RU2772716C2 - Antibody against il-5, its antigen-binding fragment and its medical use - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention relates to the field of biotechnology and medicine, in particular to a monoclonal antibody or its fragment against IL-5; to a pharmaceutical composition for the treatment of a disease associated with human IL-5; to an isolated nucleic acid molecule encoding a monoclonal antibody or its antigen-binding fragment against IL-5; to a recombinant expression vector containing the above-mentioned nucleic acid molecule; to an expressing host cell transformed by the above-mentioned vector; to a method for the production of a monoclonal antibody or its antigen-binding fragment against IL-5; to a method and means for the detection or determination of human IL-5; to a method for the treatment of a disease associated with human IL-5; as well as to the use of the above-mentioned antibody or its antigen-binding fragment, a pharmaceutical composition, an isolated nucleic acid molecule, or a combination thereof in the production of a drug for the prevention or treatment of a disease associated with human IL-5.

EFFECT: obtaining an antibody against IL-5.

20 cl, 5 dwg, 19 tbl, 6 ex

Description

FIELD OF THE INVENTION

The present disclosure relates to antibodies against IL-5 (interleukin-5) and their antigen-binding fragments. In addition, the present disclosure also relates to chimeric antibodies, humanized antibodies containing CDR regions (hypervariable region) of anti-IL-5 antibodies, and the present disclosure also relates to a pharmaceutical composition containing an anti-IL-5 antibody and an antigen-binding fragment thereof, and to its use as a diagnostic and therapeutic agent for diseases associated with IL-5.

PRIOR ART

Interleukin-5 (IL-5) is one of the important members of the interleukin family, also known as T-cell replacement factor (TRF), B-cell growth factor-II (BCGF-II), IgA enhancing factor (IgA-EF) or eosinophil differentiation factor (EDF). It is a homodimeric glycoprotein secreted primarily by the helper T cell 2 (Th2). Human IL-5 consists of 134 amino acid residues, including a signal peptide consisting of 22 amino acids and two glycosylation sites. Human IL-5 shares 70% identity with mouse IL-5 at the amino acid level. Active IL-5 is in the form of an oligodimer with two peptide chains linked to each other via disulfide(s) bond(s) and in an anti-parallel configuration, while IL-5 monomers are not biologically active (Adv Immunol. 1994; 57:145 -90).

Eosinophils (EOS) are associated with a variety of inflammatory diseases in the lung, including allergic diseases associated with anaphylactic reaction. Among these diseases, asthma is a chronic respiratory inflammatory disease affecting approximately 300 million patients worldwide, with an incidence of 10%. Its pathogenesis is associated with a number of cytokines, and IL-5 and its receptor IL-5R play an important role in the pathogenesis of asthma. There are a large number of inflammatory cells infiltrating the bronchopulmonary tissue of patients with asthma, among which eosinophils are most significantly increased in number. Many studies have shown that eosinophils are one of the main cells leading to airway inflammation in asthma (Curr Opin Pulm Med. 2005 Jan; 11(1): 1-6). IL-5 plays a critical role in the differentiation, maturation, adhesion, infiltration, and apoptosis of EOS. A large number of animal and clinical studies have demonstrated that IL-5 can activate EOS precursor cells in the bone marrow and initiate EOS aggregation in the peripheral blood and airways, leading to chronic inflammation and airway hypersensitivity (J Immunol. 2014 Oct 15;193(8):4043-52). In addition, IL-5 can prolong the survival of EOS, enhance their degranulating response to specific stimulatory factors (such as IgA or IgG), and mediate the chemotactic activity of eosinophils (J Asthma Allergy. 2015 Nov 3; 8: 125-34). Increased expression of IL-5 was detected both in patients with asthma and in human models induced by bronchial antigens (Greenfeder et al., Respiratory Research, 2: 71-79, 2001). Recombinant human IL-5 protein taken by asthma patients will result in increased eosinophil counts, bronchial hypersensitivity, and release of toxic particles by eosinophils, indicating that IL-5 is a key factor in the pathogenesis of asthma.

Currently, the most effective way to treat asthma is to inhibit the expression of several key mediators (including IL-5) in asthma through nasal or oral administration of sterols to alleviate inflammation in the lung. However, long-term use of sterols has many side effects. Therefore, it is necessary to find new pharmaceutical targets for the treatment of asthma. Studies have demonstrated that, by inhibiting the binding of IL-5 to its receptor, anti-IL-5 antibodies can significantly reduce the accumulation of eosinophils in the lung, reduce the level of eosinophils in blood, tissue, and sputum, reduce the eosinophil-mediated inflammatory response, improve lung function, and show good efficacy. for the treatment of severe eosinophilic asthma and recurrent asthma (Drugs. 2017 May; 77(7): 777-784). Currently, only the anti-IL-5 antibodies mepolizumab from GSK and reslizumab from Teva Pharma are commercially available. Other antibodies against the target - IL-5 - are in the preclinical phase. Related patents are e.g. However, there is still a need to improve IL-5 induced eosinophil clearance and improve lung function. Therefore, it is necessary to continue to develop antibodies with high selectivity, high affinity and good potency to provide more preferred treatment regimens against IL-5 in asthma.

SUMMARY OF THE INVENTION

The present disclosure provides monoclonal antibodies or antigen-binding fragments (also referred to as IL-5 binding molecules) that specifically bind to an IL-5 amino acid sequence or three-dimensional structure.

In one aspect, this disclosure provides a monoclonal antibody, or antigen-binding fragment thereof, that binds to human IL-5, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein:

(i) the heavy chain variable region comprises an HCDR1, HCDR2, and HCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 16-18, or HCDR variants having 3, 2, or 1 amino acid difference(s) from the HCDR1, HCDR2, and HCDR3 region , as defined in SEQ ID NO: 16-18, respectively; and the light chain variable region comprises an LCDR1, LCDR2, and LCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 19-21, or LCDR variants having 3, 2, or 1 amino acid difference(s) from the LCDR1, LCDR2, and LCDR3 region, as defined in SEQ ID NO: 19-21, respectively; or

(ii) the heavy chain variable region comprises an HCDR1, HCDR2, and HCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 22-24, or HCDR variants having 3, 2, or 1 amino acid difference(s) from the HCDR1, HCDR2, and HCDR3 region , as defined in SEQ ID NOs: 22-24, respectively, and the light chain variable region comprises an LCDR1, LCDR2, and LCDR3 region, as defined in the amino acid sequences of SEQ ID NOs: 25-27, or LCDR variants having 3, 2, or 1 amino acid difference(s) from the LCDR1, LCDR2 and LCDR3 region as defined in SEQ ID NO: 25-27, respectively; or

(iii) the heavy chain variable region comprises an HCDR1, HCDR2, and HCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 28-30, or HCDR variants having 3, 2, or 1 amino acid difference(s) from the HCDR1, HCDR2, and HCDR3 region , as defined in SEQ ID NO: 28-30, respectively; and the light chain variable region comprises an LCDR1, LCDR2, and LCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 31-33, or LCDR variants having 3, 2, or 1 amino acid difference(s) from the LCDR1, LCDR2, and LCDR3 region as defined in SEQ ID NO: 31-33, respectively; or

(iv) the heavy chain variable region comprises an HCDR1, HCDR2, and HCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 34-36, or HCDR variants having 3, 2, or 1 amino acid difference(s) from the HCDR1, HCDR2, and HCDR3 region , as defined in SEQ ID NO: 34-36, respectively; and the light chain variable region comprises an LCDR1, LCDR2, and LCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 37-39, or LCDR variants having 3, 2, or 1 amino acid difference(s) from the LCDR1, LCDR2, and LCDR3 region, as defined in SEQ ID NO: 37-39, respectively; or

(v) the heavy chain variable region comprises an HCDR1, HCDR2, and HCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 40-42, or HCDR variants having 3, 2, or 1 amino acid difference(s) from the HCDR1, HCDR2, and HCDR3 region , as defined in SEQ ID NO: 40-42, respectively; and the light chain variable region comprises an LCDR1, LCDR2, and LCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 43-45, or LCDR variants having 3, 2, or 1 amino acid difference(s) from the LCDR1, LCDR2, and LCDR3 region, as defined in SEQ ID NO: 43-45, respectively; or

(vi) the heavy chain variable region comprises an HCDR1, HCDR2, and HCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 34-36, or HCDR variants having 3, 2, or 1 amino acid difference(s) from the HCDR1, HCDR2, and HCDR3 region , as defined in SEQ ID NO: 34, 82 and 36, respectively; and the light chain variable region comprises an LCDR1, LCDR2, and LCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 37-39, or LCDR variants having 3, 2, or 1 amino acid difference(s) from the LCDR1, LCDR2, and LCDR3 region, as defined in SEQ ID NO: 37-39, respectively.

In some embodiments, monoclonal antibody or antigen binding fragment CDR variants (comprising 3 heavy chain CDRs and 3 light chain CDRs) having 3, 2, or 1 amino acid difference(s) are monoclonal antibody or antigen binding fragment CDR variants that are produced by affinity maturation methods. .

In some embodiments, the monoclonal antibodies or antigen-binding fragments bind to IL-5 with an affinity (KD) of less than 10 ^-8 M, less than 10 ^-9 M, less than 10 ^-10 M, or less than 10 ^-11 M.

In some embodiments, the monoclonal antibody or antigen-binding fragment specifically binds to human IL-5, and the monoclonal antibody contains a heavy chain variable region and a light chain variable region, where:

(vii) the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 region as defined in SEQ ID NOs: 16-18 and the light chain variable region contains the LCDR1, LCDR2 and LCDR3 region as defined in SEQ ID NOs: 19-21; or

(viii) the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 region as defined in SEQ ID NOs: 22-24, and the light chain variable region contains the LCDR1, LCDR2 and LCDR3 region as defined in SEQ ID NOs: 25-27; or

(ix) the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 region as defined in SEQ ID NOs: 28-30 and the light chain variable region contains the LCDR1, LCDR2 and LCDR3 region as defined in SEQ ID NOs: 31-33; or

(x) the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 region as defined in SEQ ID NOs: 34-36 and the light chain variable region contains the LCDR1, LCDR2 and LCDR3 region as defined in SEQ ID NOs: 37-39; or

(xi) the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 region as defined in SEQ ID NOs: 40-42 and the light chain variable region contains the LCDR1, LCDR2 and LCDR3 region as defined in SEQ ID NOs: 43-45; or

(xii) the heavy chain variable region contains the HCDR1, HCDR2 and LCDR3 region as defined in SEQ ID NOs: 34, 82 and 36, and the light chain variable region contains the LCDR1, LCDR2 and LCDR3 region as defined in SEQ ID NOs: 37- 39.

In some embodiments, the monoclonal antibody is a recombinant antibody.

In some embodiments, the monoclonal antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a recombinant humanized antibody, or an antigen-binding fragment thereof.

In some embodiments, the light and heavy chain FR (framework) region sequences on the light and heavy chain variable region of the humanized antibody are derived from the human germline light and heavy chain, respectively, or mutated sequences thereof.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, the humanized antibody comprises the heavy chain variable region of SEQ ID NO: 49, 57, 63, 69, or 75, or a variant thereof; wherein the variant has 1-10 amino acid(s) mutation(s) on the heavy chain variable region as defined in SEQ ID NOs: 49, 57, 63, 69, or 75.

In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, variant has 1-10 amino acid backmutations on the FR region of the heavy chain variable region as defined in SEQ ID NO: 49, 57, 63, 69, or 75; preferably, the backmutation is selected from the group consisting of S49T, V93T and K98S or a combination thereof on the heavy chain variable region of SEQ ID NO: 49, or the backmutation is selected from the group consisting of S49T, V93T and K98T or a combination thereof on the heavy chain variable region chain of SEQ ID NO: 57, or a back mutation selected from the group consisting of R38K, M48I, R67K, V68A, M70L, R72V, T74K, and L83F, or a combination thereof on the heavy chain variable region of SEQ ID NO: 63, or a back mutation selected from the group consisting of F29I, R38K, V48I, R72A, T97F and N55V or combinations thereof on the heavy chain variable region of SEQ ID NO: 69, or the back mutation is selected from the group consisting of R38K, M48I, R67K, V68A, R72A, T74K, M81L, L83F and D89E or combinations thereof on the heavy chain variable region of SEQ ID NO: 75.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, the humanized antibody comprises the heavy chain variable region of SEQ ID NO: 50 or 51, or comprises the heavy chain variable region of SEQ ID NO: 58 or 59, or comprises a heavy chain variable region selected from any of SEQ ID NOs: 64, 65 and 66, or contains a heavy chain variable region of SEQ ID NOs: 70 or 71, or contains a heavy chain variable region selected from any of SEQ ID NOs: 76-79.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, the humanized antibody comprises the light chain variable region of SEQ ID NO: 46, 54, 60, 67, or 72, or a variant thereof; this variant has 1-10 amino acid substitutions on the light chain variable region as defined in SEQ ID NOs: 46, 54, 60, 67, or 72.

In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, variant has 1-10 amino acid backmutations on the FR region of the light chain variable region as defined in SEQ ID NOs: 46, 54, 60, 67, or 72; preferably, said backmutation is selected from the group consisting of A43S, L47V, G66R, T69S, F71Y, and Y87F, or combinations thereof, on the light chain variable region of SEQ ID NO: 46; or the reverse mutation is selected from the group consisting of A43S, L47M, F71Y, and Y87F, or a combination thereof, on the light chain variable region of SEQ ID NO: 54; or the reverse mutation is selected from the group consisting of E1D, I2T, I57V, V84T and Y86F or combinations thereof on the light chain variable region of SEQ ID NO: 60; or the reverse mutation is selected from the group consisting of M4L, A42S, L45P and L46W, or combinations thereof, on the light chain variable region of SEQ ID NO: 67; or the reverse mutation is selected from the group consisting of A43S, I48V and F71Y or a combination thereof on the light chain variable region of SEQ ID NO: 72.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, the humanized antibody comprises the light chain variable region of SEQ ID NO: 47 or 48; or contains a light chain variable region of SEQ ID NO: 55 or 56; or contains a light chain variable region of SEQ ID NO: 61 or 62; or contains a light chain variable region of SEQ ID NO: 68; or contains a light chain variable region of SEQ ID NO: 73 or 74.

In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, the humanized antibody comprises:

a heavy chain variable region selected from any of SEQ ID NOs: 49-51 and a light chain variable region selected from any of SEQ ID NOs: 46-48; or

a heavy chain variable region selected from any of SEQ ID NOs: 57-59 and a light chain variable region selected from any of SEQ ID NOs: 54-56; or

a heavy chain variable region selected from any of SEQ ID NOs: 63-66 and a light chain variable region selected from any of SEQ ID NOs: 60-62; or

a heavy chain variable region selected from any of SEQ ID NOs: 69-71 and a light chain variable region selected from any of SEQ ID NOs: 67-68; or

a heavy chain variable region selected from any of SEQ ID NOs: 75-79; and a light chain variable region selected from any of SEQ ID NOs: 72-74.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, the antibody is a full-length antibody, further comprises a human antibody constant region, where the heavy chain constant region is preferably a heavy chain constant region of a human IgG1, IgG2, IgG3, and IgG4 antibody. More preferably, the full length antibody comprises a human antibody heavy chain constant region as defined in SEQ ID NO: 52 and a human antibody light chain constant region as defined in SEQ ID NO: 53.

In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single chain antibody (scFv), dimerized V region (diabody), disulfide stabilized V region (dsFv), and a peptide containing a CDR .

The present disclosure also provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that competes for binding to human IL-5 with the monoclonal antibody, or antigen-binding fragment thereof, described above.

The present disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof according to the present disclosure and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients. The amount of the monoclonal antibody or antigen-binding fragment thereof contained in a unit dose of the pharmaceutical composition is preferably 0.1 to 2000 mg, more preferably 1 to 1000 mg.

The present disclosure also provides an isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof according to the present disclosure.

The present disclosure also provides a recombinant vector containing the nucleic acid molecule described above.

The present disclosure also provides a host cell transformed with the recombinant vector of the present disclosure, the host cell being selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells.

The present disclosure also provides a method for producing a monoclonal antibody or an antigen-binding fragment thereof according to the present disclosure, comprising culturing the above-described host cell in culture to form and accumulate the above-described monoclonal antibody or antigen-binding fragment thereof, and isolating the monoclonal antibody or antigen-binding fragment thereof from the culture.

The present disclosure also provides a method for detecting or detecting human IL-5, the method comprising the use of the above-described monoclonal antibody or antigen-binding fragments thereof.

The present disclosure also provides an agent for detecting or detecting human IL-5, which comprises a monoclonal antibody, or an antigen-binding fragment thereof, according to any of those described above.

The present disclosure also provides a diagnostic agent for a disease associated with human IL-5, the diagnostic agent comprising a monoclonal antibody or an antigen-binding fragment thereof as described above.

The present disclosure also provides a method for diagnosing a disease associated with human IL-5, comprising detecting or detecting human IL-5 or IL-5 positive cells using the above-described monoclonal antibody or antigen-binding fragment thereof.

The present disclosure also provides the use of the above-described monoclonal antibody, or antigen-binding fragment thereof, for the production of a diagnostic agent for a disease associated with human IL-5.

The present disclosure also provides a medicament for the treatment of a disease associated with human IL-5, comprising the above-described monoclonal antibody or antigen-binding fragment thereof, or containing the above-described pharmaceutical composition, or containing the above-described nucleic acid molecule.

The present disclosure also provides a method for treating a disease associated with human IL-5, comprising administering to a subject a pharmaceutically effective amount of the above-described monoclonal antibody or antigen-binding fragment thereof, or a pharmaceutical composition containing them, or the above-described nucleic acid molecule for the prevention or treatment of a disease associated with human IL-5.

The present disclosure also provides the use of the above-described monoclonal antibody or antigen-binding fragment thereof, or a pharmaceutical composition containing them, or the above-described nucleic acid molecule for the preparation of a therapeutic agent for a disease associated with human IL-5.

The above disease or condition is preferably selected from the group consisting of asthma, malignant asthma attack, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocirrhotic dermatitis, episodic angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, helminth infections, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, eosinophilic hyperplastic bronchitis, nodular arteritis, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocirrhotic dermatitis, endometriosis, and steroid-dependent eosinophilic bronchitis.

The anti-IL-5 monoclonal antibodies or antigen-binding fragments of the present disclosure have high specificity and high affinity for IL-5. The humanized antibodies have significantly reduced immunogenicity and completely retain the specificity of the mouse antibody, and exhibit high affinity and excellent in vitro and in vivo activities.

The anti-IL-5 monoclonal antibodies or antigen-binding fragments of the present disclosure have good selectivity for simply specific recognition of IL-5.

Monoclonal antibodies or antigen-binding fragments against IL-5 according to the present disclosure have good characteristics of metabolic dynamics in rats, show a long half-life and high bioavailability.

The humanized anti-IL-5 antibody molecules of the present disclosure have good long-term stability, no obvious abnormal chemical modification, no obvious aggregation at high concentration, and high purity and thermal stability.

In addition to reducing eosinophil proliferation, the anti-IL-5 monoclonal antibodies or antigen-binding fragments of the present disclosure have good properties in improving lung function.

DESCRIPTION OF GRAPHICS

Fig. 1: Anti-IL-5 antibodies block IL-5 binding to the IL-5 receptor in a FACS (fluorescent cell sorting) experiment;

Fig. 2: detection of the binding specificity of antibodies against IL-5 with cytokine Th2;

Fig. 3: Anti-IL-5 antibodies increase respiratory periodic value (Penh). G1: normal control group (PBS (phosphate buffered saline)); G2: model group (IgG); G3: group with 10 mpk (milligrams per kilogram) h1705-008 antibody; G4: group with 2 mpk h1705-008 antibody; G5: group with 10 mpk of h1706-009 antibody; G6: group with 2 mpk h1706-009 antibody; G7: group with 10 mpk Hu39D10; *p less than 0.05, ** less than 0.01 (compared to G2 group by ANOVA/Bonferroni correction);

Fig. 4A: Level of eosinophils BALF (bronchoalveolar lavage) in the lung of asthmatic mice; Fig. 4B: Asthma mouse tracheal mucosal thickness scores. G1: normal control group; G2: model group; G3: group with 10 mpk h1705-008 antibody; G4: group with 2 mpk h1705-008 antibody; G5: group with 10 mpk of h1706-009 antibody; G6: group with 2 mpk h1706-009 antibody; G7: group with 10 mpk Hu39D10; Fig. 4C: Percentage of BALF eosinophils in the lung of asthmatic mice;

On FIG. 5A and FIG. 5B shows the ability of anti-IL5 mAb (monoclonal antibody) to reduce the level of eosinophils in BALF.

DETAILED DESCRIPTION OF THE INVENTION

1. Terminology

In order to make the present disclosure easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used in this document have the meaning generally understood by one of ordinary skill in the art to which this disclosure belongs.

The three letter codes and one letter amino acid codes used in the present disclosure are as described in J. biol. chem, 243, p3558 (1968).

The term "antibody" as used herein refers to an immunoglobulin, a structure of four peptide chains linked to each other by a disulfide bond between two identical heavy chains and two identical light chains. Different immunoglobulin heavy chain constant regions exhibit different amino acid compositions and orders of rank, hence representing different antigenicity. Accordingly, immunoglobulins can be divided into five types, or they are referred to as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, with heavy chain μ, δ, γ, α and ε respectively. According to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds, the same type of Ig can be further subdivided into different subtypes, for example, IgG can be subdivided into IgG1, IgG2, IgG3 and IgG4. The light chain can be subdivided into κ and λ chain based on different constant region. Each of the five Ig types can have a κ or λ chain.

In the present disclosure, the antibody light chain referred to in the present disclosure further comprises a light chain constant region that contains a human or murine κ, λ chain, or a variant thereof.

In the present disclosure, the heavy chain of the antibody referred to in the present disclosure further comprises a heavy chain constant region that contains a human or mouse IgG1, IgG2, IgG3, IgG4, or a variant thereof.

The approximately 110 amino acid sequences adjacent to the N-terminus of the heavy and light chains of an antibody are highly variable, known as the variable region (Fv region); the remaining amino acid sequences close to the C-terminus are relatively stable, known as the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions that determine the specificity of an antibody are also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and each heavy chain variable region (HCVR) consists of three CDRs and four FR regions, in sequential order from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three light chain CDR regions are LCDR1, LCDR2 and LCDR3, and the three heavy chain CDR regions are HCDR1, HCDR2 and HCDR3. The number and position of CDR amino acid residues in the LCVR and HCVR regions of an antibody or antigen-binding fragments herein are consistent with the known Kabat numbering criteria (LCDR1-3, HCDR1-3).

An antibody of the present disclosure includes a mouse antibody, a chimeric antibody, and a humanized antibody, preferably it is a humanized antibody.

The term "mouse antibody" in the present disclosure refers to a monoclonal antibody against human IL-5, obtained according to knowledge and skill in this field. During preparation, an IL-5 antigen can be injected into the analyzed subject, and then a hybridoma is isolated expressing an antibody that has the desired sequence or functional characteristics. In a preferred embodiment of the present disclosure, the mouse anti-IL-5 antibody, or antigen-binding fragment thereof, further comprises a mouse κ, λ light chain constant region, or a variant thereof, or further comprises a mouse IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a variant thereof.

A "chimeric antibody" is an antibody obtained by fusing a variable region of a mouse antibody with a constant region of a human antibody, and the chimeric antibody can alleviate the immune response induced by the mouse antibody. To obtain a chimeric antibody, a hybridoma secreting a specific mouse monoclonal antibody can be created and the variable region gene cloned from the mouse hybridoma. The desired human antibody constant region gene can then be cloned and coupled to the mouse variable region gene to form a chimeric gene, which can then be inserted into an expression vector. Finally, the chimeric antibody molecule will be expressed in a eukaryotic or prokaryotic system. In a preferred embodiment of the present disclosure, the light chain of the chimeric anti-IL-5 antibody further comprises a light chain constant region derived from the human κ, λ chain, or a variant thereof. The heavy chain of the chimeric anti-IL-5 antibody further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3, IgG4, or a variant thereof, preferably comprises a heavy chain constant region derived from human IgG1, IgG2, or IgG4, or contains a constant region variant heavy chain human IgG1, IgG2 or IgG4 with amino acid(s) mutation(s), such(s) as mutation(s) YTE or reverse(s) mutation(s).

The term "humanized antibody" refers to an antibody obtained by grafting murine CDR sequences into a human antibody variable region framework, i.e. an antibody produced in different types of human germline antibody backbone sequences. The humanized antibody can overcome the heterologous responses induced by the large number of mouse protein components carried by the chimeric antibody. Such framework sequences can be obtained from a public DNA database covering germline antibody gene sequences and published references. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the VBase human germline sequence database (available online: www.mrccpe.com.ac.uk/vbase) and also in Kabat, EA, et al. 1991 Sequences of Proteins of Immunological Interest, 5th Ed. In order to avoid a decrease in activity caused by reduced immunogenicity, framework sequences in the variable region of a human antibody can be subjected to minimal reverse mutations or back mutations to maintain activity. The humanized antibody of the present disclosure also includes a humanized antibody on which CDR affinity maturation is performed by phage display. In a preferred embodiment of the present disclosure, the CDR sequence of the humanized anti-IL-5 antibody is selected from the group consisting of SEQ ID NOs: 16-21, 22-27, 28-33, 34-39 and 40-45. A human antibody variable region framework is designed and selected in which the FR region sequence on the antibody heavy chain variable region is derived from a human germline heavy chain sequence and a human germline light chain sequence. In order to avoid a decrease in activity caused by reduced immunogenicity, the variable region of a human antibody can be subjected to minimal reverse mutations (back mutations, that is, amino acid residues of the FR region derived from a human antibody are replaced with amino acid residues corresponding to the original antibody) to maintain activity.

CDR grafting can lead to a decrease in the affinity of the resulting anti-IL-5 antibody or its antigen-binding fragment for the antigen due to alteration of the scaffold residues in contact with the antigen. Such interactions may be the result of highly somatic mutations. Therefore, it may still be necessary to transfer donor scaffold amino acids into a humanized antibody scaffold. Amino acid residues derived from a non-human anti-IL-5 antibody or antigen-binding fragment thereof that are involved in antigen binding can be identified by checking the sequence and structure of the variable region of a mouse monoclonal antibody. Amino acid residues in the framework of the donor CDR that differ from amino acid residues in the germline can be considered related. If it is not possible to determine the most closely related germline, the sequence can be compared to the common sequence that the subtypes have, or to the common sequence of mouse sequences that have a high percentage of similarity. It is believed that the rare scaffold residues are the result of a high mutation in somatic cells that plays an important role in binding.

The term "antigen-binding fragment" or "functional fragment" as used herein refers to one or more antibody fragments that retain the ability to bind to an antigen (eg, IL-5). It has been shown that full length antibody fragments can be used to achieve antigen binding function. Examples of binding fragments in the term "antigen binding fragment" include: (i) a Fab fragment - a monovalent fragment consisting of a VL domain, VH, CL and CH1; (ii) fragment F(ab') ₂ - divalent fragment containing two fragments of Fab linked by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of an incomplete antibody; (v) one dAb domain or fragment (Ward et al. (1989) Nature 341:544-546) consisting of a VH domain; and (vi) a single complementarity determining region (CDR), and (vii) a combination of two or more than two separate CDRs, optionally linked by a synthetic linker. In addition, although the VL domain and the VH domain of the Fv fragment are encoded by two separate genes, they can be linked by a synthetic linker through the use of genetic engineering methods, thereby generating one protein chain of a single-shaft molecule formed by the pairing of the VL domain and VH (called a single-stranded Fv (scFv), see, for example, Bird et al (1988) Science 242: 423-426 and Huston et al (1988) Proc Natl Acad Sci USA 85:5879-5883). It is also meant that a given single chain antibody is also included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are prepared using conventional techniques known in the art and screened for functional fragments using the same method as for an intact antibody. The antigen-binding portions can be obtained by genetic engineering or by enzymatic or chemical cleavage of intact immunoglobulin. Antibodies can be in the form of different isotypes, for example, an IgG antibody (eg, a subtype of IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM.

The antigen binding fragment in the present disclosure includes Fab, F(ab')2, Fab', single chain antibody (scFv), dimerized V region (diabody), disulfide stabilized V region (dsFv), and a CDR-containing peptide.

A Fab is an antibody fragment obtained by treating an IgG antibody molecule with papain (which cleaves the amino acid residue at position 224 of the H chain). A Fab fragment in which about half of the N-terminal side of the H chain and the entire L chain are disulfide bonded to each other has a molecular weight of about 50,000 and has antigen-binding activity.

The Fab of the present disclosure can be obtained by treating with papain a monoclonal antibody of the present invention that specifically recognizes human IL-5 and binds to its extracellular amino acid sequence or three-dimensional structure. Also, a Fab can be obtained by inserting a DNA encoding an antibody Fab into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryote or eukaryote to express the Fab.

F(ab')2 is an antibody fragment having a molecular weight of approximately 100,000, having antigen-binding activity, and containing two Fab regions that bind at the hinge position, F(ab')2 is prepared by pepsin cleavage of an underlying portion with two disulfide bonds at the hinge IgG regions.

The F(ab')2 of the present disclosure can be obtained by treating with pepsin a monoclonal antibody of the present invention that specifically recognizes human IL-5 and binds to its extracellular amino acid sequence or three-dimensional structure. Also, F(ab')2 can be obtained by linking Fab', described below, via a thioether bond or a disulfide bond.

Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity. Fab' is obtained by cleavage of the disulfide bond in the hinge region of the above F(ab')2. The Fab' of the present disclosure can be obtained by treatment with a reducing agent such as dithiothreitol, F(ab')2 of the present invention, which specifically recognizes IL-5 and binds to its extracellular amino acid sequence or three-dimensional structure.

Also, a Fab' can be obtained by inserting a DNA encoding a Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing this vector into a prokaryote or eukaryote to express the Fab'.

The term "single chain antibody", "single chain Fv", or "scFv" refers to a molecule containing an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules have the general structure NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. A suitable linker in the prior art consists of the repeat amino acid sequence GGGGS or a variant thereof, e.g. using a 1-4 repeat variant (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) . Other linkers that can be used for the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Euro. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

The scFv of the present disclosure can be obtained through the following steps: obtaining a cDNA encoding VH and VL of a monoclonal antibody of the present disclosure that specifically recognizes human IL-5 and binds to its extracellular amino acid sequence or three-dimensional structure, construction of a DNA encoding scFv, insert this DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then inserting this expression vector into a prokaryote or eukaryote to express the scFv.

A diabody is an antibody fragment in which scFv dimerizes and is an antibody fragment having bivalent antigen-binding activity. With bivalent binding activity, the two antigens may be the same or different.

The diabody of the present disclosure can be obtained through the following steps: obtaining cDNAs encoding VH and VL of a monoclonal antibody of the present disclosure that specifically recognizes human IL-5 and binds to its extracellular amino acid sequence or three-dimensional structure, constructing a DNA encoding scFv such such that the length of the linker peptide is 8 amino acid residues or less, inserting this DNA into a prokaryotic expression vector or eukaryotic expression vector, and then inserting this expression vector into a prokaryote or eukaryote to express the diabody.

dsFvs are prepared by replacing one amino acid residue in each VH and VL with a cysteine residue and then linking these polypeptides with the replacement via a disulfide bond between the two cysteine residues. The amino acid residue to be replaced by the cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody according to known methods (Protein Engineering, 7, 697 (1994)).

The dsFv of the present disclosure can be obtained through the following steps: obtaining cDNAs encoding VH and VL of a monoclonal antibody of the present disclosure that specifically recognizes human IL-5 and binds to its extracellular amino acid sequence or three-dimensional structure, construction of a DNA encoding dsFv, insert this DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then inserting this expression vector into a prokaryote or eukaryote to express dsFv.

The CDR-containing peptide is constructed by one or more VH and VL CDR regions. Peptides containing multiple CDRs can be linked directly or through a suitable peptide linker.

The peptide containing the CDR of the present disclosure can be obtained by the following steps: constructing a DNA encoding the VH and VL CDRs of the monoclonal antibody of the present disclosure that specifically recognize human IL-5 and bind to its extracellular amino acid sequence or three-dimensional structure, inserting this DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introducing this expression vector into a prokaryote or eukaryote to express this peptide. The CDR-containing peptide can also be prepared by a chemical synthesis method such as the Fmoc method or the tBoc method.

The term "antibody scaffold" as used herein refers to the portion of the variable domain, either VL or VH, that serves as the scaffold for the antigen binding loops (CDRs) of that variable domain. Essentially, it is a variable domain without a CDR.

The term "amino acid difference" refers to differences in one or more amino acid positions along the length of a polypeptide fragment between a polypeptide and a variant thereof, where the variant can be produced by substitution, insertion, or deletion of one or more amino acids on the polypeptide.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on an IL-5 molecule). Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique tertiary conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, ed. G.E. Morris (1996).

The term "specifically binds to", "selectively binds to", "selectively binds to" or "specifically binds to" refers to the binding of an antibody to a given epitope on an antigen. Typically, the antibody binds with an affinity (KD) of less than about 10 ^-8 M, such as less than about 10 ^-9 M, 10 ^-10 M, or 10 ^-11 M, or even less.

The term "KD" refers to the equilibrium dissociation constant with respect to a particular antibody-antigen interaction. Typically, an antibody of the present disclosure binds to IL-5 with an equilibrium dissociation constant (KD) of less than about 10 ^-7 M, such as less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M, or even less, for example, when determined using surface plasmon resonance (SPR) techniques on the BIACORE instrument.

When used in the context of antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) that compete for the same epitope, the term "competition" means that competition occurs among antigen-binding proteins, as determined by the following assays: the antigen-binding protein to be assay (eg, an antibody or immunologically functional fragment thereof) prevents or inhibits (eg, reduces) specific binding between a reference antigen-binding protein (eg, a ligand or reference antibody) and a common antigen (eg, an IL-5 antigen or fragment thereof). Many types of competitive binding assays are available to determine if an antigen-binding protein competes with another antigen-binding protein. These assays are, for example, solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), competitive sandwich assay (see, for example, Stahli et al, 1983, Methods in Enzymology 9: 242-253) ; biotin-avidin solid phase direct EIA (see e.g. Kirkland et al, 1986, J. Immunol. 137: 3614-3619), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see e.g. Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct RIA labeled with I-125 label (see, for example, Morel et al, 1988, Molec. Immunol. 25: 7-15); biotin-avidin solid phase direct EIA (see, for example, Cheung, et al, 1990, Virology 176: 546-552); and direct labeling RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82). Typically, this assay involves the use of a purified antigen (either on a solid surface or on a cell surface) capable of binding to both the unlabeled antigen-binding protein to be assayed and the labeled reference antigen-binding protein. Competitive inhibition is determined by measuring the amount of label bound to a solid surface or cell in the presence of the antigen-binding protein to be analyzed. Typically, the antigen-binding protein to be analyzed is present in excess. Antigen-binding proteins identified by competitive analysis (competing with antigen-binding protein) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to an epitope that is sufficiently close to the epitope to which the reference antigen-binding protein binds, where the two epitopes interfere spatially with difficulty in binding. Additional details regarding methods for determining competitive binding are provided herein in the Examples. Typically, when a competing antigen-binding protein is present in excess, it will inhibit (eg, reduce) specific binding between the reference antigen-binding protein and the total antigen by at least 40-45%, 45-50%, 50-55%, 55-60% , 60-65%, 65-70%, 70-75% or 75%, or even more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97% or 97%, or even more.

The term "nucleic acid molecule" as used herein refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single or double stranded, but is preferably double stranded DNA. A nucleic acid "binds in a functional manner" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer binds operatively to a coding sequence if it affects the transcription of that sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded loop of DNA into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. The vectors disclosed herein are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial replicator and mammalian episomal vectors) or can be integrated into the host cell genome when introduced into the host cell. and, thereby, are replicated along with the host genome (eg, non-episomal mammalian vectors).

Methods for making and purifying antibodies and antigen-binding fragments are well known in the art and can be found in, for example, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, chapters 5-8 and 15. For example, mice can be immunized with human IL-5 or fragments thereof, and the resulting antibodies can then be annealed, purified and sequenced into amino acid sequences using conventional methods well known in the art. Antigen binding fragments can also be obtained by conventional methods. The antibodies or antigen-binding fragments of the present disclosure are designed to contain one or more human FR regions per CDR derived from a non-human antibody. Human germline FR sequences can be obtained by alignment using the human germline antibody variable region gene database and the MOE program from ImMunoGeneTics (IMGT) via their website http://imgt.cines.fr, or from The Immunoglobulin Facts Book , 2001, ISBN 012441351.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include microbial (eg, bacterial), plant, or animal cells. Bacteria that are susceptible to transformation include members of Enterobacteria such as strains of Escherichia coli or Salmonella; bacilli such as Bacillus subtilis; pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary cell line), HEK cells (as non-limiting examples, HEK293E cells), and NS0 cells.

The engineered antibodies or antigen-binding fragments of the present disclosure may be prepared and purified using known methods. For example, a cDNA sequence encoding a heavy chain and a light chain can be cloned and constructed into a GS expression vector. The expression vector for the recombinant immunoglobulin can then be stably transfected into CHO cells. As a more preferred method, well known in the art, mammalian cell-based expression systems will result in glycosylation of the antibody, typically at the highly conserved N-terminal sites of the Fc region. Stable clones can be confirmed to express an antibody that specifically binds to human IL-5. Positive clones can be expanded in serum-free culture medium in bioreactors to produce antibodies. The culture medium into which the antibody has been secreted can be purified by conventional techniques. For example, purification may be carried out on a Protein A or G Sepharose FF column that has been equilibrated with buffer added. The column is washed to remove non-specifically binding components, and then the bound antibody is eluted with a pH gradient and the antibody fractions are detected by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and then collected. These antibodies can be filtered and concentrated using conventional techniques. Suitable mixtures and polymers can be removed by conventional techniques such as gel filtration or ion exchange. The resulting product is then immediately frozen, for example at -70°C, or it can be lyophilized.

The term "administering", "administering", or "treating", as applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refers to bringing into contact an exogenous pharmaceutical, therapeutic, diagnostic agent, or compositions with an animal, human, subject, cell, tissue, organ or biological fluid. The term "administration", "administration" or "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Processing a cell encompasses bringing a reagent into contact with a cell, as well as bringing a reagent into contact with a liquid, where the liquid is in contact with the cell. The term "administration", "administration", or "treatment" also means in vitro and ex vivo treatments of a cell with a reagent, diagnostic, binding composition, or other cell. The term "treatment", as applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

"Treat" means the administration of a therapeutic agent, such as a composition containing any of the antibodies or fragments thereof according to the present disclosure, internally or externally to a patient who has one or more symptoms of a disease for which the agent has a known therapeutic activity. Typically, the agent is administered in an effective amount to alleviate one or more symptoms of the disease in the patient or population being treated by inducing regression or inhibiting the progression of such symptom(s) to any clinically measurable level. The amount of a therapeutic agent that is effective in alleviating any particular symptom of a disease (also referred to as a "therapeutically effective amount") may vary according to such factors as the condition of the disease, the age, weight of the patient, and the ability of the drug to elicit a desired response in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other qualified health care providers to assess the severity or progression status of a given symptom. While an embodiment of the present disclosure (e.g., a method of treatment or an article of manufacture) may not be effective in alleviating the target symptom(s) of the disease in each patient, it should alleviate the target symptom(s) of the disease in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, Chi-square test, Mann and Whitney U-test, Kruskal-Wallis test (H-test), Jonkheer-Terpstra test, and Wilcoxon test .

The term "conservative modification" or "conservative substitution" refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), thus that changes can often be made without changing the biological activity of the protein. It is known to those skilled in the art that, in general, a single amino acid change in non-essential regions of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p.224 (4th Ed.)). In addition, substitutions with structurally or functionally similar amino acids are less likely to impair biological activity.

An "effective amount" encompasses a sufficient amount to reduce or prevent a symptom or sign of a medical condition. An effective amount also means a sufficient amount to provide or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary, depending on such factors as the condition being treated, the general health of the patient, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or a dosing protocol that avoids significant side effects or toxic effects.

The term "exogenous" refers to substances that are produced outside the body, cell, or human body, depending on the context. The term "endogenous" refers to substances that are produced within a cell, an organism, or the human body, depending on the context.

The term "homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences. When a position in both of the two compared sequences is occupied by the same monomeric base or amino acid subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions that divide the two sequences, divided by the number of positions to be compared, and then multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, with optimal data alignment sequences, then the two given sequences have 60% homology; if 95 out of 100 positions in two sequences are identical or homologous, then the two sequences have 95% homology. Typically, comparison is made by aligning two sequences to obtain the maximum percentage of homology.

The terms "cell", "cell line" and "cell culture" as used herein are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include cells of the primary subject and cultures derived from them, regardless of the number of passages. It should also be understood that all progeny may not be exactly identical in DNA content due to intentional or accidental mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cells are included. When distinct designations are intended, this will be clear from the context.

The term "polymerase chain reaction" or "PCR", as used herein, refers to a procedure or technique in which tiny amounts of a specific portion of a nucleic acid, RNA and/or DNA are amplified, as described, for example, in US Pat. No. 4,683,195. In general, sequence information should be available near the ends of the region of interest, or outside the region of interest, so that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to the opposite strands of the template to be amplified. The 5'-terminal nucleotides of the two primers are consistent with the ends of the substance to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See, in general, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). The PCR assay used in the present disclosure is considered one, but not the only, example of a polymerase reaction method for amplifying a nucleic acid prototype. This method includes the use of known nucleic acid sequences as primers and a nucleic acid polymerase to amplify or obtain a specific portion of the nucleic acid.

The term "possible" or "possible" means that the event or situation that follows may, but does not necessarily occur, and this description includes examples in which the event or circumstance does or does not occur. For example, the phrase "possibly contains 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region with a specific sequence may be present, but not necessarily present.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds according to the present disclosure or its physiologically/pharmaceutically acceptable salt or prodrug and other chemical components such as physiologically/pharmaceutically acceptable carriers and excipients. This pharmaceutical composition is aimed at promoting the introduction into the body, facilitating the absorption of the active ingredient and thereby producing a biological effect.

In addition, the present disclosure includes an agent for the treatment of diseases associated with IL-5, and this agent contains the monoclonal antibody of the present disclosure or its antibody fragment as an active ingredient.

There is no limitation of diseases associated with IL-5, provided that they are associated with IL-5. For example, therapeutic responses induced by molecules of the present disclosure can be obtained by binding to human IL-5 and thereby repressing or inhibiting eosinophil-induced stimulation. Thus, when found in formulations and compositions suitable for therapeutic applications, the molecules of the present disclosure are very useful in individuals who suffer from allergic and/or atopic or eosinophil-associated responses, e.g. asthma attack, chronic pneumonia, allergic rhinitis, perennial allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocirrhotic dermatitis, episodic angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, helminth infections, Hodgkin's disease, nasal polyps, syndrome Leffler, urticaria, eosinophilic hyperplastic bronchitis, nodular arteritis, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocirrhotic dermatitis, endometriosis, steroid dependent eosinophilic bronchitis, etc. similar, but not limited to. In a preferred embodiment, such treatment inhibits or reduces infiltrating eosinophils in lung tissue. Antibodies or fragments thereof can be administered from three times a day to once every six months and can be administered intravenously, subcutaneously, intramuscularly, parenterally or topically.

In addition, the present disclosure relates to a method for immunodetection or immunoassay of IL-5, reagents for immunodetection or immunoassay of IL-5, immunodetection or immunoassay of cells expressing IL-5, and a diagnostic tool for diagnosing a disease associated with IL-5, which contains a monoclonal antibody or antibody fragment of the present disclosure specifically recognizing human IL-5 and binding to its extracellular amino acid sequence or three-dimensional structure, as an active ingredient.

In the present disclosure, the method for detecting or quantifying IL-5 may be any method known in the art. For example, it includes immunodetection or immunoassay.

An immunodetection or immunoassay is a method for detecting or quantifying an antibody or antigen by using a labeled antigen or antibody. Examples of the immunodetection or immunoassay include a radioactive labeling method for an immunological antibody (RIA), an enzyme immunoassay (EIA or ELISA), a fluorescence immunoassay (FIA), a luminescent immunoassay, a Western blot, physicochemical assays, and the like.

The aforementioned IL-5 associated diseases can be diagnosed by detecting or identifying cells expressing IL-5 using a monoclonal antibody or antibody fragment of the present disclosure.

In order to detect cells expressing a polypeptide, known immunoassays, preferably immunoprecipitation, fluorescent staining of cells, immunohistochemical staining, and the like, can be used. In addition, a fluorescent antibody staining method using FMAT8100HTS (Applied Biosystem) and the like can be used.

In the present disclosure, the live sample used to detect or detect IL-5 is not particularly limited, as long as it likely contains cells expressing IL-5, for example, tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture medium.

A diagnostic agent comprising the monoclonal antibody or antibody fragment of the present disclosure may further comprise an agent for performing an antigen-antibody reaction or an agent for detecting a reaction, depending on the desired diagnostic method. The agent for carrying out the antigen-antibody reaction includes components such as a buffer and salts. The agent for detecting a reaction includes reagents commonly used in an immunodetection or immunoassay method, such as, for example, a labeled secondary antibody that recognizes a monoclonal antibody, an antibody fragment thereof, or a conjugate containing it, and a labeled substrate.

2. Examples and experimental examples

The following examples are provided to further describe the present disclosure, but are not intended to limit the scope of this disclosure. Experimental methods for which specific conditions are not specifically stated are generally carried out according to conventional conditions, see Molecular Cloning, Laboratory Manual of antibody technology, Cold Spring Harbor Laboratory; or according to the conditions recommended by the substance or product manufacturer. Reagents for which sources are not specifically indicated are commercially available reagents.

Example 1 Preparation of IL-5 Antigens and Proteins for Detection

Construction and expression of the IL-5 antigen

Sequences encoding His-tagged human IL-5, rhesus monkey IL-5, mouse IL-5, rat IL-5, or the extracellular domain of the human IL-5Rα receptor fused to a human IgG1-Fc fragment were inserted into the phr vector to construct expression expressions. plasmids, which were then transfected into HEK293. On day 6 post-transfection, samples were centrifuged at 4500 rpm for 10 minutes and cell supernatants were collected. The supernatant containing the recombinant IL-5 or IL-5α receptor protein was purified using a nickel column, and the recombinant human IL-5-Fc fusion protein was purified using a protein A affinity chromatography column. The purified protein can be used in the following examples. The following antigen protein sequences are shown:

1. Amino acid sequence of human IL-5 with His tag (rhIL-5-his)

Note: The text in italics shows the His6 tag.

SEQ ID NO: 1

2. Amino acid sequence of His-tagged cynomolgus monkey IL-5

Note: The text in italics shows the His6 tag.

3. Amino acid sequence of His-tagged mouse IL-5

Note: The text in italics shows the His6 tag.

4. Amino acid sequence of His-tagged rat IL-5

Note: The text in italics shows the His6 tag.

5. Amino acid sequence of human IL-5α receptor fused to human Fc fragment

Note: Text in italics shows human Fc label.

Example 2: Construction and identification of cell lines with recombinant IL-5α receptor and IL-5α/β receptor

For screening functional antibodies, the present disclosure constructs a CHO-S/IL-5α cell line expressing IL-5α and a CHO-S/IL-5α/IL-5β cell line expressing both IL-5α and IL-5β.

Specifically, the full-length human IL-5α gene (Q01344) was cloned into a mammalian cell expression vector, pTargeT, and the linearized plasmid was transfected into CHO-S cells by electroporation. Screening was performed under G418 for 2 weeks, followed by two cycles of limiting dilutions. The cell surface expressed IL-5α gene was detected by FACS (fluorescent cell sorting), CHO-S/IL-5α cell lines with high IL-5α expression were selected and transfected into linearized pcDNA3.1-IL-5β by electroporation. Carried out screening with G418 and Zeocin for 2 weeks, followed by two cycles of limiting dilutions. The gene for IL-5α and IL-5β expressed on the cell surface was detected by FACS, and CHO-S/IL-5α/IL-5β cell lines with high expression of IL-5α and IL-5β were selected.

Example 3: Production of a mouse anti-human IL-5 monoclonal antibody

Two groups of Balb/c mice (5 mice/group) and four groups of SJL mice (5 mice/group) were immunized with recombinant rhIL-5-his protein and Freund's adjuvant CFA (Sigma, lot no. SLBQ1109V); or IFA (Sigma, Lot # SLBJ2845V) at two dosages of 100 g/50 g/50 g (high dose group) and 25 g/12.5 g/12.5 g (low dose group), respectively. Specific immune response to IL-5 was determined by serum ELISA titer detection, receptor ligand blocking assay and TF-1 proliferation inhibition assay. Mice with the best specific immune response were selected, sacrificed, spleen cells were selected and fused with myeloma cells.

Primary screening was performed using an anti-human IL-5 ELISA binding assay. Hybridoma cells were transferred to 24-well plates and supernatants were rescreened by anti-human, cynomolgus or mouse IL-5 ELISA binding assay, by anti-IL-5 receptor ELISA-based blocking assay and by TF-1 proliferation inhibition assay. After such screening, the resulting positive clones were subjected to two rounds of subcloning to obtain hybridoma clones for antibody production. The resulting antibodies were purified by affinity chromatography.

Purified antibodies were subjected to the following assays: SEC-HPLC (high performance liquid chromatography gel filtration), endotoxin detection, Biacore affinity assay for various IL-5s, FACS-based anti-IL-5 receptor blocking assay, TF- 1, Eosinophil adhesion analysis and performance evaluation of a mouse model of asthma and an in vivo guinea pig neutralization model. The mAb1705, mAb1706, mAb1780, mAb1773 and mAb1779 monoclonal hybridoma cell lines were selected because of their excellent in vitro and in vivo activities.

Cloned sequences from a positive hybridoma as follows. Log phase hybridoma cells were selected, RNA was isolated using Trizol (Invitrogen cat. no. 15596-018) according to the manufacturer's instructions or kit, and reverse transcription was performed using the PrimeScript™ reverse transcriptase kit (Takara, cat. no. 2680A). cDNAs obtained by reverse transcription were subjected to PCR amplification using a mouse Ig primer set (Novagen, TB326 Rev. B 0503) and the resulting products sequenced. Amino acid sequences were obtained corresponding to the mAb1705, mAb1706, mAb1780, mAb1773 and mAb1779 heavy and light chain variable region DNA sequences (VH/VL CDR amino acid residues were determined and annotated using the Kabat numbering system):

mouse mAb1705 heavy chain variable region sequence

mouse mAb1705 light chain variable region sequence

mouse mAb1706 heavy chain variable region sequence

mouse mAb1706 light chain variable region sequence

mouse mAb1780 heavy chain variable region sequence

mouse mAb1780 light chain variable region sequence

mouse mAb1773 heavy chain variable region sequence

mouse mAb1773 light chain variable region sequence

mouse mAb1779 heavy chain variable region sequence

mouse mAb1779 light chain variable region sequence

The light and heavy chain CDR sequences of each antibody are shown in Table 1.

The activity results of the Biacore assay are shown in Table 2.

These results indicate that the mouse antibodies of the present disclosure have high affinity for this antigen.

Example 4 Purification of Recombinant IL-5 Related Proteins and Purification of Hybridoma Antibodies and Recombinant Antibodies

4.1 Steps for purification of recombinant IL-5-Flag-His proteins:

Samples were centrifuged at high speed to remove impurities and concentrated to an appropriate volume. An affinity column NI-NTA (QIAGEN, Cat #30721) was equilibrated with PBS (phosphate buffered saline) and washed with 2-5 column volumes. Cell-expressed, uncontaminated supernatants were loaded onto the column, which was then washed with PBS until the A280 reading (absorbance at 280 nm) dropped back to baseline. The column was then washed with PBS to remove the crude protein. The protein of interest was eluted with wash buffer (20 mM imidazole) and then with elution buffer (300 mM imidazole) and the eluted peak was collected.

The withdrawn eluate was further purified by ion exchange (Hiload 16/600 superdex 200 column). This column was equilibrated with about 2 column volumes of PBS to provide a pH of 7.4. The elution buffer containing the protein of interest was concentrated and loaded onto a column for subsequent purification. These samples were collected, identified by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and LC-MS (liquid chromatography-mass spectrometry), and then aliquoted for use.

4.2. Purification of hybridoma-expressed antibody and Fc-fusion proteins

Cell-expressed supernatant samples were centrifuged at high speed to remove impurities, and then hybridoma-expressed supernatants were purified using a protein G column, supernatants expressing an Fc fusion protein were purified using a protein A column. The column was washed with PBS until the A280 reading dropped to initial level. Proteins of interest were eluted with 100 mM acetic acid, pH 3.0 and neutralized with 1 M Tris-HCl, pH 8.0. The eluted samples were suitably concentrated and further purified by size exclusion chromatography on Superdex 200 (GE) pre-equilibrated with PBS. Peaks devoid of aggregates were collected and aliquoted for use.

Example 5: Construction of humanized anti-human IL-5 monoclonal antibodies

Humanization of mouse monoclonal antibodies against human IL-5 was performed as disclosed in the literature in this field. Briefly, mouse antibody constant regions were replaced with human constant regions, mouse antibody CDRs were grafted onto a human template having the highest FR homology, and certain amino acid residues in the FR region that have a key effect on maintaining antibody conformation and affecting antibody-antigen binding were reversed. mutations.

By aligning with the IMGT database the human antibody heavy and light chain variable region germline genes as templates, respectively, the human heavy and light chain variable region genes that have high amino acid sequence identity with mAb-1705, mAb-1706 antibody were selected. , mAb1780, mAb1773 and mAb1779. The CDRs of these mouse antibodies were separately grafted onto appropriate templates of human origin to form the variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Amino acid residues were identified and annotated using the Kabat numbering system.

Selection of human FR region and backmutation of key amino acids

Based on the typical CDR structure of the VH/VL of the resulting mouse antibody, homologous light chain variable region (VL) and heavy chain variable region (VH) sequences were selected from the human germline database. The resulting human germline VL and VH sequences were ranked from high score to low based on FR homology, and the germline sequences with the highest FR homology were selected as master templates. Mouse antibody CDRs were grafted onto human matrices. And then, using the software and based on the three-dimensional structure of mouse antibodies, the inserted residues, the residues that directly interact with the CDR regions, and the residues that have a significant effect on the VL and VH conformation were backmutated. In addition, chemically unstable amino acid residues have been optimized to produce final humanized molecules.

5.1 Selection of humanized scaffold for mAb1705 hybridoma clone

IGHV3-23*04 was chosen as the template for VH h1705 and IGKV1-12*01 was chosen as the template for VL. The mouse mAb1705 CDR was grafted onto a human template. Inserted residues and residues that interact directly with CDR regions were detected by the program and were backmutated. The light and heavy chain variable regions of the humanized antibodies were constructed as shown in Table 3.

Specific Variable Region Sequences

humanized h1705 antibodies are as follows:

>h1705_VL.1 (SEQ ID NO: 46)

>h1705_VL.1A (SEQ ID NO: 47)

>h1705_VL.1B (SEQ ID NO: 48)

>h1705_VH.1 (SEQ ID NO: 49)

>h1705_VH.1A (SEQ ID NO: 50)

>h1705_VH.1B (SEQ ID NO: 51)

Each of the above light chain variable regions was combined with a light chain constant region as defined in SEQ ID NO: 53 to form the final intact light chain sequences. Each heavy chain variable region was combined with a heavy chain constant region as defined in SEQ ID NO: 52 to form the final heavy chain sequences.

Humanized antibody constant region sequence

>IgG1 heavy chain constant region:

Note: Underlined text represents engineered mutation M252Y, S254T, or T256E

>Constant region of kappa light chain:

5.2 Selection of humanized scaffold for mAb1706 hybridoma clone

IGHV3-23*04 was chosen as the template for VH h1706 and IGKV1-12*01 was chosen as the template for VL. The mouse mAb1706 CDR was grafted onto a human template. Inserted residues and residues that interact directly with CDR regions were detected by the program and were backmutated. The light and heavy chain variable regions of the humanized antibodies were constructed as shown in Table 5.

The following specific variable region sequences of the humanized h1706 antibody are shown:

>h1706_VL.1 (SEQ ID NO: 54)

>h1706_VL.1A (SEQ ID NO: 55)

>h1706_VL.1B (SEQ ID NO: 56)

>h1706_VH.1 (SEQ ID NO: 57)

>h1706_VH.1A (SEQ ID NO: 58)

>h1706_VH.1B (SEQ ID NO: 59)

Each of the above light chain variable regions was combined with a light chain constant region as defined in SEQ ID NO: 53 to form the final intact light chain sequences. Each heavy chain variable region was combined with a heavy chain constant region as defined in SEQ ID NO: 52 to form the final heavy chain sequences.

5.3 Selection of humanized scaffold for mAb1780 hybridoma clone

IGHV1-2*02 was selected as the template for VH h1780 and IGKV3-11*01 was selected as the template for VL. The mouse mAb1780 CDR was grafted onto a human template. Inserted residues and residues that interact directly with CDR regions were detected by the program and were backmutated. The light and heavy chain variable regions of the humanized antibodies were constructed as shown in Table 7.

The following specific variable region sequences of the humanized h1780 antibody are shown:

>h1780_VL.1 (SEQ ID NO: 60)

>h1780_VL.1A (SEQ ID NO: 61)

>h1780_VL.1B (SEQ ID NO: 62)

>h1780_VH.1 (SEQ ID NO: 63)

>h1780_VH.1A (SEQ ID NO: 64)

>h1780_VH.1B (SEQ ID NO: 65)

>h1780_VH.1C (SEQ ID NO: 66)

Each of the above light chain variable regions was combined with a light chain constant region as defined in SEQ ID NO: 53 to form the final intact light chain sequences. Each heavy chain variable region was combined with a heavy chain constant region as defined in SEQ ID NO: 52 to form the final heavy chain sequences.

5.4 Selection of humanized scaffold for mAb1773 hybridoma clone

IGHV3-73*01 was chosen as the template for VH h1773, and IGKV1-39*01 was chosen as the template for VL. The mouse mAb1773 CDR was grafted onto a human template. Inserted residues and residues that interact directly with CDR regions were detected by the program and were backmutated. The light and heavy chain variable regions of the humanized antibodies were designed as shown in Table 9. In the meantime, in order to remove isomerized sites in the CDR regions, N located in HCDR2 (RIDPANGDTK HGPKFQG) h1773 was replaced with V (i.e., N55V ) to form a heavy chain variable region and an antibody containing the HCDR2 variant (the sequence of the mutated HCDR2 is shown as SEQ ID NO: 82: RIDPAVGDTKHGPKFQG).

The following specific variable region sequences of the humanized h1773 antibody are shown:

>h1773_VL.1 (SEQ ID NO: 67)

>h1773_VL.1A (SEQ ID NO: 68)

>h1773_VH.1 (SEQ ID NO: 69)

>h1773_VH.1A (SEQ ID NO: 70)

>h1773_VH.1B (SEQ ID NO: 71)

Each of the above light chain variable regions was combined with a light chain constant region as defined in SEQ ID NO: 53 to form the final intact light chain sequences. Each heavy chain variable region was combined with a heavy chain constant region as defined in SEQ ID NO: 52 to form the final heavy chain sequences.

5.5 Selection of humanized scaffold for mAb1779 hybridoma clone

IGHV1-2*02 was chosen as the template for VH h1779, and IGKV1-33*01 was chosen as the template for VL. The mouse h1773 CDR was grafted onto a human template. Inserted residues and residues that interact directly with CDR regions were detected by the program and were backmutated. The light and heavy chain variable regions of the humanized antibodies were constructed as shown in Table 11.

The engineered humanized molecules were combined with various molecules as shown in Table 12.

The following specific variable region sequences of the humanized h1779 antibody are shown:

>h1779_VL.1 (SEQ ID NO: 72)

>h1779_VL.1A (SEQ ID NO: 73)

>h1779_VL.1B (SEQ ID NO: 74)

>h1779_VH.1 (SEQ ID NO: 75)

>h1779_VH.1A (SEQ ID NO: 76)

>h1779_VH.1B (SEQ ID NO: 77)

>h1779_VH.1C (SEQ ID NO: 78)

>h1779_VH.1D (SEQ ID NO: 79)

Each of the above light chain variable regions was combined with a light chain constant region as defined in SEQ ID NO: 53 to form the final intact light chain sequences. Each heavy chain variable region was combined with a heavy chain constant region as defined in SEQ ID NO: 52 to form the final heavy chain sequences.

Meanwhile, the anti-IL-5 antibody Hu39D10 disclosed in WO 2012083370 A1 was used in the present disclosure as a positive control, and its heavy and light chain sequences are shown in SEQ ID NO: 80 and SEQ ID NO: 81, respectively.

Hu39D10 heavy chain sequence

Hu39D10 light chain sequence

Example 6 Preparation of Recombinant Chimeric Antibodies and Humanized Antibodies

1. Molecular cloning of recombinant chimeric antibodies

Positive antibody molecules obtained by hybridoma screening were sequenced to obtain gene sequences encoding variable regions. Based on the obtained sequences, forward and reverse primers were designed, and the VH/VK gene fragment of each antibody was constructed by PCR using the gene sequence as a template. The VH/VK gene fragment was then homologously recombined with the pHr expression vector (containing the signal peptide and the hIgG1/hkappa (CH1-Fc/CL) constant region gene fragment to construct a full-length expression plasmid for the recombinant VH-CH1-Fc-pHr/VL chimeric antibody -CL-pHr to form five chimeric antibodies Ch1705, Ch1706, Ch1780, Ch1773 and Ch1779.

2. Molecular cloning of humanized antibodies

On the sequences of the engineered humanized antibody, codon optimization was performed to obtain coding sequences for genes with a preference for human codons. PCR primers were designed to construct the VH/VK gene fragment of each antibody, and then the VH/VK gene fragment was subjected to homologous recombination with the pHr expression vector (containing the signal peptide and the hIgG1/hkappa (CH1-Fc/CL) constant region gene fragment) to construct full length expression plasmid of the humanized antibody VH-CH1-Fc-pHr/VL-CL-pHr.

3. Expression and purification of recombinant chimeric antibodies and humanized antibodies

Plasmid expressing the light or heavy chain of the antibody was transfected into HEK293E cells at a ratio of 1:1.2. After 6 days, expression supernatants were collected, centrifuged at high speed to remove impurities, and purified via a protein A column. The column was washed with PBS until the A280 reading dropped to baseline. The protein of interest was eluted with an acidic elution buffer - pH 3.0-pH 3.5 - and neutralized with 1 M Tris-HCl, pH 8.0-9.0. The eluted samples were suitably concentrated and further purified by size exclusion chromatography on Superdex 200 (GE) pre-equilibrated with PBS to remove aggregates. Monomer peaks were collected and aliquoted for use.

The efficacy and beneficial effects of the antibodies of the present disclosure were confirmed by the following assay methods.

Assessment of biological activity in vitro

Test Example 1: Binding of Mouse Anti-IL-5 Antibodies to Different Species of IL-5 Determined by Biacore Assay

Affinity between experimental mouse anti-IL-5 antibodies and human IL-5 was determined using a Biacore T200 instrument (GE).

Molecules to be analyzed were affinity captured by the protein A biosensor chip, and then the antigen (recombinant human, simian, and mouse IL-5 prepared in Example 1) flowed through the surface of the chip, and reaction signals were detected in real time by using the Biacore instrument. T200 to obtain binding and dissociation curves. After dissociation, each cycle of the experiment was completed, the biosensor chip was washed and regenerated with a regenerating solution based on glycine-hydrochloric acid (pH 1.5). The data were fitted to the Langmuir model (1:1) using the BIAevaluation software version 4.1 GE, and affinity values were obtained as shown in Table 13.

This example demonstrates that mAb 1705, mAb 1706, mAb 1780, mAb 1773 and mAb 1779 of the present disclosure all have high affinity for IL-5 of different species (human, monkey).

Experimental Example 2: Affinity of humanized anti-IL-5 antibodies to IL-5 of different species, determined by Biacore analysis

The affinity between the test humanized anti-IL-5 antibodies and human IL-5 was determined using a Biacore T200 (GE) instrument.

Molecules to be analyzed were affinity captured by a protein A biosensor chip, and then the antigen (obtained in Example 1) flowed through the surface of the chip, and reaction signals were detected in real time by using a Biacore T200 instrument to obtain binding and dissociation curves. After dissociation, each cycle of the experiment was completed, the biosensor chip was washed and regenerated with a regenerating solution based on glycine-hydrochloric acid (pH 1.5). The data were fitted to the Langmuir model (1:1) using the BIAevaluation software version 4.1 GE, and affinity values were obtained as shown in Table 14.

These results indicate that humanized anti-IL-5 antibodies still have high affinity for human IL-5 (only representative data has been given for humanized variants of other mouse antibodies, except for each humanized h1705 variant).

Experimental Example 3: Mouse anti-IL-5 antibodies block binding between IL-5 and IL-5α receptor as determined by ELISA assay

To demonstrate the ability of anti-IL-5 antibodies to prevent IL-5 from binding to the extracellular region of recombinantly expressed IL-5α receptor protein, an ELISA plate was coated with IL-5 (5 μg/ml in PBS), incubated at 37°C for 1 hour, liquid was discarded, then a blocking solution with 5% skimmed milk diluted with PBS was added in a volume of 200 μl/well and blocked at 37°C for 2.5 hours in an incubator. After blocking was complete, the blocking solution was discarded, the plate was washed 5 times with PBST buffer (PBS containing 0.05% Tween-20, pH 7.4), 25 μl of 10 μg/ml IL-5Rα (in 1% BSA (bovine serum) was added). albumin)), which was labeled with a biotin labeling kit (Dojindo Chemical, LK03), and then 25 µl of a gradient diluted antibody that competed with IL-5Rα for binding to IL-5 was added and incubated at 37°C for 1 hour . After incubation, the reaction solution in the plate was discarded, the plate was washed 5 times with PBST, streptavidin-peroxidase polymer (Sigma, S2438-250UG) diluted 1:600 with sample dilution solution was added in a volume of 50 µl/well and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, 50 µl/well of chromogenic substrate TMB (tetramethylbenzidine) (KPL, 52-00-03) was added, incubated at room temperature for 3-10 min, and 50 µl/well of 1 M H ₂ SO ₄ was added to stop the reaction. The absorbance value was read at 450 nm using a NOVOStar microplate reader. IC50 values (half-maximal inhibition) were calculated for anti-IL-5 antibody blocking binding between IL-5 and IL-5Rα. The results are shown in Table 15, both antibodies of the present disclosure can effectively inhibit the binding of IL-5 to its receptor.

Test Example 4 Anti-IL-5 Antibodies Block Binding Between IL-5 and IL-5 Receptor as Detected by FACS

In order to identify nascent anti-IL-5 antibodies that can block the IL-5 receptor on the cell surface, we constructed a recombinant CHOS cell line that strongly expresses both IL-5Rα/β receptors. This experiment identified that anti-IL-5 antibodies can prevent IL-5 from binding to the recombinant IL-5α/β receptor on the surface of the CHOS cell line, respectively.

Specific Method: CHO-S-IL-5Rα and β were cultured with CD-CHO containing 100 ng/ml G418 and 25 ng/ml Zeocin, and the concentration during cell culture was no more than 3×10 ⁶ cells/ml. IL-5Rα/β-CHOS cells in good condition were centrifuged (1000 rpm, 5 min), washed once with 10% FBS (fetal calf serum) in PBS, counted, cell concentration was adjusted to 4×10 ⁶ cells/ml , and 25 µl of which were added to a 96-well round bottom plate. The antibodies to be analyzed were diluted with a PBS solution containing 10% FBS at an initial concentration of 200 μg/ml, and 8 gradients were obtained by a 1:10 dilution. 25 μl of 100 ng/ml IL-5 labeled with biotin labeling kit (Dojindo Chemical, LK03) was uniformly mixed with 50 μl of each diluted antibody, added to the 96-well plate in which the cells had been added, and incubated at 4°C within 1 hour. After incubation, centrifugation at 4°C (400 g, 5 min), the supernatant was discarded, centrifuged and washed with 200 μl of pre-chilled PBS, repeated twice; a secondary antibody conjugated with PE(phycoerythrin)-avidin, diluted 1:1333, was added and incubated in the dark at 40°C for 40 min, the supernatant was discarded after centrifugation at 4°C (400 g, 5 min), 200 μl was added pre-chilled PBS, the cells were dissociated, centrifuged and washed at 4°C, repeated three times and 100 μl of PBS was added. The plate was read on the instrument and IC50 values of blocking antibodies against IL-5 binding between IL-5 and IL-5Rα/β were calculated based on the fluorescent signal values. The results are shown in Table 16 and in FIG. one.

The results show that antibodies h1705-008, h1706-009, h1780-017, h1773-007 and h1779-014 showed a stronger ability to block the binding of IL-5 to the IL-5 receptor on the cell surface.

Experimental Example 5 Anti-IL-5 Antibodies Inhibit IL5-Induced Proliferation of TF1 Cells

IL-5 can induce proliferation of TF-1 cells, and antibodies against IL-5 can prevent IL-5 from stimulating proliferation of TF-1 cells.

Specifically, TF-1 cells (ATCC, CRL-2003) were cultured in RPMI1640 containing 10% FBS and 2 ng/mL rhGM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor) (Lianke Bio, cat. no. 96-AF-300 -03-20) were incubated in an incubator at 37° C., 5% CO ₂ , with a cell density not greater than 1×10 ⁶ cells/ml. For detection of antibodies, cells in logarithmic growth phase were washed three times with PBS by centrifugation at 800 rpm for 5 min, and the cell density was adjusted to 6000 cells/well/90 μl using RPMI1640 (FBS: 2%, recombinant human IL-5: 10 ng/ml). Added to a 96-well plate 10 μl of gradient dilutions of antibodies to be analyzed, and cultured for 3 days. 30 μl of cell titer was added and mixed. IC50 values were calculated based on readings. The results are shown in Table 17 below.

Experimental Example 6 Anti-IL-5 Antibodies Inhibit IL-5-Induced Eosinophil Adhesion

IL-5 can induce differentiation, maturation, migration and activation of eosinophils, induce inflammation in the respiratory system and lead to asthma. According to the principle that the cytokine IL-5 can stimulate eosinophil adhesion and activate eosinophils, in this test case, the effects of IL-5-specific antibodies on blocking the IL-5 mediated pathway were determined by collecting and purifying eosinophils from human peripheral blood and detecting the effects in vitro anti-IL-5 antibodies to block IL-5 mediated eosinophil adhesion.

Specifically, human peripheral blood was diluted 5-fold with PBS containing 2 mM EDTA (ethylenediaminetetraacetic acid), monocytes and granulocytes were separated by Percoll™ (with a density gradient of 1.088), the erythrocyte layer containing granulocytes was gently aspirated, and the erythrocytes were removed using lysis buffer for erythrocytes. Remaining cells were counted, magnetic bead separators coated with human anti-CD16 antibody (Miltenyi cat. no. 130-045-701) were added, incubated for 30 min and passed through a magnetic bead column. The eluent of the cell fraction, which mainly consisted of eosinophils, was directly collected. The resulting eosinophils were counted and added to a 96-well cell culture plate pre-coated with anti-IgG antibody, approximately 1 x 10 ⁴ cells per well, human IL-5 (20 ng/ml) and different concentrations of anti-IL-5 antibody molecules ( starting at 10 µg/mL, 3-fold dilution, 10 concentration levels). The cell culture plate was incubated in an incubator at 37° C., 5% CO ₂ for 1 hour and then 0.3% CTAB (cetyltrimethylammonium bromide) was added to lyse the cells. Finally, TMB peroxidase reaction substrate was added to develop the color. OD450 absorbance values were read on a microplate reader. The maximum absorbance value was read in the well with the addition of IL-5 only, and wells containing neither IL-5 nor antibody preparation were taken as background control. The inhibition value of each concentration of antibody preparations relative to the maximum absorbance value was calculated as (maximum absorbance value - [antibody preparation] / (maximum absorbance value - background control value) × 100%, and IC50 was calculated. The results are shown in Table 18.

These results indicate that the humanized antibodies of the present disclosure exhibit a strong ability to inhibit IL-5 mediated eosinophil adhesion.

Test Example 7: Evaluation of the Specificity of Humanized Anti-IL-5 Antibodies to Th2 Cytokines

IL-5 is one of the Th2 cytokines. To confirm that anti-IL-5 antibodies specifically target only IL-5 without cross-reactivity with other cytokines, 12 types of Th2 cytokines and related cytokines were used in the Fortebio assay, including IL2 (R&D, 202-IL-010/CF), IL4(R&D, 204-IL-050/CF), IL-5(R&D, 205-IL-025/CF), IFNgamma, IL6(R&D, 7270-IL-025/CF), IL9(R&D, 209-IL -010/CF), IL10(R&D, 217-IL-025/CF), IL13(R&D, 213-ILB-025/CF), IL25(R&D, 8134-IL-025/CF), IL31(R&D, 2824 -IL-010/CF), IL3 sharing α receptor with IL-5 (203-IL-050/CF), and GMCSF (R&D, 215-GM-010/CF).

In particular, antibodies were captured using a protein A biosensor (PALL Fortebio, 18-5010), the capture signal was recorded, then 40 nM of each cytokine was added and new binding signals were recorded. Finally, the binding signal for IL-5 was determined to be 100%, and binding signals between antibodies and other cytokines were observed. The results are shown in FIG. 2.

These results indicate that among the 12 types of related cytokines, the humanized anti-IL-5 antibodies h1705-008 and h1706-009 simply bind specifically to IL-5 and show no cross-reactivity with other Th2 cytokines.

Pharmacokinetic evaluation

Test Example 8: Pharmacokinetic Evaluation of Humanized Anti-IL-5 Antibodies in Rats

Experimental SD rats (provided by Sipple-BK Lab Animal Co., Ltd.), 18 males, were divided into 6 groups with 3 rats in each group. Hu39D10, h1705-008 and h1706-009 were administered intravenously and subcutaneously. The other 9 SD rats received h1773-007, h1779-014 or h1780-017 intravenously only. For the intravenous injection group, 0.2 ml of whole blood was taken without the introduction of an anticoagulant before administration and after 5 minutes, 8 hours, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 21 day and 28 days. after the introduction. Blood samples were placed at 4°C for 30 min, centrifuged at 1000 g for 15 min, supernatants (serum) were placed in EP tubes and stored at -80°C. For the subcutaneous injection group, whole blood was collected before and after 1 hour, 2 hours, 4 hours, 8 hours, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days. and 28 days. after the introduction. Serum antibody concentration was determined by ELISA.

The results indicate that the humanized antibody molecules of the present disclosure have a long half-life and high bioavailability in rats.

Biological evaluation in vivo

Experimental Example 9 Evaluation of Anti-IL-5 Antibody Efficacy in a Mouse Model of OVA Induced Asthma

This pilot example is designed to evaluate the efficacy of anti-IL-5 antibodies in the ovalbumin aerosol (OVA) induced asthma model in BALB/c mice based on airway inflammation and airway remodeling.

Mice were randomly divided into 7 groups according to body weight, 10 mice in each group: normal control group (G1); model group (G2); treatment groups for antibody h1705-008 (G3 and G4 for two dosages of 10 mpk and 2 mpk, respectively) and h1706-009 (G5 and G6 for two dosages of 10 mpk and 2 mpk, respectively); and a control group for positive Hu39D10 antibody (G7, 10 mpk). On day 1 and day 14, all mice were sensitized by intraperitoneal injection of the sensitizing solution. On days 28, 29 and 30, six groups of mice, excluding group 1, were stimulated with an OVA aerosol stimulating solution for 30 minutes. Two hours prior to challenge, group 2 (G2) was intraperitoneally injected with phosphate buffer, and group 3-7 (G3-G7) mice were intraperitoneally injected with different doses of different antibodies, once a day for three consecutive days. Fresh solutions of antibodies to be analyzed were prepared immediately prior to each injection and all injections were completed within half an hour. As a normal control, 2 hours after the intraperitoneal injection of phosphate buffer, mice in group 1 were stimulated with an aerosol of PBS for 30 minutes, once a day for three consecutive days.

On day 31, animals were analyzed for airway hypersensitivity using the WBP system. All animals were inhaled with a spray of methacholine at a doubly increasing concentration: 1.5625; 3.125; 6.25; 12.5; 25, 50 mg / ml to determine the repeated value of the forced exhalation at the appropriate concentration.

On day 31, 1 hour after analysis of the airway response using the WBP system, inserted into the trachea and fixed a tracheal cannula with a diameter of 1.2 mm, twice performed lung lavage sampling, each time using 0.8 ml of phosphate buffer containing 1% BSA and 0.6 mM EDTA. The volume of lavage fluid recovered was recorded.

BALF was centrifuged at 300 g at 4° C. for 5 minutes and the supernatant was saved for cytokine analysis. After centrifugation, cells were resuspended in 1.5 ml PBS (containing 1% BSA and 0.6 mM EDTA) for cell count. The total number of cells in BALF was counted using a hemocytometer and trypan blue staining assay. Cells were smeared, stained with Wright's stain for one minute, and then stained with Giemsa for 7 minutes to distinguish between eosinophils, neutrophils, macrophages, and lymphocytes. Cells were counted under an optical microscope.

After lavage, lung tissues were taken, immersed in 10% neutral formaldehyde solution, and then fixed in 10% neutral formaldehyde solution. The fixed tissues were then subjected to paraffin embedding, trimming, H&E (hematoxylin and eosin) staining, and scoring. The results of the analysis are shown in Fig. 3 and FIG. 4A and 4B.

These results indicate that the h1705-008 and h1706-009 antibody molecules of the present disclosure can significantly improve lung function in a dose-dependent manner, while a high dose (10 mpk) of the positive compound cannot improve lung function (see Fig. 3) . At the same time, these two antibodies significantly reduced eosinophil levels and mucosal thickness, and showed a stronger ability to reduce eosinophils than the positive antibody at the same dosage (10 mpk) (see Fig. 4A and 4B). By repeating the experiment in the same type of mouse asthma model, the inventors of the present invention also confirmed that 1 mg/ml of all of h1705-008, h1706-009 and h1780-017 significantly reduced the level of eosinophils in BalF compared to positive antibody (see Fig. .4C).

Test Example 10: Evaluation of In Vivo Efficacy of Anti-IL-5 Antibodies in Exogenous Human IL-5 Induced Acute Asthma in a Guinea Pig Model

In this experimental example, male guinea pigs were used to create a model of acute asthma induced by human IL-5, and hu39D10 was used as a positive antibody to assess whether the five humanized anti-IL-5 monoclonal antibodies of the present disclosure have inhibitory effects on human IL-induced IL. -5 increase in eosinophil levels in bronchoalveolar lavage fluid (BALF) in guinea pigs. Guinea pigs were divided into 9 groups, 8-10 in each group: normal control group, model group, hu39D10 group (1 mg/kg), h1705-008 group (1 mg/kg), h1706-009 group (1 mg/kg). kg), group h1780-017 (1 mg/kg), group h1773-007 (1 mg/kg), and group h1779-014 (1 mg/kg). On day 1, guinea pigs in the model and drug groups were intratracheally injected with 100 μl of human IL-5 (containing 5 μg of IL-5 antigen) for priming, and the normal control group was intratracheally injected with PBS. 2 hours after priming, the drug-treated groups were intraperitoneally injected with 1 mg/kg of the above anti-IL5 monoclonal antibodies (at an administration volume of 5 ml/kg), the corresponding IgG antibody was administered to the model group, and PBS was administered intraperitoneally to the normal control group. 24 hours after intratracheal injection, guinea pigs were anesthetized and the bronchoalveolar lavage fluid was removed. The cell concentration was adjusted to 5×10 ⁶ /ml and 15 μl of their suspension was dropped onto a glass slide, dried and fixed for HE staining, and the total number of cells and the number of eosinophils were counted under a microscope at 400× magnification. The percentage of eosinophils was calculated. The results are shown in FIG. 5A and 5B indicating that all five humanized antibodies of the present disclosure can significantly reduce eosinophil levels in BALF.

Stability assessment

Test Example 11: Stability of Humanized Anti-IL-5 Antibodies

1. Chemical stability of antibodies

Deamidation modification is a common chemical modification of antibodies that can affect long-term stability. In particular, a high degree of modification in the form of deamidation, oxidation or isomerization of a portion of the amino acids in the CDR regions should generally be avoided or should be reduced by mutation. 100 μg samples taken at different time points were dissolved in 100 μl of a solution of 0.2 M His-HCl, 8 M Gua-HCl, pH 6.0, 3 μl of 0.1 g/ml DTT (dithiothreitol) was added, incubated in water bath at 50°C for 1 hour and then ultrafiltered twice with 0.02 M His-HCl, pH 6.0. Added 3 μl of 0.25 mg/ml trypsin, incubated in a water bath at 37°C overnight for enzymatic digestion. LC-MS analysis (liquid chromatography-mass spectrometry) was performed using an Agilent 6530 Q-TOF. The results show that the chemical stability of the humanized antibodies of the present disclosure was good, and these antibodies did not show abnormal modification after the accelerating reaction in the 529 buffer system at 40° C. for one month.

2. Study on the degree of antibody aggregation under conditions of high concentration

The stability of the experimental antibodies was evaluated at high concentrations under different buffer systems and different temperatures for one month. Stability was studied at a concentration of 50 mg/ml in three buffer systems: PB9, His and 529 at 40°C, 25°C, 4°C and -80°C with repeated freezing and thawing. The degree of aggregation was monitored by SEC-HPLC. A Waters e2695 chromatograph was used with a Waters Xbridge BEN 200A SEC column and the mobile phase was PBS (pH adjusted to 6.8 with dilute hydrochloric acid). 50 μg of protein was loaded and isocratic elution was performed at a flow rate of 0.5 ml/min. None of the humanized anti-IL-5 antibodies were observed to significantly aggregate under high concentration conditions. After 4 weeks of accelerating reaction at 40°C, the purity of the antibody monomer was greater than 95% in all three systems.

3. Study of the purity of antibodies under conditions of high concentration

The stability of the analyzed antibodies was evaluated at high concentrations and under conditions of different buffer systems and different temperatures for one month. Stability was studied at a concentration of 50 mg/ml in the His system and at 40°C. Purity was monitored by CE-SDS (sodium dodecyl sulfate capillary electrophoresis). 100 μg of protein was taken and sample buffer was added to reach 95 μl. For analysis in recovery mode, 5 µl of dimercaptoethanol was added; for analysis in non-reducing mode, 5 μl of IAA was added. Incubated in a water bath at 70°C for 10 min, centrifuged and loaded the supernatant. Data were acquired and analyzed using a Beckman PA800plus electrophoresis instrument. It was observed that the antibody proteins have good stability in terms of purity under high concentration conditions, and after 28 days of accelerating reaction at 40° C., CE-SDS analysis showed that the main antibody peak was reduced by only 2%.

4. Detection of thermal stability of different antibodies by means of UNIT

The samples were dissolved in an appropriate buffer (PBS buffer) and the concentration of these samples was controlled at about 1 mg/ml. Loaded 9 µl. Setting parameters: initial temperature - 20°С; incubation - 0 s; heating rate - 0.3°C/min; retention on the tile - 5 s; final temperature - 95°C. The Tm values of the antibodies were determined. These antibodies have a high Tm value and show good thermal stability.

The above invention has been described in detail with the accompanying drawings and examples. However, this description and examples should not be construed as limiting the scope of this disclosure. All patent and scientific literature disclosures cited herein are expressly incorporated by reference in their entirety.

--->

Sequence listing

<110> JIANGSU HENGRUI MEDICINE CO., LTD.; SHANGHAI HENGRUI PHARMACEUTICAL CO., LTD.

<120> ANTIBODY AGAINST IL-5, ITS ANTIGEN-BINDING FRAGMENT AND ITS

MEDICAL APPLICATION

<130> 370337CG

<160> 82

<170> PatentIn version 3.3

<210> 1

<211> 140

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of human IL-5 with his

labeled (rhIL-5-his)

<400> 1

Met Arg Met Leu Leu His Leu Ser Leu Leu Ala Leu Gly Ala Ala Tyr

1 5 10 15

Val Tyr Ala Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu

20 25 30

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

35 40 45

Thr Leu Arg Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr

50 55 60

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

65 70 75 80

Gly Gly Thr Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys

85 90 95

Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly Glu Glu Arg Arg Arg Val

100 105 110

Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Asn Thr

115 120 125

Glu Trp Ile Ile Glu Ser His His His His His His His

130 135 140

<210> 2

<211> 140

<212> PRT

<213> Artificial sequence

<220>

<223> Cynomolgus monkey IL-5 amino acid sequence with his

label

<400> 2

Met Arg Met Leu Leu His Leu Ser Leu Leu Ala Leu Gly Ala Ala Tyr

1 5 10 15

Val Tyr Ala Ile Pro Thr Glu Ile Pro Ala Ser Ala Leu Val Lys Glu

20 25 30

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

35 40 45

Thr Leu Arg Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr

50 55 60

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

65 70 75 80

Gly Gly Thr Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys

85 90 95

Tyr Ile Gly Gly Gln Lys Lys Lys Cys Gly Glu Glu Glu Arg Arg Arg Val

100 105 110

Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Asn Thr

115 120 125

Glu Trp Ile Ile Glu Ser His His His His His His His

130 135 140

<210> 3

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse IL-5 amino acid sequence with his tag

<400> 3

Met Glu Ile Pro Met Ser Thr Val Val Lys Glu Thr Leu Thr Gln Leu

1 5 10 15

Ser Ala His Arg Ala Leu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro

20 25 30

Val Pro Thr His Lys Asn His Gln Leu Cys Ile Gly Glu Ile Phe Gln

35 40 45

Gly Leu Asp Ile Leu Lys Asn Gln Thr Val Arg Gly Gly Thr Val Glu

50 55 60

Met Leu Phe Gln Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp Arg Gln

65 70 75 80

Lys Glu Lys Cys Gly Glu Glu Arg Arg Arg Thr Arg Gln Phe Leu Asp

85 90 95

Tyr Leu Gln Glu Phe Leu Gly Val Met Ser Thr Glu Trp Ala Met Glu

100 105 110

Gly His His His His His His

115

<210> 4

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Rat IL-5 amino acid sequence with his tag

<400> 4

Met Glu Ile Pro Met Ser Thr Val Val Lys Glu Thr Leu Ile Gln Leu

1 5 10 15

Ser Thr His Arg Ala Leu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro

20 25 30

Val Pro Thr His Lys Asn His Gln Leu Cys Ile Gly Glu Ile Phe Gln

35 40 45

Gly Leu Asp Ile Leu Lys Asn Gln Thr Val Arg Gly Gly Thr Val Glu

50 55 60

Ile Leu Phe Gln Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp Gly Gln

65 70 75 80

Lys Glu Lys Cys Gly Glu Glu Arg Arg Lys Thr Arg His Phe Leu Asp

85 90 95

Tyr Leu Gln Glu Phe Leu Gly Val Met Ser Thr Glu Trp Ala Met Glu

100 105 110

Val His His His His His His His

115

<210> 5

<211> 560

<212> PRT

<213> Artificial sequence

<220>

<223> human IL-5 alpha fused to a human Fc fragment

<400> 5

Asp Leu Leu Pro Asp Glu Lys Ile Ser Leu Leu Pro Pro Val Asn Phe

1 5 10 15

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

20 25 30

Asn Pro Asp Gln Glu Gln Arg Asn Val Asn Leu Glu Tyr Gln Val Lys

35 40 45

Ile Asn Ala Pro Lys Glu Asp Asp Tyr Glu Thr Arg Ile Thr Glu Ser

50 55 60

Lys Cys Val Thr Ile Leu His Lys Gly Phe Ser Ala Ser Val Arg Thr

65 70 75 80

Ile Leu Gln Asn Asp His Ser Leu Leu Ala Ser Ser Trp Ala Ser Ala

85 90 95

Glu Leu His Ala Pro Pro Gly Ser Pro Gly Thr Ser Ile Val Asn Leu

100 105 110

Thr Cys Thr Thr Asn Thr Thr Glu Asp Asn Tyr Ser Arg Leu Arg Ser

115 120 125

Tyr Gln Val Ser Leu His Cys Thr Trp Leu Val Gly Thr Asp Ala Pro

130 135 140

Glu Asp Thr Gln Tyr Phe Leu Tyr Tyr Arg Tyr Gly Ser Trp Thr Glu

145 150 155 160

Glu Cys Gln Glu Tyr Ser Lys Asp Thr Leu Gly Arg Asn Ile Ala Cys

165 170 175

Trp Phe Pro Arg Thr Phe Ile Leu Ser Lys Gly Arg Asp Trp Leu Ala

180 185 190

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

195 200 205

Gln Leu Phe Ala Leu His Ala Ile Asp Gln Ile Asn Pro Pro Leu Asn

210 215 220

Val Thr Ala Glu Ile Glu Gly Thr Arg Leu Ser Ile Gln Trp Glu Lys

225 230 235 240

Pro Val Ser Ala Phe Pro Ile His Cys Phe Asp Tyr Glu Val Lys Ile

245 250 255

His Asn Thr Arg Asn Gly Tyr Leu Gln Ile Glu Lys Leu Met Thr Asn

260 265 270

Ala Phe Ile Ser Ile Ile Asp Asp Leu Ser Lys Tyr Asp Val Gln Val

275 280 285

Arg Ala Ala Val Ser Ser Met Cys Arg Glu Ala Gly Leu Trp Ser Glu

290 295 300

Trp Ser Gln Pro Ile Tyr Val Gly Asn Asp Glu His Lys Pro Leu Arg

305 310 315 320

Glu Trp Ile Glu Gly Arg Met Asp Glu Pro Lys Ser Cys Asp Lys Thr

325 330 335

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

340 345 350

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

355 360 365

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

370 375 380

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

385 390 395 400

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

405 410 415

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

420 425 430

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

435 440 445

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

450 455 460

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

465 470 475 480

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

485 490 495

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

500 505 510

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

515 520 525

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

530 535 540

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

545 550 555 560

<210> 6

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> murine heavy chain variable region sequence

mAb1705

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Gln Thr Leu Arg Glu Ser Phe Asp Tyr Trp Gly Gln Gly Val

100 105 110

Met Val Thr Val Ser Ser

115

<210> 7

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> murine light chain variable region sequence

mAb1705

<400> 7

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

1 5 10 15

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

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Ile Ala Arg Ser Pro Lys Leu Val Ile

35 40 45

Tyr Gly Thr Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Asn Thr Leu Glu Ser

65 70 75 80

Glu Asp Thr Gly Ile Tyr Phe Cys Leu Gln Asp Lys Glu Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Arg Leu Glu Leu Lys

100 105

<210> 8

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> murine heavy chain variable region sequence

mAb1706

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Thr Glu Thr Leu Arg Glu Ser Leu Asp Tyr Trp Gly Gln Gly Val

100 105 110

Met Val Thr Val Ser Ser

115

<210> 9

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> murine light chain variable region sequence

mAb1706

<400> 9

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

1 5 10 15

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

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Leu Gly Lys Ser Pro Lys Leu Met Ile

35 40 45

His Ser Ala Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Asn Thr Leu Glu Ser

65 70 75 80

Glu Asp Pro Gly Ile Tyr Phe Cys Leu Gln His Lys Gln Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 10

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> murine heavy chain variable region sequence

mAb1780

<400> 10

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Asp Tyr Thr Phe Thr Glu Tyr

20 25 30

Leu Ile His Trp Val Lys Gln Ser Gln Gly Arg Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Ser Gly Gly Thr Val Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Phe Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Phe Arg Arg Leu Thr Phe Glu Asp Ser Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Gly Tyr Ser Asp Pro Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Val Met Val Thr Val Ser Ser

115

<210> 11

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> murine light chain variable region sequence

mAb1780

<400> 11

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

1 5 10 15

Arg Val Ser Ile Ser Cys Arg Ala Ser Glu Gly Leu Thr Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile Tyr

35 40 45

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

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala Asp

65 70 75 80

Asp Ala Ala Thr Tyr Phe Cys Gln Gln Asn Trp Asn Asp Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 12

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> murine heavy chain variable region sequence

mAb1773

<400> 12

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

1 5 10 15

Ser Val Thr Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asn Thr

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asp Thr Lys His Gly Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

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

85 90 95

Phe Arg Tyr Gly Ile Tyr Pro Asp His Trp Gly Gln Gly Thr Pro Leu

100 105 110

Thr Val Ser Ser

115

<210> 13

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> murine light chain variable region sequence

mAb1773

<400> 13

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Leu Thr Ala Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Glu

100 105

<210> 14

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> murine heavy chain variable region sequence

mAb1779

<400> 14

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

1 5 10 15

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

20 25 30

Ile Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Phe Asn Pro Asn Ser Gly Gly Ser Asn Tyr Asn Glu Asn Phe

50 55 60

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

65 70 75 80

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

85 90 95

Gly Arg Arg Ile Ala Trp Asp His Trp Tyr Phe Asp Phe Trp Gly Pro

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 15

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> murine light chain variable region sequence

mAb1779

<400> 15

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

1 5 10 15

Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp

20 25 30

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

35 40 45

Tyr Ala Ala Ser Arg Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Arg Tyr Phe Phe Lys Ile Ser Gly Met Gln Pro

65 70 75 80

Glu Asp Glu Ala Asp Tyr Phe Cys Gln Gln Gly Tyr Lys Thr Pro Leu

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 16

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR1 mAb1705

<400> 16

His Tyr Tyr Met Ala

fifteen

<210> 17

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR2 mAb1705

<400> 17

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

1 5 10 15

gly

<210> 18

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR3 mAb1705

<400> 18

Gln Thr Leu Arg Glu Ser Phe Asp Tyr

fifteen

<210> 19

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR1 mAb1705

<400> 19

Arg Ala Ser Gln Asp Ile Ala Asn Tyr Leu Ser

1 5 10

<210> 20

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR2 mAb1705

<400> 20

Gly Thr Ser Asn Leu Glu Val

fifteen

<210> 21

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR3 mAb1705

<400> 21

Leu Gln Asp Lys Glu Phe Pro Arg Thr

fifteen

<210> 22

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR1 mAb1706

<400> 22

His Tyr Tyr Met Ala

fifteen

<210> 23

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR2 mAb1706

<400> 23

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

1 5 10 15

gly

<210> 24

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR3 mAb1706

<400> 24

Glu Thr Leu Arg Glu Ser Leu Asp Tyr

fifteen

<210> 25

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR1 mAb1706

<400> 25

Arg Ala Ser Gln Asp Ile Gly Asn Tyr Leu Ser

1 5 10

<210> 26

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR2 mAb1706

<400> 26

Ser Ala Ser Asn Leu Glu Val

fifteen

<210> 27

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR3 mAb1706

<400> 27

Leu Gln His Lys Gln Phe Pro Arg Thr

fifteen

<210> 28

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR1 mAb1780

<400> 28

Glu Tyr Leu Ile His

fifteen

<210> 29

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR2 mAb1780

<400> 29

Tyr Ile Asn Pro Tyr Ser Gly Gly Thr Val Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ser

<210> 30

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR3 mAb1780

<400> 30

Asp Gly Gly Tyr Ser Asp Pro Leu Asp Tyr

1 5 10

<210> 31

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR1 mAb1780

<400> 31

Arg Ala Ser Glu Gly Leu Thr Ser Tyr Met His

1 5 10

<210> 32

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR2 mAb1780

<400> 32

Lys Ala Ser Asn Leu Ala Ser

fifteen

<210> 33

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR3 mAb1780

<400> 33

Gln Gln Asn Trp Asn Asp Pro Trp Thr

fifteen

<210> 34

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR1 mAb1773

<400> 34

Asn Thr Tyr Ile His

fifteen

<210> 35

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR2 mAb1773

<400> 35

Arg Ile Asp Pro Ala Asn Gly Asp Thr Lys His Gly Pro Lys Phe Gln

1 5 10 15

gly

<210> 36

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR3 mAb1773

<400> 36

Tyr Gly Ile Tyr Pro Asp His

fifteen

<210> 37

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR1 mAb1773

<400> 37

Ser Ala Ser Ser Ser Val Asn Tyr Ile Tyr

1 5 10

<210> 38

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR2 mAb1773

<400> 38

Leu Thr Ala Thr Leu Ala Ser

fifteen

<210> 39

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR3 mAb1773

<400> 39

Gln Gln Trp Asn Ser Tyr Pro Tyr Thr

fifteen

<210> 40

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR1 mAb1779

<400> 40

Asp Tyr Ile Ile His

fifteen

<210> 41

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR2 mAb1779

<400> 41

Tyr Phe Asn Pro Asn Ser Gly Gly Ser Asn Tyr Asn Glu Asn Phe Lys

1 5 10 15

Arg

<210> 42

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR3 mAb1779

<400> 42

Arg Ile Ala Trp Asp His Trp Tyr Phe Asp Phe

1 5 10

<210> 43

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR1 mAb1779

<400> 43

Leu Ala Ser Glu Gly Ile Ser Asn Asp Val Ala

1 5 10

<210> 44

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR2 mAb1779

<400> 44

Ala Ala Ser Arg Leu Gln Asp

fifteen

<210> 45

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR3 mAb1779

<400> 45

Gln Gln Gly Tyr Lys Thr Pro Leu Thr

fifteen

<210> 46

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1705_VL.1

<400> 46

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

1 5 10 15

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

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Lys Glu Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 47

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1705_VL.1A

<400> 47

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Gly Thr Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Lys Glu Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 48

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1705_VL.1B

<400> 48

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Gly Thr Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Leu Gln Asp Lys Glu Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 49

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> h1705_VH.1

<400> 49

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Gln Thr Leu Arg Glu Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 50

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> h1705_VH.1A

<400> 50

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Gln Thr Leu Arg Glu Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 51

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> h1705_VH.1B

<400> 51

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Gln Thr Leu Arg Glu Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 52

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> IgG1 heavy chain constant region

<400> 52

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

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

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 53

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Kappa light chain constant region

<400> 53

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

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 54

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1706_VL.1

<400> 54

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

1 5 10 15

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

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Lys Gln Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 55

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1706_VL.1A

<400> 55

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Ala Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Lys Gln Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 56

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1706_VL.1B

<400> 56

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

1 5 10 15

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

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Met Ile

35 40 45

Tyr Ser Ala Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Leu Gln His Lys Gln Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 57

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> h1706_VH.1

<400> 57

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Ile Asn Tyr Glu Gly Asn Ser Ala Tyr Tyr Gly Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Glu Thr Leu Arg Glu Ser Leu Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser

115

<210> 58

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> h1706_VH.1A

<400> 58

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Ile Asn Tyr Glu Gly Asn Ser Ala Tyr Tyr Gly Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Thr Glu Thr Leu Arg Glu Ser Leu Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser

115

<210> 59

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> h1706_VH.1B

<400> 59

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Thr Glu Thr Leu Arg Glu Ser Leu Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser

115

<210> 60

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1780_VL.1

<400> 60

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Gly Leu Thr Ser Tyr

20 25 30

Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Trp Asn Asp Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 61

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1780_VL.1A

<400> 61

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Gly Leu Thr Ser Tyr

20 25 30

Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Trp Asn Asp Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 62

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1780_VL.1B

<400> 62

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Gly Leu Thr Ser Tyr

20 25 30

Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Asn Trp Asn Asp Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 63

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> h1780_VH.1

<400> 63

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

1 5 10 15

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

20 25 30

Leu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Ser Gly Gly Thr Val Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asp Gly Gly Tyr Ser Asp Pro Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 64

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> h1780_VH.1A

<400> 64

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

1 5 10 15

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

20 25 30

Leu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Ser Gly Gly Thr Val Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asp Gly Gly Tyr Ser Asp Pro Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 65

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> h1780_VH.1B

<400> 65

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

1 5 10 15

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

20 25 30

Leu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Ser Gly Gly Thr Val Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asp Gly Gly Tyr Ser Asp Pro Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 66

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> h1780_VH.1C

<400> 66

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

1 5 10 15

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

20 25 30

Leu Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Ser Gly Gly Thr Val Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asp Gly Gly Tyr Ser Asp Pro Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 67

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> h1773_VL.1

<400> 67

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

1 5 10 15

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

20 25 30

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

35 40 45

Leu Thr Ala Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 68

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> h1773_VL.1A

<400> 68

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

1 5 10 15

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

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Leu Thr Ala Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 69

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> h1773_VH.1

<400> 69

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Asp Pro Ala Val Gly Asp Thr Lys His Gly Pro Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Tyr Gly Ile Tyr Pro Asp His Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 70

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> h1773_VH.1A

<400> 70

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Asp Pro Ala Val Gly Asp Thr Lys His Gly Pro Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Phe Arg Tyr Gly Ile Tyr Pro Asp His Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 71

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> h1773_VH.1B

<400> 71

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Lys Gln Ala Ser Gly Lys Gly Leu Glu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Val Gly Asp Thr Lys His Gly Pro Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Phe Arg Tyr Gly Ile Tyr Pro Asp His Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 72

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1779_VL.1

<400> 72

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp

20 25 30

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

35 40 45

Tyr Ala Ala Ser Arg Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Lys Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 73

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1779_VL.1A

<400> 73

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp

20 25 30

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

35 40 45

Tyr Ala Ala Ser Arg Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Lys Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 74

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> h1779_VL.1B

<400> 74

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp

20 25 30

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

35 40 45

Tyr Ala Ala Ser Arg Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Lys Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 75

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> h1779_VH.1

<400> 75

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

1 5 10 15

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

20 25 30

Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Phe Asn Pro Asn Ser Gly Gly Ser Asn Tyr Asn Glu Asn Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg Ile Ala Trp Asp His Trp Tyr Phe Asp Phe Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 76

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> h1779_VH.1A

<400> 76

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

1 5 10 15

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

20 25 30

Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Phe Asn Pro Asn Ser Gly Gly Ser Asn Tyr Asn Glu Asn Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg Ile Ala Trp Asp His Trp Tyr Phe Asp Phe Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 77

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> h1779_VH.1B

<400> 77

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

1 5 10 15

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

20 25 30

Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Phe Asn Pro Asn Ser Gly Gly Ser Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Arg Arg Ala Thr Met Thr Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg Ile Ala Trp Asp His Trp Tyr Phe Asp Phe Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 78

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> h1779_VH.1C

<400> 78

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

1 5 10 15

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

20 25 30

Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Phe Asn Pro Asn Ser Gly Gly Ser Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Arg Arg Ala Thr Met Thr Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg Ile Ala Trp Asp His Trp Tyr Phe Asp Phe Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 79

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> h1779_VH.1D

<400> 79

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

1 5 10 15

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

20 25 30

Ile Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Phe Asn Pro Asn Ser Gly Gly Ser Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Arg Lys Ala Thr Met Thr Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg Ile Ala Trp Asp His Trp Tyr Phe Asp Phe Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 80

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Hu39D10 heavy chain sequence

<400> 80

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

Ser Val Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Leu Ile Trp Ser Asn Gly Asp Thr Asp Tyr Asn Ser Ala Ile Lys

50 55 60

Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Ser Thr Val Tyr Leu

65 70 75 80

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

85 90 95

Arg Glu Tyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

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

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

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

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 81

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Hu39D10 light chain sequence

<400> 81

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Gly Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Lys Phe Pro Asn

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 82

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> h1773 heavy chain variant HCDR2

<400> 82

Arg Ile Asp Pro Ala Val Gly Asp Thr Lys His Gly Pro Lys Phe Gln

1 5 10 15

gly

<---

Claims (31)

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to human interleukin-5 (IL-5), where said monoclonal antibody contains a heavy chain variable region and a light chain variable region, where: (i) the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 16-18, respectively; and the light chain variable region comprises the LCDR1, LCDR2, and LCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 19-21, respectively; or (ii) the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 22-24, respectively; and the light chain variable region comprises the LCDR1, LCDR2, and LCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 25-27, respectively; or (iii) the variable region of the heavy chain contains the region of HCDR1, HCDR2 and HCDR3, as defined in the amino acid sequences of SEQ ID NO: 28-30, respectively; and the light chain variable region comprises the LCDR1, LCDR2, and LCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 31-33, respectively; or (iv) the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 34-36, respectively; and the light chain variable region comprises the LCDR1, LCDR2, and LCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 37-39, respectively; or (v) the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 40-42, respectively; and the light chain variable region comprises the LCDR1, LCDR2, and LCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 43-45, respectively; or (vi) the variable region of the heavy chain contains the region of HCDR1, HCDR2 and HCDR3, as defined in the amino acid sequences of SEQ ID NO: 34, 82 and 36, respectively; and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 region as defined in the amino acid sequences of SEQ ID NOs: 37-39, respectively. 2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein said monoclonal antibody is a recombinant antibody, preferably selected from the group consisting of a mouse antibody, a chimeric antibody, and a humanized antibody. 3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein said humanized antibody contains the heavy chain variable region of SEQ ID NO: 49, 57, 63, 69, or 75, or a variant thereof, wherein said variant has 1-10 amino acid mutations on the heavy chain variable region as defined in SEQ ID NOs: 49, 57, 63, 69, or 75. 4. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein said variant has 1-10 amino acid backmutations on the FR (framework) region of the heavy chain variable region as defined in SEQ ID NO: 49, 57, 63, 69, or 75 , wherein preferably said backmutation is selected from the group consisting of S49T, V93T and K98S or a combination thereof on the heavy chain variable region of SEQ ID NO: 49, or said backmutation is selected from the group consisting of S49T, V93T and K98T or a combination thereof on the heavy chain variable region of SEQ ID NO: 57, or said backmutation is selected from the group consisting of R38K, M48I, R67K, V68A, M70L, R72V, T74K and L83F, or a combination thereof on the heavy chain variable region of SEQ ID NO: 63, or said backmutation is selected from the group consisting of F29I, R38K, V48I, R72A, T97F on the heavy chain variable region of SEQ ID NO: 69 and N55V on CDR or a combination thereof, or said backmutation is selected from the group consisting of R38K , M48I, R67K, V68A, R72A, T74K, M81L, L83F and D89E or combinations thereof on the heavy chain variable region of SEQ ID NO: 75. 5. The monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein said humanized antibody comprises the heavy chain variable region of SEQ ID NO: 50 or 51, or contains the heavy chain variable region of SEQ ID NO: 58 or 59, or contains the heavy chain variable region , selected from any of SEQ ID NOs: 64, 65 and 66, or contains a heavy chain variable region of SEQ ID NOs: 70 or 71, or contains a heavy chain variable region selected from any of SEQ ID NOs: 76-79. 6. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the humanized antibody contains the light chain variable region of SEQ ID NO: 46, 54, 60, 67, or 72, or a variant thereof, wherein said variant has 1-10 amino acid substitutions per light chain variable region as defined in SEQ ID NO: 46, 54, 60, 67, or 72. 7. The monoclonal antibody or antigen-binding fragment thereof according to claim 6, wherein said variant has 1-10 amino acid backmutations on the FR region of the light chain variable region as defined in SEQ ID NOs: 46, 54, 60, 67, or 72, wherein preferably, said backmutation is selected from the group consisting of A43S, L47V, G66R, T69S, F71Y, and Y87F, or a combination thereof on the light chain variable region of SEQ ID NO: 46, or said backmutation is selected from the group consisting of A43S, L47M , F71Y and Y87F or combinations thereof on the light chain variable region of SEQ ID NO: 54, or said backmutation is selected from the group consisting of E1D, I2T, I57V, V84T and Y86F or combinations thereof on the light chain variable region of SEQ ID NO: 60 or said backmutation is selected from the group consisting of M4L, A42S, L45P and L46W or a combination thereof on the light chain variable region of SEQ ID NO: 67, or said backmutation is selected from the group consisting of A43S, I48V and F71Y or a combination thereof on var the capable region of the light chain of SEQ ID NO: 72. 8. The monoclonal antibody or antigen-binding fragment thereof according to claim 7, wherein said humanized antibody comprises the light chain variable region of SEQ ID NO: 47 or 48, or comprises the light chain variable region of SEQ ID NO: 55 or 56, or comprises the light chain variable region of SEQ ID NO: 61 or 62, or contains the light chain variable region of SEQ ID NO: 68; or contains a light chain variable region of SEQ ID NO: 73 or 74. 9. Monoclonal antibody or antigennegative fragment according to any one of paragraphs. 1-8, where said humanized antibody contains: a heavy chain variable region selected from any of SEQ ID NOs: 49-51 and a light chain variable region selected from any of SEQ ID NOs: 46-48; or a heavy chain variable region selected from any of SEQ ID NOs: 57-59 and a light chain variable region selected from any of SEQ ID NOs: 54-56; or a heavy chain variable region selected from any of SEQ ID NOs: 63-66 and a light chain variable region selected from any of SEQ ID NOs: 60-62; or a heavy chain variable region selected from any of SEQ ID NOs: 69-71 and a light chain variable region selected from any of SEQ ID NOs: 67-68; or a heavy chain variable region selected from any of SEQ ID NOs: 75-79; and a light chain variable region selected from any of SEQ ID NOs: 72-74. 10. Monoclonal antibody or antigennegative fragment according to any one of paragraphs. 1-9, wherein said antibody is a full length antibody and further comprises a human antibody constant region, wherein preferably said full length antibody comprises a human heavy chain constant region as defined in SEQ ID NO: 52 and a human light chain constant region as defined in SEQ ID NO: 53. 11. Monoclonal antibody or antigennegative fragment according to any one of paragraphs. 1-9, wherein said antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single chain antibody (scFv), dimerized V region (diabody), disulfide stabilized V region (dsFv), and peptide The containing the CDR. 12. Pharmaceutical composition for the treatment of a disease associated with human IL-5, containing a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-11 and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients. 13. An isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-11. 14. Recombinant expression vector containing the isolated nucleic acid molecule according to claim 13. 15. An expression host cell transformed with the recombinant expression vector of claim 14, wherein said host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells. 16. The method of obtaining a monoclonal antibody or antigennegative fragment according to any one of paragraphs. 1-11, including culturing the host cell according to claim 15 in a culture medium with the formation and accumulation of a monoclonal antibody or its antigen-binding fragment according to any one of paragraphs. 1-11 and isolating the monoclonal antibody or antigen-binding fragment thereof from the culture. 17. A method for detecting or determining human IL-5, including the use of a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-11. 18. Means for detecting or determining human IL-5, which contains a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-11. 19. A method of treating a disease associated with human IL-5, said method comprising administering to a subject a therapeutically effective amount of a monoclonal antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-11, or a pharmaceutical composition according to claim 12, or an isolated nucleic acid molecule according to claim 13 for the treatment of a disease associated with human IL-5, said disease being preferably selected from the group consisting of asthma, malignant asthma attack, chronic pneumonia , allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocirrhotic dermatitis, episodic angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, helminth infections, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, eosinophilic hyperplastic arteritis, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocirrhotic dermatitis, endometriosis, and steroid-dependent eosinophilic bronchitis. 20. The use of a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-11, or a pharmaceutical composition according to claim 12, or an isolated nucleic acid molecule according to claim 13, or a combination thereof in the preparation of a medicament for the prevention or treatment of a disease or condition, wherein said disease or condition is a disease associated with human IL -5, wherein said disease or condition is preferably selected from the group consisting of asthma, malignant asthma attack, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocirrhotic dermatitis, episodic angioedema, eosinophilic myalgia syndrome , eosinophilic gastroenteritis, helminth infections, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, eosinophilic hyperplastic bronchitis, nodular arteritis, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocirrhotic dermatitis ita, endometriosis and steroid-dependent eosinophilic bronchitis.

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