Anti-human interleukin 13 monoclonal antibodies and uses thereof

Abstract

The application provides an anti-human interleukin 13 monoclonal antibody and an application thereof. The anti-human interleukin 13 monoclonal antibody of the application comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein: (a) the amino acid sequence of CDR-H1 is shown as SEQ ID NO:1; (b) the amino acid sequence of CDR-H2 is shown as SEQ ID NO:2; (c) the amino acid sequence of CDR-H3 is shown as SEQ ID NO:3; (d) the amino acid sequence of CDR-L1 is shown as SEQ ID NO:4; (e) the amino acid sequence of CDR-L2 is shown as SEQ ID NO:5; and (f) the amino acid sequence of CDR-L3 is shown as SEQ ID NO:6. The anti-human interleukin 13 monoclonal antibody is superior to Tralokinumab (prepared according to the patent US63233440P0 disclosure sequence expression) in biological activity and affinity, and is expected to exhibit good clinical effects in the prevention and treatment of related diseases.

Description

Technical Field

[0001] This application relates to the field of antibody drugs. Specifically, this application relates to a monoclonal antibody against human interleukin-13 and its applications. Background Technology

[0002] Interleukin-13 (IL-13) is a pleiotropic Th2 cytokine primarily secreted by activated ILC2 cells, Th2 cells, mast cells, macrophages, basophils, eosinophils, and B cells, playing a crucial role in allergic inflammation. The multiple functions of IL-13 are mediated by a complex receptor system, including IL-4 receptor α (IL-4Rα) and two other homologous cell membrane proteins: IL-13 receptor α1 (IL-13Rα1) and IL-13 receptor α2 (IL-13Rα2). IL-13Rα1 binds weakly to IL-13 alone, but upon forming a heterodimer with IL-4Rα, it forms a high-affinity IL-13 receptor complex, activating the Janus kinase / signal transducers and activators of transcription (JAK / STAT) signaling pathway to exert its biological functions. Current research has found that IL-13 is closely associated with various autoimmune diseases such as atopic dermatitis, fibrosis, and asthma. IL-13 also binds to IL-13Rα2, which is considered a key mediator of fibrosis. IL-13 neutralizing antibodies work by preventing IL-13 from binding to IL-13Rα1, IL-4Rα, and / or IL-13Rα2 (doi.org / 10.1016 / j.jmb. 2016.12.005).

[0003] IL-13-mediated signal transduction and biological effects are associated with a wide range of diseases, including: atopic dermatitis, arthritis (including septic arthritis), herpes, chronic primary urticaria, scleroderma, hypertrophic scars, Whipple's disease, benign prostatic hyperplasia, mild, moderate and severe asthma, allergic rhinitis, chronic sinusitis, hay fever, chronic obstructive pulmonary disease, pulmonary fibrosis, eosinophilia, psoriasis, psoriatic arthritis, inflammatory bowel disease, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, purpura aura, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, tuberculosis, ulcerative colitis and nephropathy.

[0004] Leo Pharma's Tralokinumab, a monoclonal antibody targeting IL-13, has been approved by the FDA for the treatment of moderate-to-severe atopic dermatitis in adults. Tralokinumab is a fully humanized monoclonal antibody that binds to IL-13 with high affinity, blocking the binding of IL-13 to IL-13Rα1 and IL-13Rα2, inhibiting the overexpression and effects of IL-13, significantly improving patients' clinical symptoms, and demonstrating good tolerability and safety during treatment. Summary of the Invention

[0005] The purpose of this application is to provide a novel anti-human interleukin-13 monoclonal antibody, a pharmaceutical composition comprising the monoclonal antibody, and the pharmaceutical use of the monoclonal antibody.

[0006] The technical solution of this application is as follows: 1. A monoclonal antibody against human interleukin-13, comprising three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein: (a) The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1 RYFMI; (b) The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2 IITYSGNTYYATWAKG; (c) The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3 EGDGNSYDM; (d) The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4 QASQSISNLLA; (e) The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5 DASKLAS; (f) The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6 QQGDLYGVDNT.

[0007] 2. The monoclonal antibody according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7EVQLVESGGGLVQPGGSLRLSCAASGFDLNRYFMIWVRQAPGKGLEYIGIITYSGNTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGDGNSYDMWGQGTLVTVSS. The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 8 DIQMTQSPSSVSASVGDRVTITCQASQSISNLLAWYQQKPGKAPKLLIYDASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDLYGVDNTFGGGTKVEIK.

[0008] 3. An isolated nucleic acid encoding any of the aforementioned monoclonal antibodies.

[0009] 4. A host cell comprising the nucleic acid as described in item 3.

[0010] The nucleic acid can be present on a vector. The vector can be of any type, for example, a recombinant vector such as an expression vector. Any of a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, such as *Escherichia coli*. E. coli In another embodiment, the host cell is a eukaryotic cell, such as a mammalian cell, like a Chinese hamster ovary (CHO) cell.

[0011] 5. A method for producing a monoclonal antibody, the method comprising culturing a host cell according to claim 4 to produce any of the aforementioned monoclonal antibodies.

[0012] The method includes producing the monoclonal antibody by expressing a recombinant vector encoding the anti-human interleukin-13 monoclonal antibody in a suitable host cell. In some embodiments, the method includes culturing host cells containing nucleic acids encoding the anti-human interleukin-13 monoclonal antibody to express the nucleic acids. The method may further include recovering the anti-human interleukin-13 monoclonal antibody from a host cell culture or host cell culture medium.

[0013] 6. A pharmaceutical composition comprising any of the aforementioned monoclonal antibodies and a pharmaceutically acceptable carrier.

[0014] The pharmaceutical composition may further comprise additional therapeutic agents (e.g., different anti-human interleukin-13 antibodies).

[0015] 7. The pharmaceutical composition according to item 6, for treating diseases related to interleukin-13-mediated signal transduction.

[0016] 8. The pharmaceutical composition according to claim 7, wherein the interleukin-13-mediated signal transduction-related diseases include immune system diseases, digestive system diseases, and blood and lymphatic system diseases, such as: atopic dermatitis, arthritis (including septic arthritis), herpes, chronic primary urticaria, scleroderma, hypertrophic scars, Whipple's disease, benign prostatic hyperplasia, mild, moderate and severe asthma, allergic rhinitis, chronic sinusitis, hay fever, chronic obstructive pulmonary disease, pulmonary fibrosis, eosinophilia, psoriasis, psoriatic arthritis, inflammatory bowel disease, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, purpura aura, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, tuberculosis, ulcerative colitis and nephropathy, etc.

[0017] 9. Use of any of the aforementioned monoclonal antibodies in the preparation of medicaments for the treatment of diseases related to interleukin-13-mediated signal transduction.

[0018] 10. A method for treating diseases related to interleukin-13-mediated signal transduction, comprising: Administer to the subject who requires the use of the monoclonal antibody or the pharmaceutical composition described in any of the preceding claims.

[0019] 11. The method according to item 10, wherein the treatment is for diseases related to signal transduction mediated by human interleukin-13 protein, such as immune system diseases, digestive system diseases, and blood and lymphatic system diseases, including: atopic dermatitis, arthritis (including septic arthritis), herpes, chronic primary urticaria, scleroderma, hypertrophic scars, Whipple's disease, benign prostatic hyperplasia, mild, moderate and severe asthma, allergic rhinitis, chronic sinusitis, hay fever, chronic obstructive pulmonary disease, pulmonary fibrosis, eosinophilia, psoriasis, psoriatic arthritis, inflammatory bowel disease, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, purpura aura, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, tuberculosis, ulcerative colitis and nephropathy, etc.

[0020] This application provides a novel anti-human interleukin-13 monoclonal antibody that exhibits superior biological activity and affinity compared to existing anti-human interleukin-13 monoclonal antibodies such as Tralokinumab (Tralokinumab is a monoclonal antibody drug targeting interleukin-13 developed by LEO Pharma, approved in 2021 for the treatment of moderate to severe atopic dermatitis in adults). Tralokinumab is a fully humanized anti-interleukin-13 monoclonal antibody that not only blocks the binding of IL-13 to IL-13Rα1 but also blocks the binding of IL-13 to IL-13Rα2, inhibiting the overexpression and effects of IL-13 in various cell types.

[0021] The monoclonal antibody of this application showed superior affinity to Tralokinumab (prepared by expression according to the sequence disclosed in patent: US63233440P0) in in vitro studies, and showed superior neutralizing activity to Tralokinumab at the cellular level. It is expected to show good clinical efficacy in the prevention and treatment of related diseases. Attached Figure Description

[0022] The accompanying drawings are provided to better understand this application and do not constitute an undue limitation thereof. Wherein: Figure 1 The image shows the nucleic acid electrophoresis results for constructing the HZD260-15 transient transgenic expression plasmid; 1 represents the detection result of the heavy chain variable region, 2 represents the detection result of the heavy chain plasmid, and M represents the marker.

[0023] Figure 2 This is a flowchart for expressing instantaneous transitions.

[0024] Figure 3 This is the electrophoresis detection image of the protein, specifically the electrophoresis detection image of HZD260-15.

[0025] Figure 4 HZD260-15 and Tralokinumab neutralize recombinant human IL-13 to induce TF-1 cell proliferation. Figure 4 A represents the test result for HZD260-15; Figure 4 B represents the detection result of Tralokinumab.

[0026] Figure 5 HZD260-15 and Tralokinumab neutralize recombinant human IL-13, blocking the cell signaling activity of the HEK Blue IL-4 / IL-13 reporter gene. Detailed Implementation

[0027] The following description provides exemplary embodiments of this application, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0028] The technical terms mentioned in this specification have the same meanings as those commonly understood by those skilled in the art, and in case of any conflict, the definitions in this specification shall prevail.

[0029] Generally speaking, the terms used in this specification have the following meanings.

[0030] In this specification, an “isolated” antibody is an antibody that has been separated from components of its native environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, which is determined by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0031] In this specification, "monoclonal antibody" means an antibody derived from a substantially homologous group of antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, and such variants are typically present in trace amounts, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles). Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to this application can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0032] In this specification, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K0). D Affinity can be measured using common methods known in the art.

[0033] In this specification, interleukin-13 (IL-13) is an important member of the interleukin family. It is a protein encoded by the IL-13 gene, secreted by various cells, and has a molecular weight of approximately 10 kDa. As shown in SEQ ID NO: 9, the underlined portion represents the signal peptide.

[0034] SEQ ID NO: 9: MHPLLNPLLLALGLMA LLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN.

[0035] In this specification, "anti-interleukin-13 monoclonal antibody" means a monoclonal antibody that is able to bind to interleukin-13 with sufficient affinity, such that the monoclonal antibody can be used as a diagnostic and / or therapeutic agent targeting interleukin-13.

[0036] The anti-interleukin-13 monoclonal antibody of this application may not bind to proteins unrelated to its target. Here, "unrelated protein" refers to proteins other than interleukin-13, which is the target; and "not binding" means that, when the binding ability of the anti-interleukin-13 monoclonal antibody of this application to its target interleukin-13 is taken as 100%, the binding ability of the anti-interleukin-13 monoclonal antibody of this application to the unrelated protein is less than 10%, for example less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or it can be 0.

[0037] The interleukin-13 monoclonal antibody of this application has equilibrium dissociation constants (K0) of ≤1 μM, ≤100 nM, ≤50 nM, and ≤40 nM. D ).

[0038] Experimental results show that the anti-interleukin-13 monoclonal antibody of this application can specifically bind to interleukin-13.

[0039] The anti-interleukin-13 monoclonal antibody of this application is superior to similar monoclonal antibody products on the market in many aspects of biological activity.

[0040] In one specific embodiment, the amino acid sequence of the heavy chain of the anti-interleukin-13 monoclonal antibody of this application is shown in SEQ ID NO: 10; the amino acid sequence of the light chain is shown in SEQ ID NO: 11.

[0041] SEQ ID NO: 10 EVQLVESGGGLVQPGGSLRLSCAASGFDLN RYFMI WVRQAPGKGLEYIG IITYSGNTYYATWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EGDGNSYDM WGQGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; SEQ ID NO: 11 DIQMTQSPSSVSASVGDRVTITC QASQSISNLLA WYQQKPGKAPKLLIY DASKLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGDLYGVDNT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0042] Both the heavy chain and light chain amino acid sequences were humanized. Through CDR transplantation and reversion mutations of amino acids potentially affecting the CDR region structure within the framework region, and after Fc modification, the activity was consistent with the maternal rabbit antibody, achieving a humanization rate of over 95.0%.

[0043] In this specification, "isolated" nucleic acid means a nucleic acid molecule that has been isolated from components of its native environment. Isolated nucleic acid includes nucleic acid molecules that are normally found in cells containing nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations other than their native chromosomal locations.

[0044] In this specification, "isolated nucleic acid encoding anti-human IL-13 monoclonal antibody" means one or more nucleic acid molecules encoding the antibody heavy and light chains, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in the host cell.

[0045] In this specification, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0046] In this specification, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny (regardless of passage number). Progeny may not be identical to parental cells in terms of nucleic acid contents, but may contain mutations. Mutant progeny with the same function or biological activity selected from the initially transformed cells are included in this specification.

[0047] In this specification, "pharmaceutical composition" means an article which is in a form that enables the bioactivity of the active ingredient contained therein to exert its effect, and which does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation is to be administered.

[0048] In this specification, "pharmaceuticalally acceptable carrier" means a component of the pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0049] In this application, "monoclonal antibody" can generally be a human antibody, which can be prepared using techniques known to those skilled in the art. For example, human antibodies are generally described in van Dijk, MA and van de Winkel, JG, Curr. Opin. Pharmacol. 5:368-374 (2001) and Lonberg, N., Curr. Opin. Immunol. 20:450-459 (2008).

[0050] Antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies in response to antigen challenge stimulation or to produce intact antibodies with human variable regions. These animals typically contain some or all of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or are located extrachromosomally or randomly integrated into the animal. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23:1117-1125 (2005). See also, for example, the XENOMOUSE described in U.S. Patent Nos. 6,075,181 and 6,150,584. TM Technology; HUMAB described in U.S. Patent No. 5,770,429 ® Technology; K-MMOUSE described in U.S. Patent No. 7,041,870 ® The technology and the VELOCIMOUSE described in U.S. Patent Application Publication No. US 2007 / 0061900 ® The technology allows for the extraction of human variable regions from complete antibodies generated by such animals, for example, through further modification by combining them with different human constant regions.

[0051] Human antibodies can also be prepared using hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cells used for the production of human monoclonal antibodies have been described (see, for example, Kozbor, D., J. Immunol. 133:3001-3005 (1984); Brodeur, BR et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; Boerner, P. et al., J. Immunol. 147:86-95 (1991)). Human antibodies produced via human B-cell hybridoma technology are also documented in Li, J. et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Other methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the generation of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, XiandaiMianyixue, 26(4); 265-268 (which describes human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers, HP and Brandlein, S., Histology and Histopathology 20: 927-937 (2005); Vollmers, HP and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27:185-191 (2005).

[0052] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human phage display libraries, and then such variable domain sequences can be combined with desired human constant domains.

[0053] Human antibodies can also be selected based on autoantibody libraries, meaning that human antibodies can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. For example, various methods for producing phage display libraries and screening such libraries for antibodies with desired binding characteristics are known in the art. This method is reviewed in, for example, Hoogenboom, HR et al., Methods in Molecular Biology 178:1-37 (2001), and further described in, for example, McCafferty, J. et al., Nature 348:552-554 (1990); Clackson, T. et al., Nature 352:624-628 (1991); Marks, JD et al., J. Mol. Biol. 222:581-597 (1992); Marks, JD and Bradbury, A., Methods in Molecular Biology 248:161-175 (2003); Sidhu, SS et al., J. Mol. Biol. 338:299-310 (2004); Lee, CV et al., J. Mol. Biol. 340: 1073-1093 (2004); Fellouse, FA, Proc. Natl. Acad. Sci. USA 101:12467-12472 (2004); and Lee, CV et al., J. Immunol. Methods 284:119-132 (2004).

[0054] In some phage display methods, complete sets of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then antigen-binding phages are screened from the phage library, as described in Winter, G. et al., Ann. Rev. Immunol. 12:433-455 (1994). Phages typically display antibody fragments as single-stranded Fv (scFv) fragments or as Fab fragments. Libraries derived from immunized sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, a non-immunized complete set can be cloned (e.g., from humans) to provide a single source of antibodies against a large number of non-self and self antigens in the absence of any immunization, as described by Griffiths, AD et al., EMBO J, 12:725-734 (1993). Finally, an unimmunized library can also be synthesized by cloning the unrearranged V gene segment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and then performing rearrangement in vitro, as described in Hoogenboom, HR and Winter, G., J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0055] The antibody may also be a multispecific antibody, such as a bispecific antibody. A bispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of immunoglobulin heavy-light chains with different specificities (see Milstein, C. and Cuello, AC, Nature 305:537-540 (1983); WO 93 / 08829; and Traunecker, A. et al., EMBO J.10:3655-3659 (1991)) and "segment-entry-cavity" engineering (see, for example, U.S. Patent No. 5,731,168). It can also be achieved through engineered electrostatic manipulation effects used to generate antibody Fc-heterodimeric molecules (WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan, M. et al., Science 229:81-83 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny, SA et al., J. Immunol. 148:1547-1553 (1992)); using “biantibody” techniques for generating bispecific antibody fragments (see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, for example, Gruber, M. et al., J. Immunol. 152:5368-5374(1994)); and the preparation of trispecific antibodies (as described, for example, in Tutt, A. et al., J. Immunol. 147:60-69(1991)) to generate multispecific antibodies.

[0056] The monoclonal antibodies described herein also include engineered antibodies having three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006 / 0025576).

[0057] The antibodies described in this article may also include the multispecific antibodies described in WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, WO2010 / 145793, WO2011 / 117330, WO2012 / 025525, WO2012 / 025530, WO2013 / 026835, WO2013 / 026831, WO2013 / 164325, or WO2013 / 174873.

[0058] The monoclonal antibodies described herein may also be antibody variants, for example, where improved binding affinity and / or other biological properties of the antibody may be desired. Amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding. Thus, in some embodiments, antibody variants with one or more amino acid substitutions are provided, where the sites of interest for substitution mutation include HVR and FR. For example, amino acid substitutions can be introduced into the antibody of interest and products with desired activities can be screened, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0059] Example The present invention will now be described in more detail through embodiments. It should be understood that the present invention is not limited to these embodiments.

[0060] Example 1: Preparation of anti-human interleukin-13 monoclonal antibody HZD260-15 Quanxin Biotechnology developed and expressed human interleukin-13 antigen, which was used as an immunogen to immunize New Zealand rabbits. Using B-cell cloning technology, antigen-binding specific monoclonal antibodies were obtained. These antibodies were then screened to identify those that bound human interleukin-13 and inhibited its activity. Target clones were selected through analysis and screening using Binding ELISA and reporter gene assays.

[0061] Sixteen clones were selected for recombinant expression, and sequencing confirmed their correctness. The rabbit anti-clone C260, exhibiting superior neutralizing activity compared to the control antibody Tralokinumab, was selected for humanization.

[0062] Human IgG germline sequence homology was compared using NCBI IgBlast. IGHV3-66*01 was selected as the heavy chain CDR transplantation template, and the CDR regions of the C260 clone heavy chain (i.e., CDR-H1 (SEQ ID No: 1), CDR-H2 (SEQ ID No: 2), and CDR-H3 (SEQ ID No: 3)) were transplanted into the backbone region of IGHV3-66*01. IGKV1-12*01 was selected as the light chain CDR transplantation template, and the CDR regions of the C260 clone light chain (i.e., CDR-L1 (SEQ ID No: 4), CDR-L2 (SEQ ID No: 5), and CDR-L3 (SEQ ID No: 6)) were transplanted into the backbone region of IGKV1-12*01. Reversion mutations were performed at specific sites in the backbone region to obtain the variable region of the monoclonal antibody HZD260-15 of this invention. Finally, the humanized heavy chain variable region sequence is shown in SEQ ID NO: 7; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 8.

[0063] The gene of the heavy chain variable region (SEQ ID NO: 7) was amplified by PCR to obtain the heavy chain variable region gene. The heavy chain expression plasmid pQX2.1 was digested with HindIII and NheI, and the PCR fragment was inserted into the corresponding expression plasmid using recombinase to construct the heavy chain expression plasmid pQX2.1-260VH-Hu12. The gene of the light chain variable region (SEQ ID NO: 8) was synthesized artificially to obtain the light chain expression plasmid pQX2.3-260VK-Hu1.

[0064] The results of PCR amplification of the antibody heavy chain variable region gene and double enzyme digestion of the heavy chain expression plasmid were detected by nucleic acid electrophoresis. Figure 1 The results show that the PCR amplification of the heavy chain variable region and the electrophoresis results of the double-enzyme digested heavy chain expression plasmid are shown. The heavy chain variable region is about 430 bp and the heavy chain plasmid size is about 6000 bp.

[0065] The correct heavy chain expression plasmid and light chain expression plasmid were co-transfected into ExpiCHO-S cells. One day before transfection, ExpiCHO-S cells were diluted to 3 × 10⁻⁶ cells / mL. 6 Cells were passaged at a density of 10 cells / ml before transfection. On the day of transfection, the cell density was diluted to 6 × 10⁻⁶ cells / ml. 6 Cells / ml, 25 ml of cells in a 125 ml shake flask, awaiting transfection. The transfection and expression process is as follows: Figure 2 As shown.

[0066] On day 6 post-transfection, the culture supernatant was harvested and purified in one step using Protein A. The purified antibody was detected by SDS-PAGE electrophoresis and named HZD260-15. The results of protein electrophoresis for this antibody are as follows: Figure 3 As shown. Protein electrophoresis was performed using a denaturing reducing gel. Figure 3 The results showed two bands, with sizes of approximately 50 kDa and 25 kDa, respectively, consistent with the theoretical molecular weights of the heavy chain (49.0 kDa) and light chain (23.3 kDa).

[0067] Example 2 Equilibrium dissociation constant (K) D Determination of ) The affinity of HZD260-15 for recombinant human IL-13 was detected using a Biacore T200 instrument, with all procedures performed at 25°C. A commercially available CM5 sensor chip was used, and the antibody was conjugated to the chip via amino-linked coupling at a flow rate of 10 μl / min. The antigen was serially diluted, and the flow rate was switched to 30 μl / min, flowing sequentially from low to high concentration through the reference channel and the channel containing the immobilized antibody. A buffer solution was used as a negative control. After each binding and dissociation cycle, the chip was regenerated using glycine at pH 1.5. The instrument's built-in analysis software was used to fit the 1:1 binding model in the Kinetics option, and the antibody binding rate constant k was calculated. a dissociation rate constant k d and the dissociation equilibrium constant K D value.

[0068] In addition, the affinity data of HZD260-15, the anti-human interleukin-13 monoclonal antibody Tralokinumab, and recombinant human IL-13 were compared. The affinity detection methods for both were the same, and the results are shown in Table 1. Tralokinumab was prepared by constructing an expression plasmid based on the sequence provided in patent US63233440P0 and then transiently transfecting it into CHO-K1 cells.

[0069] Table 1. Affinity of anti-human interleukin-13 monoclonal antibodies to human interleukin-13

[0070] The data in the table is the average value of three tests performed on each sample.

[0071] Example 3: HZD260-15 and Tralokinumab neutralize recombinant human IL-13-induced TF-1 cell proliferation activity The cell proliferation activity induced by HZD260-15 and Tralokinumab in neutralizing recombinant human IL-13 (rhIL-13) was measured using TF-1 cells. TF-1 cells were stored at 2 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates. HZD260-15 and Tralokinumab were diluted to concentrations ranging from 0 to 150 ng / mL and 0 to 5 μg / mL, respectively, and mixed with an equal volume of 1 ng / mL recombinant human IL-13. The mixture was incubated for 1.5 hours. After incubation, the antigen-antibody mixture was added to the cells, and the plates were cultured at 37°C and 5% CO2 for 72 hours. 100 μL of the cell suspension was collected and added to a black 96-well plate. 100 μL of a luciferase-based cell viability assay kit (Cell Counting-Lite 2.0 Luminescent Cell Viability Assay kit (Vazyme, DD1101-02)) was mixed with the cell suspension. Cell proliferation was detected using a multi-mode microplate reader, and a dose-response curve was plotted (e.g., ...). Figure 4 (as shown), and then the neutralizing activity of the antibody was analyzed.

[0072] The results showed that HZD260-15 had the activity of neutralizing the proliferation of TF-1 cells induced by recombinant human IL-13. Figure 4 Results showed that HZD260-15 neutralized the IC50 of recombinant human IL-13-induced TF-1 cell proliferation activity. 50 The value was 16.25 ng / ml; Figure 4 Results B showed that the IC50 of the control antibody Tralokinumab was significantly lower than that of Tralokinumab. 50 The value was 231.43 ng / mL. HZD260-15 showed superior hIL-13-induced TF-1 cell proliferation activity compared to the control antibody Tralokinumab.

[0073] Example 4: HZD260-15 and Tralokinumab neutralize recombinant human IL-13, blocking HEK Blue. TM IL-4 / IL-13 reporter gene cell signaling activity Leveraging HEK Blue TM IL-4 / IL-13 reporter gene assays showed that HZD260-15 and Tralokinumab neutralized recombinant human IL-13 (hIL-13) and blocked HEK Blue. TM Intracellular signaling activity of IL-4 / IL-13 reporter genes. HEKBlue was used. TM IL-4 / IL-13 cells were introduced at a rate of 4 × 10⁶ cells per well. 4Cells were seeded into 96-well plates and cultured overnight at 37°C and 5% CO2. HZD260-15 and Tralokinumab were diluted to concentrations ranging from 0 to 50 ng / mL and 0 to 2 μg / mL, respectively, and mixed with an equal volume of 1 ng / mL recombinant human IL-13, then incubated for 1.5 hours. After incubation, the antigen-antibody mixture was added to the cells, and the cells were cultured at 37°C and 5% CO2 for 24 hours. Quanti Blue was used for cell culture. TM Cells were detected using the assay solution (InvivoGen, rep-qbs), and a four-parameter curve was fitted using SoftMax to calculate the sample IC50. 50 The value was used to analyze the neutralizing activity of the antibody.

[0074] The results are as follows Figure 5 As shown, the results indicate that HZD260-15 can neutralize recombinant human IL-13 and block HEK Blue. TM IL-4 / IL-13 reporter gene cell signal transduction, HZD260-15 neutralizes recombinant human IL-13 and blocks HEK Blue TM IC50 of IL-4 / IL-13 reporter gene cell signal transduction 50 The value was 0.93 ng / mL, and the IC50 of the control antibody Tralokinumab was... 50 The value was 46.29 ng / mL, and HZD260-15 showed superior neutralizing and reporter gene blocking activity against hIL-13 compared to the control antibody Tralokinumab.

Claims

1. A monoclonal antibody against human interleukin-13, comprising three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein: (a) The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1 RYFMI; (b) The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2 IITYSGNTYYATWAKG; (c) The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3 EGDGNSYDM; (d) The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4 QASQSISNLLA; (e) The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5 DASKLAS; (f) The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6 QQGDLYGVDNT.

2. The monoclonal antibody according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7EVQLVESGGGLVQPGGSLRLSCAASGFDLNRYFMIWVRQAPGKGLEYIGIITYSGNTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGDGNSYDMWGQGTLVTVSS. The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 8 DIQMTQSPSSVSASVGDRVTITCQASQSISNLLAWYQQKPGKAPKLLIYDASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDLYGVDNTFGGGTKVEIK.

3. The monoclonal antibody according to claim 1 or 2, comprising a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain is shown in SEQ ID NO:

11.

4. An isolated nucleic acid encoding the monoclonal antibody according to claim 1.

5. A host cell comprising the nucleic acid according to claim 4.

6. A method for producing a monoclonal antibody, comprising culturing a host cell according to claim 5 to produce a monoclonal antibody according to claim 1.

7. A pharmaceutical composition comprising the monoclonal antibody according to claim 1 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, for treating diseases related to human interleukin-13 protein-mediated signal transduction.

9. The pharmaceutical composition according to claim 8, wherein, The diseases associated with the signal transduction mediated by the human interleukin-13 protein are selected from immune system diseases, digestive system diseases, respiratory system diseases, and skin diseases, such as: atopic dermatitis, arthritis (including septic arthritis), herpes, chronic primary urticaria, scleroderma, hypertrophic scars, Whipple's disease, benign prostatic hyperplasia, mild, moderate, and severe asthma, allergic rhinitis, chronic sinusitis, hay fever, chronic obstructive pulmonary disease, pulmonary fibrosis, eosinophilia, psoriasis, psoriatic arthritis, inflammatory bowel disease, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, purpura aura, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, tuberculosis, ulcerative colitis, and nephropathy.

10. Use of the monoclonal antibody according to claim 1 in the preparation of a medicament for treating diseases related to human interleukin 13 protein-mediated signal transduction.

11. The use according to claim 10, wherein, The diseases associated with the signal transduction mediated by the human interleukin-13 protein are selected from immune system diseases, digestive system diseases, respiratory system diseases, and skin diseases, such as: atopic dermatitis, arthritis (including septic arthritis), herpes, chronic primary urticaria, scleroderma, hypertrophic scars, Whipple's disease, benign prostatic hyperplasia, mild, moderate, and severe asthma, allergic rhinitis, chronic sinusitis, hay fever, chronic obstructive pulmonary disease, pulmonary fibrosis, eosinophilia, psoriasis, psoriatic arthritis, inflammatory bowel disease, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, purpura aura, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, tuberculosis, ulcerative colitis, and nephropathy.

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