阅读说明：本技术 抗IL-5Rα的单克隆抗体 (anti-IL-5R alpha monoclonal antibody ) 是由 E.V.索弗龙诺娃 A.K.米索林 A.N.多罗宁 T.A.聂曼金 A.A.索佐诺娃 G. 于 2018-10-02 设计创作，主要内容包括：本发明涉及生物工程领域,并且提供了与IL-5Rα特异性结合的抗体。本发明还涉及编码所述抗体的DNA、相应的表达载体和生产方法,以及使用所述抗体的治疗方法。(The present invention relates to the field of bioengineering and provides antibodies that specifically bind to IL-5 ra. The invention also relates to DNA encoding said antibodies, corresponding expression vectors and methods of production, and methods of treatment using said antibodies.)

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to IL-5ra, wherein the monoclonal antibody comprises:

1) a heavy chain variable domain comprising a heavy chain variable domain identical to SEQ ID NO: 3 is at least 80% homologous;

2) a light chain variable domain comprising a heavy chain variable domain identical to SEQ ID NO: 8 is at least 80% homologous to the sequence of seq id no.

2. The monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 3.

3. The monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 8.

4. The monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein the heavy chain variable domain comprises an amino acid sequence identical to SEQ ID NO: 1-3, an amino acid sequence that is at least 80% homologous.

5. The monoclonal antibody or antigen binding fragment thereof according to claim 4, wherein the heavy chain variable domain comprises a light chain variable region consisting of SEQ ID NO: 1-3, or a pharmaceutically acceptable salt thereof.

6. The monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein the light chain variable domain comprises an amino acid sequence identical to SEQ ID NO: 6-8, an amino acid sequence that is at least 80% homologous.

7. The monoclonal antibody or antigen binding fragment thereof according to claim 6, wherein the light chain variable domain comprises a light chain variable domain consisting of SEQ ID NO: 6-8.

8. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein

1) The heavy chain variable domain comprises a heavy chain variable domain identical to SEQ ID NO: 1-3, an amino acid sequence that is at least 80% homologous;

2) the light chain variable domain comprises a heavy chain variable domain identical to SEQ ID NO: 6-8, an amino acid sequence that is at least 80% homologous.

9. The monoclonal antibody or antigen-binding fragment thereof according to claim 8, wherein

1) The heavy chain variable domain comprises SEQ ID NO: 1-3;

2) the light chain variable domain comprises SEQ ID NO: 6-8.

10. The monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein said heavy chain variable domain comprises a heavy chain variable domain that differs from a light chain variable domain selected from the group consisting of SEQ ID NOs: 4-5, an amino acid sequence that is at least 90% homologous.

11. The monoclonal antibody or antigen binding fragment thereof according to claim 10, wherein said heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-5.

12. The monoclonal antibody, or antigen binding fragment thereof, according to claim 1, wherein the light chain variable domain comprises a heavy chain variable domain that differs from a light chain variable domain selected from the group consisting of SEQ ID NOs: 9-10 amino acid sequence that is at least 90% homologous.

13. The monoclonal antibody, or antigen binding fragment thereof, according to claim 12, wherein the light chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-10.

14. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein

1) The heavy chain variable domain comprises a heavy chain variable domain identical to a light chain variable domain selected from SEQ ID NOs: 4-5 amino acid sequences that are at least 90% homologous;

2) the light chain variable domain comprises a heavy chain variable domain identical to a light chain variable domain selected from the group consisting of SEQ ID NO: 9-10 amino acid sequence that is at least 90% homologous.

15. The monoclonal antibody or antigen-binding fragment thereof according to claim 14, wherein

1) The heavy chain variable domain comprises a sequence selected from SEQ ID NOs: 4-5 amino acid sequence;

2) the light chain variable domain comprises a sequence selected from SEQ ID NOs: 9-10.

16. The monoclonal antibody according to claim 1, comprising:

1) a heavy chain comprising a heavy chain sequence identical to a sequence selected from SEQ ID NOs: 11-12, an amino acid sequence that is at least 90% homologous;

2) a light chain comprising a heavy chain variable region substantially identical to a light chain variable region selected from SEQ ID NOs: 13-14, an amino acid sequence that is at least 90% homologous.

17. The monoclonal antibody according to claim 16, comprising:

1) a heavy chain comprising a sequence selected from SEQ ID NOs: 11-12;

2) a light chain comprising a sequence selected from SEQ ID NOs: 13-14.

18. The monoclonal antibody according to claim 1, wherein the IL-5 ra-specific antibody is a complete IgG antibody.

19. The monoclonal antibody of claim 18, wherein the fully IgG antibody has a human IgG1, IgG2, IgG3, or IgG4 isotype.

20. The monoclonal antibody according to claim 19, wherein said fully IgG antibody has a human IgG1 isotype.

21. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-20.

22. The nucleic acid according to claim 21, wherein said nucleic acid is DNA.

23. The nucleic acid according to claim 21, comprising:

a) a heavy chain encoding an antibody according to claim 1 and which hybridizes with a sequence selected from the group consisting of SEQ ID NO: 15-16, and/or a nucleotide sequence encoding a light chain of an antibody according to claim 1 and being at least 90% homologous to a sequence selected from SEQ ID NOs: 17-18, a nucleotide sequence that is at least 90% homologous; or

b) Selected from the group consisting of SEQ ID NO: 15-16, which encodes the heavy chain of an antibody according to claim 1, and/or a nucleotide sequence selected from SEQ ID NO: 17-18, which encodes the light chain of an antibody according to claim 1.

24. An expression vector comprising a nucleic acid according to any one of claims 21-23.

25. A method for producing a host cell for the preparation of an antibody or antigen-binding fragment thereof according to any one of claims 1-20, comprising transforming a cell with a vector according to claim 24.

26. A host cell for the preparation of an antibody or antigen-binding fragment thereof according to any one of claims 1-20, said host cell comprising a nucleic acid according to any one of claims 21-23.

27. A process for the preparation of an antibody or antigen-binding fragment thereof according to any one of claims 1-20, comprising culturing a host cell according to claim 26 in a growth medium under conditions sufficient for the production of said antibody, followed, if necessary, by isolation and purification of the obtained antibody.

28. A pharmaceutical composition intended for the prevention or treatment of a disease or disorder mediated by IL-5ra, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-20 in combination with one or more pharmaceutically acceptable excipients.

29. The pharmaceutical composition according to claim 28, intended for the prevention or treatment of a disease or condition mediated by IL-5ra, selected from the group consisting of: asthma, e.g., eosinophilic asthma (atopic asthma), e.g., severe eosinophilic asthma (atopic asthma); COPD (chronic obstructive pulmonary disease); Churg-Strauss syndrome; eosinophilic esophagitis; eosinophilic gastroenteritis.

30. A pharmaceutical combination intended for the prevention or treatment of a disease or disorder mediated by IL-5ra, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-20 and at least one different therapeutically active compound.

31. A pharmaceutical combination according to claim 30 intended for the prevention or treatment of a disease or condition mediated by IL-5ra, selected from the group consisting of: asthma, e.g., eosinophilic asthma (atopic asthma), e.g., severe eosinophilic asthma (atopic asthma); COPD (chronic obstructive pulmonary disease); Churg-Strauss syndrome; eosinophilic esophagitis; eosinophilic gastroenteritis.

32. The pharmaceutical combination according to any of claims 30-31, wherein the different therapeutically active compounds are selected from small molecules, antibodies or steroid hormones such as corticosteroids.

33. A method for inhibiting a biological activity of IL-5ra in a subject in need of such inhibition comprising administering an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-20.

34. A method for treating a disease or disorder mediated by IL-5ra, comprising administering to a subject in need of such treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-20, or a pharmaceutical composition according to claim 28.

35. The method for treating a disease or condition according to claim 34, wherein the disease or condition is selected from the group consisting of: asthma, e.g., eosinophilic asthma (atopic asthma), e.g., severe eosinophilic asthma (atopic asthma); COPD (chronic obstructive pulmonary disease); Churg-Strauss syndrome; eosinophilic esophagitis; eosinophilic gastroenteritis or hypereosinophilic syndrome.

36. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-20, or a pharmaceutical composition according to claim 28, for treating a disease or disorder mediated by IL-5ra in a subject in need of such treatment.

37. Use according to claim 36, wherein the disease or condition is selected from: asthma, e.g., eosinophilic asthma (atopic asthma), e.g., severe eosinophilic asthma (atopic asthma); COPD (chronic obstructive pulmonary disease); Churg-Strauss syndrome; eosinophilic esophagitis; eosinophilic gastroenteritis.

Technical Field

The present invention relates to biotechnology, in particular to antibodies or antigen-binding fragments thereof, and uses thereof. More specifically, the invention relates to monoclonal antibodies that specifically bind to IL-5R α (interleukin 5 receptor alpha chain). The invention also relates to nucleic acids encoding the antibodies or antigen-binding fragments thereof, expression vectors, methods for making the antibodies, and uses of the antibodies in the treatment of diseases or disorders associated with IL-5 ra.

Background

Interleukin 5 (IL-5), a proinflammatory cytokine, the granulocyte-macrophage colony stimulating factor group, is a four-helical protein. Interleukin 5 is typically produced by Th2 cells and mast cells. IL-5 stimulates proliferation and differentiation of activated B lymphocytes, inducing the switch from immunoglobulin synthesis to IgA. Many of the functions of eosinophils and basophils are mediated by the action of interleukin 5 (IL-5). IL-5 is known to promote the differentiation and activation of eosinophils and also to increase their viability by inhibiting apoptosis [ Lotvall j., Pullertis t. Treating asthma with Anti-IgE orAnti IL-5. Curr Pharm des. 1999; 5: 757-70, and Kolbeck R., Kozhich A., Koike M. et al Medi-563, a manipulated anti-IL-5 receptor alpha mAb with enhanced anti-dependent cell mediated cytotoxicity function J Allergy Clin Immunol. 2010; 125(1): 1344-53].

IL-5 acts through specific receptors (IL-5R) expressed on human eosinophils/basophil precursors and mature eosinophils/basophils. IL-5R consists of a unique alpha chain (IL-5 Ra/CD125, extracellular domain) and a beta chain (bc/CD 131) common to IL-3/GM-CSF receptors, which does not bind to ligands by itself, but increases The affinity of IL-5 for synonymous receptors and is directly involved in signal transduction [ Tetsuya A., Rafeul A. The mechanism of IL-5 signal transduction American Journal of Physiology-Cell Physiology, published 9/1/1998, Vol. 275, No. 3, C623-C633 ].

The development of eosinophilia is associated with the selective expression of IL-5R on early myeloid eosinophil precursors. Therefore, inhibition of the interaction between IL-5 and its cellular receptor appears to be most preferred for suppressing eosinophilia.

The therapeutic significance of eosinophilia suppression is due to the high levels of eosinophils in many pathological processes. Thus, an increase in eosinophil count in the respiratory tract of patients with bronchial asthma and in the esophageal epithelium of patients with eosinophilic esophagitis underlies the pathophysiology of the disease. Eosinophils release pro-inflammatory mediators, such as Eosinophil Cationic Protein (ECP) and leukotrienes.

The monoclonal antibody benralizumab is known from the prior art, which binds IL-5ra and thereby inhibits the interaction of the receptor with the ligand. The monoclonal antibody significantly depletes eosinophils in blood and lung tissue. Benralizumab is a humanized monoclonal antibody (IgG 1/k) derived from a murine antibody produced using hybridoma technology [ Koike m., Nakamura k. et al, antibodies of humanized anti-intercalary antibodies having a potential therapeutic activity, humantotides 2009, 18 (1-2): 17-27]. The antibodies bind to IL-5ra with high affinity (KD = 11 pm) and inhibit IL-5 dependent cell proliferation (IC 50 = 0.3 nm) [ Kolbeck R, Kozhich a et al MEDI-563, ahumanized anti-interleukin 5 receptor-alpha monomeric antibody, with enhanced cytotoxic-dependent cell-mediated cytotoxic function J Allergy clinimunol 2010, 125 (6): 1344-53.e2 ]. Benralizumab is produced in an afucosylated cell line and in this way The absence of fucose in The oligosaccharide core is achieved, leading to a 5-10 fold improvement in binding to soluble human Fc γ RIIIa and thereby increasing antibody-dependent cytotoxicity [ Shinkawa t., Nakamura k. et al The presence of fungal but not The expression of yeast or bioscience N-acetylglucosamine of human IgG1 complex-type oligosaccharides displays The critical role of infectious-dependent cellular cytotoxicity J l Chem 2003, 278 (5): 3466-73]. The antibodies were in phase 3 clinical trials. The antibody benralizumab has been described in international application WO9710354 (a 1).

Bispecific antibodies that bind to IL-5R and human CD3e (ND 003) are also known in the art. The antibodies are in preclinical research phase and are described in international applications WO2015172800 and WO 2015058861.

From the foregoing, it is important to generate novel antibodies that effectively bind IL-5R α and inhibit IL-5R α -mediated activation.

We have developed a fully human monoclonal antibody mAb (BCD-133) that binds human IL-5Ra with comparable affinity to benralizumab and inhibits IL-5 Ra-mediated activation. As with benralizumab, BCD-133 has enhanced antibody-dependent cytotoxicity, thus allowing activation of an immune response against cells with IL-5 receptors. Antibody BCD-133 binds selectively to IL-5R α and is a potent inhibitor of IL-5R α -mediated activation of immunocompetent cells and associated specific inflammation. Cell assays showed that antibody BCD-133 activity exceeded the effect of benazezumab. Moreover, BCD-133 is a fully human antibody obtained de novo; this fact allows to reduce the risk of immunogenicity and does not require further genetic transformation for an increase in affinity to human proteins, which may lead to a loss of binding affinity.

Summary of The Invention

The present invention relates to binding molecules, such as antibodies directed to binding to IL-5 Ra. Such antibodies may be used to treat diseases or disorders mediated by the interaction of IL-5 with its cellular receptors.

In one aspect, the invention relates to a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds IL-5ra and comprises a heavy chain variable domain comprising a heavy chain variable domain that specifically binds to the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain comprising an amino acid sequence at least 80% homologous to the sequence of SEQ ID NO: 8, i.e. the amino acid sequence of SEQ ID NO: the amino acid sequences of 3 and 8 may comprise amino acid substitutions of 1 or 2 amino acids.

In one embodiment, the monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3.

In one embodiment, the monoclonal antibody or antigen binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8.

In one embodiment, the monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable domain comprising a heavy chain variable domain that differs from SEQ ID NO: 1-3, i.e. the amino acid sequence of SEQ ID NO: the amino acid sequence of 1-3 may comprise amino acid substitutions of 1 or 2 amino acids.

In one embodiment, the monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable domain comprising a heavy chain variable domain consisting of SEQ ID NO: 1-3, or a pharmaceutically acceptable salt thereof.

In one embodiment, the monoclonal antibody or antigen binding fragment thereof comprises a light chain variable domain comprising a heavy chain variable domain that differs from the light chain variable domain of SEQ ID NO: 6-8, i.e. the amino acid sequence of SEQ ID NO: the amino acid sequence of 6-8 may comprise 1 or 2 amino acid substitutions.

In one embodiment, the monoclonal antibody or antigen binding fragment thereof comprises a light chain variable domain comprising a heavy chain variable domain consisting of SEQ ID NO: 6-8.

In one embodiment, a monoclonal antibody, or antigen binding fragment thereof, comprises a heavy chain variable domain comprising a heavy chain variable domain that differs from the amino acid sequence of SEQ ID NO: 1-3, and a light chain variable domain comprising an amino acid sequence at least 80% homologous to the sequence of SEQ ID NO: 6-8, i.e., the amino acid sequence of SEQ id no: the amino acid sequences of 1-3 and 6-8 may comprise 1 or 2 amino acid substitutions.

In one embodiment, a monoclonal antibody, or antigen binding fragment thereof, comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1-3, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 6-8.

In one embodiment, the monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable domain comprising a heavy chain variable domain that differs from a light chain variable domain selected from the group consisting of SEQ ID NOs: 4-5, an amino acid sequence that is at least 90% homologous.

In one embodiment, the monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable domain comprising a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 4-5.

In one embodiment, the monoclonal antibody or antigen binding fragment thereof comprises a light chain variable domain comprising a heavy chain variable domain identical to a light chain variable domain selected from the group consisting of SEQ ID NOs: 9-10 amino acid sequence that is at least 90% homologous.

In one embodiment, the monoclonal antibody or antigen binding fragment thereof comprises a light chain variable domain comprising a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 9-10.

In one embodiment, a monoclonal antibody, or antigen binding fragment thereof, comprises a heavy chain variable domain comprising a heavy chain variable domain that differs from a light chain variable domain selected from the group consisting of SEQ ID NOs: 4-5, and a light chain variable domain comprising an amino acid sequence at least 90% homologous to the amino acid sequence selected from SEQ ID NOs: 9-10 amino acid sequence that is at least 90% homologous.

In one embodiment, a monoclonal antibody, or antigen binding fragment thereof, comprises a heavy chain variable domain comprising a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 4-5, and the light chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 9-10.

In one embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain comprising a heavy chain variable domain identical to a sequence selected from the group consisting of SEQ ID NOs: 11-12, and the light chain comprises an amino acid sequence that is at least 90% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-14 amino acid sequence that is at least 90% homologous.

In one embodiment, a monoclonal antibody or antigen-binding fragment thereof comprises: a heavy chain comprising a sequence selected from the group consisting of SEQ ID NOs: 11-12, and the light chain comprises an amino acid sequence selected from SEQ ID NOs: 13-14.

In one embodiment, the IL-5 Ra-specific antibody is a full-length IgG antibody.

In one embodiment, the IL-5 Ra-specific full length IgG monoclonal antibody has human IgG1, IgG2, IgG3, or IgG4 isotype.

In one embodiment, the IL-5 Ra-specific full-length IgG monoclonal antibody has a human IgG1 isotype.

In one aspect, the invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof.

In one embodiment, the nucleic acid is DNA.

In one embodiment, the nucleic acid comprises a heavy chain encoding an antibody and is complementary to a light chain sequence selected from SEQ ID NOs: 15-16, and/or a nucleotide sequence encoding a light chain of an antibody that is at least 90% homologous to a sequence selected from SEQ ID NOs: 17-18, a nucleotide sequence that is at least 90% homologous.

In one embodiment, the nucleic acid comprises a nucleotide sequence encoding a heavy chain of an antibody selected from the group consisting of seq id NOs: 15-16, and/or a light chain encoding an antibody selected from SEQ ID NOs: 17-18.

In one aspect, the invention relates to an expression vector comprising said nucleic acid.

In one aspect, the invention relates to a method for producing a host cell for producing said antibody or antigen-binding fragment thereof, said method comprising transforming a cell with said vector.

In one aspect, the invention relates to a host cell for making the antibody or antigen-binding fragment thereof, and comprising the nucleic acid.

In one aspect, the invention relates to a method for producing said antibody or antigen-binding fragment thereof, and comprises culturing said host cell in a growth medium under conditions sufficient for production of said antibody, followed, if necessary, by isolation and purification of the obtained antibody.

In one aspect, the invention relates to a pharmaceutical composition for preventing or treating a disease or disorder mediated by IL-5ra, comprising said antibody or antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition is intended for the prevention or treatment of a disease or disorder mediated by IL-5ra, selected from asthma, e.g. eosinophilic asthma (atopic asthma), e.g. severe eosinophilic asthma (atopic asthma); COPD (chronic obstructive pulmonary disease); Churg-Strauss syndrome; eosinophilic esophagitis; eosinophilic gastroenteritis.

In one aspect, the invention relates to a pharmaceutical combination comprising said antibody or antigen-binding fragment thereof and at least one different therapeutically active compound, intended for the prevention or treatment of a disease or disorder mediated by IL-5 ra.

In one embodiment, the pharmaceutical combination is intended for the prevention or treatment of a disease or disorder mediated by IL-5ra, selected from asthma, e.g. eosinophilic asthma (atopic asthma), e.g. severe eosinophilic asthma (atopic asthma); COPD (chronic obstructive pulmonary disease); Churg-Strauss syndrome; eosinophilic esophagitis; eosinophilic gastroenteritis.

In one embodiment, the pharmaceutical combination comprises a therapeutically active compound selected from small molecules, antibodies or steroid hormones, such as corticosteroids.

In one aspect, the invention relates to a method for inhibiting the biological activity of IL-5ra in a subject in need of such inhibition comprising administering an effective amount of the antibody or antigen-binding fragment thereof.

In one aspect, the invention relates to a method for treating a disease or disorder mediated by IL-5ra, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof or the pharmaceutical composition in a subject in need of such treatment.

In one embodiment, the disease or disorder is selected from asthma, e.g., eosinophilic asthma (atopic asthma), e.g., severe eosinophilic asthma (atopic asthma); COPD (chronic obstructive pulmonary disease); Churg-Strauss syndrome; eosinophilic esophagitis; eosinophilic gastroenteritis, hypereosinophilic syndrome.

In one aspect, the invention relates to the use of the antibody or antigen-binding fragment thereof or the pharmaceutical composition for the treatment of a disease or disorder mediated by IL-5ra in a subject in need of such treatment.

In one embodiment, the disease or disorder is selected from asthma, e.g., eosinophilic asthma (atopic asthma), e.g., severe eosinophilic asthma (atopic asthma); COPD (chronic obstructive pulmonary disease); Churg-Strauss syndrome; eosinophilic esophagitis; eosinophilic gastroenteritis, hypereosinophilic syndrome.

Brief Description of Drawings

FIG. 1. plasmid map for transient generation of antigen with Fc fusion protein.

FIG. 2 is a plasmid map for transient generation of an antigen bearing the C-terminal signature Perwhereby A (FE) is present.

FIG. 3. plasmid map for transient generation of His-b tagged antigens.

FIG. 4 is an electrophoretogram of antigen under reducing conditions of 10% SDS-PAGE.

FIG. 5 is an electrophoretogram of antigen under reducing conditions 10% SDS-PAGE.

FIG. 6 shows electrophoretograms of antigen and antibody under non-reducing conditions (7.5% SDS-PAGE).

FIG. 7. synthetic scheme of the first experimental human library combined.

FIG. 8 phagemid map used to clone the Fab phage display library.

FIG. 9 expression plasmid map for Fab production.

FIG. 10 immunoenzymatic analysis of polyclonal phages of the post-selection library for specific and non-specific antigens.

FIG. 11 is a sensorgram of antibody interaction with IL-5Ra on an Octet RED 384 device (BCD 133-03-002).

FIG. 12 is a sensorgram of antibody interaction with IL-5Ra on an Octet RED 384 device (BCD 133-03-020).

FIG. 13 is a sensorgram of the interaction of antibodies with IL-5Ra on an Octet RED 384 device (BCD 133-03-021).

FIG. 14 dependence of antibody-dependent cellular cytotoxicity on antibody concentration.

Figure 15 values of half maximal effective concentration (EC 50) in comparative analysis with the antibody benralizumab.

FIG. 16 is a sensorgram of antibody interaction with cynomolgus IL-5Ra using an Octet RED 384 device (BCD 133-03-002).

FIG. 17 is a sensorgram of antibody interaction with cynomolgus IL-5Ra on an Octet RED 384 device (BCD 133-03-020).

FIG. 18 is a sensorgram of the interaction of antibodies with cynomolgus IL-5Ra on an Octet RED 384 device (BCD 133-03-021).

FIG. 19 3D spatial modeling of BCD-133 and IL-5Ra complexes.

FIG. 20 3D spatial modeling of BCD-133 and IL-5Ra complexes.

FIG. 21 is a graph of average particle size (Z-average) versus temperature.

FIG. 22 is a graph of average particle size (Z-average) versus temperature.

FIG. 23 is a combined chromatogram of BCD-133 on a reduced scale. Blue-intact, red-incubated at 50 ℃ for 72 hours in 20mM acetate, pH = 5.0. The wavelength is 220 nm.

FIG. 24. Combined chromatogram of BCD-133 on a larger scale. Blue-intact, red-incubated at 50 ℃ for 72 hours in 20mM acetate, pH = 5.0. The wavelength is 220 nm.

Examples

The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

All publications, patents, and patent applications cited in this specification are herein incorporated by reference. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments.

Materials and general methods

General information on the nucleotide sequences of human immunoglobulin light and heavy chains is given in: kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health service, National Institutes of Health, Bethesda, Md. (1991). The amino acids of the antibody chain are numbered according to EU numbering and are mentioned (Edelman, G.M. et al, Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A. et al, Sequences of Proteins of immunologicalcaleInterest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD, (1991).

Recombinant DNA technology

Such as Sambrook, j, et al, Molecular cloning: a laboratory manual; standard methods are used to manipulate DNA as described in Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

Gene synthesis

The desired gene segments are prepared from oligonucleotides prepared by chemical synthesis. A300-4000 kb long gene segment flanked by a single restriction site was assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequently cloned via the indicated restriction sites. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing.

DNA sequencing

DNA sequence was determined by sanger sequencing.

DNA and protein sequence analysis and sequence data management

Vector NT1 Advance suite version 8.0 of Infomax is used for sequence creation, mapping, analysis, annotation and specification.

Expression vector

For the expression of said antibodies and antigens, variants of expression plasmids intended for expression in prokaryotic cells (E.coli), transient expression in eukaryotic cells (e.g.in CHO cells) are applied. In addition to the antibody expression cassette, the vector contains: an origin of replication which allows the plasmid to replicate in E.coli, a gene conferring resistance to various antibiotics (e.g., ampicillin and kanamycin) in E.coli.

Fusion genes comprising the antibody chains as described below are generated by PCR and/or gene synthesis and assembled by ligating the corresponding nucleic acid segments using known recombinant methods and techniques, e.g., using unique restriction sites in the corresponding vectors. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfection, larger numbers of plasmids were prepared by plasmid preparations from transformed E.coli cultures.