Anti-human TSLP monoclonal antibody and its use

Abstract

The present application provides an anti-human thymic stromal lymphopoietin (TSLP) monoclonal antibody and uses thereof, which has high affinity and neutralizing activity against human TSLP and can be used for the prevention or treatment of associated diseases mediated by TSLP.

Description

[Technical Field]

[0001] The present application relates to the field of antibody pharmaceuticals. Specifically, the present application relates to monoclonal antibodies against human thymic stromal lymphopoietin (TSLP) and uses thereof. [Background technology]

[0002] Cytokines and immune cells mediate specific physiological mechanisms and pathways, including those that lead to various inflammatory diseases. Human thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine produced by human epithelial cells that promotes B cell differentiation and can also costimulate thymocytes and mature T cells. TSLP binds human CD11c + TSLP binds to a specific heterodimeric receptor on dendritic cells (DCs). This receptor heterodimer is composed of a heterodimer of the common gamma-like receptor chain (TSLP receptor; TSLPR) and the IL-7R-α chain. See, for example, Tonozuka et al., Cytogenet. Cell Genet. 93:23-25, 2001; Pandey et al., Nat. Immunol. 1:59-64, 2000; L.Spark et al., J. Exp. Med. 192:659-670, 2000; Reche et al., J. Immunol. 167:336-343, 2001. Receptor-bound ligand induces DCs to secrete TH2-attracting chemical factors, TARC (thymus and activation-regulating chemical factor) and MDC (macrophage-derived chemical factor). TSLP also induces potent DC activation, natural CD4 expression, and IL-7R activation. 4+ It also induces T cell proliferation and subsequent polarization towards a TH2 phenotype, producing the pro-allergic cytokines interleukin 4 (IL-4), IL-5, IL-13, and tumor necrosis factor-α.

[0003] TSLP signaling has also been found to cause activation of the STAT5 transcription factor. Furthermore, TSLP has been reported to be overexpressed in skin wounds from patients with acute and chronic atopic dermatitis, indicating that TSLP expression is associated with allergic inflammation in the body. In addition to skin keratinocytes, high levels of TSLP expression have also been found in bronchial epithelial cells, smooth muscle cells, and lung fibroblasts, supporting a possible role for TSLP in respiratory allergic indications. Furthermore, IgE-activated mast cells express very high levels of TSLP, a mechanism that may be involved in maintaining a TH2 phenotype.

[0004] Approximately 20% of the population in Western countries suffers from inflammatory diseases, such as asthma, rhinitis, atopic dermatitis, and allergic diseases, including food allergies. 50% to 80% of patients with atopic dermatitis also suffer from or develop asthma or allergic rhinitis. Currently, there are no treatments available to treat allergy-induced asthma, atopic dermatitis, and allergic rhinitis. Current treatments, such as beta-2 adrenergic receptor antagonists for asthma, Elidel for atopic dermatitis, and H1 antihistamines for allergic rhinitis, target these symptoms. Therefore, there is a growing need in the art for better treatments for these inflammatory diseases, particularly allergic inflammation. This application addresses this and other problems. Summary of the Invention

[0005] The present application aims to provide a novel anti-human TSLP monoclonal antibody, a pharmaceutical composition containing the monoclonal antibody, and pharmaceutical uses of the monoclonal antibody.

[0006] The technical solution of this application is as follows:

[0007] 1. An anti-human TSLP monoclonal antibody 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), The amino acid sequence of the CDR-H1 (herein, CDR-H1 refers to heavy chain CDR1) is shown in SEQ ID NO: 1 (SYYMS); The amino acid sequence of the CDR-H2 (herein, CDR-H2 refers to heavy chain CDR2) is shown in SEQ ID NO: 2 (FISYGGSAYHATWAQG); The amino acid sequence of the CDR-H3 (herein, CDR-H3 refers to heavy chain CDR3) is shown in SEQ ID NO: 3 (EFRSMTYGAEWGI); The amino acid sequence of the CDR-L1 (herein, CDR-L1 refers to light chain CDR1) is shown in SEQ ID NO: 4 (QASESIYDTLA); The amino acid sequence of said CDR-L2 (herein, CDR-L2 refers to light chain CDR2) is set forth in SEQ ID NO: 5 (SASSLAS); and The monoclonal antibody, wherein the amino acid sequence of the CDR-L3 (herein, CDR-L3 represents light chain CDR3) is set forth in SEQ ID NO: 6 (QQGYTMPDVDKNP).

[0008] 2. Contains a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMSWVRQAPGKGLEWVGFISYGGSAYHATWAQGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAREFRSMTYGAEWGIWGQGTLVTVSS; Item 2. The monoclonal antibody according to Item 1, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8, and the amino acid sequence is AYQMTQSPSSVSASVGDRVTITCQASESIYDTLAWYQQKPGKAPKLLIYSASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYTMPDVDKNPFGGGTKVEIK.

[0009] 3. An isolated nucleic acid encoding the monoclonal antibody described in any one of the preceding claims.

[0010] 4. A host cell comprising the nucleic acid according to item 3.

[0011] 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 several host cells can be used. In one embodiment, the host cell is a prokaryotic cell, for example, E. coli. In another embodiment, the host cell is a eukaryotic cell, for example, a mammalian cell such as a Chinese hamster ovary (CHO) cell.

[0012] 5. A method for producing a monoclonal antibody, comprising culturing the host cell according to Item 4 to produce the monoclonal antibody according to any one of the preceding items.

[0013] The method includes expressing a recombinant vector encoding the anti-human TSLP monoclonal antibody in a suitable host cell, thereby producing the monoclonal antibody. In certain embodiments, the method includes culturing a host cell containing nucleic acid encoding the anti-human TSLP monoclonal antibody, thereby expressing the nucleic acid. The method can further include recovering the anti-human TSLP monoclonal antibody from the host cell culture or host cell culture medium.

[0014] 6. A pharmaceutical composition comprising the monoclonal antibody described in any one of the preceding claims and a pharmaceutically acceptable carrier.

[0015] The pharmaceutical composition may further comprise an additional therapeutic agent (e.g., a different anti-human TSLP antibody).

[0016] 7. The pharmaceutical composition according to item 6, which is used to treat a disease associated with TSLP-mediated signaling.

[0017] 8. The pharmaceutical composition according to Item 7, wherein the disease associated with TSLP-mediated signaling is allergic asthma, allergic dermatitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis fibrosis, inflammatory bowel disease, or the like.

[0018] 9. Use of the monoclonal antibody according to any preceding claim in the preparation of a medicament for treating a disease associated with TSLP-mediated signaling.

[0019] 10. The use according to item 9, wherein the disease associated with TSLP-mediated signaling is allergic asthma, allergic dermatitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis fibrosis, inflammatory bowel disease, etc.

[0020] 11. A method comprising administering the monoclonal antibody described in any one of the preceding claims or the pharmaceutical composition described in any one of the preceding claims to a subject in need thereof. Methods for treating diseases associated with TSLP-mediated signaling.

[0021] 12. The method according to item 11, wherein the disease associated with TSLP-mediated signaling is allergic asthma, allergic dermatitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis fibrosis, inflammatory bowel disease, or the like. [Effects of the Invention]

[0022] The present invention provides a novel anti-human TSLP monoclonal antibody that has comparable binding affinity to TSLP and superior neutralizing activity at the cellular level to that of a prior art anti-human TSLP monoclonal antibody (tezepelumab is a monoclonal antibody drug targeting TSLP developed by Amgen / AstraZeneca, and a Phase III clinical trial of tezepelumab for the treatment of severe asthma has been successful).

[0023] The monoclonal antibody of the present application exhibits superior neutralizing activity at the cellular level to tezepelumab (prepared according to the sequence expression disclosed in the patent), and is expected to show good clinical efficacy in the prevention and treatment of related diseases. [Brief explanation of the drawings]

[0024] The accompanying drawings are used for a better understanding of the present application and are not intended to unduly limit the present application. [Figure 1] Figure 1 shows the results of nucleic acid electrophoresis for constructing the HZD8G2-57 transient expression plasmid, where M is a marker; band 1 is the PCR product 8G2VH-Hu27; band 2 is pHZDCH, HindIII / NheI; band 3 is the PCR product 8G2VK-Hu14; and band 4 is pHZDCK, HindIII / BsiWI. [Figure 2] FIG. 2 is a flow chart of transient expression. [Figure 3] FIG. 3 is an electrophoretic detection pattern of QX008N (HZD8G2-57). [Figure 4] FIG. 4 shows the activity of QX008N and tezepelumab to neutralize human TSLP-induced STAT5 phosphorylation in SW756-STAT5-luciferase cells. [Figure 5] FIG. 5 shows the activity of QX008N and tezepelumab to neutralize native TSLP-induced STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells. [Figure 6] FIG. 6 shows the activity of QX008N and tezepelumab to neutralize STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells induced by cynomolgus monkey TSLP. [Figure 7] FIG. 7 shows the activity of QX008N and tezepelumab to neutralize human TSLP-induced TARC (CCL17) release from human whole blood. [Figure 8]FIG. 8 shows the activity of QX008N and tezepelumab to neutralize human TSLP-induced TARC (CCL17) release from human PBMC cells. Details of the invention

[0025] The following describes exemplary embodiments of the present application. For ease of understanding, various details of the embodiments of the present application are included in the description, but they should be considered as merely examples. 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 the present application. For clarity and conciseness, the following description omits descriptions of well-known functions and configurations.

[0026] Scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, except that in case of conflict, the definitions used herein shall control.

[0027] Generally speaking, the terms used herein have the following meanings:

[0028] As used herein, an "isolated" antibody refers to an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, and the purity is determined, for example, by electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0029] As used herein, a "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody in the population is identical and / or binds to the same epitope. Except for possible variant antibodies (e.g., those containing naturally occurring mutations or those arising during the preparation of a monoclonal antibody), such variants are generally present in minor amounts. Unlike a typical polyclonal antibody preparation, which contains different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring the antibody to be prepared by any particular method. For example, the monoclonal antibodies of the present application can be produced by several techniques, including, but not limited to, hybridoma, recombinant DNA, phage display, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. This specification describes such methods, as well as other exemplary methods for preparing monoclonal antibodies.

[0030] As used herein, "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 that reflects a 1:1 interaction between binding partner members (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed as an equilibrium dissociation constant (KD). Affinity can be measured by routine methods known in the art.

[0031] As used herein, human thymic stromal lymphopoietin (TSLP) refers to a cytokine derived from humans, the amino acid sequence of which is shown in SEQ ID NO: 9, where the underlined portion represents the signal peptide. SEQ ID NO:9: MFPFALLYVLSVSFRKIFILQLVGLVLTYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ

[0032] As used herein, the term "anti-human TSLP monoclonal antibody" refers to a monoclonal antibody that can bind to human TSLP with sufficient affinity so that it can be used as a diagnostic and / or therapeutic agent targeting human TSLP.

[0033] The anti-human TSLP monoclonal antibodies of the present application do not bind to target, unrelated proteins. Here, "unrelated proteins" refers to proteins other than human TSLP as the target. Here, "does not bind" means that, when the binding ability of the anti-human TSLP monoclonal antibodies of the present application to their target human TSLP is taken as 100%, the binding ability of the anti-human TSLP monoclonal antibodies of the present application to the unrelated proteins is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.

[0034] The anti-human TSLP monoclonal antibodies of the present application may not bind to TSLP from other animal species. Here, "other animal species" refers to animal species other than humans, such as marmosets, cynomolgus monkeys, pigs, dogs, rabbits, rats, mice, and guinea pigs. Here, "does not bind" refers to the binding ability of the anti-human TSLP monoclonal antibodies of the present application to TSLP from other animal species being less than 10%, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0, when the binding ability of the anti-human TSLP monoclonal antibodies of the present application to their target, human TSLP, is taken as 100%.

[0035] The human TSLP monoclonal antibody of the present application has an equilibrium dissociation constant (K D )

[0036] The experimental results demonstrate that the anti-human TSLP monoclonal antibody of the present application can specifically bind to human TSLP.

[0037] The anti-human TSLP monoclonal antibodies of the present application are comparable to or superior to commercially available similar monoclonal antibody products in many biological activities, including, for example, neutralizing STAT5 phosphorylation in human, natural, and cynomolgus monkey TSLP-induced cells, and neutralizing human TSLP-induced TARC (CCL17) release from human whole blood and human PBMC cells.

[0038] In a specific embodiment, the amino acid sequence of the heavy chain of the anti-human TSLP monoclonal antibody of the present application is set forth in SEQ ID NO:10, and the amino acid sequence of the light chain is set forth in SEQ ID NO:11. SEQ ID NO: 10 EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMSWVRQAPGKGLEWVGFISYGGSAYHATWAQGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAREFRSMTYGAEWGIWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 11 AYQMTQSPSSVSASVGDRVTITCQASESIYDTLAWYQQKPGKAPKLLIYSASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYTMPDVDKNPFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Here, SEQ ID NOs: 10 and 11 are both humanized sequences.

[0039] As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in cells that ordinarily contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0040] As used herein, "an isolated nucleic acid encoding an anti-human TSLP monoclonal antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of the antibody, including such nucleic acid molecules in a single vector or separate vectors, and including such nucleic acid molecules present in one or more locations in a host cell.

[0041] As used herein, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of nucleic acids that are operably linked to them. Such vectors are referred to herein as "expression vectors."

[0042] As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny (regardless of the number of passages). Progeny may not be entirely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0043] As used herein, "pharmaceutical composition" refers to a composition-like product that is in a form that enables the biological activity of the active ingredient contained therein and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is administered.

[0044] As used herein, the term "pharmaceutically acceptable carrier" refers to any component of a pharmaceutical composition other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0045] As used herein, a "monoclonal antibody" is generally a human antibody and can be prepared using techniques well 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).

[0046] Antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to stimulate the production of fully human antibodies or intact antibodies with human variable regions in response to antigenic challenge. These animals typically contain some or all of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci and are present extrachromosomally or randomly integrated into the animal. In such transgenic animals, 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, e.g., the XENOMOUSE™ technology described in U.S. Patent Nos. 6,075,181 and 6,150,584, the HUMAB® technology described in U.S. Patent No. 5,770,429, the K-MMOUSE® technology described in U.S. Patent No. 7,041,870, and the VELOCIMOUSE® technology described in U.S. Patent Application Publication No. US2007 / 0061900. The human variable regions from intact antibodies generated from such animals can be further modified, for example, by combination with different human constant regions.

[0047] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cells and mouse-human hybrid myeloma cells used to produce human monoclonal antibodies have been described (see, e.g., Kozbor, D., J. Immunol. 133:3001-3005 (1984); Brodeur, B. R. et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; Boerner, P. et al., Immunol. 147:86-95 (1991)). Human antibodies produced by human B cell hybridoma technology are also described 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 production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 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).

[0048] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries, and then combining such variable domain sequences with desired human constant domains.

[0049] Human antibodies can also be selected based on an autoantibody library. That is, human antibodies can be isolated by screening for antibodies with one or more desired activities from a combinatorial library. For example, various methods are known in the art for creating phage display libraries and screening for antibodies with desired binding properties from such libraries. This method is reviewed, for example, in Hoogenboom, H.R. et al., Methods in Molecular Biology 178:1-37 (2001), and further described, for example, in McCafferty, J. et al., Nature 348:552-554 (1990); Clackson, T. et al., Nature 352:624-628 (1991); Marks, J.D. et al., J.Mol.Biol. 222:581-597 (1992); Marks, J.D. and Bradbury, A., Methods in Molecular Biology 248:161-175 (2003); Sidhu, S.S. et al., J.Mol.Biol. 338:299-310 (2004); Lee, C.V. et al. 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).

[0050] In some phage display methods, complete sets of VH and VL genes are cloned by polymerase chain reaction (PCR) and randomly recombined into a phage library, which is then screened for antigen-binding phages, as described in Winter, G. et al., Ann. Rev. Immunol. 12:433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, unimmunized repertoires (e.g., from humans) can be cloned to provide a single source of antibodies against multiple non-self and self antigens in the absence of immunization, as described in Griffiths, AD et al., EMBO J, 12:725-734 (1993). Finally, non-immunized libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, encoding highly variable CDR3 regions using PCR primers containing random sequences, and rearranging them in vitro, as described by Hoogenboom, H.R. 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 Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0051] The antibody may also be a multispecific antibody, such as a bispecific antibody. Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. Techniques for producing multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305:537-540 (1983); WO93 / 08829; and Traunecker, A. et al., EMBO J. 10:3655-3659 (1991)) and "protuberance-into-cavity" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by various techniques, including engineered electrostatic steering to generate antibody Fc heterodimeric molecules (WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan, M. et al., Science 229:81-83 (1985)), using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny, S.A. et al., J. Immunol. 148:1547-1553 (1992)), using "double antibody" technology to generate bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M. et al., J. Immunol. 148:1547-1553 (1992)). al., J. Immunol. 152:5368-5374 (1994)), and preparation of trispecific antibodies (e.g., as described in Tutt, A. et al., J. Immunol. 147:60-69 (1991)).

[0052] The monoclonal antibodies described herein also include engineered modified antibodies with three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576).

[0053] The antibodies herein can also include multispecific antibodies described in WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, and WO2010 / 145793, WO2011 / 117330, WO2012 / 025525, WO2012 / 025530, WO2013 / 026835, WO2013 / 026831, WO2013 / 164325, or WO2013 / 174873.

[0054] The monoclonal antibodies described herein may be antibody variants, for example, if it is desired to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, as long as the final construct possesses the desired properties, such as antigen binding. Thus, in certain embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutations include HVRs and FRs. For example, amino acid substitutions can be introduced into a target antibody and products with the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC, can be screened. [Example]

[0055] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0056] Unless otherwise specified, all materials, reagents, etc. used in the following examples are commercially available.

[0057] Example 1 Preparation of anti-human TSLP monoclonal antibody QX008N Human thymic stromal lymphopoietin (hTSLP) was purchased from Shanghai Jin'an Technology Co., Ltd. for immunization of New Zealand rabbits. Antigen-binding specific antibody clones were obtained using B cell cloning technology, and monoclonal antibodies binding to human TSLP and with human TSLP inhibitory activity were screened. Cell supernatants were detected using binding ELISA and blocking ELISA, and target clones were selected. The above immunization and screening processes were completed by a commercial company.

[0058] Seven clones were selected for recombinant expression and sequenced. Measurements revealed that 8G2 had the best cell-neutralizing activity. Therefore, the 8G2 clone was humanized. Identity alignment of human IgG germline sequences (Germline) was performed using NCBI IgBlast. IgGHV3-66*01 was selected as the heavy chain CDR-grafting template, and the CDR regions of the 8G2 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 grafted into the IgGHV3-66*01 framework region. IGKV1-12*01 was selected as the light chain CDR-grafting template, and the CDR regions of the 8G2 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 grafted into the IGKV1-12*01 framework region. Backmutations were performed at specific sites in the framework regions to obtain the variable regions of the monoclonal antibody QX008N of the present application. Finally, the sequence of the humanized heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the humanized light chain variable region is shown in SEQ ID NO:8.

[0059] The genes for the heavy chain variable region (SEQ ID NO: 7) and the light chain variable region (SEQ ID NO: 8) were obtained by PCR amplification. The heavy chain expression plasmid pHZDCH was double-digested with HindIII and NheI, and the light chain expression plasmid pHZDCK was double-digested with HindIII and BsiWI. The PCR-amplified genes were then inserted into the corresponding expression plasmids using infusion recombinase to construct the heavy chain expression plasmid pHZDCH-8G2VH-Hu27 and the light chain expression plasmid pHZDCK-8G2VK-Hu14.

[0060] The results of detecting the PCR-amplified variable region gene fragments and the double-digested plasmids by nucleic acid electrophoresis are shown in Figure 1. As can be seen from the results in Figure 1, the PCR amplification of the antibody heavy chain variable region and light chain variable region, and the double digestion of the heavy chain and light chain expression plasmids, show that the sizes of the heavy chain and light chain plasmids are approximately 10,000 bp, the heavy chain variable region is approximately 477 bp, and the light chain variable region is approximately 447 bp.

[0061] ExpiCHO-S cells were co-transfected with the sequence-correct heavy chain expression plasmid pHZDCH-8G2VH-Hu27 (the amino acid sequence of the heavy chain expressed thereby is shown in SEQ ID NO: 10) and the sequence-correct light chain expression plasmid pHZDCK-8G2VK-Hu14 (the amino acid sequence of the light chain expressed thereby is shown in SEQ ID NO: 11). The day before transfection, 3 × 10 ExpiCHO-S cells were cultured. 6 The cells were diluted to 6 × 10 cells / ml and passaged before transfection. 6 The cells were diluted to 125 ml / ml and 25 ml of cells were placed in a 125 ml shake flask for transfection. The transfection and expression process is shown in Figure 2.

[0062] Six days after transfection, the culture supernatant was collected and purified in one step using Protein A. The purified antibody was detected by SDS-PAGE electrophoresis and designated QX008N (HZD8G2-57). The results of protein electrophoresis of this antibody are shown in Figure 3. Protein electrophoresis was performed on a denaturing reducing gel. As can be seen from the results shown in Figure 3, two bands were observed, with sizes of approximately 50 kDa and 25 kDa, respectively, which is consistent with the theoretical molecular weights of the heavy chain (49.3 kDa) and light chain (23.6 kDa).

[0063] Example 2 Equilibrium dissociation constant (K D ) measurement The affinity of QX008N (HZD8G2-57) to human TSLP was detected using a Biacore T200, and all processes were performed at 25°C. A commercially available Protein A chip was used, and an appropriate amount of antibody was immobilized by the capture method to achieve an Rmax of approximately 50 RU and a capture flow rate of 10 μl / min. The antigen was serially diluted, and the instrument flow rate was switched to 30 μl / min. The reference channel and antibody-immobilized channel were run in order of low to high concentration, with buffer flow as a negative control. After each binding and dissociation, the chip was regenerated with pH 1.5 glycine. Using the instrument's software, fitting was performed according to a 1:1 binding model in the kinetics section to determine the antibody binding rate constant, k a , dissociation rate constant k d , and the equilibrium dissociation constant K D The value of was calculated.

[0064] We also compared the affinity of QX008N (HZD8G2-57) with that of tezepelumab, a human TSLP monoclonal antibody already in Phase III clinical trials. The detection method for the known antibody was the same as that for QX008N. The results are shown in Table 1. Tezepelumab was generated by the inventors by constructing an expression plasmid based on the A5 sequence provided by patent US20110274687A1 and transiently transfecting ExpiCHO-S cells.

[0065] [Table 1]

[0066] Example 3: Activity of QX008N and tezepelumab to neutralize human TSLP-induced STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells The activity of QX008N in antagonizing human TSLP-mediated phosphorylation of the intracellular signal molecule STAT5 via the TSLPR-IL-7R was measured using the SW756-STAT5-luciferase reporter cell line. Cells were cultured at 4 × 10 per well in culture medium. 4 Cells were seeded into 96-well plates and cultured overnight at 37°C and 5% CO2. The pre-incubated antibody and human TSLP mixture was added to the cells. The final concentrations of QX008N were 0-50 ng / ml, those of tezepelumab were 0-400 ng / ml, and the final concentration of TSLP was 0.5 ng / ml. The cells were then cultured for 24 hours at 37°C and 5% CO2. The cell culture supernatant was collected, and 120 μl of ONE-Glo-Luciferase Reagent was added to each well. After 30 minutes of incubation, 100 μl of each well was transferred to a white 96-well plate. The fluorescent signal was measured to generate a dose-response curve for antibody antagonist activity. The dose-response curve is shown in Figure 4.

[0067] As can be seen from the results shown in Figure 4, QX008N can inhibit human TSLP-induced STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells, and the IC value of QX008N's activity in inhibiting human TSLP-induced STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells was 0.01. 50 The IC50 of tezepelumab's activity in inhibiting human TSLP-induced STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells was 0.837 ng / ml. 50 was 3.8ng / ml.

[0068] Example 4: Activity of QX008N and tezepelumab to neutralize native TSLP-induced STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells The SW756-STAT5-luciferase reporter cell line was used to measure the activity of QX008N in antagonizing native TSLP-mediated phosphorylation of the intracellular signaling molecule STAT5 via the TSLPR-IL-7R. Cells were cultured at 4 × 10 per well in culture medium. 4 Cells were seeded into 96-well plates and cultured overnight at 37°C and 5% CO2. The pre-incubated antibody and native TSLP mixture was added to the cells. The final concentrations of QX008N ranged from 0 to 50 ng / ml, tezepelumab ranged from 0 to 400 ng / ml, and the final concentration of native TSLP was diluted 62.5-fold from the stock solution. The cells were then cultured for 24 hours at 37°C and 5% CO2. The cell culture supernatant was collected, and 120 μl of ONE-Glo-Luciferase Reagent was added to each well. After 30 minutes of incubation, 100 μl of each well was transferred to a white 96-well plate. The fluorescent signal was measured to generate a dose-response curve for antibody antagonist activity. The dose-response curve is shown in Figure 5.

[0069] As can be seen from the results shown in Figure 5, QX008N can inhibit native TSLP-induced STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells, and the IC of QX008N's activity in inhibiting native TSLP-induced STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells was 0.01. 50 The IC50 of tezepelumab's activity in inhibiting STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells induced by native TSLP was 0.462 ng / ml. 50 was 1.45ng / ml.

[0070] Example 5: Activity of QX008N and tezepelumab to neutralize STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells induced by cynomolgus monkey TSLP The activity of QX008N in antagonizing cynomolgus monkey TSLP-mediated phosphorylation of the intracellular signaling molecule STAT5 via the TSLPR-IL-7R was measured using the SW756-STAT5-luciferase reporter cell line. Cells were cultured at 4 × 10 per well in culture medium. 4 Cells were seeded into 96-well plates and cultured overnight at 37°C and 5% CO2. The pre-incubated antibody and cynomolgus TSLP mixture was added to the cells at final concentrations ranging from 0 to 50 ng / ml for QX008N, 0 to 400 ng / ml for tezepelumab, and 0.5 ng / ml for cynomolgus TSLP. The cells were then cultured for 24 hours at 37°C and 5% CO2. The cell culture supernatant was collected, and 120 μl of ONE-Glo-Luciferase Reagent detection reagent was added to each well. After 30 minutes of incubation, 100 μl of each well was transferred to a white 96-well plate. The fluorescent signal was measured to generate a dose-response curve for antibody antagonist activity. The dose-response curve is shown in Figure 6.

[0071] As can be seen from the results shown in Figure 6, QX008N can inhibit cynomolgus monkey TSLP-induced STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells, and the IC value of QX008N's activity in inhibiting cynomolgus monkey TSLP-induced STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells was 0.01. 50 The IC value of tezepelumab's activity in inhibiting STAT5 phosphorylation in SW756-STAT5-luciferase reporter gene cells induced by cynomolgus monkey TSLP was 0.889 ng / ml. 50 was 1.88ng / ml.

[0072] Example 6 Activity of QX008N and tezepelumab in neutralizing human TSLP-induced TARC (CCL17) release from human whole blood The activity of QX008N in antagonizing human TSLP-induced TARC (CCL17) release via the TSLPR-IL-7R was measured using human whole blood. Whole blood was added to a 96-well plate at 100 μl per well and temporarily stored at 37°C and 5% CO2. A pre-incubated antibody and human TSLP mixture was added to the whole blood. The antibody concentrations ranged from 0 to 10 μg / ml, human TSLP was 0.5 ng / ml, and IL-33 was added at a final concentration of 0.5 ng / ml. The cells were then cultured for 48 hours at 37°C and 5% CO2. Cell culture supernatants were collected, and TARC (CCL17) expression in the supernatant was detected using a sandwich ELISA to generate a dose-response curve for antibody antagonism. The dose-response curve is shown in Figure 7.

[0073] As can be seen from the results shown in Figure 7, QX008N can inhibit human TSLP-induced TARC (CCL17) release from human whole blood, and the IC 50 The IC50 of tezepelumab's activity in inhibiting human TSLP-induced TARC (CCL17) release from human whole blood was 0.839 ng / ml, whereas the IC50 of tezepelumab's activity in inhibiting human TSLP-induced TARC (CCL17) release from human whole blood was 0.839 ng / ml. 50 was 23.9ng / ml.

[0074] Example 7 Activity of QX008N and tezepelumab to neutralize human TSLP-induced TARC (CCL17) release from human PBMC cells The activity of QX008N in antagonizing human TSLP-induced TARC (CCL17) release via the TSLPR-IL-7R was measured using human PBMC cells. PBMCs were isolated by density gradient centrifugation and added to a 96-well plate at 300,000 cells / well. PBMCs were temporarily stored at 37°C and 5% CO2. A pre-incubated antibody and human TSLP mixture was added to the PBMCs. The antibody concentrations ranged from 0 to 10 μg / ml, human TSLP at a final concentration of 0.5 ng / ml, and IL-33 at a final concentration of 0.5 ng / ml. The cells were then cultured for 48 hours at 37°C and 5% CO2. The cell culture supernatants were collected, and TARC (CCL17) expression in the supernatants was detected using a sandwich ELISA to generate a dose-response curve for antibody antagonism. The dose-response curve is shown in Figure 8.

[0075] As can be seen from the results shown in Figure 8, QX008N can inhibit human TSLP-induced TARC (CCL17) release from PBMC cells, and the IC 50 The IC50 of tezepelumab's activity in inhibiting human TSLP-induced TARC (CCL17) release from PBMC cells was 77.1 ng / ml, whereas the IC50 of tezepelumab's activity in inhibiting human TSLP-induced TARC (CCL17) release from PBMC cells was 77.1 ng / ml. 50 was 216ng / ml.

[0076] Although the embodiments of the present application have been described above, the present application is not limited to the above specific embodiments and application fields, and the above specific embodiments are not limiting but merely illustrative and teaching. Under the teachings of this specification, a person skilled in the art may create many forms without departing from the scope of protection of the claims of the present application, and all of them are covered by the protection of the present application.

Claims

1. An anti-human thymic stromal lymphopoietin (TSLP) monoclonal antibody 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), The amino acid sequence of the CDR-H1 is shown in SEQ ID NO: 1; The amino acid sequence of the CDR-H2 is shown in SEQ ID NO:2; The amino acid sequence of the CDR-H3 is shown in SEQ ID NO:3; The amino acid sequence of the CDR-L1 is shown in SEQ ID NO:4; The amino acid sequence of said CDR-L2 is set forth in SEQ ID NO:5; and The monoclonal antibody, wherein the amino acid sequence of the CDR-L3 is set forth in SEQ ID NO:

6.

2. comprising a heavy chain variable region and a light chain variable region, The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:7; The monoclonal antibody of claim 1, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:

8.

3. An isolated nucleic acid encoding the monoclonal antibody of claim 1 or 2.

4. A host cell comprising the nucleic acid of claim 3.

5. A method for producing a monoclonal antibody, comprising culturing the host cell of claim 4 to produce the monoclonal antibody of claim 1 or 2.

6. A pharmaceutical composition comprising the monoclonal antibody of claim 1 or 2 and a pharmaceutically acceptable carrier.

7. 7. The pharmaceutical composition according to claim 6, for use in the treatment of diseases associated with TSLP-mediated signaling.

8. 8. The pharmaceutical composition according to claim 7, wherein the diseases associated with TSLP-mediated signaling are allergic asthma, allergic dermatitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis fibrosis, and inflammatory bowel disease.

9. 10. Use of the monoclonal antibody of claim 1 or 2 in the preparation of a medicament for treating a disease associated with TSLP-mediated signaling.

10. 10. The use according to claim 9, wherein the diseases associated with TSLP-mediated signaling are allergic asthma, allergic dermatitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis fibrosis, and inflammatory bowel disease.

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