RU2588462C2 - Monoclonal antibodies against interleukin-31 - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to biotechnology. Disclosed is an isolated antibody that specifically binds to at least one of canine interleukin-31 (IL-31), or feline IL-31. Such antibodies may be in form of diagnostic and/or veterinary compositions useful for treatment accompanied by itching and/or allergic condition in dogs or cats.

EFFECT: invention widens range of treatments for animals.

27 cl, 23 dwg, 5 tbl, 12 ex

Description

FIELD OF THE INVENTION

The present invention relates to the field of recombinant monoclonal antibodies and their uses in clinical and scientific methods, including diagnostic methods. The present invention also provides isolated antibodies against IL-31 (interleukin-31) in the form of veterinary compositions useful for treating an itchy or allergic condition in dogs or cats.

BACKGROUND OF THE INVENTION

Atopic dermatitis is defined by a special board of the American College of Veterinary Dermatology as “inflammatory and itchy allergic skin disease with a genetic predisposition with characteristic clinical features” (Olivry et al., Veterinary Immunology and Immunopathology, 2001, 81: 143-146). The Special Commission also recognized that this disease in canines is 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 visible and disturbing symptoms for pet owners.

The prevalence of atopic dermatitis is not exactly known due to insufficient and conflicting epidemiological data, but it is estimated to be 10% of the total population of canines (Marsella & Olivry, 2003, above; Scott et al., Canadian Veterinary Journal, 2002, 43: 601-603 ; Hillier Veterinary Immunology and Immunopathology, 2001, 81: 147-151). Altogether, approximately 4.5 million dogs are affected by this chronic and lifelong condition. Apparently, the incidence rate is increasing. It was believed that there is a predisposition to breed and gender, but there may be great differences depending on the geographical region (Hillier, 2001, supra; Picco et al., Vet. Dermatol., 2008, 19: 150-155).

The possible factors involved in allergic dermatitis are numerous and poorly understood. Atonic dermatitis can be triggered by components present in food (Picco, 2008, above), 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 a predisposition to the breed, 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).

Interleukin-31 (IL-31) is a cytokine cloned in 2004. It is produced mainly by activated T-helper (Th) 2 cells (Dillon et al., Nat. Immunol., 2004, 5: 752-60), but is also produced in mast cells and macrophages. IL-31 binds to a coreceptor consisting of IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR) (Dillon et al., 2004, supra, and Bilsborough et al., J. Allergy Clin. Immunol., 2006, 117 (2): 418-25). Receptor activation causes phosphorylation of STAT (signal transducer and transcriptional activator) involving JAK receptor (s) (Janus kinase). Coreceptor expression has been demonstrated in macrophages, keratinocytes and in the dorsal radicular ganglia. Not so long ago, IL-31 was found to be involved in dermatitis, itchy skin lesions, allergies, and airway hypersensitivity. See FIG. 1.

Stimulation of T cells with anti-CD3 and anti-CD28 antibodies immediately increases IL-31 mRNA expression (Dillon et al., 2004, supra). Microarray analysis showed that IL-31 induces certain genes of chemotactic agents, such as CXCL1 (CXC chemokine ligand 1), CCL17 (CC chemokine ligand 17) (thymic chemokine activated by activation (TARC)), CCL19 (macrophage inflammatory protein (MIP) 3β), CCL22 (monocytic chemokine (MDC)), CCL23 (MIP3) and CCL4 (MIPβ) (Dillon et al., 2004, supra).

Transgenic mice that overexpress IL-31 exhibit skin inflammation, pruritus, severe dermatitis and alopecia (Dillon et al., 2004, supra). Subcutaneous injection of IL-31 in mice initiates infiltration by inflammatory cells, neutrophils, eosinophils, lymphocytes and macrophages and leads to epidermal thickening and acanthosis of the skin. In NC / Nga mice with atopic dermatitis (AD) due to natural causes, IL-31 is overexpressed at the skin lesion sites and its level correlates with prurite (Takaoka et al., Eur. J. Pharmacol., 2005, 516, 180-181; Takaoka et al., Exp. Dermatol., 2006, 15, 161-167). In addition, in mouse models it was shown that IL-31 induces fast-moving prurite (Raap et al., J. Allergy Clin. Immunol., 2008, 122 (2): 421-3).

Other studies have shown that IL-31 is associated with atopic dermatitis-induced skin inflammation and pruritis in humans. In human patients with AD, IL-31 mRNA expression is significantly greater in skin lesions than in unaffected areas of the skin, and expression in unaffected areas of the skin exceeds that in normal skin of healthy patients (Sonkoly et al., J. Allergy Clin Immunol., 2006, 117: 411-7). Another study reported that CD45RO + T cells (memory cells) that are positive for cutaneous lymphocytic antigen (CLA) express mRNA and IL-31 protein in the skin of patients with AD (Bilsborough et al., 2006, supra). Overexpression of IL-31 mRNA in the skin of patients or allergic contact dermatitis has also been reported to correlate with expression of IL-4 and IL-13 mRNA, rather than expression of interferon (IFN) -γ mRNA (Neis et al., J. Allergy Clin. Immunol., 2006, 118, 930-937). In addition, it was shown that serum levels of IL-31 in patients with chronic spontaneous urticaria are elevated and even higher in patients with AD (Raap et al., Exp. Dermatol., 2010, 19 (5): 464-6 ) In addition, correlation between the severity of AD and serum IL-31 levels was observed in humans (Rapp et al., 2008, supra). It was also shown that IL-31 secretion was enhanced in mast cells after IgE cross-linking and in response to staphylococcal superantigen in individuals with atopy. In addition, IL-31 has been shown to stimulate the production of certain pro-inflammatory mediators, including IL-6, IL-8, CXCL1, CC17 and numerous metalloproteinases, in human colon myofibroblasts (Yagi et al., International Journal of Molecular Medicine, 2007, 19 (6): 941-946).

Type I hypersensitivity to environmental allergens is believed to be the main AD mechanism in canines, and the levels of Th2-mediated cytokines, such as IL-4, are elevated in areas of skin lesion in dogs with AD (Nuttall et al., Vet. Immunol. Immunopathol., 2002, 87, 379-384). In addition, infiltration by inflammatory cells, lymphocytes and neutrophils is an important mechanism underlying the exacerbation of skin lesions; overexpression of genes of chemotactic agents such as CCL17 / TARC, CCR4, and CCL28 / mucosal epithelial chemokine (MEC) contributes to exacerbation of skin lesions in dogs with AD (see Maeda et al., Vet. Immunol. Immunopathol., 2005, 103, 83-92; Maeda et al., Vet. Immunol. Immunopathol., 2002b, 90, 145-154; and Maeda et al., J. Vet. Med. Sci., 2008, 70, 51-55 )

Recent evidence suggests that IL-31 may be involved in the stimulation of allergic inflammation and the airway epithelial response characteristic of allergic asthma (Chattopadhyay et al., J. Biol. Chem., 2007, 282: 3014-26; and Wai et al., Immunology, 2007, 122, 532-541).

These observations support the hypothesis that IL-31 plays a significant role in both pruritus and allergic conditions. It would be desirable to have a therapeutic anti-IL-31 antibody useful for treating an itchy condition and / or an allergic condition in dogs or cats.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an isolated antibody that specifically binds to at least one of a canine IL-31 or a feline IL-31. In some embodiments, the antibody is a monoclonal antibody. In one embodiment, the monoclonal antibody is chimeric. In another embodiment, the antibody is caninized or felinized.

In some embodiments, the antibody reduces, inhibits, or neutralizes the activity of IL-31 in a dog or cat. In preferred embodiments, the antibody attenuates, suppresses, or neutralizes the pruritic or allergic condition. Pruritic conditions include, for example, atopic dermatitis, eczema, psoriasis, scleroderma, and pruritis. Allergic conditions include, for example, allergic dermatitis, summer eczema, urticaria, fever, inflammatory airway disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease and inflammatory processes that result from an autoimmune reaction such as irritable bowel syndrome ( IBS). In one embodiment, the present invention provides an isolated antibody or antigen binding site thereof comprising at least one of the following:

complementarity determining region (CDR) 1, variable heavy domain (V _H) chain having the amino acid sequence YYDIN (SEQ ID NO: 1; 11E12-VH-CDR1), SYDMS (SEQ ID NO: 2; 19D07-VH-CDR1) or NYGMS (SEQ ID NO: 3; 34D03-VH-CDR1);

The heavy chain variable domain CDR2 having the amino acid sequence WIFPGDGGTKYNETFKG (SEQ ID NO: 4; 11E12-VH-CDR2), TITSGGGYTYSADSVKG (SEQ ID NO: 5; 19D07-VH-CDR2) or TISYGGSYTYYPDHG (6); -CDR2);

The heavy chain variable domain CDR3 having the amino acid sequence ARGGTSVIRDAMDY (SEQ ID NO: 7; 11E12-VH-CDR3), ARQNWWGLAY (SEQ ID NO: 8; 19D07-VH-CDR3) or VRGYGYDTMDY (SEQ ID NO: 9; 34D03-VH -CDR3); and

their variant having one or more than one conservative amino acid substitution in at least one of CDR1, CDR2 or CDR3.

In another embodiment, the invention provides an isolated antibody or antigen binding site thereof comprising at least one of the following group:

the variable domain of the light (V _L ) chain containing the complementarity determining region (CDR) 1 having the amino acid sequence RASESVDNYGISFMH (SEQ ID NO: 10; 11E12-VL-CDR1), KSSQSLLNSGNQKNYLA (SEQ ID NO: 11; 19D07-VL-CDR1) or KASQSVSFAGTGLMH (SEQ ID NO: 12; 34D03-VL-CDR1);

CDR2 of the light chain variable domain having the amino acid sequence RASNLES (SEQ ID NO: 13; 11E12-VL-CDR2), GASTRES (SEQ ID NO: 14; 19D07-VL-CDR2) or RASNLEA (SEQ ID NO: 15; 34D03-VL -CDR2);

CDR3 of the light chain variable domain having the amino acid sequence QQSNKDPLT (SEQ ID NO: 16; 11E12-VL-CDR3), QNDYSYPYT (SEQ ID NO: 17; 19D07-VL-CDR3) or QQSREYPWT (SEQ ID NO: 18; 34D03-VL -CDR3); and

their variant having one or more than one conservative amino acid substitution in at least one of CDR1, CDR2 or CDR3.

In other embodiments, an antibody having at least one of the CDRs of the light chain variable domain described above may further comprise at least one of the following CDRs of the heavy chain variable domain:

the complementarity determining region (CDR) 1 of the heavy chain variable domain having the amino acid sequence YYDIN (SEQ ID NO: 1; 11E12-VH-CDR1), SYDMS (SEQ ID NO: 2; 19D07-VH-CDR1) or NYGMS (SEQ ID NO : 3; 34D03-VH-CDR1);

The heavy chain variable domain CDR2 having the amino acid sequence WIFPGDGGTKYNETFKG (SEQ ID NO: 4; 11E12-VH-CDR2), TITSGGGYTYSADSVKG (SEQ ID NO: 5; 19D07-VH-CDR2) or TISYGGSYTYYPDHG (6); -CDR2);

The heavy chain variable domain CDR3 having the amino acid sequence ARGGTSVIRDAMDY (SEQ ID NO: 7; 11E12-VH-CDR3), ARQNWWGLAY (SEQ ID NO: 8; 19D07-VH-CDR3) or VRGYGYDTMDY (SEQ ID NO: 9; 34D03-VH -CDR3); and

their variant having one or more than one conservative amino acid substitution in at least one of CDR1, CDR2 or CDR3.

In some embodiments, the antibody may comprise at least one of the following:

a) the variable domain of the light chain containing

b) the variable domain of the heavy chain containing

c) variants thereof having one or more than one conservative amino acid substitution.

In one embodiment, the invention provides a monoclonal antibody that specifically binds to a region between the amino acid residues 95 and 125 of the amino acid sequence of canine IL-31 of SEQ ID NO: 32, or to the corresponding region of feline IL-31. In some embodiments, the antibody specifically binds to the region between the amino acid residues 102 and 122 of the canine IL-31 amino acid sequence (SEQ ID NO: 32) or the corresponding region of feline IL-31.

The present invention also provides a veterinary composition comprising a therapeutically effective amount of at least one antibody described above.

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

In other embodiments, the invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding at least one of the following:

complementarity determining region (CDR) 1 of the heavy domain (V _H ) variable domain having the amino acid sequence YYDIN (SEQ ID NO: 1; 11E12-VH-CDR1), SYDMS (SEQ ID NO: 2; 19D07-VH-CDR1) or NYGMS (SEQ ID NO: 3; 34D03-VH-CDR1);

The heavy chain variable domain CDR2 having the amino acid sequence WIFPGDGGTKYNETFKG (SEQ ID NO: 4; 11E12-VH-CDR2), TITSGGGYTYSADSVKG (SEQ ID NO: 5; 19D07-VH-CDR2) or TISYGGSYTYYPDHG (6); -CDR2);

The heavy chain variable domain CDR3 having the amino acid sequence ARGGTSVIRDAMDY (SEQ ID NO: 7; 11E12-VH-CDR3), ARQNWWGLAY (SEQ ID NO: 8; 19D07-VH-CDR3) or VRGYGYDTMDY (SEQ ID NO: 9; 34D03-VH -CDR3); and

their variant having one or more than one conservative amino acid substitution in at least one of CDR1, CDR2 or CDR3.

In other embodiments, the isolated nucleic acid described above may further comprise a nucleic acid sequence encoding at least one of the following:

the variable domain of the light (V _L ) chain containing the complementarity determining region (CDR) 1 having the amino acid sequence RASESVDNYGISFMH (SEQ ID NO: 10; 11E12-VL-CDR1), KSSQSLLNSGNQKNYLA (SEQ ID NO: 11; 19D07-VL-CDR1) or KASQSVSFAGTGLMH (SEQ ID NO: 12; 34D03-VL-CDR1);

CDR2 of the light chain variable domain having the amino acid sequence RASNLES (SEQ ID NO: 13; 11E12-VL-CDR2), GASTRES (SEQ ID NO: 14; 19D07-VL-CDR2) or RASNLEA (SEQ ID NO: 15; 34D03-VL -CDR2);

CDR3 of the light chain variable domain having the amino acid sequence QQSNKDPLT (SEQ ID NO: 16; 11E12-VL-CDR3), QNDYSYPYT (SEQ ID NO: 17; 19D07-VL-CDR3) or QQSREYPWT (SEQ ID NO: 18; 34D03-VL -CDR3); and

their variant having one or more than one conservative amino acid substitution in at least one of CDR1, CDR2 or CDR3.

The present invention also provides a vector comprising at least one of the nucleic acids described above.

In other embodiments, the present invention provides a method for producing an antibody, comprising culturing a host cell as described above under conditions that produce an antibody and isolating the antibody from the host cell or culture medium of the host cell.

Also provided is a method of treating a condition or disorder selected from an itchy condition or an allergic condition, comprising administering a therapeutically effective amount of an antibody described above. In some embodiments, the pruritic condition is selected from atopic dermatitis, eczema, psoriasis, scleroderma, and prurite. In other embodiments, the allergic condition being treated is selected from allergic dermatitis, summer eczema, urticaria, fuse, inflammatory airway disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, and inflammatory processes resulting from an autoimmune reaction such as syndrome irritable bowel (IBS).

In addition, a method of inhibiting the activity of IL-31 in a dog or cat is provided, comprising administering the antibody described above.

Also provided is a method for detecting or quantifying IL-31 in a sample, comprising incubating a clinical or biological sample containing IL-31 in the presence of the antibody described above and detecting an antibody that is bound to IL-31 in the sample. In one embodiment, the antibody is detectably labeled. In another embodiment, the antibody is unlabeled and is used in combination with a secondary antibody that is labeled.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Figure 1 is a schematic representation of an IL-31 pathway.

Figure 2 is a schematic representation of the overall structure of a mouse immunoglobulin G (IgG) molecule with an isolated antigen binding site.

Figure 3 is a schematic representation of the overall structure of a chimeric mouse: canine IgG.

Figure 4 is an illustration showing speciation or "caninization" of mouse IgG; murine CDRs are grafted onto canine Ig scaffolds identified from sequence databases.

Figure 5 is an illustration of a "heterochemical" monoclonal antibody with a chimeric light chain paired with a fully caninized heavy chain.

6 shows ELISA titers for IL-31 immunized mice (CF-1 MU No. 1-4) relative to mice prior to immunization and mice as a positive control.

7 is an illustration of the variable domains of antibody chains, which shows primers for constant regions and degenerate primers in the direction of the variable regions of mouse Ig.

Fig. 8 is a graph of the efficacy of a chimeric 11E12 in a pilot, placebo-controlled study with s / c (subcutaneous) single dose (76A60).

Fig. 9 is a table showing individual scores for the severity of pruritus in the dogs scores recorded in study 76A60.

Figure 10 presents Western blots demonstrating the binding of chimeric (blot No. 1), caninized (blot No. 2) and heterochemical (blots No. 3 and 4) variants 11E12 with canine IL-31. The heterocimera on blot No. 3 has a caninized light chain paired with a chimeric heavy chain. The heterocimera on blot No. 4 has a chimeric light chain paired with a cannulated heavy chain. Each nitrocellulose blot contains: left lane — pre-stained protein standards (Seeblue plus 2, Invitrogen Corp., Carlsbad, CA), and right lane — 800 ng canine IL-31.

Figure 11 shows a schematic representation of the result of the replacement in the frame of the light chain cannized 11E12.

12 is a Western blot showing the binding of canned variants 11E12 containing single reverse mutations in residues of mouse Ig light chain framework 2. Each nitrocellulose blot contains: left lane — pre-stained protein standards (Seeblue plus 2, Invitrogen Corp., Carlsbad, CA), and right lane — 800 ng canine IL-31.

13 shows western blots using full-length and truncated canine IL-31 proteins. Individual nitrocellulose blots were probed using antibodies A) against His, B) 34D03 and C) 11E12. Lines 1-9 of blots correspond to the following: lane 1 - pre-stained protein standards (Seeblue plus 2, Invitrogen Corp., Carlsbad, CA); lane 2 is a full-sized canine IL-31; lane 3 - structure shortened at the N-end, -20N; lane 4 - structure shortened at the N-end, -40N; lane 5 - structure shortened at the N-end, -60N; lane 6 - structure shortened at the C-end of -20 ° C; lane 7 - structure shortened at the C-end of -40 ° C; lane 8 - structure shortened at the C-end of -60 ° C; and lane 9 - beta-galactosidase (lacZ). Note: for full-length IL-31 and C-terminated proteins (-20C, -40C and -60C) no detectable expression was detected under these conditions.

On Fig presents Western blots for truncated proteins of canine IL-31. Individual nitrocellulose blots were probed using antibodies A) against His, B) 11E12 and C) 34D03. Lines 1-5 of blots correspond to the following: lane 1 - pre-stained protein standards (Seeblue plus 2, Invitrogen Corp., Carlsbad, CA); lane 2 - shortened at the C-end of the structure, corresponding to the provisions of 20-122; lane 3 - shortened at the C-end of the structure, corresponding to the provisions of 20-100; lane 4 - shortened at the C-end of the structure, corresponding to the provisions of 20-80; and lane 5 - beta-galactosidase (lacZ).

15 is a portion of western blots for lysates of E. coli strains expressing canine IL-31 with alanine substitution for each amino acid position (76-122). Individual nitrocellulose blots were probed with antibodies against His, 11E12 and 34D03, as shown in this FIG.

16 is a portion of Western blots for double and triple mutation lysates in canine IL-31. The -20N protein lysate was used as a positive control.

17 is a graph showing scores of itch severity in points in dogs that were injected with a subcutaneously canned antibody 34D03 (1.0 mg / kg). The severity of pruritus was scored on each day of the study before (initial response) and after (response over a period of 2 hours) administration of canine IL-31 (1.5 μg / kg).

Fig. 18 is a polyacrylamide 4-12% gel electrophoresis picture in bis-Tris buffer in the presence of sodium dodecyl sulfate (4-12% Bis-Tris SDS PAGE) for purified canine and feline IL-31 proteins. Panel A shows the staining of proteins using Coomassie, the analysis of which was carried out under reducing conditions. Panel B shows the staining of proteins using Coomassie, the analysis of which was carried out in non-reducing conditions. Panels C and D are western blots of gels identical to A and B, respectively, probed with anti-His antibody. Lane 1 - canine IL-31; lane 2 - feline IL-31; lane 3 — pre-stained protein standards (Seeblue plus 2, Invitrogen Corp., Carlsbad, CA); lane 4 - canine IL-31; and lane 5 is feline IL-31.

19 is a graph of pSTAT signal transmission in canine monocytes DH-82, which is induced by canine and feline IL-31 produced in E. coli. Canine IL-31 (CHO) is a reference protein used for all previous cell-based assays, a dog prurite model, and as an immunogen for initial antibody identification.

FIG. 20 is an alignment showing sequence conservation among feline and canine IL-31 in the region of this protein involved in the binding of antibodies 11E12 and 34D03 (marked with a plus sign).

On Fig presents Western blots using proteins of IL-31. Individual nitrocellulose blots were probed with antibodies A) against His, B) 11E12 and C) 34D03. Note: canine IL-31 (CHO) does not contain 6-His tags.

FIG. 22 is a graph showing inhibition of canine IL-31 induced pSTAT signaling in canine DH82 monocytes with a comparison of felinized and caninized 34D03 antibodies.

23 is a western blot of feline and canine IL-31 under reducing conditions, probed with felinated antibody 34D03.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is a heavy chain variable domain CDR1 designated herein as 11E12-VH-CDR1;

SEQ ID NO: 2 represents a heavy chain variable domain CDR1 designated herein as 19D07-VH-CDR1;

SEQ ID NO: 3 is a heavy chain variable domain CDR1 designated herein as 34D03-VH-CDR1;

SEQ ID NO: 4 is a heavy chain variable domain CDR2 designated herein as 11E12-VH-CDR2;

SEQ ID NO: 5 is a heavy chain variable domain CDR2 designated herein as 19D07-VH-CDR2;

SEQ ID NO: 6 is a heavy chain variable domain CDR2, designated herein as 34D03-VH-CDR2;

SEQ ID NO: 7 is a heavy chain variable domain CDR3 designated herein as 11E12-VH-CDR3;

SEQ ID NO: 8 is a heavy chain variable domain CDR3, designated herein as 19D07-VH-CDR3;

SEQ ID NO: 9 is a heavy chain variable domain CDR3 designated herein as 34D03-VH-CDR3;

SEQ ID NO: 10 represents CDR1 of the variable domain of the light chain, indicated in this description as 11E12-VL-CDR1;

SEQ ID NO: 11 is a light chain variable domain CDR1 designated herein as 19D07-VL-CDR1;

SEQ ID NO: 12 is a light chain variable domain CDR1, designated herein as 34D03-VL-CDR1;

SEQ ID NO: 13 represents a light chain variable domain CDR2 designated herein as 11E12-VL-CDR2;

SEQ ID NO: 14 is a light chain variable domain CDR2, designated herein as 19D07-VL-CDR2;

SEQ ID NO: 15 is a light chain variable domain CDR2, designated herein as 34D03-VL-CDR2;

SEQ ID NO: 16 is a light chain variable domain CDR2 designated herein as 11E12-VL-CDR3;

SEQ ID NO: 17 is a CDR3 of the variable domain of the light chain, indicated in this description as 19D07-VL-CDR3;

SEQ ID NO: 18 is a CDR3 of the variable domain of the light chain, indicated in this description as 34D03-VL-CDR3;

SEQ ID NO: 19 represents the sequence of the variable domain of the light chain, referred to in this description as MU-11E12-VL;

SEQ ID NO: 20 represents the sequence of the variable domain of the light chain, indicated in this description as CAN-11E12-VL-cUn-FW2;

SEQ ID NO: 21 represents the sequence of the variable domain of the light chain, referred to in this description as CAN-11E12-VL-cUn-13;

SEQ ID NO: 22 represents the sequence of the variable domain of the light chain, referred to in this description as MU-19D07-VL;

SEQ ID NO: 23 represents the sequence of the variable domain of the light chain, indicated in this description as CAN-19D07-VL-998-1;

SEQ ID NO: 24 represents the sequence of the variable domain of the light chain, referred to in this description as MU-34D03-VL;

SEQ ID NO: 25 represents the sequence of the variable domain of the light chain, indicated in this description as CAN-34D03-VL-998-1;

SEQ ID NO: 26 represents the sequence of the variable domain of the heavy chain, referred to in this description as MU-11E12-VH;

SEQ ID NO: 27 represents the sequence of the variable domain of the heavy chain, referred to in this description as CAN-11E12-VH-415-1;

SEQ ID NO: 28 represents the sequence of the variable domain of the heavy chain, referred to in this description as MU-19D07-VH;

SEQ ID NO: 29 represents the sequence of the variable domain of the heavy chain, indicated in this description as CAN-19D07-VH-400-1;

SEQ ID NO: 30 represents the sequence of the variable domain of the heavy chain, referred to in this description as MU-34D03-VH;

SEQ ID NO: 31 represents the sequence of the variable domain of the heavy chain, indicated in this description as CAN-34D03-VH-568-1;

SEQ ID NO: 32 is the amino acid sequence corresponding to Access No. in GenBank C7GOW1, and corresponds to a full-length dog protein IL-31;

SEQ ID NO: 33 represents the nucleotide sequence corresponding to Access No. in GenBank C7G0W1, and corresponds to the nucleotide sequence encoding the full-length protein of IL-31 dogs;

SEQ ID NO: 34 is the nucleotide sequence encoding the sequence of the variable domain of the light chain, referred to in this description as MU-11E12-VL;

SEQ ID NO: 35 is a nucleotide sequence encoding a sequence of a heavy chain variable domain, designated herein as MU-11E12-VH;

SEQ ID NO: 36 is the nucleotide sequence encoding the sequence of the variable domain of the light chain, referred to in this description as MU-19D07-VL;

SEQ ID NO: 37 is the nucleotide sequence encoding the sequence of the variable domain of the heavy chain, referred to in this description as MU-19D07-VH;

SEQ ID NO: 38 is the nucleotide sequence encoding the sequence of the variable domain of the light chain, referred to in this description as MU-34D03-VL;

SEQ ID NO: 39 is the nucleotide sequence encoding the sequence of the variable domain of the heavy chain, referred to in this description as MU-34D03-VH;

SEQ ID NO: 40 is the amino acid sequence for the constant region of the canine Ig heavy chain, designated herein as HC-64 (GenBank Accession No. AF354264);

SEQ ID NO: 41 is a nucleotide sequence encoding a constant region of a canine Ig heavy chain designated herein as HC-64 (GenBank Accession No. AF354264);

SEQ ID NO: 42 is the amino acid sequence for the constant region of the canine Ig heavy chain, designated herein as HC-65 (GenBank Accession No. AF354265);

SEQ ID NO: 43 is a nucleotide sequence encoding a constant region of a canine Ig heavy chain designated herein as HC-65 (GenBank Accession No. AF354265);

SEQ ID NO: 44 is the amino acid sequence for the constant region of the canine Ig light chain, designated as kappa in this description (GenBank Accession No. XP_532962);

SEQ ID NO: 45 is a nucleotide sequence encoding a canine Ig light chain constant region designated as kappa (GenBank Accession No. XP_532962);

SEQ ID NO: 46 is the nucleotide sequence encoding the sequence of the variable domain of the light chain, referred to in this description as CAN-19D07-VL-998-1;

SEQ ID NO: 47 is a nucleotide sequence encoding a sequence of the variable domain of a heavy chain designated herein as CAN-19D07-VH-998-1;

SEQ ID NO: 48 is a nucleotide sequence encoding the sequence of the variable domain of the light chain, referred to in this description as CAN-34D03-VL-998-1;

SEQ ID NO: 49 is the nucleotide sequence encoding the sequence of the variable domain of the heavy chain, referred to in this description as CAN-34D03-VH-568-1;

SEQ ID NO: 50 is the nucleotide sequence encoding the sequence of the variable domain of the light chain, referred to in this description as CAN-11E12-VL-cUn-FW2;

SEQ ID NO: 51 is the nucleotide sequence encoding the sequence of the variable domain of the heavy chain, referred to in this description as CAN-11E12-VH-415-1;

SEQ ID NO: 52 is a nucleotide sequence encoding the sequence of the variable domain of the light chain, referred to in this description as CAN-11E12-VL-cUn-13;

SEQ ID NO: 53 represents the sequence of the variable domain of the light chain, indicated in this description as CAN-11E12_VL_cUn_1;

SEQ ID NO: 54 is a nucleotide sequence encoding the sequence of the variable domain of the light chain, referred to in this description as CAN-11E12-VL-cUn-1;

SEQ ID NO: 55 corresponds to the amino acid sequence of the full-length canine IL-31 construct used herein for expression in E. coli;

SEQ ID NO: 56 is the nucleotide sequence corresponding to the full-length canine IL-31 construct used herein for expression in E. coli;

SEQ ID NO: 57 is the amino acid sequence of the canine IL-31 construct -20N for expression in E. coli;

SEQ ID NO: 58 is the nucleotide sequence corresponding to the -20N construct of canine IL-31 for expression in E. coli;

SEQ ID NO: 59 is the amino acid sequence of the canine IL-31 construct -40N for expression in E. coli;

SEQ ID NO: 60 is the nucleotide sequence corresponding to the -40N construct of canine IL-31 for expression in E. coli;

SEQ ID NO: 61 is the amino acid sequence of the -60N construct of canine IL-31 for expression in E. coli;

SEQ ID NO: 62 is the nucleotide sequence corresponding to the -60N construct of canine IL-31 for expression in E. coli;

SEQ ID NO: 63 is the amino acid sequence of a canine IL-31 construct 20-122 for expression in E. coli;

SEQ ID NO: 64 represents the nucleotide sequence corresponding to the construction of 20-122 canine IL-31, for expression in E. coli;

SEQ ID NO: 65 represents the amino acid sequence of a 20-100 canine IL-31 construct for expression in E. coli;

SEQ ID NO: 66 represents the nucleotide sequence corresponding to the construction of 20-100 canine IL-31, for expression in E. coli;

SEQ ID NO: 67 represents the amino acid sequence of the construction of 20-80 canine IL-31, for expression in E. coli;

SEQ ID NO: 68 represents the nucleotide sequence corresponding to the construction of 20-80 canine IL-31, for expression in E. coli;

SEQ ID NO: 69 is the nucleotide sequence corresponding to the full-length feline IL-31 construct for expression in E. coli;

SEQ ID NO: 70 is the amino acid sequence corresponding to the full-length feline IL-31 construct for expression in E. coli;

SEQ ID NO: 71 represents the sequence of the variable domain of the light chain, indicated in this description as FEL-34D03-VL-021-1;

SEQ ID NO: 72 is a nucleotide sequence encoding the sequence of the variable domain of the light chain, referred to in this description as FEL-34D03-VL-021-1;

SEQ ID NO: 73 represents the sequence of the variable domain of the heavy chain, indicated in this description as FEL-34D03-VH-035-1;

SEQ ID NO: 74 is the nucleotide sequence encoding the sequence of the variable domain of the heavy chain, referred to in this description as FEL-34D03-VH-035-1;

SEQ ID NO: 75 represents the amino acid sequence for the constant region of the feline Ig heavy chain, designated herein as HC-A of cats (GenBank Accession No. AB016710.1);

SEQ ID NO: 76 is a nucleotide sequence encoding a constant region of a feline Ig heavy chain designated herein as HC-A of cats (GenBank Accession No. AB016710.1);

SEQ ID NO: 77 represents the amino acid sequence for the constant region of the feline Ig light chain, designated herein as feline LC-kappa (GenBank Accession No. AF198257.1);

SEQ ID NO: 78 is a nucleotide sequence encoding a feline Ig light chain constant region designated herein as feline LC-kappa (GenBank Accession No. AF198257.1).

DEFINITIONS

Before a detailed description of the present invention, certain terms used in the context of the present invention will be defined. In addition to these terms, other terms are defined herein, if necessary. Unless expressly stated otherwise in this description, the terms adopted in the art, which are used in this description, will have their own meanings recognized in the art.

As used in the description and claims, singular forms include plural references unless the context clearly dictates otherwise. For example, a reference to an “antibody” includes many such antibodies.

The term "comprising" as used herein is intended to mean that the compositions and methods include the listed elements, but not excluding other elements.

An epitope, as used herein, refers to an antigenic determinant recognized by an antibody CDR. In other words, an epitope refers to that part of any molecule that is able to recognize and to which an antibody can bind. 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 portion of a molecule that an antibody can bind to, which can also recognize and that can bind an antibody (the corresponding antibody binding region may be called a paratope). In general, epitopes consist of chemically active surface groupings of molecules, for example, amino acid or sugar sugar side chains, and have certain spatial structural characteristics, as well as specific charge characteristics.

The term "specifically" as applied to binding to an antibody refers to a highly avid and / or high affinity binding of an antibody to a particular antigen, i.e. a polypeptide or epitope. In many embodiments, the particular antigen is the antigen (or fragment or subfraction of the antigen) used to immunize an animal host from which 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 be able to bind other polypeptides at a low, but detectable level (for example, 10% or less of the binding shown for the polypeptide of interest). Such weak binding or background binding is readily distinguishable from the specific binding of an antibody to a test polypeptide, for example by using appropriate controls. In general, specific antibodies bind to an antigen with a binding affinity of K _{D of} 10 ^-7 M or less, for example 10 ^-8 M or less (for example 10 ^-9 M or less, 10 ^-10 or less, 10 ^-11 or less, 10 ^-12 or less, or 10 ^-13 or less, 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 heterochemical antibody, a cannibalized antibody, or a felinized antibody. The term "antibody" preferably refers to monoclonal antibodies and their fragments, and their immunologically binding equivalents that can bind to the IL-31 protein and its fragments. The term “antibody” is used to mean 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, made up of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds in the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has correctly located intrachain disulfide bridges. Each heavy chain has a variable domain (V _H ) at one end, 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 constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that certain amino acid residues form a contact surface between the variable domains of the light and heavy chains. Figure 2 is an example of the overall structure of a native mouse immunoglobulin G (IgG) with an isolated antigen binding site.

The term “antibody fragment” refers to a portion of the structure of an intact antibody, including, without limitation, an isolated single antibody chain, an Fv construct, a Fab construct, an Fc construct, a sequence of a light chain variable domain or complementarity determining region (CDR), etc. .

The term "variable" region includes the framework and CDR (also known as hypervariable region) and refers to the fact that some parts of the variable domains differ significantly in sequence among antibodies and are used to bind each specific antibody to its specific antigen and determine the specificity of this antibody. However, the variability is not evenly distributed across all variable domains of antibodies. It is concentrated in three segments of the variable domains of both the light chain and heavy chain, called the hypervariable regions. The more highly conserved regions of the variable domains are called the framework region (FR). Each of the variable domains of the native heavy and light chains contains numerous FRs, in most cases adopting a β-folded configuration, connected by three hypervariable regions that form loops connecting and, in some cases, forming part of the α-folded structure. The hypervariable regions in each chain are held together in close proximity to each other by FR and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647-669). The constant domains are not directly involved in the binding of the antibody to the antigen, however, they demonstrate various effector functions, such as the 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 binding to an antigen. The hypervariable region contains amino acid residues from the "complementarity determining region" or "CDR" (Kabat et al. (1991), above) and / or amino acid residues from the "hypervariable loop" (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987)). "Frame" or "FR" residues are residues of the variable domain, other than residues of the hypervariable region defined in this description.

As a result of papain digestion of antibodies, two identical antigen-binding fragments are formed, called “Fab” fragments, each of which has an antigen-binding site, and the remaining “Fc” fragment, the name of which reflects its ability to crystallize easily. Pepsin treatment results in an F (ab ′) ₂ fragment having two antigen-binding sites and still capable of cross-linking with the antigen.

“Fv” is a minimal antibody fragment that contains the entire antigen-recognizing and β-binding site. This region consists of a dimer of the variable domain of one heavy chain and one light chain, which are in dense non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact with the establishment of an antigen-binding site on the surface of the V _H -V _L dimer. Together, six hypervariable regions confer antigen binding specificity to the antibody. However, even one variable domain (or half of the Fv, containing only three hypervariable regions specific for the antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the whole binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′-fragments differ from Fab-fragments in that they have added several residues at the carboxyl end of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody. Fab′-SH in this description is the designation for Fab ′, in which the cysteine residue (s) of the constant domains carries (ut) a free thiol group. F (ab ′) ₂ fragments of antibodies are initially obtained as pairs of Fab ′ fragments that contain articulated cysteines. In addition, other methods for chemically combining antibody fragments are known.

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

Based on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. Five main classes of immunoglobulins are currently known: IgA, IgD, IgE, IgG and IgM, and some of them can be further subdivided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA, IgA2 (as defined by for mice and humans). The heavy chain constant domains corresponding to different classes of immunoglobulins are designated as alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and spatial configurations of immunoglobulins of different classes are well known for many species. The prevalence of individual isotypes and the functional activities associated with these constant domains are species-specific and must be determined experimentally.

A "monoclonal antibody," as defined herein, is an antibody produced by cells of one clone (specifically, hybridoma cells of one 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.

Monoclonal antibodies as used herein include, in particular, “chimeric” antibodies (immunoglobulins) in which the heavy and / or light chain region is identical or homologous to the corresponding sequences in antibodies originating from a particular species, while the rest 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, provided that they exhibit the desired biological activity. Typically, chimeric antibodies are antibodies for which light and heavy chain genes were constructed, typically by a genetic engineering method, from different types of genes of the variable and constant regions of an antibody. For example, variable segments of genes from a murine monoclonal antibody can be attached to constant segments of canine Ig. Figure 3 is a schematic representation of the overall structure of one of the embodiments of mouse: canine IgG. In this embodiment, the antigen binding site is derived from murine Ig, while the F _C region is from canine Ig.

"Caninized" forms of non-canine (eg, murine) antibodies are genetically modified antibodies that contain a minimal sequence derived from a non-canine immunoglobulin. Caninized antibodies are canine immunoglobulin (recipient antibody) sequences in which the residues of the reciprocal region of the recipient are replaced by residues of the hypervariable region of Ig from a non-family of canine species (donor antibody), such as a mouse having the desired specificity, affinity and capacity. In some cases, residues of frame regions (FR) of canine immunoglobulin sequences are replaced by corresponding non-canine Ig residues. In addition, caninized antibodies may contain residues that are not present in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. In general, a caninized antibody will comprise essentially the entire at least one variable domain and typically two variable domains in which all or substantially all of the hypervariable regions correspond to those of a non-canine immunoglobulin sequence, and all or substantially all of the FRs are those of a canine immunoglobulin sequence. A caninized antibody may also contain the entire constant region of an immunoglobulin (Fc) or at least a portion thereof, usually one of a canine immunoglobulin sequence. Figure 4 is an illustration of one of the embodiments, which shows the process of "speciation" or "caninization" of mouse IgG. In this embodiment, murine CDRs are grafted onto canine Ig scaffolds.

"Felinated" forms of non-feline (eg, murine) antibodies are genetically modified antibodies that contain a minimal sequence derived from a non-feline immunoglobulin. Felinated antibodies are feline immunoglobulin (recipient antibody) sequences in which the residues of the recipient's hypervariable region are replaced by residues of the hypervariable region of Ig from a non-family of feline species (donor antibody), such as a mouse having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of feline immunoglobulin sequences are replaced by corresponding non-feline Ig residues. In addition, felinated antibodies may contain residues that are not present in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. In general, a felinized antibody will comprise essentially all of at least one and usually two variable domains in which all or substantially all of the hypervariable regions correspond to those of a non-feline immunoglobulin sequence, and all or essentially all FRs are those of feline immunoglobulin sequences. A felined antibody may also contain the entire constant region of an immunoglobulin (Fc) or at least a portion thereof, usually one of a feline immunoglobulin sequence.

The term “heterochemical”, as defined herein, refers to an antibody in which one of the chains of the antibody (heavy or light) is caninized, while the other is chimeric. Figure 5 shows one of the embodiments of a heterochemical molecule. In this embodiment, a canned heavy chain variable domain (where all CDRs are mouse CDRs and all FRs are canine FRs) are paired with a light chain chimeric variable domain (where all CDRs are mouse CDRs and all FRs are mouse FR ) In this embodiment, both the variable domain of the heavy and the variable domain of the light chains are fused to the constant region of canine Ig.

The term “variant” anti-IL-31 antibody as used herein refers to a molecule that has differences in amino acid sequence compared to the amino acid sequence of a “parental” anti-IL-31 antibody due to the addition, deletion and / or replacement of one or more than one amino acid residue 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 signal transmission in a cell based assay. In one embodiment, the variant comprises one or more amino acid substitutions in one or more than one hypervariable and / or framework region of the parent antibody. For example, a variant may contain at least one substitution, for example, from about one to about ten, and preferably from about two to about five, in one or more than one hypervariable and / or frame region of the parent antibody. Typically, the variant will have an amino acid sequence having at least 50% amino acid sequence identity with the variable domain sequences of the heavy or light chains of the parent antibody, more preferably at least 65%, 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% identity is followed lnosti. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the residues of the parent antibody, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. None of the N-terminal, C-terminal or internal extensions, deletions or insertions in the sequence of antibodies should not be construed as affecting the identity or homology of the sequence. The variant retains the ability to bind to IL-31 and preferably has the desired activities superior to the activity of the parent antibody. For example, a variant may have higher binding affinity, improved ability to reduce, inhibit, or neutralize IL-31 activity in an animal, and / or improved ability to inhibit IL-31, a mediated pSTAT signal transmission in a cell-based assay.

The term "variant" nucleic acid as used herein refers to a molecule that has sequence differences from a "parent" nucleic acid. Mismatch of polynucleotide sequences may be due to mutational changes, such as deletions, substitutions or additions of one or more than one nucleotide. Each of these changes can occur individually or in combination one or more times in a given sequence.

A “parent” antibody as used herein is an antibody that is encoded by the amino acid sequence used to produce the variant. Preferably, the parent antibody has a canine Ig framework region and, if it is present, has a canine antibody constant region (s). For example, the parent antibody may be a caninized or canine antibody. As another example, the parent antibody may be a felinized or feline antibody. As another example, the parent antibody is a murine monoclonal antibody.

The term "isolated" means that a substance (for example, an antibody or nucleic acid) is separated from and extracted from a component of its natural environment. The polluting components of its natural environment are substances that will interfere with the diagnostic or therapeutic uses of the substance, and may include enzymes, hormones, and other protein or non-protein dissolved substances. As for nucleic acid, an isolated nucleic acid may include one isolated from 5′-3′-sequences with which it is usually associated on the chromosome. In preferred embodiments, the substance will be purified to more than 95% by weight of the substance, and most preferably to more 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 not be present. Normally, however, the recovered material will be obtained by at least one purification step.

The word "label" when used in this description refers to a detectable compound or composition that is conjugated directly or indirectly with an antibody or nucleic acid. The label itself can be detected by it itself (for example, radioisotope labels or fluorescent labels), or, in the case of an enzymatic label, it can catalyze a chemical change that is a substrate of a compound or composition that can be detected.

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

“Subject” or “patient” refers to an animal in need of treatment that can be affected by the molecules of the invention. Animals that can be treated according to the invention include vertebrates, with mammals such as canine, feline and equine families being particularly preferred.

A “therapeutically effective amount” (or “effective amount”) refers to an amount of an active ingredient, for example, an agent of the invention, sufficient to achieve useful or desired results when administered to a subject or patient. An effective amount may be administered in one or more than one administration, application, or in the form of one or more than one dose. A therapeutically effective amount of a composition of the invention can be readily determined by one of ordinary skill 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 itchy or allergic condition, including clinical improvement in symptoms. Undoubtedly, a therapeutically effective amount will vary depending on the particular subject being treated and the condition, weight and age of the subject, the severity of the disease state, the particular compound selected, the dosage regimen to be observed, timing of the administration, route of administration and the like, all of which can be easily determined by one of ordinary skill in the art.

Used in this description, the term "therapeutic" covers the full range of treatments for a disease or disorder. The action of the “therapeutic” agent according to the invention can be prophylactic or preventive, including when using techniques designed for target animals, which can be defined as risky (pharmacogenetics); or enhancing or curative in nature; or may lead to a decrease in the rate or degree of progression of at least one symptom of the disease or disorder being treated.

The terms "treatment", "treatment" and the like refer to both therapeutic treatment and prophylactic or preventive measures. Animals in need of treatment include animals already having the disorder, as well as those in which the disorder should be prevented. The term “treating” or “treating” a disease or disorder includes preventing or protecting against the disease or disorder (that is, taking measures to prevent the development of clinical symptoms); inhibiting a disease or disorder (i.e., delaying or suppressing the development of clinical symptoms); and / or alleviating the disease or disorder (i.e., implementing measures that cause regression of clinical symptoms). Obviously, it is not always possible to distinguish between the "prevention" and the "suppression" of a disease or disorder, since the main triggering event (s) may / may be unknown (s) or hidden (s). Accordingly, it is apparent that the term “prevention” constitutes a type of “treatment” that encompasses 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 aero allergens such as pollen, dust, molds, dust mite proteins, saliva injected with insect bites, etc. Examples of allergic conditions include, but are not limited to, the following: allergic dermatitis, summer eczema, urticaria, fuse, inflammatory airway disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease and inflammatory processes resulting from an autoimmune reaction, such as irritable bowel syndrome (IBS).

The term “pruritic condition” is defined herein as a disease or disorder characterized by an intense sensation of itching that causes the urge to rub or scratch the skin for relief. Examples of pruritic conditions include, but are not limited to, the following: atopic dermatitis, eczema, psoriasis, scleroderma, and pruritis.

Used in this description, the terms "cell", "cell line" and "cell culture" can be used interchangeably. All these terms also include their offspring, which represents any and all subsequent generations. It is understood that all offspring may not be identical due to intentional or accidental mutations. As applied to the expression of a heterologous nucleic acid sequence, a “host cell” refers to a prokaryotic or eukaryotic cell (eg, bacterial cells, yeast cells, mammalian cells and insect cells) regardless of whether they are localized in vitro or in vivo. For example, host cells can be localized in a transgenic animal. A host cell can be used as a recipient for vectors and can include any transformative organism, that is, capable of replicating a vector and / or expressing a heterologous nucleic acid encoded by a vector.

The term “composition” is intended to mean a combination of an active agent and other compounds or compositions that may be inert (eg, label) or active, such as an adjuvant.

The pharmaceutically acceptable carriers defined herein that are suitable for use in this invention are well known to those skilled in the art. Such carriers include, but are not limited to, water, saline, buffered saline, phosphate buffer, alcohol / water solutions, emulsions or suspensions. Other conventional diluents, adjuvants and excipients may be added according to conventional methods. 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, but are not limited to salts derived from organic acids or bases. Typical buffers include, but are not limited to, buffers based on salts of organic acids such as citric acid salts such as citrates, ascorbic acid, gluconic acid, histidine-HCl, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid, Tris, tromethamine hydrochloride or phosphate. Parenteral carriers may include sodium chloride solution, dextrose in Ringer's solution, dextrose, trehalose, sucrose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous vehicles may include liquid and nutrient excipients, electrolyte excipients, for example, based on dextrose in Ringer's solution and the like. Preservatives and other excipients, such as, for example, antimicrobials, antioxidants, chelating agents (e.g. EDTA), inert gases and the like, may also be present in pharmaceutical carriers. The present invention is not limited to this choice of carrier. The manufacture of such pharmaceutically acceptable compositions from the above components having suitable pH, isotonicity, stability and other traditional characteristics is within the competence of a person skilled in the art. See, for example, documents such as Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott Williams & Wilkins, publ., 2000; and The Handbook of Pharmaceutical Excipients, 4 ^th edit., eds. RC Rowe et al., APhA Publications, 2003.

The term "conservative amino acid substitution" refers to any amino acid substitution for a given amino acid residue, in which the replaced residue is so chemically similar to the given residue that it does not cause any significant weakening of the function of the polypeptide (for example, enzymatic activity). Conservative amino acid substitutions are generally known in the art and examples thereof are described, for example, in US Pat. Nos. 6,790,639, 6774107, 6194167 or 5350576. In a preferred embodiment, a conservative amino acid substitution will be any substitution that occurs within one of the following six groups :

- 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 (Gln; His); Asp (Glu); Gln (Asn); Glu (Asp); Gly (Pro); His (Asn; Gln); Ile (Leu; Val); Leu (Ile; Val); Lys (Arg; Gln; Glu); Met (Leu; Ile); Phe (Met; Leu; Tyr); Ser (Thr); Thr (Ser); Trp (Tyr); Tyr (Trp; Phe); and Val (Ile; Leu).

Just as a polypeptide may contain a conservative amino acid (s) substitution (s), the polynucleotide of the present invention may contain a conservative (s) substitution (s) of codons. Codon substitution is considered conservative if, upon expression, it gives a conservative amino acid substitution as described above. Substitution of degenerate codons, which does not lead to any amino acid substitution, is also useful in the polynucleotides of the present invention. So, for example, a polynucleotide encoding a selected polypeptide useful in one embodiment of the present invention can be mutated by replacing degenerate codons in order to approach the frequency of use of the codons shown by the expressing host cell to be transformed, or otherwise improve its expression.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to the specific methodology, protocols and reagents, etc. set forth in this description, and which, as such, may vary. The terminology used in this description is used only to describe specific embodiments and is not intended to limit the scope of the present invention, which is defined solely by the claims.

Unless otherwise specified, the scientific and technical terms used with reference to the antibodies described herein will have meanings that are generally understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, terms used in the singular will include the plural, and terms in the plural will include the singular. In most cases, the nomenclatures used in relation to the cultivation of cells and tissues, molecular biology, chemistry of proteins and oligo or polynucleotides and hybridization, and the methods related to all of the above described in this description, are methods well known and widely used in this area.

To work with recombinant DNA, the synthesis of oligonucleotides, tissue culture and transfection using standard methods (e.g. electroporation, lipofection). Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions or are carried out as is customary in the art or as set forth herein. The above methods and techniques are usually carried out in accordance with traditional methods well known in the art, and as described in various general and more specialized references that are cited and discussed throughout the description of the present invention. See, for example, Sambrook et al. MOLECULAR CLONING: LAB. MANUAL (3rd ed., Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY, 2001), and Ausubel et al., Current Protocols in Molecular Biology (New York: Greene Publishing Association / Wiley Interscience), 1993. Nomenclatures used in relation to analytical chemistry, synthetic organic chemistry and medical and pharmaceutical chemistry, and laboratory techniques and methods related to all of the above, described in this description, are well known and widely used in this field. For carrying out chemical syntheses, chemical analyzes, obtaining, manufacturing and delivering pharmaceuticals and treating patients, standard methods are used.

With the exception of working examples or where not otherwise indicated, it should be understood that all numbers expressing the amounts of ingredients or reaction conditions used in this description, in all cases, are corrected using the term "about".

All patents and other publications found are expressly incorporated into this description by reference for the purpose of exposition and disclosure, for example, of the methodologies described in such publications that can be used in relation to the present invention. These publications are provided solely in view of their disclosure prior to the filing date of this application.

The present invention provides recombinant monoclonal antibodies and peptides and their use in clinical and scientific methods, including diagnostic methods.

With the advent of molecular biology methods and recombinant DNA technology, it has become possible to obtain antibody molecules and molecules similar to antibodies using recombinant DNA methods and, at the same time, to create genetic sequences encoding specific amino acid sequences found in the polypeptide structure of antibodies. Such antibodies can be obtained either by cloning genetic sequences encoding the polypeptide chains of these antibodies, or by direct synthesis of these polypeptide chains with the assembly of synthesized chains to form active tetrameric (H ₂ L ₂ ) structures with affinity for specific epitopes and antigenic determinants. This made it easy to obtain antibodies having sequences characteristic of neutralizing antibodies from different species and sources.

Regardless of the source of the antibodies or whether they are obtained by recombinant construction either by synthesis, in vitro or in vivo, using transgenic animals, large-scale cell cultures of laboratories or industrial plants, using transgenic plants, or by direct chemical synthesis without using living organisms at any stage of the method, all antibodies have a similar overall spatial structure. This structure is often given in the form of H ₂ L ₂ , and this means that antibodies usually contain two light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions capable of interacting with a structurally complementary antigenic target. The regions interacting with the target are called “variable” or “V regions” and are characterized by differences in the amino acid sequence of antibodies with different antigenic specificity. The variable regions of either H or L chains contain amino acid sequences capable of specific binding to antigenic targets.

Used in this description, the term "antigennegative region" refers to that part of the antibody molecule that contains amino acid residues that interact with the antigen and give the antibody its specificity and affinity for the antigen. The antibody binding region contains “framework” amino acid residues necessary to maintain the proper conformation of antigen binding residues.

Within the variable regions of the H- or L-chains, in which antigen-binding regions are provided, there are smaller sequences, called "hypervariable" due to their extreme variability among antibodies with different specificities. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDR" regions. These CDR regions are responsible for the main specificity of the antibody for the structure of a particular antigenic determinant.

CDRs are non-contiguous sections of amino acids within the variable regions, however, it has been found that regardless of the species, these important amino acid sequences within the variable regions of the heavy and light chains have the same arrangement within the amino acid sequences of the variable domain domains. Each of the variable domains of the heavy and light chains of all antibodies has three regions of CDR, each of which is non-adjacent to the other.

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

As for the antigenic determinant recognized by the regions of the CDR of an antibody, it is also referred to as an “epitope”. In other words, an epitope refers to that part of any molecule that is able to recognize and with which an antibody can bind (the corresponding antibody binding region may be called a paratope).

An “antigen” is a molecule or portion of a molecule that an antibody can bind to, which can also induce an animal to produce an antibody capable of binding to an epitope of that antigen. An antigen may have one or more than one epitope. The above reference to a specific reaction indicates that the antigen will interact in a highly selective manner with its corresponding antibody, and not with many other antibodies that can be induced by other antigens.

The term “antibody” is intended to include both intact immunoglobulin molecules and their regions, fragments, peptides and derivatives, such as, for example, Fab, Fab ′, F (ab ′) ₂ , Fv, Fse, CDR regions, paratopes or any portion or peptide sequence of an antibody that is capable of binding to an antigen or epitope. An antibody is said to be “capable of binding” to a molecule if it is able to specifically interact with a given molecule, whereby the molecule is bound to the antibody.

An antibody also includes chimeric antibodies, heterochemical antibodies, caninized antibodies, or felinized antibodies, as well as fragments, regions, peptides or derivatives thereof, obtained by any known method, such as, but not limited to, enzymatic cleavage, peptide synthesis or recombinant DNA methods . Such antibodies of the present invention are capable of specifically binding to at least one of canine IL-31 or feline IL-31. Antibody fragments or regions may not contain an Fc fragment of an intact antibody, more rapidly excreted from the bloodstream, and may have less nonspecific binding in tissues than an intact antibody. Examples of antibody fragments can be obtained from intact antibodies using methods well known in the art, for example, by proteolytic cleavage by 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). Antibody sites can be obtained by any of the methods mentioned above or can be obtained by expression of a recombinant molecule site. For example, the CDR region (s) of the recombinant antibody may (gut) be isolated (s) and subcloned (s) into a suitable expression vector. See, for example, US patent No. 6680053.

The nucleotide and amino acid sequences of clones 11E12, 34D03 and 19D07

In some embodiments, the present invention provides new monoclonal antibodies that specifically bind to at least one of canine IL-31 or feline IL-31. In one embodiment, the monoclonal antibody of the invention binds to canine IL-31 or feline IL-31 and prevents its binding to its coreceptor complex containing IL-31 receptor A (IL-31Ra) and an oncostatin-M specific receptor (OsmR or IL -31Rb), and the activation of this complex. The monoclonal antibodies of the present invention are identified herein as “11E12”, “34D03” and “19D07”, which refers to the number assigned to their hybridoma clone. In this description, the designation "11E12", "34D03" or "19D07" also refers to a portion of a monoclonal antibody, a paratope or CDR that specifically binds to an epitope of IL-31 identified as 11E12, 34D03 or 19D07 due to its ability to bind to antibodies 11E12, 34D03 or 19D07, respectively. The various recombinant, chimeric, heterochemical, caninized and / or felinated forms 11E12, 34D03 and 19D07 described herein may have the same name.

In one embodiment, the present invention provides an isolated antibody or antigen binding site thereof comprising at least one of the following:

complementarity determining region (CDR) 1 of the heavy domain (V _H ) variable domain having the amino acid sequence YYDIN (SEQ ID NO: 1; 11E12-VH-CDR1), SYDMS (SEQ ID NO: 2; 19D07-VH-CDR1) or NYGMS (SEQ ID NO: 3; 34D03-VH-CDR1);

The heavy chain variable domain CDR2 having the amino acid sequence WIFPGDGGTKYNETFKG (SEQ ID NO: 4; 11E12-VH-CDR2), TITSGGGYTYSADSVKG (SEQ ID NO: 5; 19D07-VH-CDR2) or TISYGGSYTYYPDHG (6); -CDR2);

The heavy chain variable domain CDR3 having the amino acid sequence ARGGTSVIRDAMDY (SEQ ID NO: 7; 11E12-VH-CDR3), ARQNWWGLAY (SEQ ID NO: 8; 19D07-VH-CDR3) or VRGYGYDTMDY (SEQ ID NO: 9; 34D03-VH -CDR3); and

their variant having one or more than one conservative amino acid substitution in at least one of CDR1, CDR2 or CDR3.

In another embodiment, the invention provides an isolated antibody or antigen binding site thereof comprising at least one of the following group:

the variable domain of the light (V _L ) chain containing the complementarity determining region (CDR) 1 having the amino acid sequence RASESVDNYGISFMH (SEQ ID NO: 10; 11E12-VL-CDR1), KSSQSLLNSGNQKNYLA (SEQ ID NO: 11; 19D07-VL-CDR1) or KASQSVSFAGTGLMH (SEQ ID NO: 12; 34D03-VL-CDR1);

CDR2 of the light chain variable domain having the amino acid sequence RASNLES (SEQ ID NO: 13; 11E12-VL-CDR2), GASTRES (SEQ ID NO: 14; 19D07-VL-CDR2) or RASNLEA (SEQ ID NO: 15; 34D03-VL -CDR2);

CDR3 of the light chain variable domain having the amino acid sequence QQSNKDPLT (SEQ ID NO: 16; 11E12-VL-CDR3), QNDYSYPYT (SEQ ID NO: 17; 19D07-VL-CDR3) or QQSREYPWT (SEQ ID NO: 18; 34D03-VL -CDR3); and

their variant having one or more than one conservative amino acid substitution in at least one of CDR1, CDR2 or CDR3.

In yet another embodiment, an antibody having at least one of the CDRs of the light chain variable domain described above may further comprise at least one of the following CDRs of the heavy chain variable domain:

the complementarity determining region (CDR) 1 of the heavy chain variable domain having the amino acid sequence YYDIN (SEQ ID NO: 1; 11E12-VH-CDR1), SYDMS (SEQ ID NO: 2; 19D07-VH-CDR1) or NYGMS (SEQ ID NO : 3; 34D03-VH-CDR1);

The heavy chain variable domain CDR2 having the amino acid sequence WIFPGDGGTKYNETFKG (SEQ ID NO: 4; 11E12-VH-CDR2), TITSGGGYTYSADSVKG (SEQ ID NO: 5; 19D07-VH-CDR2) or TISYGGSYTYYPDHG (6); -CDR2);

The heavy chain variable domain CDR3 having the amino acid sequence ARGGTSVIRDAMDY (SEQ ID NO: 7; 11E12-VH-CDR3), ARQNWWGLAY (SEQ ID NO: 8; 19D07-VH-CDR3) or VRGYGYDTMDY (SEQ ID NO: 9; 34D03-VH -CDR3); and

their variant having one or more than one conservative amino acid substitution in at least one of CDR1, CDR2 or CDR3.

In some embodiments, the antibody may comprise at least one of the following:

a) the variable domain of the light chain containing

b) the variable domain of the heavy chain containing

c) variants thereof having one or more than one conservative amino acid substitution.

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

The present invention also includes, within its scope, nucleotide sequences encoding the variable regions of the light and heavy chains of an anti-IL-31 antibody of the present invention. Any nucleotide sequence that encodes the amino acid sequence of antibodies 11E12, 34D03 or 19D07 or their peptides is also included within the scope of the invention.

In some embodiments, the invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding at least one of the following:

complementarity determining region (CDR) 1 of the heavy domain (V _H ) variable domain having the amino acid sequence YYDIN (SEQ ID NO: 1; 11E12-VH-CDR1), SYDMS (SEQ ID NO: 2; 19D07-VH-CDR1) or NYGMS (SEQ ID NO: 3; 34D03-VH-CDR1);

The heavy chain variable domain CDR2 having the amino acid sequence WIFPGDGGTKYNETFKG (SEQ ID NO: 4; 11E12-VH-CDR2), TITSGGGYTYSADSVKG (SEQ ID NO: 5; 19D07-VH-CDR2) or TISYGGSYTYYPDHG (6); -CDR2);

The heavy chain variable domain CDR3 having the amino acid sequence ARGGTSVIRDAMDY (SEQ ID NO: 7; 11E12-VH-CDR3), ARQNWWGLAY (SEQ ID NO: 8; 19D07-VH-CDR3) or VRGYGYDTMDY (SEQ ID NO: 9; 34D03-VH -CDR3); and

their variant having one or more than one conservative amino acid substitution in at least one of CDR1, CDR2 or CDR3.

In other embodiments, the isolated nucleic acid described above may further comprise a nucleic acid sequence encoding at least one of the following:

the variable domain of the light (V _L ) chain containing the complementarity determining region (CDR) 1 having the amino acid sequence RASESVDNYGISFMH (SEQ ID NO: 10; 11E12-VL-CDR1), KSSQSLLNSGNQKNYLA (SEQ ID NO: 11; 19D07-VL-CDR1) or KASQSVSFAGTGLMH (SEQ ID NO: 12; 34D03-VL-CDR1);

CDR2 of the light chain variable domain having the amino acid sequence RASNLES (SEQ ID NO: 13; 11E12-VL-CDR2), GASTRES (SEQ ID NO: 14; 19D07-VL-CDR2) or RASNLEA (SEQ ID NO: 15; 34D03-VL -CDR2);

CDR3 of the light chain variable domain having the amino acid sequence QQSNKDPLT (SEQ ID NO: 16; 11E12-VL-CDR3), QNDYSYPYT (SEQ ID NO: 17; 19D07-VL-CDR3) or QQSREYPWT (SEQ ID NO: 18; 34D03-VL -CDR3); and

their variant having one or more than one conservative amino acid substitution in at least one of CDR1, CDR2 or CDR3.

The present invention also provides a vector comprising at least one of the nucleic acids described above.

Since the genetic code is degenerate, more than one codon can be used to encode a particular amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which will be able to encode a given amino acid. The likelihood that a particular oligonucleotide will indeed constitute a specific XXX coding sequence can be estimated taking into account the ratios of abnormal base pairings and the frequency with which a particular codon is actually used (to encode a specific amino acid) in eukaryotic or prokaryotic cells expressing an anti-IL-31 antibody or plot. Such “codon usage rules” are described in Lathe et al., 183, J. Molec. Biol. 1-12 (1985). Using the Lathe “codon rules”, a single nucleotide sequence or a set of nucleotide sequences can be identified containing the theoretically “most likely” nucleotide sequence capable of encoding anti-IL-31 antibody sequences.

In addition, it is understood that the regions encoding the antibody for use in the present invention can also be obtained by modifying the genes of existing antibodies using standard molecular biology methods, resulting in the variants (agonists) of antibodies and peptides described herein. Such variants include, but are not limited to, deletions, insertions and substitutions in the amino acid sequence of antibodies or peptides against IL-31.

For example, one class of substitutions are conservative amino acid substitutions. Such substitutions are substitutions as a result of which a given amino acid in an anti-IL-31 antibody peptide is replaced with another amino acid with similar characteristics. Usually conservative substitutions are considered substitutions of one type among the aliphatic amino acids Ala, Val, Leu and Ile, interchange of hydroxyl residues Ser and Thr, permutation of acid residues Asp and Glu, substitution between amide residues Asn and Gin, permutation of the main residues Lys and Arg, substitutions among aromatic residues Phe, Tyr and the like. Guidance on which amino acid substitutions are likely to be phenotypically silent is found in Bowie et al., 247, Science, 1306-10 (1990).

A variant or agonist of antibodies or peptides against IL-31 may be fully functional or it may lack one or more than one kind of functional activity. Fully functional variants usually contain only conservative variations or variations in non-critical residues or in non-critical areas. Functional options may also include the replacement of similar amino acids, which does not cause any change or leads to a slight change in function. Alternatively, such substitutions may, to some extent, positively or negatively affect the function. Non-functional variants typically comprise one or more than one non-conservative amino acid substitution, deletion, insertion, inversion or shortening, or substitution, insertion, inversion or deletion in a critical residue or critical region.

Amino acids that are essential for functioning 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). Using the latter technique, single mutations are introduced with alanine substitution in each residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as epitope binding or in vitro ADCC activity. Sites critical for ligand-receptor binding can also be determined by structural analysis, such as crystallography, nuclear magnetic resonance, or photoaffinity labeling. Smith et al., 224, J. Mol. Biol., 899-904 (1992); de Vos et al., 255, Science, 306-12 (1992).

In addition, polypeptides often contain amino acids other than twenty “natural” amino acids. In addition, many amino acids, including terminal amino acids, can undergo modification as a result of natural processes, such as processing and other post-translational modifications, or as a result of applying chemical modification methods well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme group, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of transfatidylinositol , disulfide bond formation, demethylation, covalent crosslinking, cystine formation, images pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation (glycosylphosphatidylinositol), hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, amino acid transfer, sulfate transfer like arginylation, and ubiquitinylation.

Such modifications are well known to specialists in this field and are described in great detail in the scientific literature. Some particularly common modifications, glycosylation, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation are described, for example, in most basic documents, such as Proteins - Structure and Molecular Properties (2nd ed., TE Creighton, WH Freeman & Co., NY, 1993). Many reviews are available 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 antibodies and peptides of the present invention also encompass derivatives or analogs in which the substituted amino acid residue is not a residue encoded by a genetic code.

Similarly, additions and substitutions in the amino acid sequence, as well as variations and modifications just described, can equally be applied to the amino acid sequence of the antigen and / or epitope of IL-31 or their peptides, and thus, they are covered by the present invention. As mentioned above, the genes encoding the monoclonal antibody of the present invention are particularly effective in recognizing IL-31.

Derivatives of Antibodies

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

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

Another derivative bifunctional antibody of the present invention is a bispecific antibody obtained by combining parts of two separate antibodies that recognize two different antigenic groups. This can be achieved by cross-linking or recombinant DNA technology. In addition, moieties can be added to the antibody or its region to increase the half-life in vivo (for example, by increasing the time it takes to remove from the bloodstream). Such methods include, for example, the addition of PEG moieties (polyethylene glycol) (also called pegylation) and are well known in the art. See U.S. Patent Application Publication No. 2000031671.

Recombinant Antibody Expression

In some embodiments, nucleic acids encoding a monoclinal antibody to be tested are introduced directly into the host cell and the cell is incubated under conditions sufficient to induce expression of the encoded antibody. After the test nucleic acids are introduced into the cell, the cell is typically incubated usually at 37 ° C, sometimes under selection conditions, for about 1-24 hours to allow expression of the antibody. In one embodiment, the antibody is secreted into the supernatant of the media in which the cell grows.

Monoclonal antibodies are traditionally prepared as native molecules in murine hybridoma lines. In addition to this technology, the present invention provides expression of recombinant DNA monoclonal antibodies. This allows one to obtain caninized and felinated antibodies, as well as a spectrum of derivatives of antibodies and fusion proteins in the selected form of the host.

The nucleic acid sequence encoding at least one anti-IL-31 antibody, region or polypeptide of the present invention, can be recombined with the DNA of the vector in accordance with traditional methods, including the use of blunt or "step" ends for ligation, digestion with restriction enzymes to obtain suitable ends, if necessary, completion of the sticky ends, treatment with alkaline phosphatase to avoid undesired attachment, and ligation with suitable ligases. Methods for performing such manipulations are described, for example, in Maniatis et al., MOLECULAR CLONING, LAB. MANUAL (Cold Spring Harbor Lab. Press, NY, 1982 and 1989) and Ausubel et al., 1993, supra, and can be used to construct nucleic acid sequences encoding a monoclonal antibody molecule or its antigen binding region.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences containing transcriptional and translational regulation information, and such sequences are "operably linked" to nucleotide sequences that encode the polypeptide. A functional link is a link through which the regulatory DNA sequences and the DNA sequence that needs to be expressed are linked in such a way that gene expression can be carried out in the form of peptides or regions of an anti-IL-31 antibody in an extractable amount. The exact nature of the regulatory regions necessary for gene expression can vary from organism to organism, as is well known in the related art. See, for example, Sambrook et al., 2001, supra; Ausubel et al., 1993, supra.

Accordingly, the present invention encompasses the expression of an anti-IL-31 antibody or peptide in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including bacteria, yeast, insects, fungi, bird and mammalian cells, either in vivo or in situ, or host cells derived from a mammal, insect, bird or yeast. A mammalian cell or tissue may be derived from a human, primate, hamster, rabbit, rodent, 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 inserted into a plasmid or viral vector capable of autonomously replicating in the recipient host. For this purpose, any of a wide variety of vectors can be used. See, for example, Ausubel et al., 1993, above. Important factors when choosing a particular plasmid or viral vector include: the simplicity with which recipient cells containing a given vector can be recognized and selected among those recipient cells that do not contain this vector; the number of copies of the vector, which is necessary for a particular host; and, when desired, the ability to implement a “shuttle” movement of the vector between host cells of different species.

Typical prokaryotic vectors known in the art include plasmids, such as those capable of replication in E. coli (such as, for example, pBR322, ColE1, pSC101, pACYC 184, .pi.VX). Such plasmids are described, for example, in Maniatis et al., 1989, supra; Ausubel et al., 1993, supra. Bacillary plasmids include pC194, pC221, pT127, etc. Such plasmids are described by Gryczan in THE MOLEC. BIO OF THE BACILLI, 307-329 (Academic Press, NY, 1982). Suitable plasmids for streptomycetes include pIJ101 (Kendall et al., 169, J. Bacteriol., 4177-83 (1987)) and bacteriophages for streptomycetes such as φC31 (Chater et al. In SIXTH INT′L SYMPOSIUM ON ACTINOMYCETALES BIO. 45. -54 (Akademiai Kaido, Budapest, Hungary 1986)). A review of plasmids for pseudomonads is given in 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 antibodies or peptides against IL-31 include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 , Mol. Cell. Biol., 280 (1983)), LTR (long terminal repeats) of Routh sarcoma virus (German et al., 79, Proc. Natl. Acad. Sci., USA, 6777 (1982)) and LTR virus mouse Moloney leukemia (Grosschedl et al., 41, Cell, 885 (1985)); (b) splicing regions and polyadenylation sites, such as those originating from the late region of the SV40 genome (Okayarea et al., 1983), and (c) polyadenylation sites, such as those in SV40 (Okayama et al., 1983).

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

For immunoglobulin genes, consisting partly of cDNA, partly of genomic DNA (Whittle et al., 1, Protein Engin., 499 (1987)), the human cytomegalovirus promoter can be a transcriptional promoter, cytomegalovirus and mouse / human immunoglobulin enhancers can be enhancers , and the regions of mRNA splicing and polyadenylation may be the natural chromosome sequences of immunoglobulins.

In one embodiment, for the expression of cDNA genes in rodent cells, the transcriptional promoter is a viral LTR sequence, the transcriptional promoter enhancers are one of two or both enhancers: a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, the splicing region contains an intron larger than 31 bp and the polyadenylation and termination of transcription region come from a natural 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 express proteins in mammalian cells.

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

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

In order to transfect with expression vectors and produce a chimeric antibody, the recipient cell line may be a myeloma cell line. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by the immunoglobulin genes used for transfection, and may have an immunoglobulin glycosylation mechanism. Myeloma cells can grow in a culture or in the abdominal cavity of a mouse, from where secreted immunoglobulin can be obtained from ascites fluid. Other suitable recipient cells include lymphoid cells, such as human or non-human B cells, hybrid cells of human or non-human origin, or interspecific heterohybrid cells.

An expression vector carrying the chimeric, caninized, or felinated antibody construct or the anti-IL-31 polypeptide of the present invention can be introduced into a suitable host cell by any of a number of suitable methods, including biochemical methods such as transformation, transfection, conjugation, protoplast fusion, precipitation of calcium phosphate and the use of polycations, such as diethylaminoethyl (DEAE) -dextran, and mechanical methods such as electroporation, direct microinjection and microparticle bombardment. Johnston et al., 240, Science, 1538 (1988).

Yeast can give significant advantages over bacteria in relation to the production of H and L chains of immunoglobulins. Post-translational modifications of peptides, including glycosylation, occur in yeast. There are currently a number of recombinant DNA strategies that use sequences of strong promoters and high copy plasmids that can be used to produce the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secrete peptides carrying leader sequences (i.e., prepeptides). Hitzman et al., 11th Int′l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

Yeast gene expression systems can be evaluated according to standard methods for the levels of production, secretion and stability of anti-IL-31 peptides, antibodies and assembled murine and chimeric, heterochemical, caninized or felinated antibodies, fragments and regions thereof. Any of a number of yeast gene expression systems may be used, including a promoter and termination elements from actively expressed genes encoding glycolytic enzymes produced in large quantities when the yeast is grown in glucose rich media. Known glycolytic enzyme genes can also provide very effective transcriptional control signals. For example, the signals of the promoter and terminator of the phosphoglycerate kinase (PGK) gene can be used. A number of approaches can be used to evaluate expression plasmids that are optimal for the expression of cloned immunoglobulin cDNAs 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 or peptide molecules described in this invention. Together with these 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 can provide phenotypic selection of transformed cells. A number of approaches can be used to evaluate expression plasmids for the production of murine, chimeric, heterochemical, caninized or felinated antibodies, antibody fragments and regions or chains encoded by cloned immunoglobulin cDNAs in bacteria (see Glover, 1985, supra; Ausubel, 1993, supra; Sambrook, 2001, supra; Colligan et al., Eds. Current Protocols in Immunology, John Wiley & Sons, NY, NY (1994-2001); Colligan et al., Eds. Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001)).

Mammalian host cells can grow in vitro or in vivo. Mammalian cells allow for post-translational modifications of protein molecules of immunoglobulins, including removal of the leader peptide, folding and assembly of H and L chains, glycosylation of antibody molecules and secretion of the functional antibody protein.

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

Many vector systems are available for expression of the cloned H- and L-chain genes of anti-IL-31 peptides in mammalian cells (see Glover, 1985, supra). To obtain full-sized antibodies H ₂ L ₂ you can follow different approaches. In order to achieve intracellular association and association of H and L chains into full-sized tetrameric antibodies H ₂ L ₂ and / or anti-IL-31 peptides, H and L chains can be co-expressed in the same cells. Co-expression can be carried out using either the same or different plasmids in the same host. The genes for both H and L chains and / or anti-IL-31 peptides can be placed in the same plasmid, which is further transfected with cells, directly selecting cells that express both chains. Alternatively, you can first transfect the cells with a plasmid encoding a single strand, for example an L chain, then transfect the resulting cell line using an H chain-carrying plasmid containing a second selectable marker. Cell lines producing anti-IL-31 peptides and / or H ₂ L ₂ molecules via one of these two pathways can be transfected with plasmids encoding additional copies of the peptides, H, L or H plus L chains along with additional selectable markers to create cell lines with improved properties, such as a higher level of production of assembled H ₂ L ₂ antibody molecules or increased stability of transfected cell lines.

For the long-term production of recombinant antibodies in high yield, stable expression can be used. For example, cell lines stably expressing an antibody molecule can be constructed. Instead of using expression vectors that contain viral origin of replication, host cells can be transformed with immunoglobulin-expressing cassettes and a selectable marker. After the introduction of foreign DNA, the constructed cells can be left to grow for 1-2 days on enriched media and then transferred to selective media. The selectable marker in the recombinant plasmid confers resistance to selection and enables cells to stably integrate the plasmid into the chromosome and grow with the formation of centers, which in turn can be cloned and expanded into cell lines. Such engineered cell lines may be particularly useful in screening and evaluating compounds / components that directly or indirectly interact with an antibody molecule.

Once the antibody of the invention is obtained, it can be purified by any method known in the art for purification of an immunoglobulin molecule, for example chromatography (for example, ion exchange, affinity, in particular with affinity for a specific antigen after purification based on protein A, and column chromatography with separation by size), centrifugation, a method based on the difference in solubility or any other standard method used for protein purification. In many embodiments, antibodies are secreted from the cell into the culture medium and are collected from the culture medium.

Pharmaceutical applications

The anti-IL-31 antibodies or peptides of the present invention can be used, for example, in the treatment of pruritic and / or allergic conditions in domestic animals, such as dogs and cats. More specifically, the invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as an active ingredient, an antibody or peptide of the invention. The antibody may be a chimeric, heterocimeric, caninized, or felinized antibody of the present invention. Intact immunoglobulins or their binding fragments, such as Fab, are also contemplated. The antibody and the pharmaceutical compositions of this invention are useful for parenteral administration, for example subcutaneously, intramuscularly or intravenously.

The anti-IL-31 antibodies and / or peptides of the present invention can be administered either as separate therapeutic agents, or in combination with other therapeutic agents. They can be administered individually, but typically they are administered with a pharmaceutical carrier selected based on the intended route of administration and standard pharmaceutical practice.

The administration of the antibodies described herein can be carried out by any suitable method, including parenteral injection (such as intraperitoneal, subcutaneous or intramuscular injection), orally or by topical administration of antibodies (typically contained in a pharmaceutical composition) to the surface of the respiratory tract. Topical administration to the surface of the respiratory tract can be accomplished by intranasal administration (for example, through the use of a dropper, swab or inhaler). Topical administration of antibodies to the surface of the respiratory tract can be accomplished by inhalation, for example by forming an aerosol suspension of respirable particles of a pharmaceutical composition (including both solid particles and liquid particles) containing antibodies, and then initiating inhalation by the subject of these respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical compositions are well known, and any conventional method can be used. Oral administration may be prescribed, for example, in a form suitable for ingestion of a liquid or solid composition.

In some desired embodiments, the antibodies are administered by parenteral injection. As for parenteral administration, anti-IL-31 antibodies or peptides can be prepared in the form of a solution, suspension, emulsion or lyophilized powder together with a pharmaceutically acceptable parenteral excipient. For example, the excipient may be an antibody solution or mixture thereof dissolved in an acceptable carrier, such as an aqueous carrier; such excipients are water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine and the like. You can also use liposomes and non-aqueous fillers, such as fixed oils. These solutions are sterile and, as a rule, do not contain solid particles. These compositions may be sterilized by conventional, well-known sterilization methods. The compositions may contain pharmaceutically acceptable excipients that are necessary to approximate physiological conditions, such as pH adjusting agents and buffering agents, toxicity correction agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and t .d. The concentration of antibodies in these compositions can vary within wide limits, for example, from less than about 0.5%, usually from 1% or at least about 1% to 15% or 20% by weight, and will be selected primarily taking into account volumes liquids, viscosity values, etc. in accordance with a specifically selected route of administration. The filler or lyophilized powder may contain excipients that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The composition is sterilized by conventional methods.

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

The antibodies of this invention can be lyophilized for storage and subsequent dilution in a suitable vehicle before use. It is shown that this method is effective when using traditional immunoglobulins. Any suitable lyophilization and re-dilution methods may be used. It will be apparent to those skilled in the art that lyophilization and re-dilution can lead to a loss of antibody activity to varying degrees and that levels should be adjusted if possible to compensate for this.

Compositions containing the antibodies of the present invention or a mixture thereof can be administered to prevent relapse and / or for therapeutic treatments of an existing disease. Suitable pharmaceutical carriers are described in the most recent edition of REMINGTON′S PHARMACEUTICAL SCIENCES, a standard document referenced in the art.

In therapeutic use, the compositions are administered to a subject already suffering from a disease in an amount sufficient to treat or at least partially suppress or alleviate the disease and its complications. An amount acceptable for such an exercise is defined as a “therapeutically effective dose” or “a therapeutically effective amount”. Amounts effective for this use will depend on the severity of the disease and the general condition of the subject's own immune system, but will usually range from about 0.1 mg of antibody per kg body weight to about 10 mg of antibody per kg body weight, preferably from about 0 3 mg of antibody per kg body weight up to about 5 mg of antibody per kg body weight. Due to the minimal content of foreign substances and the decrease in the probability of rejection of a “foreign substance”, which is achieved as a result of the presence of antibodies of this invention, such as dogs and cats, it is possible to introduce significant excesses of these antibodies.

The dosage administered will undoubtedly vary depending on known factors, such as the pharmacodynamic characteristics of the particular agent and the method and route of administration; age, state of health and mass of the recipient; the nature and severity of the symptoms of concomitant treatment, the frequency of treatment and the desired effect.

As a non-limiting example, treatment of IL-31-related pathologies in dogs or cats can be carried out by administering the anti-IL-31 antibodies of the present invention once every two weeks or once a month in the dosage range described above.

Examples of antibodies for therapeutic use in dogs or cats are high affinity (they can also have high avidity) antibodies and their fragments, regions and derivatives having, according to the present invention, high in vivo anti-IL-31 activity.

Single or multiple administrations of the compositions may be performed, with the dose level and schedule being chosen by the attending veterinarian. In any case, the pharmaceutical compositions must ensure the receipt of such a quantity of antibodies according to this invention, which is sufficient for effective treatment of the subject.

Diagnostic applications

The present invention also provides the aforementioned anti-IL-31 antibodies and peptides for use in diagnostic methods for detecting IL-31 in domestic animals having or suspected to have an itchy and / or allergic condition.

The anti-11-31 antibodies and / or peptides of the present invention are useful for immunoassays that detect or quantify IL-31 or anti-IL-31 antibodies in a sample. An immunoassay for IL-31 typically involves incubating a clinical or biological sample in the presence of a labeled, detectable high affinity (or highly avid) anti-IL-31 antibody or polypeptide of the present invention capable of selectively binding to IL-31, and detecting a labeled peptide or antibody that bind in the sample. Various clinical assay techniques are well known in the art. See, for example, IMMUNOASSAYS FOR THE 80′S (Voller et al., Eds., Univ. Park, 1981). Such samples include biopsy-derived tissue samples, blood, serum, and feces or fluid samples taken from animal subjects and analyzed by ELISA as described below.

In some embodiments, the detection of antigen binding to an antibody is carried out without using a solid support. For example, detection of antigen binding to an antibody can be carried out in a liquid format.

In other embodiments, the anti-IL-31 antibody or polypeptide may be fixed, for example, to a nitrocellulose or other solid support on which the immobilization of cells, cell particles or soluble proteins can be carried out. The substrate can then be washed with suitable buffers and then treated with detectable IL-31-specific peptide or antibody. The solid phase support can then be washed with buffer a second time to remove unbound peptide or antibody. Further, the amount of bound label on a solid substrate can be determined using the steps of the known method.

A "solid phase support" or "carrier" refers to a support having the ability to bind a peptide, antigen or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride (PVDF), dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses and magnetite. For the purposes of the present invention, the carrier in nature may be either soluble to some extent or insoluble. The support material can have almost any possible structural configuration, provided that the molecule attached to it is capable of binding to IL-31 or an anti-IL-31 antibody. Thus, the configuration of the substrate can be spherical, like a ball, or cylindrical, like the inner surface of the tube or the outer surface of the rod. Alternatively, the surface may be flat, such as that of a leaf, culture dish, test strip, etc. For example, the substrates may include polystyrene beads. Many other suitable carriers for binding an antibody, peptide or antigen will be known to those skilled in the art, or they can be determined by routine experimentation.

Using well-known process steps, the binding activity of the anti-IL-31 peptide and / or antibody from a given series can be determined. Specialists in the art can determine the working and optimal conditions for the analysis by routine experimentation.

Detection of an IL-31-specific peptide and / or antibody can be accomplished by binding it to an enzyme for use in an enzyme-linked immunosorbent assay (EIA) or enzyme-linked immunosorbent assay (ELISA). The bound enzyme interacts with an accessible substrate so that a chemical moiety is formed, the detection of which can be accomplished, for example, by spectrophotometric, fluorimetric or visual means. Enzymes that can be used to detect detectable IL-31-specific antibodies of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta 5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogen , triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

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

You can also tag IL-31-specific antibodies with a fluorescent compound. If a fluorescently labeled antibody is exposed to light of an appropriate wavelength, then its presence can then be detected by fluorescence. Among the most commonly used fluorescent compounds for labeling are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine.

IL-31 specific antibodies can also be labeled with detectability using fluorescent light emitting metals such as ¹²⁵ Eu or other metals from the lanthanide family. These metals can be attached to an IL-31 specific antibody using metal chelating groups such as diethylene triamine pentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

IL-31-specific antibodies can also be labeled with detection as a result of combination with a chemiluminescent compound. The presence of a chemiluminescent-labeled antibody is then determined by detecting the presence of luminescence that occurs during a chemical reaction. Examples of useful compounds for chemiluminescent labeling are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalic ester.

Similarly, a bioluminescent compound can be used to label an IL-31-specific antibody, region, fragment, polypeptide or derivative of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytically active protein increases the effectiveness of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the appearance of luminescence. Important bioluminescent compounds used for labeling are luciferin, luciferase and equorin.

Detection of an IL-31-specific antibody, region, fragment, polypeptide or derivative can be carried out using a scintillation counter, for example, if the detectable label is a gamma-ray emitter, or using a fluorimeter, for example, if the label is a fluorescent substance. In the case of an enzymatic label, detection can be carried out by colorimetric methods that use a substrate for the enzyme. Detection can also be done by visually comparing the extent of the enzymatic reaction with the substrate relative to similarly prepared standards.

For the purposes of the present invention, IL-31, the detection of which is carried out in the above analyzes, can be represented in a biological sample. Any sample containing IL-31 may be used. For example, such a sample is a biological fluid, such as, for example, blood, serum, lymph, urine, feces, inflammatory exudates, cerebrospinal fluid, amniotic fluid, tissue extract or homogenate, and the like. This invention is not limited to analyzes that use only these samples, however, the average person skilled in the art, in accordance with the present description, will be able to determine suitable conditions under which it is possible to use other samples.

In situ detection can be performed by extracting a histological sample from an animal subject and introducing a combination of labeled antibodies of the present invention into such a sample. An antibody (or portion thereof) can be introduced by applying or applying a labeled antibody (or portion) to a biological sample. By applying this technique, it becomes possible to determine not only the presence of IL-31, but also the distribution of IL-31 in the test tissue. 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 techniques) can be modified to allow such in situ detection.

An 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, a certain amount of unlabeled antibody (or antibody fragment) is bound to a solid support that is insoluble in the liquid to be tested, and a certain detectably labeled antibody is added to detect and / or quantify the triple complex formed between solid phase antibody, antigen and labeled antibody.

Antibodies can be used to quantify or qualitatively determine IL-31 in a sample or to detect the presence of cells expressing IL-31. This can be accomplished by immunofluorescence methods that use a fluorescently labeled antibody (see below) in combination with fluorescence microscopy, flow cytometric or fluorimetric detection. For diagnostic purposes, antibodies can be either labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (secondary antibodies) capable of interacting with the antibody, such as antibodies that are specific for the constant regions of a canine or feline immunoglobulin. Alternatively, antibodies can be directly labeled. A wide variety of labels can be used, such as radionuclides, fluorescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (in particular haptens), etc. Numerous types of immunoassays, such as those previously discussed, are available and well known to those skilled in the art.

In one embodiment, the diagnostic method for detecting IL-31 is a test that uses lateral flow immunoassay. It is also known as immunochromatographic analysis, rapid immunomigration (RIM ™), or test strip analysis. Lateral flow immunoassays are essentially immunoassays adapted to run along one axis in accordance with the format of the test strips. A number of variants of this technology have been developed, implemented in commercially available products, but they all work on the same basic principle. A typical test strip consists of the following components: (1) zone for the sample - the absorbing zone on which the test sample is applied; (2) zone for conjugate or reagent — it contains antibodies specific for the target analyte conjugated to colored particles (usually colloidal gold particles or latex microspheres); (3) a membrane for conducting the reaction — typically a membrane of hydrophobic nitrocellulose or cellulose acetate, on which antibodies against the target analyte are immobilized, one line across the membrane as a capture zone or test line (there may also be a control zone containing antibodies, specific for conjugated antibodies); and (4) a swab or reservoir for waste - another absorbent zone designed to move the sample across the membrane to carry out the reaction under the action of capillary forces and its collection. As a rule, the components of the strip are fixed on an inert substrate material, and they can be presented in the form of a simple test strip or in a plastic case with a hole for the sample and a reaction window showing the capture zone and the control zone.

There are two main types of lateral flow immunoassay used in microbiological testing: sandwich assays with two antibodies and competitive assays. In a two antibody sandwich assay format, the sample migrates from the sample zone through the conjugate zone, where any target analyte present in the sample will bind to the conjugate. The sample continues to migrate further along the membrane until it reaches the capture zone, where the target / conjugate complex will bind to immobilized antibodies, forming a visible line on the membrane. The sample migrates even further along the strip until it reaches the control zone, where excess conjugate will bind and form a second visible line on the membrane. This control line indicates that the sample migrated across the membrane as planned. Two distinct lines on the membrane indicate a positive result. The presence of one line in the control zone indicates a negative result. Competitive assays differ from the two antibody sandwich assay format in that the conjugate zone contains antibodies that are already bound to the target analyte or its analogue. If the target analyte is present in the sample, then as a result, it will not bind to the conjugate and will remain unlabeled. After the sample moves along the membrane and reaches the capture zone, an excess of unlabeled analyte will bind to immobilized antibodies and block the capture of the conjugate, so no visible line is formed. The unbound conjugate will then bind to antibodies in the control zone, forming a visible control line. A single control line on the membrane indicates a positive result. Two visible lines in the capture zone and the control zone indicate a negative result. However, if there is no excess of unlabeled target analyte, a weak line may form in the capture zone, indicating an uncertain result. There are a number of options for lateral flow technology. Depending on the target analyte, the capture zone on the membrane may contain immobilized antigens or enzymes rather than antibodies. Multiple capture zones can also be used for multiple testing. For example, commercially available test strips have been developed that can detect both EHES ST1 and ST2 shiga toxins individually in the same sample.

It is important that the antibodies of the present invention may be useful in the diagnosis of pruritus and / or allergies in dogs or cats. More specifically, using the antibodies of the present invention, overexpression of IL-31 in domestic animals can be identified. Thus, the antibody of the present invention may be an important immunohistochemical agent.

The antibodies of the present invention can be used in microarrays with antibodies that are extremely suitable for determining gene expression profiles.

Sets

Kits for practicing the methods of the invention are also included within the scope of the present invention. The kits contain at least one or more than one antibody of the present invention, a nucleic acid encoding the same, or a cell containing the same. In one embodiment, the antibody of the present invention may be provided in a container, usually in lyophilized form. Antibodies that can be conjugated to a label or toxin or to be unconjugated are typically included in kits along with buffers such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, such as serum albumin, or the like like that. Typically, these substances will be present in an amount of less than 5 wt.% Based on the amount of active antibody, and usually their total amount is at least about 0.001 wt.% Again based on the concentration of the antibody. Often it will be desirable to include an inert excipient or excipient for reconstitution of the active ingredients, wherein the excipient may be present in an amount of from about 1% to 99% by weight. from the whole composition. If a secondary antibody capable of binding to the primary antibody is used in the assay, it will usually be in a separate vial. A secondary antibody is typically conjugated to a label and prepared analogously to the antibody compositions described above. Typically, a kit will also include a set of instructions for use.

In one embodiment, the kit of the present invention is a test strip kit (lateral flow immunoassay kit) useful for detecting canine or feline IL-31 protein in a sample. Such a test strip will typically include a zone for the sample onto which the test sample is applied; an area for conjugate or reagent containing an antibody specific for canine or feline IL-31, wherein the antibody is conjugated to colored particles (usually colloidal gold particles); a membrane for conducting a reaction on which anti-IL-31 antibodies are immobilized along one line across the membrane as a capture zone or test line (a control zone may also be present containing antibodies specific for conjugated antibodies); and another absorbent zone, designed to move the sample along the membrane for the reaction under the action of capillary forces and its collection. Typically, a set of test strips will also include instructions for use.

The invention will now be described further in the following non-limiting examples.

EXAMPLES

Example 1. Identification of murine monoclinal antibodies that recognize canine interleukin 31 (IL-31)

Recombinant canine IL-31 was created in CHO (Chinese Hamster Ovary) cells using the ACE (Artificial Chromosome Expression) system CHROMOS (Chromos Molecular Systems, Inc., Burnaby, British Columbia) to synthesize a secreted dog IL-31 protein with the sequence SEQ ID NO: 32. This protein is encoded by the nucleotide sequence of SEQ ID NO: 33. A conditioned medium was prepared from 400 ml of cell culture (CHO cell line) and dialyzed against 10 volumes of QA buffer (20 mM Tris pH 8.0; 20 mM NaCl) for 4.5 hours. The dialyzed medium was filtered (0.2 μm) and loaded at a speed of 1 ml / min onto a SOURSE ™ Q column (GE Healthcare, Uppsala, Sweden), pre-equilibrated with QA buffer. Protein was eluted using a multi-step linear gradient. Most IL-31 remained in the flow fraction (FT), a small amount of IL-31 was eluted at the beginning of the gradient. The identity of the protein was previously confirmed by Western immunoblotting and mass spectrometric (MS) analysis of tryptic digest. Protein in the FT fraction was concentrated 4-5 times and dialyzed overnight against phosphate buffered saline (PBS) at 4 ° C. Protein stability was checked after dialysis in PBS. No precipitation and no proteolysis were observed after several days at 4 ° C. After conducting deglycosylation experiments using N-glycosidase F, a protein compacted into one zone corresponding to ~ 15 kDa was obtained according to SDS-PAGE. Protein concentration was determined using bicinchoninic acid analysis (BCA analysis) with bovine serum albumin (BSA) as a standard (ThermoFisher Scientific, Inc., Rockford, IL). The protein solution was aliquoted, quickly frozen (liquid N ₂ ) and stored at -80 ° C.

Mouse monoclonal antibodies were identified using standard immunizations of female CF-1 mice with recombinant canine IL-31 generated in CHO cells. Titers from immunized mice were determined using enzyme-linked immunosorbent assay (ELISA). Canine IL-31 (50 ng / well) was immobilized on polystyrene microplates and used as capture antigen. Serum from immunized mice was diluted in phosphate buffered saline with 0.05% Tween-20 (PBST). Detection of the presence of murine anti-canine IL-31 antibodies was performed using horseradish peroxidase conjugated (HRP) secondary goat anti-mouse antibody (Kirkegard & Perry Laboratories, Inc. (KPL, Inc.), Gaithersburg, MD). After adding a chromogenic substrate (SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate, KPL, Inc., Gaithersburg, MD) and ten minutes of incubation at room temperature (CT), the reaction was stopped by adding 100 μl of 0.1 N. HCl. The absorbance in each well was determined by optical density (OD) at 450 nm. Figure 6 summarizes the results of the antibody response of individual mice immunized with canine IL-31. A pool of donor splenocytes from mice 3 and 4 was used for fusion. After fusion and screening for binding to IL-31, 100 wells were selected for direct propagation and secondary screening for anti-IL-31 activity by direct ELISA. Secondary screening results confirmed that the 81 fusion construct retained the ability to produce anti-IL-31 antibodies. Frozen cell masses and supernatants of these 81 candidates were stored for further evaluation.

To identify candidates with inhibitory activity, all 81 supernatants were evaluated for their ability to act on IL-31-mediated pSTAT signal transmission in a cell-based assay. In this cell-based assay, pSTAT signal transmission was measured in dog DH-82 monocytic cells pretreated for 24 hours with canine gamma interferon (R&D Systems, Minneapolis, MN) at a concentration of 10 ng / ml and depleted serum for 2 hours before treatment with IL-31 to enhance expression of the IL-31 receptor. After this pretreatment, recombinant canine IL-31 was added at a concentration of 1 μg / ml over 5 minutes and STAT phosphorylation was evaluated using Alpha Screen technology (Perkin Elmer, Waltham, MA). Since the concentration and purity of antibodies in hybridoma supernatants are unknown, a qualitative determination was made of the ability of these supernatants to inhibit STAT phosphorylation 1 hour after co-incubation with IL-31 (1 μg / ml) using 1: 2 or 1:20 dilutions of supernatants. In this experiment, 31 supernatants were identified that inhibit STAT phosphorylation by more than 50% compared to untreated wells, which was the basis for the purification and subsequent characterization.

After purification and quantification of each monoclonal antibody (mAb), IC ₅₀ values for all 31 antibodies were evaluated in a DH-82 cell assay. Based on the obtained IC ₅₀ values and competitive ELISA data for determining antibody classes based on the epitope bins, the three antibodies described in Table 1, 11E12, 19D07 and 34D03, were put forward for further research.

TABLE 1 Antibody Isotype NS Isotype LC 11E12 G1 / 2b kappa 19D07 2b kappa 34D03 G1 kappa

Example 2. DNA sequences. coding antibodies 11E12, 19D07 and 34D03

Ribonucleic acid (RNA) was isolated from 11E12, 19D07, and 34D03 hybridoma cells using an Rneasy mini-kit (Qiagen, Inc., Germantown, MD) as described by the manufacturer. One million frozen cells of each hybridoma were collected by centrifugation and RNA was purified from cell lysates using an Rneasy spin column according to the method described in the protocol. RNA was eluted from each column and immediately used to quantify and prepare cDNA. RNA was analyzed for yield and purity by measuring its absorbance at 260 nm and 280 nm using a GeneQuant pro spectrophotometer (GE Healthcare, Uppsala, Sweden). After isolation, the remaining RNA was stored at -80 ° C for further use.

Oligonucleotide primers designed to amplify the variable domains of mouse immunoglobulin (Ig) were used in accordance with the manufacturer's instructions (EMD Chemicals, Inc., Gibbstown, NJ). Received cDNA from total hybridoma RNA by reverse transcription (RT) using the ThermoScript RT kit (Invitrogen Corp., Carlsbad, CA) in accordance with the manufacturer's instructions. 200-400 ng of RNA from each hybridoma was added to a separate reaction tube containing a 3′-primer for the constant region of Ig. This 3′-primer for the Ig constant domain is located proximal to the variable region of Ig, and transcription of the first cDNA chain corresponding to the variable region of the murine antibody will occur with its participation. An individual RT reaction was performed for each hybridoma RNA using a 3′-primer for the constant domain of the heavy chain and a 3′-primer for the constant domain of the light Kappa chain.

cDNA from each hybridoma was used as a template in the polymerase chain reaction (PCR) to amplify the cDNA of the variable domain of the heavy chain and light Kappa chain of IgG to determine the sequence. For each PCR sample, multiple reactions were performed using a degenerate 5′-primer or primer pools designed for annealing with regions encoding the signal sequence of the murine Ig variable domain. Separate PCR reactions were carried out using a degenerate primer or primer pools to amplify the variable regions of the mouse Ig heavy and light chains (Figure 7). PCR was performed for 1 μl of the cDNA-containing reaction mixture using the Expand High Fidelity DNA Polymerase Kit (Roche Diagnostics Corp., Indianapolis, IN) according to the manufacturer's protocol. The following thermal cycling parameters were established for PCR: 94 ° С for 2 min, 35 cycles (94 ° С / 15 s, 55 ° С / 30 s, 72 ° С / 1 min), 72 ° С / 7 min. PCR amplified fragments were separated by 1% agarose gel electrophoresis and purified using a Qiagen gel extraction kit (Qiagen, Inc., Germantown, MD). Direct primers for the variable region of the heavy and light chains contain EcoRI or Sall sites (New England Biolabs (NEB), Inc., Ipswich, MA), and reverse primers for the variable domain of the heavy and light chains contain the HindIII site (NEB Inc., Ipswich, MA) to facilitate cloning into the plasmid pUC19. The purified PCR fragments and plasmid pUC19 were digested with the above restriction endonucleases at 37 ° C for 1-2 hours. After digestion, the PCR fragments were purified using a Qiaquick PCR purification kit (Qiagen, Inc., Germantown, MD). The digested plasmid was separated by 1% agarose gel electrophoresis and purified using a Qiagen gel extraction kit. Purified PCR fragments, which are the DNA of the variable domains of the heavy chain and light Kappa chain of IgG, were ligated into plasmid pUC19 using T4 DNA ligase and ligation buffer (NEB, Inc., Ipswich, MA), at 4 ° C for nights. 3 l of each reaction mixture after ligation was used to transform E. coli TOP 10 cells (Invitrogen Corp., Carlsbad, CA).

Plasmids were isolated from the positive clones of each hybridoma presenting variable regions using the Qiagen Miniprep kit (Qiagen 27106) according to the manufacturer's protocol. To amplify the DNA sequence for each cloned insert, M13 forward and reverse primers were used using the BigDye sequencing reaction kit (Applied Biosystems represented by Life Technologies Corp., Carlsbad, Calif.) According to the manufacturer's protocol. After sequencing, the reaction mixtures were purified using a 96-well plate cleaning kit (Zymo Research, Irvine, Calif.) According to the manufacturer's protocol. Samples were loaded onto an ABI-3730 capillary sequencer and the resulting sequence records were analyzed using a Sequencher (GeneCodes v. 4.2) for complete open reading frames. The sequences of the variable domains of murine anti-canine IL-31 antibodies defined for each antibody are as follows: 11E12 light chain variable domain (MU-11E12-VL with SEQ ID NO: 19, the corresponding nucleotide sequence of which is SEQ ID NO: 34); the variable domain of the heavy chain 11E12 (MU-11E12-VH with SEQ ID NO: 26, the corresponding nucleotide sequence of which is SEQ ID NO: 35); the variable domain of the light chain 19D07 (MU-19D07-VL with SEQ ID NO: 22, the corresponding nucleotide sequence of which is SEQ ID NO: 36); the variable domain of the heavy chain 19D07 (MU-19D07-VH with SEQ ID NO: 28, the corresponding nucleotide sequence of which is SEQ ID NO: 37); the variable domain of the light chain 34D03 (MU-34D03-VL with SEQ ID NO: 24, the corresponding nucleotide sequence of which is SEQ ID NO: 38) and the variable domain of the heavy chain 34D03 (MU-34D03-VH with SEQ ID NO: 30, corresponding the nucleotide sequence for which is SEQ ID NO: 39).

To confirm the correctness of the cDNA sequence corresponding to the variable domains of the heavy and light chains of each antibody, the N-terminal sequence was analyzed on a purified mAb protein using Edman degradation on a Model 494 gas phase protein sequencer, Applied Biosystems. The following Table 2 shows the supporting data for the light chain variable domain sequences for antibodies 11E12 and 34D03 and the heavy chain variable domain sequences for 34D03. According to the translation of the cDNA sequence, it was determined that the N-terminal amino acid of the variable domain of the heavy chain for antibody 11E12 is glutamine. Glutamine as the amino-terminal residue of a protein can spontaneously undergo cyclization to pyroglutamic acid, interfering with sequencing using Edman degradation (Chelius et al., Anal. Chem., 2006, 78 (7): 2370-6).

Table 2* Light chain variable domain Antibody Translated cDNA Sequence N-terminal sequence 11E12 Divlt Divlt 19D07 Divs Not tested 34D03 Dillt Dillt The variable domain of the heavy chain Antibody Translated cDNA Sequence N-terminal sequence 11E12 QVQLQ Blocked 19D07 EVKLV Not tested 34D03 EVQLV EVQLV

* In Table 2, "DIVLT" corresponds to residues 1-5 in SEQ ID NO: 19, "DIVMS" corresponds to residues 1-5 in SEQ ID NO: 22, "DILLT" corresponds to residues 1-5 in SEQ ID NO: 24, " QVQLQ "corresponds to residues 1-5 in SEQ ID NO: 26," EVKLV "corresponds to residues 1-5 in SEQ ID NO: 28, and" EVQLV "corresponds to residues 1-5 in SEQ ID NO: 30.

Example 3. Construction of chimeric antibodies 11E12, 19D07 and 34D03

As described above, antibodies are a homodimer formed by the pairing of two heterodimeric proteins. Each protein chain (one heavy and one light) of the heterodimer consists of a variable domain and a constant domain. Each variable domain contains three complementarity determining regions (CDRs) contributing to antigen binding. CDRs are separated in the variable domain by framework regions that provide a backbone for proper spatial representation of antibody binding sites. Together, CDRs and framework regions contribute to the ability of antibodies to bind their cognate antigen (Figure 2).

As also described above, the chimeric antibody consists of the variable domain sequence (both CDR and scaffold) of a mouse antibody (according to the above sequence analysis) grafted onto the corresponding constant regions of the heavy and light chains of the canine IgG molecule (Figure 3). Since the variable domain is responsible for binding to the antigen, it is expected that grafting a whole mouse variable domain on the constant region of canine IgG will have little or no effect on the ability of the antibody to bind to the IL-31 immunogen.

In order to simultaneously confirm the correct identification of the sequence of the variable regions of the heavy and light chains and to obtain a recombinant homogeneous substance, expression vectors were created to produce chimeric antibodies in mammalian expression systems. Direct and reverse primers were designed to amplify the sequence of the variable region of the heavy and light chains of murine antibodies derived from 11E12, 19D07 and 34D03 hybridomas. A unique restriction site for endonuclease, a Kozak consensus sequence, and a secretory leader sequence were introduced into each direct primer to facilitate expression and secretion of the recombinant antibody from a mammalian cell line. Each reverse primer was designed to amplify each corresponding variable domain of the heavy and light chains, and a unique restriction site was introduced to facilitate cloning. For amplification of each heavy and light chains, PCR was performed using the cloned hybridoma DNA of the variable domain of the antibody chains as a template for each reaction. Each PCR product was cloned into a mammalian expression plasmid containing constant region DNA of either the heavy chain (designated as HC-64 or HC-65 in this description) or the light chain (designated as kappa) of canine IgG based on the sequences with access numbers in GenBank AF354264 or AF354265 and XP_532962, respectively. Amino acid and nucleotide sequences of HC-64 are represented by SEQ ID NO: 40 and 41, respectively. Amino acid and nucleotide sequences of HC-65 are represented by SEQ ID NO: 42 and 43, respectively. The amino acid and nucleotide sequences of the kappa chain are represented by SEQ ID NO: 44 and 45, respectively. Plasmids encoding each heavy and light chain under the control of the CMV promoter (cytomegalovirus promoter) co-transfected HEK 293 cells using standard lipofectamine methods. After six days of expression, chimeric mAbs were purified from 30 ml of supernatants of transiently transfected HEK293FS cells using Protein A-based MabSelect SuRe resin (GE Healthcare, Uppsala, Sweden) according to standard protein purification methods. The eluted fractions were combined, concentrated to ~ 500 μl using a Nanosep Omega centrifuge (Pall Corp., Port Washington, NY) with a nominal molecular weight cut-off (MM) of 10,000 Da, dialyzed overnight at 4 ° C in 1 × PBS (pH 7.2) and stored at 4 ° C for future use.

The expression of chimeric canine IgG was evaluated using polyacrylamide gel electrophoresis in the presence of SDS (sodium dodecyl sulfate) (SDS-PAGE) under native and denaturing conditions. Monoclonal antibodies (mAb) after each transfection were separated in a 4-12% bis-Tris gel using SDS-MES working buffer according to the manufacturer's protocol (Invitrogen Corp., Carlsbad, CA). After electrophoresis, proteins were visualized using a simple Coomassie Blue dye (Invitrogen Corp., Carlsbad, Calif.) To verify proper pairing and give an approximate estimate of protein homogeneity. To determine whether recombinant mAbs retain the ability to bind canine IL-31, the mAbs were evaluated for their ability to bind canine IL-31 using Western blotting. Protein standards and recombinant canine IL-31 (800 ng), separated by SDS-PAGE, were transferred to a nitrocellulose membrane using an iBlot device from Invitrogen (Invitrogen Corp., Carlsbad, CA). After the transfer, the membranes were washed with distilled deionized water and blocked with 5% skimmed milk powder (NFDM) in phosphate buffered saline containing 0.05% Tween-20 (PBST) for 1 hour at room temperature (CT). After blocking, the membranes were washed in PBST and incubated with either the diluted supernatant obtained after transient expression or purified chimeric antibodies. Chimeric antibody binding was evaluated using peroxidase-conjugated goat anti-dog IgG antibodies (Bethyl Laboratories Inc., Montgomery, TX or Rockland, Immunochemicals, Inc., Gilbertsville, PA) at a dilution of 1: 5000 in PBST for 1 hour at RT. Confirmation of binding to IL-31 was determined by the presence of a stained zone (apparent molecular weight of 15 kDa) corresponding to the glycosylated form of canine IL-31 after adding TMB (tetramethylbenzidine) substrate to the blot (KPL, Inc., Gaithersburg, MD).

Chimeric mAbs demonstrating expression from HEK 293 cells and binding to canine IL-31 recombinant immunogen according to Western blotting were further analyzed for affinity and functionality. In order to characterize the affinity with which mAb candidates bind to IL-31, a surface plasma resonance (SPR) evaluation was performed using the Biacore system (Biacore Life Sciences (GE Healthcare), Uppsala, Sweden). In order to avoid differences in affinity associated with unequal surface preparation, which may occur when immobilizing antibodies on surfaces, a strategy was applied in which IL-31 was directly conjugated to the surface. Immobilization was carried out by the amine combination of IL-31 (5 μg / ml) using a chemical method using N-hydroxysuccinimide (NHS) / 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC). The reaction on the chips was quenched with ethanolamine and the affinity with which all mAb candidates contacted immobilized IL-31 was evaluated. All curves were approximated in accordance with a 1: 1 model. Affinities <E-11 (i.e. 10 ^-11 ) lie below the lower limit of quantification for a given device.

All mAb candidates were also evaluated for their ability to inhibit IL-31-mediated signal transmission in a cell-based assay in two independent formats. In a co-incubation format, the mAb: IL-31 complexes were preincubated for one hour to ensure that the complex was formed before the cells were added. To increase the possibility of detecting differences between mAbs, a second series of experiments was performed without co-incubation, and the mAbs were added directly to the cells for 5 minutes, after which IL-31 was added. In both cases, IL-31 stimulation occurred within 5 minutes. As shown in Table 3 below, the conversion of murine monoclonal antibodies 11E12, 19D07 and 34D03 to a canine chimeric form had a weak effect on their ability to bind to IL-31 or inhibit cell-mediated signal transmission. These results also confirm the presence of the correct sequences of the variable domains of the heavy and variable domains of the light chains originating from each mouse hybridoma.

TABLE 3 Antibody DH82 pSTAT analysis Biacore affinity Joint incubation IC ₅₀ , mcg / ml Pretreatment IC ₅₀ , μg / ml K _D (M) Mouse 11E12 1,61 2.28 8.93E-13

^▪ Chimeric 11E12 1.48 1,57 2.68E-13 Mouse 19D07 1.76 3.46 7.24E-12 ^* Chimeric 19D07 1.92 1.33 5.15E-13 Mouse 34D03 1.73 2.28 1.01E-12 ^◘ Chimeric 34D03 1.28 1,08 4.65E-12 ^▪ Chimeric 11E12: for the heavy chain, MU-11E12-VH was paired with HC-64, and for the light chain, MU-11E12-VL was paired with a kappa chain. ^* Chimeric 19D07: for the heavy chain, MU-19D07-VH was paired with HC-64, and for the light chain, MU-19D07-VL was paired with a kappa chain. ^◘ Chimeric 34D03: for the heavy chain, MU-34D03-VH was paired with HC-64, and for the light chain, MU-34D03-VL was paired with a kappa chain.

Example 4. Assessment of chimeric mAb in vivo

To confirm the correlation of the inhibition of I L-31-mediated cell signal transmission observed in the DH-82 assay with the suppression of IL-31-mediated prurite in dogs, the chimeric monoclonal antibody 11E12 described above in Table 3 was evaluated (chimeric 11E12-64 ), in a model of IL-31-induced prurite in dogs. In this model, canine IL-31 when administered intravenously (iv) at a dose of 1-1.5 μg / kg causes a rapid onset of a persistent pruritic response that can be quantified over a two-hour observation period. In order to evaluate the itchy responses of the dogs, they were placed in single shelters and measurements of the intensity of itching were carried out using video surveillance. After an acclimatization period of ≥1 hour, each dog was given baseline (BL) scores of itching in points, using real-time video surveillance using a categorical rating system in points. Specifically, during consecutive 1-minute intervals, yes / no conclusions were made as to whether each dog exhibits pruritus behavior. Demonstrations of pruritus behavior, such as, for example, licking / biting paws, flanks and / or portions of the anal area, combing the sides, neck, and / or scratching the litter, shaking the head, and moving quickly on the back of the cage floor, were enough to display the answer "yes" in the specified time interval. At the end of this period, the number of yes responses identified was summarized, giving a cumulative score for itching severity scores (PSI). Scores of itching severity in points were determined twice for each animal, with the initial score being set, determined within 30 minutes immediately before the start of the treatment period with the test product. At the end of each observation period stipulated by the plan, dogs were returned to their places of usual accommodation.

To assess the possibility of inhibiting IL-31-mediated prurite by subcutaneous (s / c) administration of chimeric 11E12-64, a pilot study was conducted (76A03), which included both the treatment group and the placebo group (N = 4 /Group). In this study, baseline responses were determined for all 8 dogs, and the dogs were randomized into groups and placed according to their PSI. It is important to note that each group consisted of two responders with increased response (PSI> 55) and two responders with moderate response (PSI = 30-55). These dogs were further administered chimeric 11E12-64 on the 7th day, and IL-31 was administered on the 8th, 14th and 22nd day. The results of this study are presented in Fig. 8. These results demonstrate that administration of the chimeric 11E12-64 reduced the average PSI by more than 75% on days 8 and 14 relative to 1 day for animals receiving the chimeric 11E12-64. This result is the opposite of a 37-51% increase in PSI values in untreated animals. PSI returned to baseline two weeks after treatment with the use of mAb, confirming that the duration of the effectiveness of the administered dose of 0.3 mg / kg is from one to two weeks.

A particular problem in assessing PSI is the diurnal changes in dog behavior characteristic of pruritus. To facilitate control of these changes, the initial PSI was determined for each dog every day for 30 minutes prior to administration of IL-31. Figure 9 shows individual scores of itch scores in dogs registered in this study (76A60). The data shown in Fig.9 illustrate that on the 8th day and on the 14th day, the initial PSI was approximately 25% after administration of IL-31 in the group receiving chimeric 11E12-64. This observation is consistent with the complete disappearance of the prurite associated with IL-31, since the time period of observation of the initial response (0.5 h) is 25% of the time period of observation after administration of IL-31 (2 h). Taken together, these in vivo data provide very rigorous evidence that: 1) chimeric monoclonal 11E12 can neutralize the ability of IL-31 to induce prurite in dogs, 2) data on the inhibition of IL-31-mediated signal transmission in a cell-based analysis correlate with in vivo efficacy and 3) the parameters needed to use a model that uses IL-31 to evaluate mAb are reasonable for evaluating other candidate antibodies.

Example 5. The cannization strategy

The production of antibody to the drug (ADA) may be associated with a loss of effectiveness against any biotherapeutic protein, including monoclonal antibodies. A comprehensive analysis of the literature showed that the process of "speciation" of monoclonal antibodies can reduce the ability of mAbs to be immunogenic, although examples of immunogenic fully human mAbs and non-immunogenic chimeric mAbs can be found. To reduce the risks associated with the formation of ADA, a cannization strategy has been applied to the murine monoclonal anti-IL-31 antibodies of the invention. This cannization strategy is based on the identification of a dog germline antibody sequence that is most suitable for CDR grafting (Figure 4). After a detailed analysis of all available dog germline antibody sequences for both heavy and light chains, germline antibody candidates were selected based on their homology with mouse mAbs, and CDRs from mouse mAb precursors were used to replace canine IgR own CDRs . The challenge was to maintain high affinity and activity in the cells when using fully canine Ig frameworks to minimize the in vivo immunogenic potential. Caninized mAbs were expressed and characterized by their ability to bind IL-31 using Western blotting. These results are described below in Example 8. Only those mAbs that retained the ability to bind IL-31 after caninization were proposed for further study. Those mAbs that have lost the ability to bind IL-31 were systematically analyzed to identify: 1) the chain responsible for the loss of function, 2) the framework responsible for the loss of function, and 3) the amino acids (s) responsible for the loss of functions.

Example 6. Caninization of antibodies 11E12, 19D07 and 34D03

Synthetic nucleotide constructs were prepared that presented canned variable domains of the heavy and light chains of mAb 11E12, 19D07, and 34D03. After subcloning the sequences of each variable chain into plasmids containing the corresponding sequences of the constant region of the heavy or kappa chain of canine Ig, plasmids co-transfected HEK 293 cells to express antibodies. In general, both 19D07 and 34D03 mAb retained the ability to bind IL-31 after caninization. The sequences of the cannulated variable domains of the anti-canine IL-31 antibody defined for each antibody are as follows: 19D07 light chain variable domain (CAN-19D07-VL-998-1 with SEQ ID NO: 23 corresponding to the nucleotide sequence of which is SEQ ID NO : 46), the variable domain of the heavy chain 19D07 (CAN-19D07-VH-400-1 with SEQ ID NO: 29, the corresponding nucleotide sequence of which is SEQ ID NO: 47), the variable domain of the light chain 34D03 (CAN-34D03-VL -998-1 with SEQ ID NO: 25, the corresponding nucleotide sequence of which is SEQ ID NO: 48) and the variable domain of the heavy chain 34D03 (CAN-34D03-VH-568-1 with SEQ ID NO: 31 corresponding to the nucleotide sequence of which is SEQ ID NO: 49).

In contrast, the use of the germline sequences used in the cannabisation of 11E12 resulted in some non-functional mAbs. With reference to FIG. 10, chimeric, heterochemical, and caninized variants of mAb 11E12 were expressed and Western blotted for their ability to bind canine IL-31. These results demonstrated that caninized 11E12 antibody did not bind to canine IL-31 (Blot No. 2). In addition, with regard to the heterocimer, the chimeric heavy chain paired with the cannulated light chain lost the ability to bind IL-31 (blot No. 3), while the cannabized heavy chain paired with the chimeric light chain retained the ability to bind IL-31 (blot No. 4 ) Based on the results obtained for the heterocimer, it was concluded that the caninized light chain is responsible for the loss of activity.

In an attempt to reconnect the caninized variant 11E12 binding to canine IL-31, the caninized light chain was modified by rearranging the framework sequences. Figure 11 shows a General view of the result of the replacement in the frame of the light chain 11E12. As a result of this work, an antibody was identified with the replacement of carcass II framework (FWH) of canine Ig with mouse Ig framework II with restoration of the ability to bind to canine IL-31 (variable domain of the light chain 11E12 (CAN-11E12-VL-cUn-FW2 with SEQ ID NO: 20, the corresponding nucleotide sequence for which is SEQ ID NO: 50), the variable domain of the heavy chain 11E12 (CAN-11E12-VH-415-1 with SEQ ID NO: 27, the corresponding nucleotide sequence for which is SEQ ID NO: 51)) .

Thanks to further optimization of these reverse mutations, an antibody with a single reverse mutation of arginine to leucine (R50L) in frame II was identified, which could restore the ability to bind to IL-31, according to Western blot analysis (light chain variable domain 11E12 (CAN-11E12 -VL-cUn-13 with SEQ ID NO: 21, the corresponding nucleotide sequence of which is SEQ ID NO: 52), the variable domain of the heavy chain 11E12 (CAN-11E12-VH-415-1 with SEQ ID NO: 27)) ( Fig. 12). After the caninized variants of each potential candidate were identified, the mAbs were purified and dialyzed against PBS for further evaluation.

Table 4 summarizes the results of both affinity measurements and inhibition assays using cells. These data demonstrate that both caninized derivatives of both 11E12 and 34D03 retain excellent inhibitory activity in the cell-based assay and affinity for IL-31 from measurements using Biacore. Also noteworthy is the observation that although the molecule of the initial canned 19D07 retained superior efficacy as measured using Biacore, the ability to inhibit IL-31-mediated signal transmission in a cell-based assay appears to be impaired compared to its murine predecessor. There was a slight loss or no loss of affinity when the mAbs were converted from their mouse isotype to a canine Ig derivative.

TABLE 4 Antibody DH82 pSTAT analysis Biacore affinity Joint incubation IC ₅₀ , mcg / ml Pretreatment IC ₅₀ , μg / ml K _D (M) Mouse 11E12 1,61 2.28 8.93E-13 Sewer 11E12 not active not active 5.06E-07 Heterocimera 11E12 2.67 3.35 4.97E-12 Sewer 11E12 FW2 2.7 5.31 1.47E-10 Sewer 11E12 13 5.49 5.18 5.16E-12 Mouse 19D07 1.76 3.46 7.24E-12 Caninized 19D07 inc. curve inc. curve 9.23E-10 Mouse 34D03 1.73 2.28 1.01E-12 Sewer 34D03 2.42 2.25 2.91E-11 Antibody Variable chain domain Heavy Easy Sewer 11E12 CAN-11E12-VH-415-1 CAN-11E12-VL-cUn-1 Heterocimera 11E12 CAN-11E12-VH-415-1 Chimeric 11E12

Sewer 11E12 FW2 CAN-11E12-VH-415-1 CAN-11E12-VL-cUn-FW2 Sewer 11E12 13 CAN-11E12-VH-415-1 CAN-11E12-VL-cUn-13 Caninized 19D07 CAN-19D07-VH-400-1 CAN-19D07-VL-998-1 Sewer 34D03 CAN-34D03-VH-568-1 CAN-34D03-VL-998-1

Heavy chains: all cannized and heterohemeric forms of 11E12 contained the V _H sequence CAN-11E12-VH-415-1 (SEQ ID NO: 27) and a constant region called HC-64 (SEQ ID NO: 40); canned 19D07 contained the V _H sequence CAN-19D07-VH-400-1 (SEQ ID NO: 29) and HC-64; cannabisized 34D03 contained the V _H sequence CAN-34D03-VH-568-1 (SEQ ID NO: 31) and HC-64.

Light chains: cannulated 11E12 contained the V _L sequence of CAN-11E12-VL-cUn-1 (SEQ ID NO: 53) and a constant region called kappa (SEQ ID NO: 44); heterochemical 11E12 contained the V _L sequence of MU-11E12-VL (SEQ ID NO: 19) and kappa; cannabisized 11E12 FW2 contained the V _L sequence of CAN-11E12-VL-cUn-FW2 (SEQ ID NO: 20) and kappa; cannabisized 11E12 13 contained the V _L sequence of CAN-11E12-VL-cUn-13 (SEQ ID NO: 21) and kappa; canned 19D07 contained the V _L sequence of CAN-19D07-VL-998-1 (SEQ ID NO: 23) and kappa; cannabisized 34D03 contained the V _L sequence of CAN-34D03-VL-998-1 (SEQ ID NO: 25) and kappa.

Example 7. The study of the binding of canine IL-31 with antibodies 11E12 and 34D03

To determine the amino acid residues of antibodies 11E12 and 34D03 involved in binding to canine IL-31, a mutagenesis strategy was used that included: 1) shortening of the IL-31 protein from both the N- and C-terminus and 2) replacing individual amino acids per alanine (alanine scan) to determine the effect on mAb binding. PCR primers were designed to amplify the canine IL-31 gene, codon-optimized for expression in the E. coli host. The sequence of this full-length canine IL-31 construct codon-optimized for expression in E. coli is shown in SEQ ID NO: 55, the corresponding nucleotide sequence of which is SEQ ID NO: 56. Primers were designed to amplify a full-sized gene and to create a protein shortened by 20 amino acids in the direction from the N- and from the C-terminus. For such shortening at the N-terminus, position 1 immediately after the N-terminal 6-His tag in the codon-optimized design corresponded to the glycine residue. PCR amplification products were cloned into pET101D (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's protocol. The use of plasmid pET101D enables the fusion of a recombinant protein with an N-terminal 6-His-epitope tag necessary to confirm expression. Sequenced plasmids were used to transform E. coli BL21 Star TOP10 cells (Invitrogen Corp., Carlsbad, Calif.) And recombinant protein expression was induced using 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) under standard culture conditions. After induction, cells were harvested by centrifugation and lysed using bacterial protein extraction reagents (abbreviated B-PER, ThermoFisher Scientific Inc., Rockford, IL). The crude lysates were subjected to SDS-PAGE, and Western blotting was performed as previously described. All western blotting for analysis using mutagenesis was performed using murine variants 11E12 and 34D03, due to the availability of the necessary purified antibodies and reagents. Each antibody was tested for its ability to bind to the crude protein lysate transferred to this blot, representing full-length and shortened IL-31. Control blots also probed anti-His mAbs to confirm expression of each protein. All proteins shortened at the N-terminus (-20N, -40N and -60N) showed strong expression in E. coli and were able to bind to 11E12 and 34D03 (Figure 13). The amino acid and nucleotide sequences corresponding to the -20N construct are SEQ ID NOs: 57 and 58, respectively. Amino acid and nucleotide sequences corresponding to the -40N construct are SEQ ID NOs: 59 and 60, respectively. Amino acid and nucleotide sequences corresponding to the -60N construct are SEQ ID NO: 61 and 62, respectively. However, full-sized IL-31 and proteins shortened at the C-terminus (-20С, -40С and -60С) were not expressed under these conditions.

It was noted that full-length protein IL-31 was expressed at a very low level. However, the design with the first 20 amino acids (-20N) removed from the N-terminus showed strong expression. Both antibodies, 11E12 and 34D03, bind to the -20N protein. Therefore, further work was carried out using this -20N design. Structures shortened at the C-terminus were prepared corresponding to the provisions of 20-122 (MM 15.3 kDa with His tag), 20-100 (MM 12.9 kDa with His tag) and 20-80 (MM 10.4 kDa with His label) to evaluate the binding of mAb to these regions on IL-31 protein. Amino acid and nucleotide sequences corresponding to constructs 20-122 are SEQ ID NO: 63 and 64, respectively. Amino acid and nucleotide sequences corresponding to constructs 20-100 are SEQ ID NOs: 65 and 66, respectively. Amino acid and nucleotide sequences corresponding to constructs 20-80 are SEQ ID NOs: 67 and 68, respectively. 14 shows western blots of crude protein lysates containing these truncated proteins that were probed using mAb 11E12 (blot B) and 34D03 (blot C). As shown in this figure, mAb 11E12 and 34D03 bind to truncated proteins IL-31 20-122 and 20-100, but did not bind to 20-80. These results indicated that amino acids between positions 80 and 100 of the full-length canine IL-31 construct with SEQ ID NO: 55 (with "SSHMA" as the N-terminus) were involved in the binding of these antibodies. This region corresponds to amino acid residues 102-122 of the full-length canine IL-31 protein sequence with SEQ ID NO: 32. On a control blot where anti-His mAbs were used (Blot A), it was shown that all truncated proteins were expressed. Additionally, pET101D-lacZ protein was used as a control to confirm the absence of nonspecific binding of mAbs to host proteins.

In order to further identify amino acids in canine IL-31 involved in binding to mAb 11E12 and 34D03, alanine scanning mutagenesis was carried out in accordance with known methods. Individual constructs were prepared (in plasmid -20 N), replacing alanine at each position, from the 76th to the 122nd, of the canine IL-31 amino acids. After sequence confirmation, protein expression was performed and crude protein lysates were analyzed using Western blotting. On Fig summarized results indicating the position in canine IL-31, which, according to the results of mutations in alanine in them, affect the binding of mAb 11E12 and 34D03. As shown in this figure, all positions 77, 78, 81, and 85 in this full-length IL-31 construct have an effect on the binding of 11E12 or 34D03 antibodies. They correspond to amino acid residues 99, 100, 103 and 107, respectively, of the sequence of SEQ ID NO: 32 of the full-length canine IL-31 protein.

Expression plasmids with double (D82A, I85A) and triple (181A, D82A, I85A) alanine substitutions were constructed to study the effect of multiple mutations in IL-31 regions important for binding to 11E12 and 34D03 antibodies. Lysates of E. coli expressing canine IL-31 with these double and triple mutations, along with the -20 N control, were blotted with 11E12 and 34D03 antibodies (Figure 16). Obviously, these three canine IL-31 amino acids are involved in the recognition of 11E12 and 34D03, since when they are replaced with alanine, binding is completely lost. These three amino acids correspond to amino acid residues 103, 104 and 107 of the full-length protein sequence of SEQ ID NO: 32 canine IL-31.

So, analysis of shortened canine IL-31 constructs revealed that amino acid residues (marked in FIG. 15 between positions 80 and 122) are involved in the binding of antibodies 11E12 and 34D03. In addition, an improved analysis using mutagenesis using alanine scanning revealed that Asp77, Lys78, Ile81, Asp82 and Ile85 in this full-length IL-31 construct all affect 11E12 or 34D03 binding, which indicates that this region with the greatest probability determines the epitope responsible for recognition by these antibodies. Interestingly, this region of the human IL-31 protein has been shown to be involved in binding to the GPL subunit of its coreceptor (Le Saux S. et al., Biol. Chem. 2010, Jan 29, 285 (5): 3470-7. Epub, 2009, Nov. 17). These observations, along with the ability of mAb 11E12 and 34D03 to neutralize IL-31-mediated pSTAT activity in monocytes, confirm the hypothesis that these mAbs bind to canine IL-31 residues that are essential for the binding of this cytokine to its receptor, thereby inhibiting its ability to induce signal transmission.

Example 8. Obtaining caninized antibodies 34D03 using plasmids. glutamine synthetase (GS) gene-bearing

Genes encoding caninized mAb 34D03 (heavy and light chains, Table 4, above) 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 one plasmid for expression in mammals. Each plasmid was used to transfect HEK 293 cells, and expression was carried out in 20 L of culture medium. Protein was isolated from conditioned HEK medium using protein A affinity chromatography in accordance with standard protein purification methods. The medium was loaded onto a chromatographic resin and eluted by changing the pH. The pH was adjusted in the eluted protein solution, and it was dialyzed and sterilized before use. The resulting antibody was more than 99 percent monomeric, according to analytical gel permeation chromatography, without any visible high molecular weight aggregates. This antibody was further used in a dog prurite model to evaluate in vivo efficacy.

Example 9. Evaluation of caninized 34D03 antibody in a dog prurite model

The antipruritic effect of caninized 34D03 (CAN 34D03-65 represented by SEQ ID NO: 31 (V _H ) paired with SEQ ID NO: 25 (V _L ) on SEQ ID NO: 42 (HC-65) and SEQ ID NO: 44 was evaluated. (LC-kappa)) using a model of IL-31-induced prurite in dogs. According to this model, an intravenous provocative dose (1.5 μg / kg) of recombinant canine IL-31, known for inducing a temporary period of behavior characteristic of pruritus in beagle dogs (administration of IL-31, prurite duration less than 24 hours) was administered repeatedly to animals before and within 63 days after the introduction of a single sc dose of 1.0 mg / kg CAN 34D03-65. In each of the periods of IL-31 administration, real-time video surveillance was used for 0.5 hours prior to cytokine administration (initial period before IL-31 administration), followed by a similar measurement for 2 hours, to assess the behavior characteristic of pruritus. 20 minutes after cytokine injection (period of 2 hours after administration of IL-31). Scores of itching severity were scored at each time period of the study, giving yes / no conclusions as to whether the behavior characteristic of pruritus was detected during consecutive 1 minute intervals (the maximum score for severity of itching in scores is: 30 for each baseline; 120 for the period after administration of IL-31). On Fig summarized estimates of the severity of itching in points obtained before and after treatment using CAN 34 D03-65, which was performed on the 0 day of the study. Seven days before treatment with mAb, the mean score for the severity of pruritus in dogs scored after IL-31 administration was 68 ± 13 (SE, n = 4). For comparison, on the 7th, 14th, 21st day of the study, the average score for the severity of itching in points after the administration of IL-31 decreased to 5 ± 2, 8 ± 4 and 9 ± 5, respectively. These changes in the assessment of the severity of pruritus in scores between the 7th and 7th-21st days represent a decrease of ≥85% in the total ability of pruritus to respond to IL-31.

In fact, the degree of inhibition of IL-31-induced prurite during these periods can be closer to 100%, given that in the interval between 0 and 21 days the initial estimates in points for the period 0.5 h before administration IL-31 averaged 1.6 ± 0.6, a level that would be extrapolated to an estimate of 6-7 the severity of itching in points for a 2 h period. After some time, the ability of dogs receiving treatment to respond with an itch to exogenous IL-31 was gradually restored. On the 63rd day after treatment with CAN D03-65, the average score for the severity of itching in points for a period of 2 hours after administration of IL-31 increased to 57 ± 8 or approximately 84% of the responses to the administration of IL-31 before treatment using mAb observed on the 7th day. Thus, in the IL-31-induced pruritus model, a single SC bolus injection of CAN 34D03-65 for several weeks provided protection against pruritus for the treated dogs.

Example 10. The study of feline IL-31

The feline IL-31 sequence was identified by looking for similarities between the feline genome and canine IL-31 using the genomic resources of NCBI (www.ncbi.nlm.nih.gov). The feline IL-31 gene was synthesized for optimal expression in E. coli. Expressing constructs with full-length canine and feline IL-31 genes were created containing an N-terminal 6-His tag for detection and purification. The design of the full-sized gene of cats used for expression in E. coli is represented by the nucleotide sequence of SEQ ID NO: 69 and the protein sequence of SEQ ID NO: 70. Sequenced plasmids were used to transform E. coli BL21 Star ™ (Invitrogen Corp., Carlsbad, CA) and expression was carried out at 30 ° C. for 5 hours. After lysis of the cell pellets after centrifugation, it was found that the insoluble lysate is highly enriched in the immunoreactive protein. These cell pellets were solubilized in 6 M urea, and recombinant proteins were purified under denaturing conditions using a nickel-cobalt resin (Thermo Fisher Scientific Inc., Rockford, IL). Pooled elution fractions showing a positive reaction for the presence of His tags were stepwise dialyzed against 0.8 M urea in PBS, then against PBS and analyzed using SDS-PAGE (Fig. 18). As previously observed, the yield of recombinant canine IL-31 after induction from E. coli was low. However, after purification, a protein was obtained that migrated to SDS-PAGE in accordance with the expected molecular weight.

To study the biological activity of canine and feline IL-31 produced in E. coli, each protein was analyzed for its ability to induce pSTAT signal transmission in an analysis using DH82 cells. Since recombinant IL-31 from mammalian cells (canine IL-31 (CHO)) is highly glycosylated, it was unclear whether the non-glycosylated form would retain biological activity. 19 shows that feline IL-31 has comparable biological activity with reference IL-31 produced in CHO cells.

By alanine scanning mutagenesis of canine IL-31, the region in the protein necessary for binding to antibodies 11E12 and 34D03 was determined. Given the conservatism of the sequences in this area (FIG. 20), it was hypothesized that these mAbs would cross-react with feline IL-31.

21 shows that mAb 11E12 and 34D03 have the ability to bind to canine IL-31 (E. coli) and are also capable of cross-reacting with feline IL-31 protein. According to these data, the process of speciation of the 34D03 antibody in the feline direction (felinization) was carried out.

Example 11. Felination of antibody 34D03

Similarly to the described cannization strategy, the corresponding germline antibody sequences were identified from all available feline sequences for inoculating CDRs from mAb 34D03. The variable domain of the light chain (FEL-34D03-VL-021-1 with SEQ ID NO: 71, the corresponding nucleotide sequence of which is SEQ ID NO: 72) and the variable domain of the heavy chain (FEL-34D03-VH-035-1 with SEQ ID NO: 73, the corresponding nucleotide sequence of which is SEQ ID NO: 74), was selected based on the highest homology with their corresponding canine Ig frameworks in cannized 34D03. Recombinant felinated 34D03 was prepared using selected variable regions linked to their corresponding IgG1 heavy chain contant domain sequence (feline HC-A with SEQ ID NO: 75, the corresponding nucleotide sequence of which is SEQ ID NO: 76 with GenBank Accession No. AB016710. 1) and the sequence of the constant domain of the light Kappa chain (LC-kappa of cats with SEQ ID NO: 77, the corresponding nucleotide sequence for which is SEQ ID NO: 78 with GenBank Accession No. AF198257.1). The antibody was obtained from HEK cells and purified as previously described. On Fig shows the ability of feline 34D03 to neutralize pSTAT signal transmission comparable to the option for canine Ig IC ₅₀ .

The felinated 34D03 was evaluated for its ability to bind both feline and canine IL-31. 23 shows Western blots for felinated 34D03 using purified protein from both mammals and E. coli, used as sources. Convincing binding was observed for both dog and cat proteins, indicating complete cross-reactivity of the felinized form 34D03 and confirming binding to the feline protein. Taken together, these results confirm that the conserved feline epitope of feline IL-31 may be a suitable target for inhibiting this cytokine in cats.

Example 12. Detection of the cytokine IL-31 in dogs with natural atopic dermatitis

In the present example, the level of IL-31 protein in serum collected from dog populations, including dogs with atopic dermatitis, was evaluated using a quantitative immunoassay method.

Serum samples were prepared for the following dog populations and frozen until serum IL-31 measurements were taken.

1) Twenty-four specially grown hounds (Marshall BioResourses, North Rose, NY) before and after sensitization to the tick-borne house dust allergen (HDM) (Dermatophagoides farina, Greer Labs). All animals were approximately 9 months old. Both sexes were represented approximately equally.

2) Thirty flea allergy dogs (Youngs Veterinary Research Services, Turlock, CA) prior to infection with fleas or approximately one week after infection with adult cat fleas (Ctenocephalides felis). Most dogs in this colony were of mixed breed. The average age was approximately 10.5 years. Both sexes were represented approximately equally.

3) Eighty-seven owners of dogs with subclinical periodontitis, but otherwise identified as healthy. Samples were collected at 18 US veterinary clinics. The animals were typical representatives of the dog population in the United States in terms of gender and breed, and their ages ranged from two to five years.

4) Two hundred and twenty-four owner-owned animals diagnosed with chronic off-season atopic dermatitis for at least 1 year (based on modified Willems criteria and Prelo publication (Willemse T. J. Small Anim. Pract, 1986, 27: 771-778 and Prelaud et al. Revue de Medecine Veterinaire, 1998, 149: 1057-1064) with a minimum "moderate pruritus" rating according to the owner and a minimum 25-point skin lesion score according to CADESI-02 (severity and size index of atopic dermatitis in dogs) according to the veterinarian’s assessment. US veterinary dermatology hospitals, approximately 75% of the dogs were purebred and ~ 25% of the general population were retrievers (Labrador (17.3%) and Golden Retriever (8.2%)). age (~ 6 years). Both sexes were represented approximately equally.

To quantify the levels of cIL-31 (canine IL-31) in the blood serum of dogs, a sandwich immunoassay was used. Serum samples were diluted 1: 2 in Rexxip buffer (Gyrolab, Warren, NJ) and analyzed on a Bioaffy CD (1000 nl) (Gyrolab) using a Gyrolab xP workstation. The capture of cIL-31 was performed using the biotin-labeled monoclonal anti-IL-31 antibody of the present invention, and detection was performed using the Alexafluor 647-labeled monoclonal anti-IL-31 antibody of the present invention. The cIL-31 concentrations in the samples were extrapolated based on a standard 8-point curve with a dynamic range of 0.013-250 ng / ml using a 5-parameter curve fitting model with Gyrolab Evaluator software.

CIL-31 levels could be detected in serum samples of 57% of dogs with natural atopic dermatitis (≥13 pg / ml), but could not be detected (<13 pg / ml) in serum samples from specially grown hounds +/- sensitized to HDM, dogs of mixed breeds +/- with infection by fleas or belonging to owners of dogs with periodontitis, but otherwise defined as healthy, regardless of breed. For dogs with natural atopic dermatitis, 53% of the analyzed samples showed serum levels of IL-31 in the range of 13-1000 pg / ml, and 4% showed levels above 1000 pg / ml (Table 5).

Table 5 Serum IL-31 levels in various dog populations Dog populations The number of animals examined The number of animals with detectable serum ^a IL-31 The percentage of animals with detectable serum IL-31 Specially grown hounds 24 0 0% Specially bred hounds sensitized to HDM 24 0 0% Mixed-breed dogs - no fleas thirty 0 0% Mixed Breed Dogs - Infected with Fleas thirty 0 0% Healthy Owner-Owned Animals - Various Breeds 87 0 0% Owned animals with natural atopic dermatitis - diverse breeds 224 128 57% ^and Less than 13 pg / ml is below the limits of quantitation.

The results of this example demonstrate that the level of IL-31 protein is elevated in a significant number of dogs with dog atopic dermatitis. Without being bound by any theory, it is believed that the IL-31-mediated pathway plays a role in the pathobiology of pruritic allergic skin conditions, such as, but not limited to, dog atopic dermatitis, and represents a new pathway for therapeutic intervention using an IL antagonist -31, such as, including, but not limited to, olacitnib (olacitnib) and / or an anti-IL-31 antibody that specifically binds canine IL-31.

Claims (27)

1. The selected monoclonal antibody or antigennegative site that specifically binds to at least one of the canine IL-31 (interleukin-31) or feline IL-31, containing at least one of the following sequence combinations determining the complementarity of the region (CDR):
1) 11E12: CDR1 of the heavy chain variable domain (VH) from SEQ ID NO: 1, VH-CDR2 from SEQ ID NO: 4, VH-CDR3 from SEQ ID NO: 7, CDR1 of the light chain variable domain (VL) from SEQ ID NO: 10, VL-CDR2 from SEQ ID NO: 13 and VL-CDR3 from SEQ ID NO: 16;
2) 19D07: VH-CDR1 from SEQ ID NO: 2, VH-CDR2 from SEQ ID NO: 5, VH-CDR3 from SEQ ID NO: 8, VL-CDR1 from SEQ ID NO: 11, VL-CDR2 from SEQ ID NO: 14 and VL-CDR3 from SEQ ID NO: 17; or
3) 34D03: VH-CDR1 from SEQ ID NO: 3, VH-CDR2 from SEQ ID NO: 6, VH-CDR3 from SEQ ID NO: 9, VL-CDR1 from SEQ ID NO: 12, VL-CDR2 from SEQ ID NO: 15 and VL-CDR3 from SEQ ID NO: 18. 2. The antibody or antigen binding site thereof according to claim 1, which is chimeric. 3. The antibody or its antigennegative plot according to claim 1, which is caninized or felinized. 4. The antibody or antigen binding site of claim 1, which reduces, inhibits, or neutralizes the activity of IL-31 in a dog or cat. 5. The antibody according to claim 4, which weakens, suppresses, or neutralizes an itchy condition or an allergic condition. 6. The antibody or antigen binding site of claim 4, which attenuates the clinical manifestations of atopic dermatitis. 7. The antibody or antigen binding site of claim 6, wherein the clinical manifestations of atopic dermatitis are selected from the group consisting of pruritus, skin lesions, and combinations thereof. 8. The antibody or its antigennegative plot according to claim 1, containing at least one of the group consisting of:
a) the variable domain of the light chain containing DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVETDDVATYYCQQSNKDPLTFGAGTKLELK-11
DIVMTQTPLSLSVSPGEPASISCRASESVDNYGISFMHWYQQKPGQPPKLLIYRASNLESGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCQQSNKDPLTFGAGTKLEIK (SEQ ID NO: 20; CAN FW-2U12 -12E12)
DIVMTQTPLSLSVSPGEPASISCRASESVDNYGISFMHWFQQKPGQSPQLLIYRASNLESGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCQQSNKDPLTFGAGTKLEIK (SEQ ID NO: 21; CAN-11En-11Un-11Un-VUn
DIVMSQSPSSLSVSAGDKVTMSCKSSQSLLNSGNQKNYLAWYQQKPWQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIK (SEQ ID 19: 22):
EIVMTQSPASLSLSQEEKVTITCKSSQSLLNSGNQKNYLAWYQQKPGQAPKLLIYGASTRESGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQNDYSYPYTFGQGTKLEIK (SEQ ID NO: 23 -19; 19L-19L-19L-19L CAN
DILLTQSPASLAVSLGQRAIISCKASQSVSFAGTGLMHWYQQKPGQQPKLLIYRASNLEAGVPTRFSGSGSRTDFTLNIHPVEEEDAATYFCQQSREYPWTFGGGTKLEIK (SEQ ID NO: 24; MU 34D-MU 34D-MU 34; -MU 34)
EIVMTQSPASLSLSQEEKVTITCKASQSVSFAGTGLMHWYQQKPGQAPKLLIYRASNLEAGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQSREYPWTFGQGTKLEIK (SEQ ID NO: 25; CAN-34D03 and
b) the variable domain of the heavy chain containing
QVQLQQSGAELVKPGASVKLSCKASGYTFKYYDINWVRQRPEQGLEWIGWIFPGDGGTKYNETFKGKATLTTDKSSSTAYMQLSRLTSEDSAVYFCARGGTSVIRDAMDYWGQGTSVTVSS (SEQ ID NO: 26; SEQ ID NO: 26;
EVQLVQSGAEVKKPGASVKVSCKTSGYTFKYYDINWVRQAPGAGLDWMGWIFPGDGGTKYNETFKGRVTLTADTSTSTAYMELSSLRAGDIAVYYCARGGTSVIRDAMDYWGQGTLVTVSS 27 -115 -1 CANQENE1-SEQ ID NO-1
EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQIPEKRLEWVATITSGGGYTYSADSVKGRFTISRDNARNTLYLQMSSLRSEDTAVYYCARQNWVVGLAYWGQGTLVTVSA SEQ ID 28: MU-19D07-VH),
EVQLVESGGDLVKPGGSLRLSCVASGFTFSSYDMSWVRQAPGKGLQWVATITSGGGYTYSADSVKGRFTISRDNARNTLYLQMNSLRSEDTAVYYCARQNWVVGLAYWGQGTLVTVSS (SEQ ID NO -1: 29; -19-NO07: 29QD NO07:
EVQLVESGGDLVKPGGSLKLSCAASGFSFSNYGMSWVRQTPDKRLEWVATISYGGSYTYYPDNIKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCVRGYGYDTMDYWGQGTSVTVSS (SEQ ID NO-30; MQH NOD03; 30; NOU.D03);
EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYGMSWVRQAPGKGLQWVATISYGGSYTYYPDNIKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRGYGYDTMDYWGQGTLVTVSS (SEQ ID NO: -31; 38-DQD NO-38: CANQ ID NO: 38DQD 56: NOQD NO: 38; NO.38: 38QD NO: 38) 9. A veterinary composition for the treatment of an itchy or allergic condition, comprising a therapeutically effective amount of an antibody according to any one of claims. 1-8 and a pharmaceutically acceptable carrier. 10. A host cell that produces an antibody according to any one of claims. 1-8. 11. The selected nucleic acid to obtain the antibody according to any one of paragraphs. 1-8, containing a nucleic acid sequence encoding at least one of the following sequence combinations of the complementarity determining region (CDR):
1) 11E12: heavy chain variable domain (VH) CDR1 from SEQ ID NO: 1, VH-CDR2 from SEQ ID NO: 4 and VH-CDR3 from SEQ ID NO: 7;
2) 19D07: VH-CDR1 from SEQ ID NO: 2, VH-CDR2 from SEQ ID NO: 5 and VH-CDR3 from SEQ ID NO: 8; or
3) 34D03: VH-CDR1 from SEQ ID NO: 3, VH-CDR2 from SEQ ID NO: 6 and VH-CDR3 from SEQ ID NO: 9. 12. The nucleic acid according to claim 11, further comprising a nucleic acid sequence encoding at least one of the following combinations of CDR sequences:
1) 11E12: CDR1 of the light chain variable domain (VL) from SEQ ID NO: 10, VL-CDR2 from SEQ ID NO: 13 and VL-CDR3 from SEQ ID NO: 16;
2) 19D07: VL-CDR1 from SEQ ID NO: 11, VL-CDR2 from SEQ ID NO: 14 and VL-CDR3 from SEQ ID NO: 17; or
3) 34D03: VL-CDR1 from SEQ ID NO: 12, VL-CDR2 from SEQ ID NO: 15 and VL-CDR3 from SEQ ID NO: 18. 13. The selected nucleic acid to obtain the antibody according to any one of paragraphs. 1-8, containing a nucleic acid sequence encoding the following sequence sequence complementarity determining region (CDR):
34D03: VL-CDR1 from SEQ ID NO: 12, VL-CDR2 from SEQ ID NO: 15 and VL-CDR3 from SEQ ID NO: 18. 14. The expression vector for producing antibodies according to any one of paragraphs. 1-8, containing the nucleic acid according to any one of paragraphs. 11-13. 15. A method of producing an antibody, comprising culturing a host cell according to claim 10 under conditions that lead to the production of an antibody, and isolating the antibody from the host cell or culture medium of the host cell. 16. A method of treating a condition or disorder selected from an itching condition or an allergic condition, comprising administering a therapeutically effective amount of an antibody according to any one of claims. 1-8. 17. The method of claim 16, wherein the itching condition is selected from the group consisting of atopic dermatitis, eczema, psoriasis, scleroderma, and prurite. 18. The method according to p. 17, where the condition accompanied by itching is atopic dermatitis. 19. The method according to p. 18, including the weakening of the clinical manifestations of atopic dermatitis. 20. The method of claim 19, wherein the clinical manifestations of atopic dermatitis are selected from the group consisting of itching, skin lesions, and combinations thereof. 21. The method according to p. 16, where the allergic condition is selected from the group consisting of allergic dermatitis, summer eczema, urticaria, fuse, inflammatory airway disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease and inflammatory processes that occur as a result of an autoimmune reaction. 22. A method of inhibiting the activity of IL-31 in a dog or cat, comprising administering an antibody according to any one of claims. 1-8. 23. A method for detecting or quantifying IL-31 in a sample, comprising:
(a) incubating a clinical or biological sample containing IL-31 in the presence of an antibody according to any one of claims. 1-8; and
(b) detecting an antibody that is bound to IL-31 in the sample. 24. The method of claim 23, wherein the antibody is detectably labeled. 25. The method of claim 23, wherein the antibody is unlabeled and is used in combination with a secondary antibody that is detectably labeled. 26. The antibody according to any one of paragraphs. 1-8, which specifically binds to the region between approximately amino acid residues 95 and 125 of the amino acid sequence of canine IL-31 of SEQ ID NO: 32 or to the region in feline IL-31 corresponding to the region between approximately amino acid residues of 95 and 125 in SEQ ID NO : 32. 27. The antibody according to claim 26, which specifically binds to the region between approximately amino acid residues 102 and 122 of the canine IL-31 sequence of SEQ ID NO: 32, or to the region in feline IL-31 corresponding to the region indicated between approximately amino acid residues 102 and 122 in SEQ ID NO: 32.

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