replaced by alanine (A) at position number 138. The ZTS-563 peptide also includes N and C terminal cysteines to facilitate conjugation using free thiol groups. The ZTS-564 peptide contains the amino acid sequence N-TEISVPADTFERKSFILT-C, which corresponds to positions 121-138 of SEQ ID NO: 155. The ZTS-564 peptide also includes N and C terminal cysteines to facilitate conjugation using free thiol groups. A multi-step CLIPS process (Timmerman et al. J Mol Recognit. 2007; 20(5): 283-299) was used to identify and optimize these four paratope-binding peptides of mAb 15H05 (Pepscan, Lelystad Netherlands). To create immunogens, these four peptides (depicted in Fig. 13A) were independently conjugated to a carrier protein that is an inactive mutant (non-toxic) form of diphtheria toxin (CRM 197) using standard cross-linking chemistry. For affinity assessment, each peptide was independently immobilized on the surface of the biacore chip and KD was determined for the felinized anti-IL-31 mAb 15H05 (ZTS-927) (FIG. 13B). All four peptides bound ZTS-927 with affinities in the nanomolar range, indicating that they are a close mapping of the binding site on full-length IL-31. To evaluate the performance of each peptide in the FCWF-4 (feline macrophage-like cell) assay, conjugated or unconjugated peptides at various concentrations were incubated at 37°C for 1 hour with 0.2 μM (6.5 μg/ml) mAb ZTS-361 and then feline IL-31 was added. IC50 values were calculated by plotting the percentage effect (y-axis), defined as the ability of the peptide to bind and block the inhibition of mAb ZTS-361 of IL-31-mediated STAT3 phosphorylation in feline macrophages FCWF-4, as a function of increasing peptide concentrations (x-axis). The ZTS-564 peptide had reduced solution solubility, which likely resulted in inefficient conjugation, low epitope density, and low efficiency. The ZTS-561 peptide had low potency in the conjugated form but retained good potency in the unconjugated form (IC60 about 1.7 μg/mL). ZTS-562 and ZTS-563 showed excellent efficacy when unconjugated, with IC50 values of 1.046 and 1.742 µg/mL, respectively. After conjugation, their potency decreased by approximately 3-fold, with IC50 values for ZTS-562 and ZTS-563 being 3.024 µg/mL and 3.384 µg/mL, respectively (FIG. 13B). The ability of these peptides to block the high affinity binding of the ZTS-361 mAb to the IL-31 protein was very promising and provided additional evidence for their use as mimetics of the corresponding epitope on IL-31 (hereinafter referred to as mimotopes IL-31 15H05). These IL-31 15H05 mimotopes were further investigated for their use as immunogens to induce an immune response against IL-31.

2.3. Study design for determination of serum titers to IL-31 after immunization of beagle dogs with mimotopes of canine and feline IL-31 15H05 and full-length feline IL-31 protein

An immunogenicity assay was performed to assess the ability of CRM-197-conjugated IL-31 15H05 mimotopes to elicit an epitope-specific immune response directed to the appropriate region of the IL-31 protein to which the 15H05 antibody binds and neutralizes the ability of cytokines to activate the IL-31Ra:OSMR co-receptor . The study scheme is shown in Fig. fourteen.

Purebred male beagle dogs were injected subcutaneously with IL-31 15H05 conjugated mimotopes with adjuvant ZA-01. The diagram below shows the design of the experiment in each group. Control groups treated with CRM-197 adjuvanted with ZA-01 (T01) and CRM-197 conjugated feline IL-31 (SEQ ID NO: 159; Feline_IL-31_E_coli) were included, the corresponding nucleotide sequence for which is (SEQ ID NO: 160; Feline_IL-31_E_coli). Each adjuvanted mimotope or control was administered subcutaneously at a dose of 10 μg on days 0, 28 and 56 (0.5 ml of a 20 μg/ml solution). Serum blood was taken on days 0 (before administration), 7, 12, 28 (before administration), 35, 42, 49, 56 (before administration), 63, 70, 77 and 84. In addition, on days 35 and 84 approximately 40 ml of blood was taken from each animal in lithium heparinate tubes and peripheral blood mononuclear cells (PBMCs) were isolated using a standard method. PBMCs were subjected to cryopreservation until further studies of antigen-specific b-cells.

2.4. Serum titers obtained after vaccination of dogs with canine and feline mimotopes IL-31 15H05 and feline full-length IL-31 protein

Serum titers were assessed for each animal on each day of the study as indicated in section 2.3 above. Titers were determined using an indirect ELISA where full-length IL-31 protein was used for capture in each respective study. In each study group, sera were analyzed for binding to feline IL-31 protein, feline 15H05 mutant protein, and canine, equine, and human IL-31 proteins. The challenge was to understand the immune response elicited by feline IL-31 protein (SEQ ID NO: 159; Feline_IL-31_E_coli) or 15H05 peptide mimotopes against IL-31 from several species whose amino acid sequences share a number of identities (Fig. 1A ). Treatment group 2 (full-length feline IL-31 with CRM) presents an adaptive immune response to multiple epitopes spanning the entire protein sequence. The use of a full-length protein as an immunogen will result in antibodies that are both neutralizing and non-neutralizing with respect to the biological activity of IL-31. Previous work in mice describing the identification of antibodies that neutralize IL-31 signaling showed that the percentage of these antibodies is low and therefore the majority of the polyclonal response to the full-length protein will be of the non-neutralizing type (US8790651, Bammert, et al.). With respect to a vaccine, the formation of non-neutralizing antibodies to IL-31 may have adverse effects on safety and efficacy. Non-neutralizing antibodies can cause an increase in the amount of bioactive IL-31 in the bloodstream as a result of binding to complexes of cytokines and antibodies. These complexes may allow monomeric or aggregated forms of IL-31 to exist in the circulation by allowing the receptor-binding portion of IL-31 to interact with the IL-31:OSMR co-receptor. Increased pSTAT signaling as a result of increased circulating IL-31 will exacerbate itching in a disease such as atopic dermatitis (Gonzales et al. Vet Dermatol. 2013 Feb; 24(1):48-53. ell-2).

On FIG. 15A-E are graphs showing serum titers for each IL-31 protein, presented by treatment group, showing the response on each day the serum was sampled. Serum titers in response to IL-31 from different species were examined to understand the severity of the antibody cross-reactivity (CRAR) that may occur. The maximum dilution tested for each serum sample was 1:50,000, so when the titer exceeded this value, it was designated as 50,000. For clarity, a description of the drawings will be given below, showing the response to each IL-31 protein used in enzyme-linked immunosorbent assay. capture analysis, in separate exposure groups.

Given the high percentage of identity between homologues, it was expected that in dogs vaccinated with full-length feline IL-31 (T02), the resulting polyclonal sera would bind protein from several species. On FIG. 15A shows canine antibody titers that bind to feline IL-31. Analysis of the T02 group showed that after the third dose, a moderate and sustained humoral response to full-length feline IL-31 protein was formed, which was maintained until the end of the study on day 84. When examining the response in the T02 group to the 15H05 mutant feline protein (SEQ ID NO: 163; Feline_IL-31_15H05_mutant), a similar profile was observed, with even higher titers at days 63, 70 and 77 (FIG. 15B). On FIG. 15C shows titers in response to canine IL-31. By studying the titers in the T02 group, it is possible to assess the severity of CRAR for canine IL-31 protein in vaccinated dogs. Before the introduction of the third dose of feline IL-31 with CRM, no response was observed. After 3 doses on day 56, a transient CRAR was observed from days 63 to 77, returning to near baseline by day 84. The magnitude of the response to canine protein was similar to the response to feline, but its duration was shorter. Interestingly, the CRAR for equine and human IL-31 was negligible or negligible, respectively (Figures 15D and E, 63 and 84 days for human). Thus, the immune response of dogs to feline IL-31 with CRM was the most robust and long lasting compared to the response to feline IL-31 protein itself. The amino acid sequences of feline and canine IL-31 share 76% identity, which appears to be a high enough level for the development of CRAR on the canine protein. Equine and human IL-31 are 57% and 49% identical to feline IL-31, respectively, which causes only minor CRAR in the case of the human protein.

The IL-31 mimotope 15H05 ZTS-561 is a binding site on canine IL-31 recognized by the 15H05 antibody. The humoral response in dogs vaccinated with ZTS-561 with CRM is designated as T03 in FIG. 15 A-E. The task in this case was to assess the humoral response to this specific site on IL-31, which is known to be involved in the interaction of cytokines with its receptor. A focused immune response to a specific epitope means that antibodies directed to that region of the protein will neutralize its biological activity. ZTS-561 CRM is a conformationalally restricted 20-mer representing a portion of canine IL-31 protein recognized by antibodies with a CDR identical to mouse antibody 15H05 (SEQ ID NO: 67; MU_15H05_VH), the corresponding nucleotide sequence for which is (SEQ ID NO : 68; MU_15H05_VH) paired with VL (SEQ ID NO: 69; MU_15H05_VL) whose corresponding nucleotide sequence is (SEQ ID NO: 70; MU_15H05_VL). Mimotope ZTS-561 did not elicit a significant response to feline IL-31 throughout the study (FIG. 15A), including the 15H05 feline IL-31 mutant (FIG. 15B). In contrast, ZTS-561 with CRM elicited a very strong immune response to canine IL-31 protein in dogs from day 35 after the second injection, which persisted until the end of the study at day 84 (FIG. 15C). ZTS-561 induced CRAR on equine IL-31 was negligible, and only a negligible response was observed on human at day 56 of the study (Fig. 15D and E). Interestingly, even though feline and canine proteins have a high degree of identity in this region of the protein (Figure 1), after vaccination of dogs with canine 15H05 mimotope, a species-specific immune response was directed to canine IL-31 protein.

The ZTS-561 CRM is a truncated 16-mer version of ZTS-561 with conformational restriction, also representing a portion of the canine IL-31 protein recognized by antibodies with a CDR identical to the mouse antibody 15H05 (SEQ ID NO: 67; MU_15H05_VH), corresponding to the nucleotide sequence for which is (SEQ ID NO: 68; MU_15H05_VH), paired with VL (SEQ ID NO: 69; MU_15H05_VL), the corresponding nucleotide sequence for which is (SEQ ID NO: 70; MU_15H05_VL). The results of the dog response to ZTS-562 are shown as group T04 in FIG. 15 A-E. Interestingly, the CRAR induced by this shorter version was more pronounced, resulting in moderate titers against feline protein from days 35 to 70 (FIG. 15A). Some response was also observed for the mutant IL-31 15H05 protein between days 35 and 63 (FIG. 15B). The anti-canine IL-31 response elicited by this mimotope was outstanding, beginning on day 35 after the second dose and continuing until the end of the study on day 84. ZTS-562 CRM did not elicit CRAR on equine and human proteins, which was consistent with other results with the canine ZTS-561 CRM peptide.

ZTS-563 CRM is an 18-mer with restricted conformational freedom and is the only mimotope representing a portion of the feline IL-31 protein recognized by antibodies with a CDR identical to the mouse antibody 15H05 (SEQ ID NO: 67; MU_15H05_VH), the corresponding nucleotide sequence for which is (SEQ ID NO: 68; MU_15H05_VH) paired with VL (SEQ ID NO: 69; MU_15H05_VL) whose corresponding nucleotide sequence is (SEQ ID NO: 70; MU_15H05_VL). The results of the dogs response to ZTS-563 are shown as group T05 in FIG. 15 A-E. Feline mimotope (ZTS-563) elicited a feline IL-31-specific response in dogs, consistent with previous data demonstrating a species-specific response to canine mimotope. On FIG. 15A shows the anti-feline IL-31 antibody titer in response to ZTS-563 vaccination reaching over 1:50,000 on day 35 and remaining at that level until day 77, decreasing slightly on day 84. Comparing exposure group T05 (ZTS-563) in FIG. 15A and 15B, one can clearly see the difference between the titers in response to feline IL-31 and feline IL-31 mutant 15H05. The decrease in titers to the mutant protein over time (compared to the wild-type protein) indicates that a significant part of the immune response is directed to a very specific part of the protein, represented by the mimotope ZTS-563. Notably, the response against canine IL-31 was moderate to low, further confirming the species-specific response generated by the canine immune system to minor differences in the amino acid sequences of the two species. ZTS-563 is the only mimotope that elicited CRAR for the equine IL-31 protein in dogs when vaccinated. These results indicate that minor changes in the mimotope sequence can confer species specificity and can also elicit a cross-species immune response. Understanding these properties is useful for developing an IL-31 vaccine using this technology for use in one or more species.

Finally, ZTS-564 CRM is an 18-mer with limited conformational freedom, identical to ZTS-561, which used an alternative mT2b linker (FIG. 15A). The ZTS-564 CRM is a portion of the canine IL-31 protein recognized by antibodies with a CDR identical to the mouse antibody 15H05 (SEQ ID NO: 67; MU_15H05_VH), the corresponding nucleotide sequence for which is (SEQ ID NO: 68; MU_15H05_VH) paired with VL (SEQ ID NO: 69; MU_15H05_VL), the corresponding nucleotide sequence for which is (SEQ ID NO: 70; MU_15H05_VL). The results of the dog response to ZTS-564 are shown as group T06 in FIG. 15 A-E. This mimotope elicited virtually no response to feline IL-31 in dogs (FIGS. 15A and 15B), consistent with other observations. The anti-canine IL-31 response induced by ZTS-564 was very robust. On FIG. 15C shows all treatment groups in this study, T06 (ZTS-564) being the only group that developed an immune response against canine IL-31 after a single dose. Anti-canine IL-31 antibody titers obtained after the second and third doses corresponded to the maximum response during the study (greater than 1:50,000) from day 35 until the end of the study on day 84. No CRAR was observed for equine IL-31, however this mimotope produced the only consistent response to human IL-31 observed among the treatment groups. Notably, these minor differences in linker chemistry, and perhaps the more stringent conformational constraints imposed by the mT2b linker, provide a more targeted response against IL-31, potentially reducing the need for more frequent administration.

The data from this study show that peptide mimetics representing the binding site of the anti-IL-31 neutralizing antibody 15H05 are capable of eliciting an immune response in the animal, and this immune response is directed against the epitope recognized by the CDR of the 15H05 antibody. It follows from these data that further characterization of this antisera using recombinant IL-31 co-receptors can be used to determine the neutralizing fraction of IL-31 generated during this polyclonal response. These results also suggest that this approach may be useful as a vaccine against IL-31 mediated disorders such as atopic dermatitis.

2.5. Identification of IL-31 Neutralizing Antibodies Produced by Single B Cells Isolated from Plasma Cells of Beagle Dogs Immunized with Mimotopes IL-31 15H05

As described above, blood was collected on days 35 and 84 of the study in order to isolate PBMCs. Due to the persistence of the response against the epitope recognized by 15H05 (FIGS. 15A and 15B), PBMCs from a single T05 dog vaccinated with ZTS-563 with CRM (feline Mimotope) were used to further evaluate antibody-positive B cells. Activated memory B cells were screened using anti-canine IgG Fc antibody coupled to microbeads to detect antibody-secreting cells. At the same time, the ability of secreted IgG to bind wild-type feline IL-31 and bind to 15H05 mutant feline IL-31 was evaluated. Based on the results of this primary screening, 7 variants were selected from this PBMC cell population. Of these 7 variants, 3 did not bind to the 15H05 mutant, indicating that immunization with IL-31 15H05 mimotope ZTS-563 resulted in these B cells producing antibodies most accurately recognizing the 15H05 epitope (data not shown). After variable heavy and light chain sequencing of these 7 variants, recombinant all-canine versions were constructed, expressed in HEK cells and purified as described above. Rescreening these 7 recombinant canine IgGs, a single variant (ZIL1) remained that retained binding to the feline IL-31 protein (FIG. 4). In addition, ZIL1 binding to the 15H05 IL-31 mutant is reduced by ELISA and Biacore, indicating that this antibody binds to the same epitope region as the 15H05 antibody. Additional variants derived directly from canine B cells, described in section 1.6 and in FIG. 4 (ZIL8 - ZIL171) was obtained from dogs immunized with full-length feline IL-31, as well as from other tissue sources described above in this document. Only the ZIL1 antibody originated from PBMC after vaccination with the peptide mimotope.

An important aspect of these experiments is the ability to identify B cells secreting antibodies specific for the 15H05 epitope in the bloodstream of a dog after immunization with a peptide mimotope from the IL-31 protein. These results justify the use of a peptide that mimics an epitope region on IL-31 that is known to be related to the mode of inhibitory action of the 15H05 antibody. Since antibodies with CDRs derived from mAb 15H05 are able to prevent IL-31-mediated pruritus in vivo, these results further support the concept of immunization with such a mimetic designed to elicit an epitope-targeted immune response as a vaccine to prevent IL-31-mediated diseases such as like atopic dermatitis.

2.6. Use of Mimotope IL-31 15H05 as a Capture Reagent to Determine the Authenticity and Potency of 15H05 Antibody and Its Derivatives

The 15H05 IL-31 mimotope is a binding site on the IL-31 protein that is recognized by the CDR of the mouse 15H05 precursor mAb. As previously mentioned, it is desirable to have such a reagent for use in an ELISA (or other assay) format to monitor antibody production throughout production. It is expected that such described mimotopes will be useful when a batch of produced anti-IL-31 antibody is produced and the peptide mimotope can be used in the qualitative and quantitative analysis of the resulting antibody.

2.7. Use of mimotope IL-31 15H05 as a diagnostic tool to measure antibody levels or determine IL-31 levels in a host species

Furthermore, this document proposes the use of the disclosed peptide mimotope as a reagent in an assay to measure the amount of circulating antibody in a host following treatment with a therapeutic agent for an IL-31 mediated disorder such as atopic dermatitis. The animal's body fluid is added directly to the mimotope, which is bound to the solid surface, and then the appropriate secondary detection reagents are added to quantify the antibody. In addition, an assay scheme is provided in which mimotope is used to capture an antibody labeled for detection in the assay. This captured antibody will have a lower affinity for the attached mimotope than for native IL-31 circulating in the host species. In this embodiment, a fluid obtained from a host species is incubated with a labeled antibody: mimotope complex bound to a solid surface. IL-31 present in the test fluid obtained from the host species will have a higher affinity for the antibody, thus the labeled antibody will be released from the solid surface and can be removed during the washing steps. Thus, the level of IL-31 in the test fluid may be inversely proportional to the signal generated at the surface to which the mimotop is bound. It is contemplated that such an assay could be used as a diagnostic test in research or in the clinic to measure IL-31.

2.8. Serum titers against IL-31 following immunization of beagle dogs with IL-31 mimotopes and full-length canine IL-31 protein

A second serological study was performed using purebred neutered male beagle dogs as in the study design described in section 2.3, however this study compared different mimotopes. Purebred male beagle dogs were injected subcutaneously with IL-31 conjugated mimotopes adjuvanted with ZA-01. A control group was included that received only CRM-197 conjugated canine IL-31 (SEQ ID NO: 155; Canine IL-31) whose corresponding nucleotide sequence is (SEQ ID NO: 156; Canine IL-31) in ZA-01 (T01). Each adjuvanted mimotope or control was administered subcutaneously at a dose of 10 μg on days 0, 28 and 56 (0.5 ml of a 20 μg/ml solution). Blood for serum was taken on days -1.0 (before administration), 14, 28 (before administration), 42, 56 (before administration), 70 and 84. In addition, on days -1, 35 and 63, each animal was taken approximately 40 ml of blood into lithium heparinate tubes and processed to isolate PBMCs using a standard method. PBMCs were subjected to cryopreservation until further studies of antigen-specific b-cells.

Treatment group 1 (full-length canine IL-31 with CRM) presented an adaptive immune response to multiple epitopes spanning the entire protein sequence, the intent was the same as with the full-length feline protein described in section 2.4. On FIG. 16A is a table listing the treatment groups studied. In addition to the full length protein described for T01, two mimotopes representing the 15H05 epitope in the dog (T02, ZTS-420) and human (T03, ZTS-421) were used. ZTS-420 is similar to ZTS-561 previously described in a feline serology study, however, ZTS-420 is restricted in conformational freedom by a disulfide bond formed by cysteines at the N- and C-terminus of the peptide, in contrast to ZTS-561, whose conformational freedom is restricted by a linker mT2a. ZTS-421 is a homologous region of the human IL-31 protein (SEQ ID NO: 181; HumanlL-31) whose conformational freedom is limited by N- and C-terminus binding to the mT2b linker as shown in FIG. 13A. References to key amino acid sequences involved in the binding of the 15H05 antibody can be found in FIG. 12. A fourth group was included to examine the immunogenic potential of the key antibody binding region on canine IL-31 described previously using two antibodies known to neutralize IL-31-mediated pSTAT signaling and IL-31-mediated pruritus in dogs ( US8790651, Bammert, et al.). This region on feline IL-31 is highlighted in the homology model shown in FIG. 6B. The adopted model of IL-31 is a cytokine with four antiparallel helical domains forming an up-down topology. In the following, a description of the structure of the IL-31 protein will be given for these four helices in canine IL-31 (SEQ ID NO: 155; Canine IL-31), the corresponding nucleotide sequence for which is (SEQ ID NO: 156; Canine_IL-31), taking into account the corresponding positions on the homologous IL-31 proteins of other biological species (Fig. 1). Helix A consists of a sequence of about 33 to 59 amino acids, helix B consists of a sequence of about 83 to 98 amino acids, helix C consists of a sequence of about 101 to 114 amino acids, and helix D consists of a sequence of about 129 to 156 amino acids. Approximately between 97 and 101 amino acids is the loop region. The loop following helix A is located at approximately amino acids 57 to 62, and the loop preceding helix D is approximately amino acids 126 to 129. Any sequence in between that lacks the predicted secondary structure will be referred to as a random helix. Exposure group 4 received ZTS-766, a mimotope representing the B and C helices of canine IL-31, which included an N-terminal cysteine residue to facilitate binding to the CRM-197 carrier protein. The peptide sequence alignment of this region of the IL-31 protein is shown in FIG. 16B comparing canine, feline, equine and human proteins with their respective sequence identification numbers and amino acid positions.

Titers were determined using an indirect ELISA where full-length IL-31 protein was used for capture in each respective study. In each study group, serum was analyzed for binding to canine and human IL-31 proteins (FIGS. 17A and 17B, respectively). Dogs vaccinated with CRM-197 conjugated to full-length canine IL-31 (T01) protein exhibited a modest increase in titer on day 42 following a second dose on day 28. In this group, there was a decrease in titer during the study even after the third dose on day 56. Given the overall response to all epitopes of the IL-31 protein (both neutralizing and non-neutralizing) in combination with low titers, it is unlikely that the full-length IL-31 protein is a promising candidate for vaccine development. Group 2 in this study received ZTS-420, which is a 15H05 canine mimotope similar to ZTS-561 described in section 2.4, however the conformational freedom of ZTS-420 is limited by a disulfide bond between cysteines added at the N- and C-terminus of the peptide, in in contrast to ZTS-561, whose conformational freedom is limited by the mT2a linker. This mimotope failed to elicit a sustained immune response in contrast to the mT2a-restricted form (see FIGS. 17A and 15C). There was a moderate increase in titer on day 84 after the second dose on day 56. It is possible that disulfide bond cyclization is incorrect or altered during conjugation with CRM-197, resulting in suboptimal immunogen presentation to canine immune cells. Group 4, treated with ZTS-766, representing the B and C helices of canine IL-31, showed the most sustained response, occurring after the second dose on day 28 and increasing to 84 days before the end of the study. Given the ability of antibodies recognizing this previously described sequence to neutralize IL-31, this mimotope is a promising candidate for a vaccine to prevent IL-31 mediated diseases. Treatment group 3 received ZTS-421, which is a 15H05 epitope, which used the sequence of human IL-31 in this mimotope region. Interestingly, none of the dogs vaccinated with this mimotope developed a response against canine IL-31 protein (data not shown), however, an immune response to human IL-31 protein was observed after the second and third doses (Fig. 17B). . Given the sequence similarity between the 15H05 mimotope key epitope region (FIG. 12), this specificity of the canine response against human IL-31 is excellent.

2.9. Serum titres against IL-31 following immunization of laboratory cats with feline and equine IL-31 mimotopes and full-length feline IL-31 protein

A second serological study was performed using laboratory cats as in the study design described in Section 2.3, however this study compared feline and equine mimotopes. Laboratory cats were injected subcutaneously with IL-31 conjugated mimotopes with a mixture containing the Bay R1005 glycolipid adjuvant (N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyl dodecanoylamide hydroacetate) as well as CpG oligonucleotides. A control group treated with CRM-197 conjugated feline IL-31 (SEQ ID NO: 157; Feline_IL-31_wildtype) whose corresponding nucleotide sequence is (SEQ ID NO: 158; Feline_IL-31_wildtype) in an adjuvant mixture including a glycolipid adjuvant was included. Bay R1005 (N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyl dodecanoylamide hydroacetate), as well as CpG oligonucleotides. (T01). Each adjuvanted mimotope or control was administered subcutaneously at a dose of 10 μg on days 0, 28 and 56 (0.5 ml of a 20 μg/ml solution). Serum blood was taken on days -14, 0 (before administration), 28 (before administration), 42, 56 (before administration), 70 and 84. In addition, on days 35 and 63, approximately 40 ml of blood was collected from each animal per day. tubes with lithium heparinate and processed to isolate PBMC using a standard method. PBMCs were subjected to cryopreservation until further studies of antigen-specific B cells. On FIG. 18A shows the treatment groups studied. ZTS-563 (T02) is described in this document in section 2.4 as an immunogen that was used in a previous serological study in dogs. ZTS-563 is a 15H05 mimotope conformationalally constrained by mT2a conjugated to CRM-197. T03 (ZTS-418) is a 15H05 mimotope with an equine sequence (for comparison, see the homologous canine version of ZTS-420 in Figure 16A). Exposure group 4 received ZTS-423, a mimotope peptide representing the BC helix described in section 2.8, with a feline IL-31 sequence. Treatment group 5 received ZTS-422, a 15H05 feline mimotope with an aminohexanoic acid (Ahx) mT2b linker. On FIG. 18B shows results of serum antibody titers in exposure groups T01, T02, T04, and T05 (T03 lacked CRAR for feline IL-31, data not shown) at days -14, 42, and 84 for full-length feline IL-31 obtained using indirect ELISA. Again, the conjugated form of full-length IL-31 protein (T01) showed the weakest humoral response in cats, with full-length IL-31 titers never exceeding 1:20,000 throughout the study. The response in the T02 group (ZTS-563) was moderate throughout the study, showing a dose-dependent increase up to 84 days, indicating that the 15H05 feline epitope presented in this way may be a suitable vaccine. Mean titers in cats in response to full-length feline IL-31 protein after three doses of ZTS-423 (T04) increase in a dose-dependent manner to more than 1:100,000 after the second and third doses, indicating an outstanding immune response to a very significant epitope region. ZTS-422 (T05), a 15H05 mimotope with an AhX mT2b linker, also shows a sustained immune response in cats with titers greater than 1:100,000 after the second and third doses. The 15H05 epitope in this form is clearly an appropriate representation of this region of the IL-31 protein and is a promising mimotope for a vaccine that neutralizes IL-31 activity in vivo.

2.10. Aspects of sequence and structure to consider when creating mimotopes. intended for use as vaccines

This document describes several peptide mappings of IL-31 protein epitopes (mimotopes) with unique sequences corresponding to the amino acids of the species from which they are derived. The ultimate goal of a vaccine is to replicate epitopes so that the immune system recognizes them and develops a sustained and specific response. The creation of a vaccine is facilitated by the addition of carrier proteins such as CRM-197 and the addition of adjuvants, but is not limited to this. This document describes examples of epitopes that have been identified based on the properties of the antibodies to which they bind. Key epitopes are those regions of the IL-31 protein that, when bound to an antibody, are unable to further interact with the IL-31RA:OSMR receptor complex and therefore are unable to elicit a pSTAT signaling response in cell culture or in vivo. Thus, blockade of signaling mediated by the IL-31 receptor is an approach to the prevention and/or treatment of disorders mediated by IL-31, such as atopic dermatitis.

Mapping of antibody binding sites on proteins using mutational methods is an effective way to identify key residues involved in antigen recognition by an antibody, as previously described for IL-31 in US8790651, Bammert, et al. Based on these data, it was possible to design canine and feline mimotope BC coils, which are described herein as effective immunogens that elicit robust anti-IL-31 responses in dogs and cats. Another method using the canine IL-31 GST fusion protein was recently described for mapping the anti-canine IL-31 antibody M14 (WO 2018/156367 (Kindred Biosciences, Inc). These authors sought to determine the minimum epitope sequence recognized by the M14 antibody, consisting of amino acids PSDX ₁ X ₂ KI (SEQ ID NO 155, amino acids 34-40) where X is any amino acid A description of this sequence compared to homologous IL-31 species can be found in Fig. 19A Further description including flanking sequence surrounding the above is shown in Fig. 19B. After identifying the minimal binding fragment indicated in Fig. 19B (amino acids 34-42 highlighted in gray box), the authors made substitutions for alanine at each position using this peptide fragment fused to GST. these data, the above minimal binding fragment M14 was described.The fundamental disadvantage of this approach is that the properties of the described binding fragment are determined by its structure within the GST fusion protein. Without wishing to be bound by a single theory, it is believed that the amino acid sequence recognized by the M14 antibody is part of an ordered alpha helix domain, described herein as the A helix. Alpha helices exist in peptides and proteins, which are maintained by hydrogen bonds between the oxygen of the carbonyl group and backbone amino nitrogen (Corey-Pauling Rule, Dictionary of Chemistry, 2008), but are not limited thereto. It is believed that the minimal binding fragment described for the M14 antibody is not an adequate description of the epitope, since there is no evidence of its binding properties in the absence of a GST framework. Moreover, the sequence surrounding and including the specified M14 epitope contains a large number of non-polar amino acids (I, L, V, P, G, A, M) (Fig. 19B). The physical properties of these amino acids will result in the peptide being insoluble in water and having a disordered secondary structure, with no polar or charged amino acids embedded between them. Thus, this document assumes that the properties of the minimal binding fragment of the M14 antibody described in WO 2018/156367 (Kindred Biosciences, Inc.) depend on the fusion with GST, and not inherent in the peptide itself.

Several peptide mappings of IL-31 epitopes are described herein, their properties inherent in the peptides themselves, existing independently, and not as part of a fusion protein. This is illustrated in Section 2.2 (FIG. 13B) showing the binding and inhibitory properties of 15H05 class mimotopes in both conjugated and unconjugated form. In addition to the characteristics of the secondary structure of peptides, another key aspect in the design of vaccines, necessary for proper presentation on the surface of T cells, is the primary amino acid sequence. Appropriate amino acid sequences, in combination with a carrier protein having B and T cell epitopes, will elicit an immune response directed at key regions of the IL-31 protein. Many regions of IL-31 are described herein as being suitable for generating a targeted immune response in dogs and cats. The success of a mimotope vaccine depends on the factors described herein and is ultimately determined by the effectiveness of the in vivo response. However, based on the examination of several epitope regions on IL-31 described herein, it is expected that there may be other such epitopes that may be suitable vaccine mimotopes. The 15H05 antibody recognizes the loop preceding the D helix shown in FIG. 6B as site 2. Other loops of the protein are also thought to represent antibody-accessible epitopes. For example, the loop formed by the approach of helix A to the sequence of random helix behind it has a similar position and structure to the 15H05 loop. This loop AB is described in Fig. 20, which compares the primary amino acid sequences of several species. While not wishing to be limited to this example as the only one, it is believed that other similar regions of the protein may share immunogenic characteristics.

2.11. Serum titers to mimotopes of equine IL-31 after vaccination of mice with full-length equine IL-31 protein

Mice were immunized with full-length equine IL-31 (SEQ ID NO: 165; Equine_IL-31) whose corresponding nucleotide sequence is (SEQ ID NO: 166; Canine_IL-31) conjugated to CRM-197, similar to the method described in section 1.6 of this descriptions. Biotin conjugated peptides representing the three epitope regions described herein were designed for biolayer interferometry binding studies (Octet, ForteBio). These peptides are described in FIG. 21A. Each peptide contains an N-terminal biotin with a three-amino acid spacer sequence (GSG) labeled in bold and underlined in the figure. The figure also indicates the corresponding position number in the amino acid sequence of SEQ ID NO 165. The 15H05 mimotope includes two terminal cysteine residues (also in bold and underlined) to facilitate disulfide cyclization. On FIG. 21B shows the results of biolayer interferometry where the peptides described in FIG. 21A was immobilized on a streptavidin-coated sensor surface and then used to test several dilutions of mouse anti-equine IL-31 antibodies or control mouse sera. Control mouse sera were obtained from mice vaccinated with the foreign protein. The response, described herein as the signal amplitude after 120 seconds of antiserum binding, is shown on the y-axis of FIG. Based on these data, it is possible to assess the immune response that develops as a result of the presentation of epitopes of the full-length equine IL-31 protein. In addition, the ability of these IL-31 mimotopes to be recognized during such immune responses can be assessed by binding. These data indicate that all three mimotope peptides described (15H05, BC-helix and A-helix) are recognized as proper immunogens by in vivo processing and presentation of equine IL-31 protein. When control serum was bound to each mimotope, minimal signal was observed, except for helix A, which gave some dilution dependent signal. Just as the presentation of the mimotopes described herein elicits immune responses to the full-length protein, this experiment confirmed the development of an immune response to mimotopes upon immunization with the protein by reciprocally testing these epitopes.

--->

SEQUENCE LISTING

<110> Zoetis Services LLC

Bammert, Gary F.

Dunham, Steven A.

<120> PEPTIDE VACCINES AGAINST INTERLEUKIN-31

<130> ZP000229A

<150> US 62/643,921

<151> 2018-03-16

<160> 203

<170>PatentIn version 3.5

<210> 1

<211> 5

<212> PRT

<213> Mus musculus

<400> 1

Ser Tyr Thr Ile His

fifteen

<210> 2

<211> 17

<212> PRT

<213> Mus musculus

<400> 2

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

1 5 10 15

asp

<210> 3

<211> 13

<212> PRT

<213> Mus musculus

<400> 3

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

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Mus musculus

<400> 4

Arg Ala Ser Gln Gly Ile Ser Ile Trp Leu Ser

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Mus musculus

<400> 5

Lys Ala Ser Asn Leu His Ile

fifteen

<210> 6

<211> 9

<212> PRT

<213> Mus musculus

<400> 6

Leu Gln Ser Gln Thr Tyr Pro Leu Thr

fifteen

<210> 7

<211> 5

<212> PRT

<213> Mus musculus

<400> 7

Tyr Tyr Asp Ile Asn

fifteen

<210> 8

<211> 17

<212> PRT

<213> Mus musculus

<400> 8

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

1 5 10 15

gly

<210> 9

<211> 14

<212> PRT

<213> Mus musculus

<400> 9

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

1 5 10

<210> 10

<211> 15

<212> PRT

<213> Mus musculus

<400> 10

Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met His

1 5 10 15

<210> 11

<211> 7

<212> PRT

<213> Mus musculus

<400> 11

Arg Ala Ser Asn Leu Glu Ser

fifteen

<210> 12

<211> 9

<212> PRT

<213> Mus musculus

<400> 12

Gln Gln Ser Asn Lys Asp Pro Leu Thr

fifteen

<210> 13

<211> 5

<212> PRT

<213> Canis familiaris

<400> 13

Ser Tyr Gly Met Ser

fifteen

<210> 14

<211> 17

<212> PRT

<213> Canis familiaris

<400> 14

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

1 5 10 15

gly

<210> 15

<211> 15

<212> PRT

<213> Canis familiaris

<400> 15

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

1 5 10 15

<210> 16

<211> 13

<212> PRT

<213> Canis familiaris

<400> 16

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

1 5 10

<210> 17

<211> 7

<212> PRT

<213> Canis familiaris

<400> 17

Ser Asp Gly Asn Arg Pro Ser

fifteen

<210> 18

<211> 11

<212> PRT

<213> Canis familiaris

<400> 18

Gln Ser Phe Asp Thr Thr Leu Asp Ala Tyr Val

1 5 10

<210> 19

<211> 5

<212> PRT

<213> Canis familiaris

<400> 19

Asp Tyr Ala Met Ser

fifteen

<210> 20

<211> 17

<212> PRT

<213> Canis familiaris

<400> 20

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

1 5 10 15

gly

<210> 21

<211> 8

<212> PRT

<213> Canis familiaris

<400> 21

Gly Phe Pro Gly Ser Phe Glu His

fifteen

<210> 22

<211> 13

<212> PRT

<213> Canis familiaris

<400> 22

Thr Gly Ser Ser Ser Asn Ile Gly Ser Gly Tyr Val Gly

1 5 10

<210> 23

<211> 7

<212> PRT

<213> Canis familiaris

<400> 23

Tyr Asn Ser Asp Arg Pro Ser

fifteen

<210> 24

<211> 10

<212> PRT

<213> Canis familiaris

<400> 24

Ser Val Tyr Asp Arg Thr Phe Asn Ala Val

1 5 10

<210> 25

<211> 5

<212> PRT

<213> Canis familiaris

<400> 25

Ser Tyr Asp Met Thr

fifteen

<210> 26

<211> 17

<212> PRT

<213> Canis familiaris

<400> 26

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

1 5 10 15

gly

<210> 27

<211> 9

<212> PRT

<213> Canis familiaris

<400> 27

Leu Gly Val Arg Asp Gly Leu Ser Val

fifteen

<210> 28

<211> 11

<212> PRT

<213> Canis familiaris

<400> 28

Ser Gly Glu Ser Leu Asn Glu Tyr Tyr Thr Gln

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Canis familiaris

<400> 29

Arg Asp Thr Glu Arg Pro Ser

fifteen

<210> 30

<211> 10

<212> PRT

<213> Canis familiaris

<400> 30

Glu Ser Ala Val Asp Thr Gly Thr Leu Val

1 5 10

<210> 31

<211> 5

<212> PRT

<213> Canis familiaris

<400> 31

Thr Tyr Val Met Asn

fifteen

<210> 32

<211> 17

<212> PRT

<213> Canis familiaris

<400> 32

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

1 5 10 15

gly

<210> 33

<211> 8

<212> PRT

<213> Canis familiaris

<400> 33

Ser Met Val Gly Pro Phe Asp Tyr

fifteen

<210> 34

<211> 11

<212> PRT

<213> Canis familiaris

<400> 34

Ser Gly Glu Ser Leu Ser Asn Tyr Tyr Ala Gln

1 5 10

<210> 35

<211> 7

<212> PRT

<213> Canis familiaris

<400> 35

Lys Asp Thr Glu Arg Pro Ser

fifteen

<210> 36

<211> 10

<212> PRT

<213> Canis familiaris

<400> 36

Glu Ser Ala Val Ser Ser Asp Thr Ile Val

1 5 10

<210> 37

<211> 5

<212> PRT

<213> Canis familiaris

<400> 37

Ser Tyr Ala Met Lys

fifteen

<210> 38

<211> 17

<212> PRT

<213> Canis familiaris

<400> 38

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

1 5 10 15

gly

<210> 39

<211> 15

<212> PRT

<213> Canis familiaris

<400> 39

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

1 5 10 15

<210> 40

<211> 11

<212> PRT

<213> Canis familiaris

<400> 40

Ser Gly Glu Ser Leu Asn Lys Tyr Tyr Ala Gln

1 5 10

<210> 41

<211> 7

<212> PRT

<213> Canis familiaris

<400> 41

Lys Asp Thr Glu Arg Pro Ser

fifteen

<210> 42

<211> 10

<212> PRT

<213> Canis familiaris

<400> 42

Glu Ser Ala Val Ser Ser Glu Thr Asn Val

1 5 10

<210> 43

<211> 5

<212> PRT

<213> Canis familiaris

<400> 43

Thr Tyr Phe Met Ser

fifteen

<210> 44

<211> 17

<212> PRT

<213> Canis familiaris

<400> 44

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

1 5 10 15

gly

<210> 45

<211> 7

<212> PRT

<213> Canis familiaris

<400> 45

Phe Trp Arg Ala Phe Asn Asp

fifteen

<210> 46

<211> 14

<212> PRT

<213> Canis familiaris

<400> 46

Gly Leu Asn Ser Gly Ser Val Ser Thr Ser Asn Tyr Pro Gly

1 5 10

<210> 47

<211> 7

<212> PRT

<213> Canis familiaris

<400> 47

Asp Thr Gly Ser Arg Pro Ser

fifteen

<210> 48

<211> 10

<212> PRT

<213> Canis familiaris

<400> 48

Ser Leu Tyr Thr Asp Ser Asp Ile Leu Val

1 5 10

<210> 49

<211> 5

<212> PRT

<213> Canis familiaris

<400> 49

Asp Arg Gly Met Ser

fifteen

<210> 50

<211> 17

<212> PRT

<213> Canis familiaris

<400> 50

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

1 5 10 15

gly

<210> 51

<211> 8

<212> PRT

<213> Canis familiaris

<400> 51

Trp Asp Gly Ser Ser Phe Asp Tyr

fifteen

<210> 52

<211> 16

<212> PRT

<213> Canis familiaris

<400> 52

Lys Ala Ser Gln Ser Leu Leu His Ser Asp Gly Asn Thr Tyr Leu Asp

1 5 10 15

<210> 53

<211> 7

<212> PRT

<213> Canis familiaris

<400> 53

Lys Val Ser Asn Arg Asp Pro

fifteen

<210> 54

<211> 9

<212> PRT

<213> Canis familiaris

<400> 54

Met Gln Ala Ile His Phe Pro Leu Thr

fifteen

<210> 55

<211> 5

<212> PRT

<213> Canis familiaris

<400> 55

Ser Tyr Val Met Thr

fifteen

<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

fifteen

<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

fifteen

<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

fifteen

<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

fifteen

<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> Heavy chain variable region sequence

felinized monoclonal antibody 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

heavy chain variable region of a felinized monoclonal

antibodies 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> Light chain variable region sequence

felinized monoclonal antibody 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

light chain variable region of a felinized monoclonal

antibodies 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> Light chain variable region sequence

felinized monoclonal antibody 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> Variable region encoding nucleotide sequence

light chain of a felinized monoclonal antibody 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> Light chain variable region sequence

felinized monoclonal antibody 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> Variable region encoding nucleotide sequence

light chain of a felinized monoclonal antibody 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

tccttggaac cggaagacgc agccacatac tattgccagc agagtaacaa ggaccctttg 300

acttttggcc agggtacc 318

<210> 119

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain variable region sequence

felinized monoclonal antibody 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 the variable region

light chain of a felinized monoclonal antibody 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> Heavy chain variable region sequence

felinized monoclonal antibody 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> Variable region encoding nucleotide sequence

heavy chain of a felinized monoclonal antibody 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> Heavy chain variable region sequence 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

heavy chain variable region of a felinized monoclonal

antibodies 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> Heavy chain variable region sequence 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

heavy chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 chain variable region sequence

monoclonal antibodies 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

light chain variable region of a felinized monoclonal

antibodies 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 representing feline IL-31

wild type with C-terminal histidine 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 representing the feline IL-31 gene

wild type with encoded C-terminal histidine 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 representing feline IL-31 protein

with N-terminal histidine 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 representing the feline IL-31 gene

with 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 representing the mutant feline

IL-31 11E12 protein with C-terminal histidine 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 representing the gene of the mutant

feline IL-31 11E12 encoded C-terminal histidine 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 representing the mutant feline

IL-31 15H05 protein with C-terminal histidine 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 sequencec representing the gene of the mutant

Feline IL-31 15H05 encoded C-terminal histidine 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 histidine 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 encoded

C-terminal histidine 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 extracellular domain of feline OSMR,

fused with Fc human IgG1

<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 Fc human IgG1

<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> Heavy chain amino acid sequence of feline origin,

genetically modified to alter the effector function of an antibody

<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> Nucleotide sequence encoding the feline heavy chain

origin, genetically modified to change the effector

antibody functions

<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> Feline kappa light chain amino acid sequence

origin, genetically modified to knock out 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 kappa light chain

of feline origin, genetically modified for knockout

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 light chain variable region sequence

monoclonal antibodies 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> Variable region encoding nucleotide sequence

light chain caninized monoclonal antibody from

Mus musculus 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 light chain variable region sequence

monoclonal antibodies 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 the variable region of the lung

caninized monoclonal antibody chains from Mus musculus 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> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Canine mimotope sequence IL-31 15H05

<400> 186

Ser Val Pro Ala Asp Thr Phe Glu Cys Lys Ser Phe

1 5 10

<210> 187

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Canine mimotope sequence IL-31 15H05

<400> 187

Ser Val Pro Ala Asp Thr Phe Glu Arg Lys Ser Phe

1 5 10

<210> 188

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Mimotope feline sequence IL-31 15H05

<400> 188

Ser Met Pro Ala Asp Asn Phe Glu Arg Lys Asn Phe

1 5 10

<210> 189

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Equine mimotop sequence IL-31 15H05

<400> 189

Ser Met Pro Thr Asp Asn Phe Glu Arg Lys Arg Phe

1 5 10

<210> 190

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IL-31 mimotope sequence 15H05

<400> 190

Ser Val Pro Thr Asp Thr His Glu Cys Lys Arg Phe

1 5 10

<210> 191

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IL-31 mimotope sequence 15H05

<400> 191

Ser Val Pro Thr Asp Thr His Glu Arg Lys Arg Phe

1 5 10

<210> 192

<211> 33

<212> PRT

<213> Artificial Sequence

<220>

<223> Canine IL-31 helix mimotope sequence

<400> 192

Asn Ser Ser Ala Ile Leu Pro Tyr Phe Arg Ala Ile Arg Pro Leu Ser

1 5 10 15

Asp Lys Asn Ile Ile Asp Lys Ile Ile Glu Gln Leu Asp Lys Leu Lys

20 25 30

Phe

<210> 193

<211> 33

<212> PRT

<213> Artificial Sequence

<220>

<223> Feline IL-31 helix mimotope sequence

<400> 193

Asn Gly Ser Ala Ile Leu Pro Tyr Phe Arg Ala Ile Arg Pro Leu Ser

1 5 10 15

Asp Lys Asn Thr Ile Asp Lys Ile Ile Glu Gln Leu Asp Lys Leu Lys

20 25 30

Phe

<210> 194

<211> 33

<212> PRT

<213> Artificial Sequence

<220>

<223> Equine IL-31 helix mimotope sequence

<400> 194

Asn Ser Ser Ala Ile Leu Pro Tyr Phe Lys Ala Ile Ser Pro Ser Leu

1 5 10 15

Asn Asn Asp Lys Ser Leu Tyr Ile Ile Glu Gln Leu Asp Lys Leu Asn

20 25 30

Phe

<210> 195

<211> 33

<212> PRT

<213> Artificial Sequence

<220>

<223> Mimotope sequence of human IL-31 BC helix region

<400> 195

His Ser Pro Ala Ile Arg Ala Tyr Leu Lys Thr Ile Arg Gln Leu Asp

1 5 10 15

Asn Lys Ser Val Ile Asp Glu Ile Ile Glu His Leu Asp Lys Leu Ile

20 25 30

Phe

<210> 196

<211> 24

<212> PRT

<213> Artificial Sequence

<220>

<223> Canine IL-31 helix mimotope A region sequence

<400> 196

Ala Pro Thr His Gln Leu Pro Ser Asp Val Arg Lys Ile Ile Leu

1 5 10 15

Glu Leu Gln Pro Leu Ser Arg Gly

twenty

<210> 197

<211> 24

<212> PRT

<213> Artificial Sequence

<220>

<223> Feline IL-31 helix region mimotope sequence A

<400> 197

Ala Pro Ala His Arg Leu Gln Pro Ser Asp Ile Arg Lys Ile Ile Leu

1 5 10 15

Glu Leu Arg Pro Met Ser Lys Gly

twenty

<210> 198

<211> 24

<212> PRT

<213> Artificial Sequence

<220>

<223> Equine IL-31 helix region mitope sequence

<400> 198

Gly Pro Ile Tyr Gln Leu Gln Pro Lys Glu Ile Gln Ala Ile Ile Val

1 5 10 15

Glu Leu Gln Asn Leu Ser Lys Lys

twenty

<210> 199

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> Mimotope sequence of helix A region of human IL-31

<400> 199

Leu Pro Val Arg Leu Leu Arg Pro Ser Asp Asp Val Gln Lys Ile Val

1 5 10 15

Glu Glu Leu Gln Ser Leu Ser Lys Met

20 25

<210> 200

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Canine IL-31 AB helix mimotope sequence

<400> 200

Thr Gly Val Pro Glu Ser

fifteen

<210> 201

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Mimotope sequence of AB helix region of feline IL-31

<400> 201

Ile Gly Leu Pro Glu Ser

fifteen

<210> 202

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Equine IL-31 helix mimotope sequence

<400> 202

Lys Gly Val Gln Lys Phe

fifteen

<210> 203

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Mimotope sequence of human IL-31 AB helix region

<400> 203

Lys Gly Val Leu Val Ser

fifteen

<---

Claims (40)

1. A vaccine composition for immunizing and/or protecting a mammal - dog, cat or horse - from an IL-31 mediated disease, wherein said composition contains: a combination of a carrier polypeptide and at least one IL-31 mimotope peptide, and an adjuvant, wherein the mimotope of the IL-31 peptide is from about 5 to about 40 amino acids in length and is selected from the group consisting of: 1) a canine IL-31 mimotope which is or contains as part of the amino acid sequences SVPADTFECKSF (SEQ ID NO: 186), SVPADTFERKSF (SEQ ID NO: 187), NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ ID NO: 192), APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO: 200) or variants thereof that retain binding to an anti-IL-31 agent; 2) mimotope feline IL-31 which is or contains as part of the amino acid sequences SMPADNFERKNF (SEQ ID NO: 188), NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ ID NO: 193), APAHRLQPSDIRKIILELRPMSKG (SEQ ID NO: 197), IGLPES (SEQ ID NO: 201) or variants thereof that retain binding to an anti-IL-31 agent; 3) an equine IL-31 mimotope which is or contains as part of the amino acid sequences SMPTDNFERKRF (SEQ ID NO: 189), NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ ID NO: 194), GPIYQLQPKEIQAIIVELQNLS KK (SEQ ID NO: 198), KGVQKF (SEQ ID NO: 202) or variants thereof that retain binding to an anti-IL-31 agent; and 4) a human IL-31 mimotope which is or contains as part of the amino acid sequences SVPTDTHECKRF (SEQ ID NO: 190), SVPTDTHERKRF (SEQ ID NO: 191), HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195), LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203), or variants thereof that retain binding to an anti-IL-31 agent. 2. The vaccine composition of claim 1, wherein the mimotope binds to an anti-IL-31 antibody or antigen-binding portion thereof that specifically binds to a region of the mammalian IL-31 protein involved in the interaction of the IL-31 protein with its co-receptor. 3. The vaccine composition according to claim 2, wherein the binding of said antibody to said region is affected by mutations in the region of the 15H05 binding epitope selected from the group consisting of: a) the region between amino acid residues 124 and 135 of the feline IL-31 sequence shown in SEQ ID NO: 157 (wild-type feline IL-31); b) the region between amino acid residues 124 and 135 of the canine IL-31 sequence represented by SEQ ID NO: 155 (canine IL-31); and c) the region between amino acid residues 118 and 129 of the equine IL-31 sequence shown in SEQ ID NO: 165 (equine IL-31). 4. The vaccine composition of claim 2, wherein the mimotope binds to an anti-IL-31 antibody or antigen-binding portion thereof, comprising at least one of the following sequence combinations of complementarity determining regions (CDRs): 1) 15H05 antibody: heavy chain variable region (VH) CDR1 with SYTIH sequence (SEQ ID NO: 1), VH-CDR2 with NINPTSGYTENNQRFKD sequence (SEQ ID NO: 2), VH-CDR3 with WGFKYDGEWSFDV sequence (SEQ ID NO: 3 ), a light chain variable region (VL) CDR1 with the sequence RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 with the sequence KASNLHI (SEQ ID NO: 5) and VL-CDR3 with the sequence LQSQTYPLT (SEQ ID NO: 6); 2) ZIL1 antibody: Heavy chain variable region (VH) CDR1 with the sequence SYGMS (SEQ ID NO: 13), VH-CDR2 with the sequence HINSGGSSTYYADAVKG (SEQ ID NO: 14), VH-CDR3 with the sequence VYTTLAAFWTDNFDY (SEQ ID NO: 15 ), a light chain variable region (VL) CDR1 with the sequence SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 with the sequence SDGNRPS (SEQ ID NO: 17) and VL-CDR3 with the sequence QSFDTTLDAYV (SEQ ID NO: 18); 3) ZIL8 antibody: VH-CDR1 with DYAMS sequence (SEQ ID NO: 19), VH-CDR2 with GIDSVGSGTSYADAVKG sequence (SEQ ID NO: 20), VH-CDR3 with GFPGSFEH sequence (SEQ ID NO: 21), VL-CDR1 with the sequence TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 with the sequence YNSDRPS (SEQ ID NO: 23), VL-CDR3 with the sequence SVYDRTFNAV (SEQ ID NO: 24); 4) ZIL9 antibody: VH-CDR1 with the sequence SYDMT (SEQ ID NO: 25), VH-CDR2 with the sequence DVNSGGTGTAYAVAVKG (SEQ ID NO: 26), VH-CDR3 with the sequence LGVRDGLSV (SEQ ID NO: 27), VL-CDR1 with the sequence SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 with the sequence RDTERPS (SEQ ID NO: 29), VL-CDR3 with the sequence ESAVDTGTLV (SEQ ID NO: 30); 5) ZIL11 antibody: VH-CDR1 with TYVMN sequence (SEQ ID NO: 31), VH-CDR2 with SINGGGSSPTYADAVRG sequence (SEQ ID NO: 32), VH-CDR3 with SMVGPFDY sequence (SEQ ID NO: 33), VL-CDR1 with the sequence SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 with the sequence KDTERPS (SEQ ID NO: 35), VL-CDR3 with the sequence ESAVSSDTIV (SEQ ID NO: 36); 6) ZIL69 antibody: VH-CDR1 with SYAMK sequence (SEQ ID NO: 37), VH-CDR2 with TINNDGTRTGYADAVRG sequence (SEQ ID NO: 38), VH-CDR3 with GNAESGCTGDHCPPY sequence (SEQ ID NO: 39), VL-CDR1 with the sequence SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 with the sequence KDTERPS (SEQ ID NO: 41), VL-CDR3 with the sequence ESAVSSETNV (SEQ ID NO: 42); 7) ZIL94 antibody: VH-CDR1 with TYFMS sequence (SEQ ID NO: 43), VH-CDR2 with LISSDGSGTYYADAVKG sequence (SEQ ID NO: 44), VH-CDR3 with FWRAFND sequence (SEQ ID NO: 45), VL-CDR1 with the sequence GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 with the sequence DTGSRPS (SEQ ID NO: 47), VL-CDR3 with the sequence SLYTDSDILV (SEQ ID NO: 48); 8) ZIL154 antibody: VH-CDR1 with DRGMS sequence (SEQ ID NO: 49), VH-CDR2 with YIRYDGSRTDYADAVEG sequence (SEQ ID NO: 50), VH-CDR3 with WDGSSFFDY sequence (SEQ ID NO: 51), VL-CDR1 with the sequence KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 with the sequence KVSNRDP (SEQ ID NO: 53), VL-CDR3 with the sequence MQAIHPLT (SEQ ID NO: 54); 9) ZIL159 antibody: VH-CDR1 with the sequence SYVMT (SEQ ID NO: 55), VH-CDR2 with the sequence GINSEGSRTAYADAVKG (SEQ ID NO: 56), VH-CDR3 with the sequence GDIVATGTSY (SEQ ID NO: 57), VL-CDR1 with the S sequence of GETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 with the sequence of KDTERPS (SEQ ID NO: 59), VL-CDR3 with the sequence of KSAVSIDVGV (SEQ ID NO: 60); 10) ZIL171 antibody: VH-CDR1 with TYVMN sequence (SEQ ID NO: 61), VH-CDR2 with SINGGGSSPTYADAVRG sequence (SEQ ID NO: 62), VH-CDR3 with SMVGPFDY sequence (SEQ ID NO: 63), VL-CDR1 with the sequence SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 with the sequence KDTERPS (SEQ ID NO: 65), VL-CDR3 with the sequence ESAVSSDTIV (SEQ ID NO: 66); or 11) variant 1)-10) differing from the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 or ZIL171 by the addition, deletion and/or substitution of one or more amino acid residues of at least in one of CDR1, CDR2 or CDR3 VH or VL. 5. Vaccine composition according to any one of paragraphs. 1-4, where the mimotope is a mimotope with limited conformational freedom. 6. The vaccine composition according to claim 5, wherein the mimotope with limited conformational freedom is a chemically linked cyclic peptide. 7. Vaccine composition according to any one of paragraphs. 1-6 wherein the mimotope is chemically conjugated to a carrier polypeptide. 8. Vaccine composition according to any one of paragraphs. 1-6 wherein the carrier polypeptide and the mimotope are part of a recombinant fusion protein. 9. Vaccine composition according to any one of paragraphs. 1-8, wherein the carrier polypeptide comprises a bacterial toxoid or derivative thereof, keyhole hemocyanin (KLH) or a virus-like particle. 10. The vaccine composition of claim 9, wherein the bacterial toxoid or derivative is tetanus toxoid, diphtheria toxoid, tetanus toxoid, N. meningitidis group B outer membrane protein complex, Pseudomonas exotoxin, or a non-toxic diphtheria toxin mutant (CRM197). 11. The vaccine composition according to claim 9, wherein the virus-like particle is HBsAg, HBcAg, E. coli bacteriophage Qbeta, Norwalk virus, canine distemper virus (CDV), or influenza virus hemagglutinin (HA). 12. The vaccine composition of claim 10 wherein the carrier polypeptide contains or is CRM197. 13. Vaccine composition according to any one of paragraphs. 1-12, wherein the adjuvant is selected from the group consisting of an oil in water adjuvant, a polymer and water adjuvant, a water in oil adjuvant, an aluminum hydroxide adjuvant, a vitamin E adjuvant, and combinations thereof. 14. Vaccine composition according to any one of paragraphs. 1-13, where the adjuvant is a composition containing a saponin, a sterol, a quaternary ammonium compound and a polymer. 15. The vaccine composition according to claim 14, wherein the saponin is Quil A or a purified fraction thereof, the sterol is cholesterol, the quaternary ammonium compound is dimethyldioctadecylammonium bromide (DDA) and the polymer is polyacrylic acid. 16. Vaccine composition according to any one of paragraphs. 1-13, where the adjuvant contains a combination of one or more isolated immunostimulatory oligonucleotides, a sterol and a saponin. 17. The vaccine composition according to claim 16, wherein one or more isolated immunostimulatory oligonucleotides contain CpG, the sterol is cholesterol, and the saponin is Quil A or a purified fraction thereof. 18. A method of protecting a mammal from an IL-31 mediated disease, comprising administering to the mammal a vaccine composition according to any one of paragraphs. 1-17. 19. The method of claim 18 wherein the mammal is selected from the group consisting of dog, cat and horse. 20. The method of claim 18, wherein the IL-31 mimotope contained in the vaccine composition is administered to the mammal at a dose of about 10 μg to about 100 μg.

Images