Anti-human tslp monoclonal antibody and use thereof

Abstract

Provided in the present invention are an anti-human thymic stromal lymphopoietin (TSLP) monoclonal antibody and the use thereof. The monoclonal antibody has high affinity for human TSLP, has a neutralizing activity, and can be used for preventing or treating TSLP-mediated diseases.

Description

An anti-human TSLP monoclonal antibody and its application Technical Field

[0001] The present application relates to the field of antibody drugs. Specifically, the present application relates to monoclonal antibodies against human thymic stromal lymphopoietin (TSLP) and their applications. Background Art

[0002] Cytokines and immune cells mediate specific physiological mechanisms or pathways, for example, pathways that lead to various inflammatory disorders. Human thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine produced by human epithelial cells. It promotes B cell differentiation and can also co-stimulate thymocytes and mature T cells. TSLP binds to human CD11c + A specific heterodimeric receptor on dendritic cells (DCs). This receptor heterodimer consists of a heterodimer of a common gamma-like receptor chain (TSLP receptor; TSLPR) and an IL-7R-alpha chain. See, for example, Tonozuka et al., Cytogenet. Cell Genet. 93: 23-25, 2001; Pandey et al., Nat. Immunol. 1: 59-64, 2000; LS Park et al., J. Exp. Med. 192: 659-670, 2000; Reche et al., J. Immunol. 167: 336-343, 2001. Ligand binding to the receptor induces DCs to secrete TH2-attracting chemokines, TARC (thymus and activation-regulated chemokine) and MDC (macrophage-derived chemokine). In addition, TSLP also induces efficient DC activation, natural CD 4+ T cells expand and subsequently polarize to 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 lead to activation of the STAT5 transcription factor. Furthermore, TSLP overexpression in skin wounds of patients with acute and chronic atopic dermatitis has been reported, suggesting that TSLP expression is associated with allergic inflammation in vivo. In addition to skin keratinocytes, high levels of TSLP expression have also been found in bronchial epithelial cells, smooth muscle, and lung fibroblasts, supporting a possible role for TSLP in respiratory allergic manifestations. Furthermore, IgE-activated mast cells express very high levels of TSLP, a mechanism that may be involved in maintaining the TH2 phenotype.

[0004] In Western countries, about 20% of the population suffers from inflammatory disorders, for example, allergic diseases, which include asthma, rhinitis, atopic dermatitis and food allergies. 50% to 80% of patients with atopic dermatitis have or develop asthma or allergic rhinitis. To date, allergy-induced asthma, atopic dermatitis and allergic rhinitis cannot be cured. Current treatments, such as the use of 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 to treat these inflammatory disorders, particularly allergic inflammation. The present application addresses this and other problems.

[0005] Summary of the Invention

[0006] The purpose of the present application is to provide a novel anti-human TSLP monoclonal antibody, a pharmaceutical composition comprising the monoclonal antibody, and pharmaceutical uses of the monoclonal antibody.

[0007] The technical solution of this application is as follows:

[0008] 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), wherein:

[0009] The amino acid sequence of CDR-H1 (CDR-H1 in this specification represents heavy chain CDR1) is shown in SEQ ID NO: 1 (SYYMS);

[0010] The amino acid sequence of CDR-H2 (CDR-H2 in this specification represents heavy chain CDR2) is shown in SEQ ID NO: 2 (FISYGGSAYHATWAQG);

[0011] The amino acid sequence of CDR-H3 (CDR-H3 in this specification represents heavy chain CDR3) is shown in SEQ ID NO: 3 (EFRSMTYGAEWGI);

[0012] The amino acid sequence of CDR-L1 (CDR-L1 in this specification represents light chain CDR1) is shown in SEQ ID NO: 4 (QASESIYDTLA);

[0013] The amino acid sequence of CDR-L2 (CDR-L2 in this specification represents light chain CDR2) is shown in SEQ ID NO: 5 (SASSLAS);

[0014] The amino acid sequence of CDR-L3 (CDR-L3 in this specification represents light chain CDR3) is shown in SEQ ID NO: 6 (QQGYTMPDVDKNP).

[0015] 2. The monoclonal antibody according to item 1, comprising a heavy chain variable region and a light chain variable region, wherein:

[0016] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, which has an amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMSWVRQAPGKGLEWVGFISYGGSAYHATWAQGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAREFRSMTYGAEWGIWGQGTLVTVSS;

[0017] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, which has the amino acid sequence of AYQMTQSPSSVSASVGDRVTITCQASESIYDTLAWYQQKPGKAPKLLIYSASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYTMPDVDKNPFGGGTKVEIK.

[0018] 3. An isolated nucleic acid encoding any one of the aforementioned monoclonal antibodies.

[0019] 4. A host cell comprising the nucleic acid according to item 3.

[0020] 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, for example, Escherichia coli (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.

[0021] 5. A method for producing a monoclonal antibody, the method comprising culturing the host cell according to item 4 to thereby produce any one of the aforementioned monoclonal antibodies.

[0022] The method comprises 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 comprises culturing a host cell containing a nucleic acid encoding the anti-human TSLP monoclonal antibody, thereby expressing the nucleic acid. The method may further comprise recovering the anti-human TSLP monoclonal antibody from the host cell culture or host cell culture medium.

[0023] 6. A pharmaceutical composition comprising any one of the aforementioned monoclonal antibodies and a pharmaceutically acceptable carrier.

[0024] The pharmaceutical composition may further comprise an additional therapeutic agent (eg, a different anti-human TSLP antibody).

[0025] 7. The pharmaceutical composition according to item 6, which is used to treat diseases associated with TSLP-mediated signal transduction.

[0026] 8. The pharmaceutical composition according to item 7, wherein the disease associated with TSLP-mediated signal transduction is allergic asthma, allergic dermatitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis fibrosis, and inflammatory bowel disease.

[0027] 9. Use of any of the aforementioned monoclonal antibodies in the preparation of a medicament for treating diseases associated with TSLP-mediated signal transduction.

[0028] 10. The use according to item 9, wherein the diseases associated with TSLP-mediated signal transduction are allergic asthma, allergic dermatitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis fibrosis, and inflammatory bowel disease.

[0029] 11. A method for treating a disease associated with TSLP-mediated signal transduction, comprising:

[0030] The monoclonal antibody according to any one of the preceding items or the pharmaceutical composition according to any one of the preceding items is administered to a subject in need thereof.

[0031] 12. The method according to item 11, wherein the diseases associated with TSLP-mediated signal transduction are allergic asthma, allergic dermatitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis fibrosis, and inflammatory bowel disease.

[0032] The present application provides a new anti-human TSLP monoclonal antibody, which has a comparable affinity for binding to TSLP as the prior art anti-human TSLP monoclonal antibody (Tezepelumab is a monoclonal antibody drug targeting TSLP developed by Amgen / AstraZeneca, and the Phase III NAVIGATOR clinical trial of Tezepelumab for the treatment of severe asthma was successful), and has better neutralizing activity at the cellular level than Tezepelumab.

[0033] The monoclonal antibody of the present application shows neutralizing activity superior to that of Tezepelumab (prepared by expression according to the sequence disclosed in the patent) at the cellular level, and is expected to show good clinical effects in preventing and treating related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings are provided to facilitate a better understanding of the present application and do not constitute an undue limitation on the present application.

[0035] Figure 1 shows the results of nucleic acid electrophoresis for the construction of the HZD8G2-57 transient expression plasmid. M: Marker; Band 1: PCR product 8G2VH-Hu27; Band 2: pHZDCH, HindIII / NheI; Band 3: PCR product 8G2VK-Hu14; Band 4: pHZDCK, HindIII / BsiWI.

[0036] Figure 2 is a flowchart of transient expression.

[0037] FIG3 is an electrophoresis detection diagram of QX008N (HZD8G2-57).

[0038] FIG4 is a graph showing the activity of QX008N and Tezepelumab in neutralizing human TSLP-induced STAT5 phosphorylation in SW756-STAT5-Luciferase cells.

[0039] FIG5 is a graph showing that QX008N and Tezepelumab neutralize the natural TSLP-induced STAT5 phosphorylation activity in SW756-STAT5-Luciferase reporter gene cells.

[0040] FIG6 is a graph showing that QX008N and Tezepelumab neutralize STAT5 phosphorylation activity induced by TSLP in cynomolgus monkey SW756-STAT5-Luciferase reporter gene cells.

[0041] FIG7 is a graph showing that QX008N and Tezepelumab neutralize the activity of human TSLP-induced TARC (CCL17) release from human whole blood.

[0042] FIG8 is a graph showing that QX008N and Tezepelumab neutralize the activity of human TSLP-induced TARC (CCL17) release from human PBMC cells. DETAILED DESCRIPTION

[0043] The following description of exemplary embodiments of the present application includes various details of the embodiments of the present application to facilitate understanding, and should be considered as merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0044] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0045] Generally speaking, the terms used in this specification have the following meanings.

[0046] As used herein, an "isolated" antibody is one that has been separated from components of its natural environment. In certain embodiments, the antibody is purified to greater than 95% or 99% purity, as 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. B 848: 79-87 (2007).

[0047] In this specification, " monoclonal antibody " represents the antibody derived from the colony of substantially homologous antibody, that is, each antibody constituting the colony is identical and / or in conjunction with identical epi-position, except possible variant antibody (for example, containing naturally occurring mutation or producing in the production process of monoclonal antibody product), such variant is usually present in trace.Different from the polyclonal antibody product that generally includes the different antibodies for different determinants (epi-positions), every kind of monoclonal antibody of monoclonal antibody product is for the single determinant on antigen.Thus, the modifier " monoclonal " indicates that the antibody derives from the feature of substantially homologous antibody colony, and should not be construed as needing to produce the antibody by any ad hoc method.For example, the monoclonal antibody to be used according to the application can be prepared by various techniques, and the technology includes, but is not limited to hybridoma method, recombinant DNA method, phage display method and the method for using all or part of the transgenic animal comprising human immunoglobulin locus, and such method and other exemplary methods for preparing monoclonal antibody are described herein.

[0048] 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 indicated, "binding affinity" as used in this specification refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D Affinity can be measured by common methods known in the art.

[0049] In this specification, human thymic stromal lymphopoietin (TSLP) refers to a cytokine derived from humans, and its amino acid sequence is shown in SEQ ID NO: 9, wherein the underlined portion represents a signal peptide.

[0050] SEQ ID NO: 9:

[0051]

[0052] In the present specification, "anti-human TSLP monoclonal antibody" refers to a monoclonal antibody that can bind to human TSLP with sufficient affinity so that the monoclonal antibody can be used as a diagnostic agent and / or therapeutic agent targeting human TSLP.

[0053] The anti-human TSLP monoclonal antibodies of the present application do not bind to proteins unrelated to their target. Here, "unrelated proteins" refers to proteins other than the target human TSLP; "no binding" means that, taking the binding ability of the anti-human TSLP monoclonal antibodies of the present application to the target human TSLP 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.

[0054] 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 macaques, pigs, dogs, rabbits, rats, mice, guinea pigs, etc.; and "not binding" means that, with the binding ability of the anti-human TSLP monoclonal antibodies of the present application to human TSLP as its target being taken as 100%, the binding ability of the anti-human TSLP monoclonal antibodies of the present application to TSLP from other animal species is less than 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.

[0055] The human TSLP monoclonal antibodies of the present application have equilibrium dissociation constants (K D ).

[0056] The experimental results show that the anti-human TSLP monoclonal antibody of the present application can specifically bind to human TSLP.

[0057] The anti-human TSLP monoclonal antibody of the present application is comparable to or superior to similar marketed monoclonal antibody products in many biological activities, such as neutralization of STAT5 phosphorylation in human, natural, and cynomolgus monkey TSLP-induced cells, and neutralization of TARC (CCL17) release induced by human TSLP in human whole blood and human PBMC cells.

[0058] In a specific embodiment, the amino acid sequence of the heavy chain of the anti-human TSLP monoclonal antibody of the present application is shown in SEQ ID NO: 10; the amino acid sequence of the light chain is shown in SEQ ID NO: 11.

[0059] SEQ ID NO: 10

[0060]

[0061] SEQ ID NO: 11

[0062]

[0063] Among them, SEQ ID NOs: 10 and 11 are both humanized sequences.

[0064] In this specification, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0065] In this specification, "isolated nucleic acid encoding an anti-TSLP monoclonal antibody" refers to 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 in one or more locations in a host cell.

[0066] As used herein, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term encompasses vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0067] In this specification, "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and the progeny derived therefrom (regardless of the number of generations). Progeny may not be identical to the parental cell in terms of nucleic acid content, but may contain mutations. Mutant progeny with the same function or biological activity that have been screened or selected for the initially transformed cells are included in this specification.

[0068] In this specification, "pharmaceutical composition" means a preparation that is in a form that allows the biological activity of the active ingredient contained therein to take effect, and that contains no additional components that are unacceptably toxic to the subject to which the formulation is to be administered.

[0069] In this specification, "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0070] In this application, "monoclonal antibody" is generally 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).

[0071] Antibodies can be prepared by administering immunogens to transgenic animals that have been modified to stimulate the production of complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. These animals typically contain a portion or all of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or are present outside the chromosomes 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; US Patent No. 5,770,429 describes Technology; US Patent No. 7,041,870 describes Technology, and U.S. Patent Application Publication No. US 2007 / 0061900 Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0072] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cells for producing 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 described in Li, J. et al., Proc. Natl. Acad. Sci. USA 103: 3557-3562 (2006). Other methods include those described in, for example, U.S. Pat. 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).

[0073] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries; these variable domain sequences can then be combined with the desired human constant domains.

[0074] Human antibodies can also be selected based on self-antibody libraries, that is, human antibodies can be isolated by screening combinatorial libraries for antibodies with desired one or more activities. For example, a variety of 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 is 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).

[0075] In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which is then screened for antigen-binding phage, as described in Winter, G. et al., Ann. Rev. Immunol. 12: 433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, non-immunized repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a large number of non-self and also self-antigens in the absence of any immunization, as described by Griffiths, AD et al., EMBO J, 12: 725-734 (1993). Finally, unimmunized libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described by 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 Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0076] The antibody can also be a multispecific antibody, such as a bispecific antibody. Bispecific antibodies are monoclonal antibodies with binding specificity to at least two different sites. The technology for generating multispecific antibodies includes but is not limited to the recombinant co-expression of two pairs of immunoglobulin heavy chain-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 "node-into-hole" engineering (see, for example, U.S. Patent No. 5,731,168). It can also be achieved by engineering electrostatic manipulation effects for generating antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., 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, e.g., Kostelny, SA et al., J. Immunol. 148:1547-1553 (1992)); using "diabody" technology for generating 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. 152:5368-5374 (1994)); and preparing trispecific antibodies (as described, e.g., in Tutt, A. et al., J. Immunol. 147:60-69 (1991)) to generate multispecific antibodies.

[0077] The monoclonal antibodies described herein also include engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies" (see, eg, US 2006 / 0025576).

[0078] The antibodies herein may also include the multispecific antibodies described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792, and WO 2010 / 145793, WO 2011 / 117330, WO 2012 / 025525, WO 2012 / 025530, WO 2013 / 026835, WO 2013 / 026831, WO 2013 / 164325, or WO 2013 / 174873.

[0079] The monoclonal antibodies described herein may also be antibody variants, for example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Suitable modifications may be introduced into the nucleotide sequence encoding the antibody, or amino acid sequence variants of the antibody may be prepared by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, as long as the final construct has the desired characteristics, such as antigen binding. Therefore, in certain embodiments, antibody variants with one or more amino acid substitutions are provided, and the sites of interest for substitution mutations include HVR and FR, for example, amino acid substitutions can be introduced into the antibody of interest and screened for products with the desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0080] Example

[0081] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0082] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0083] Example 1 Preparation of anti-human TSLP monoclonal antibody QX008N

[0084] Human thymic stromal lymphopoietin (hTSLP) was purchased from Shanghai Jinan Technology Co., Ltd. and used to immunize New Zealand rabbits. Using B cell cloning technology, antigen-binding specific antibody clones were obtained. Monoclonal antibodies that bind to human TSLP and exhibit inhibitory activity were then screened. Cell supernatants were analyzed using binding ELISA and blocking ELISA to identify target clones. The immunization and screening processes were outsourced to a commercial company.

[0085] Seven clones were selected for recombinant expression and sequencing. 8G2 was found to have the highest neutralizing activity in cells. Therefore, the 8G2 clone was humanized. Human IgG germline sequences were aligned using NCBI IgBlast. IGHV3-66*01 was selected as the heavy chain CDR grafting template, and the CDR regions of the heavy chain of the 8G2 clone (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 framework region of IGHV3-66*01. IGKV1-12*01 was selected as the light chain CDR grafting template, and the CDR regions of the light chain of the 8G2 clone (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 framework region of IGKV1-12*01. Specific sites in the framework region were backmutated 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; the amino acid sequence of the humanized light chain variable region is shown in SEQ ID NO: 8.

[0086] The heavy chain variable region gene (SEQ ID NO: 7) and the light chain variable region gene (SEQ ID NO: 8) were amplified by PCR. 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 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.

[0087] The results of nucleic acid electrophoresis for detecting the PCR-amplified variable region gene fragments and the double-enzyme digested plasmids are shown in Figure 1. As can be seen from the results in Figure 1, the PCR amplification results of the antibody heavy and light chain variable regions, as well as the double-enzyme digested heavy and light chain expression plasmids, show that the heavy and light chain plasmids are approximately 10,000 bp in size, with the heavy chain variable region being approximately 477 bp and the light chain variable region being approximately 447 bp.

[0088] The heavy chain expression plasmid pHZDCH-8G2VH-Hu27 (the amino acid sequence of the expressed heavy chain is shown in SEQ ID NO: 10) and the light chain expression plasmid pHZDCK-8G2VK-Hu14 (the amino acid sequence of the expressed light chain is shown in SEQ ID NO: 11) with the correct sequence were co-transfected into ExpiCHO-S cells. One day before transfection, ExpiCHO-S cells were diluted to 3×10 6On the day of transfection, the cell density was diluted to 6×10 6 cells / ml, 25ml of cells were placed in a 125ml shake flask and ready for transfection. The transfection and expression process is shown in Figure 2.

[0089] On day 6 after transfection, the culture supernatant was harvested and purified using Protein A. The purified antibody, designated QX008N (HZD8G2-57), was detected by SDS-PAGE electrophoresis. The results of protein electrophoresis for this antibody are shown in Figure 3. Protein electrophoresis was performed on a denaturing reducing gel. Figure 3 shows two bands, approximately 50 kDa and 25 kDa, respectively, which are consistent with the theoretical molecular weights of the heavy chain (49.3 kDa) and light chain (23.6 kDa).

[0090] Example 2 Equilibrium dissociation constant (K D )

[0091] Biacore T200 was used to detect the affinity of QX008N (HZD8G2-57) to human TSLP, and all processes were carried out at 25°C. A commercial ProteinA chip was used, and an appropriate amount of antibody was fixed by the capture method so that Rmax was around 50RU, and the capture flow rate was 10μl / min. The antigen was gradient diluted, and the instrument flow rate was switched to 30μl / min. The flow was passed through the reference channel and the channel of the fixed antibody in order from low to high concentration, and the buffer solution was used as a negative control. After each binding and dissociation was completed, the chip was regenerated with pH1.5 glycine. Use the instrument's own analysis software to select the 1:1 binding model in the Kinetics option for fitting, and calculate the binding rate constant k of the antibody. a , the dissociation rate constant k d and the dissociation equilibrium constant K D value.

[0092] In addition, the affinity of QX008N (HZD8G2-57) was compared with that of tezepelumab, a monoclonal antibody against human TSLP currently in Phase III clinical trials. The detection method for the known antibody was the same as that used for QX008N, and the results are shown in Table 1. Tezepelumab was produced in-house by transiently transfecting ExpiCHO-S cells with an expression plasmid constructed based on the A5 sequence provided in patent US20110274687A1.

[0093] Table 1 Affinity of anti-human TSLP monoclonal antibodies binding to human TSLP

[0094] Sample name k a (10 6 M -1 S-1 )k d (10 -5 S -1 )K D (10 -11 M)QX008N1.742.991.75Tezepelumab2.706.462.38

[0095] The data in the table are: each sample was tested three times and the average value was calculated.

[0096] Example 3 QX008N and Tezepelumab Neutralize Human TSLP-Induced STAT5 Phosphorylation Activity in SW756-STAT5-Luciferase Reporter Cells

[0097] The SW756-STAT5-Luciferase reporter gene cell line was used to determine the activity of QX008N in antagonizing the phosphorylation of the intracellular signaling molecule STAT5 mediated by human TSLP through TSLPR-IL-7R: cells in the culture medium were plated at 4×10 4 Cells were added to 96-well plates and incubated overnight at 37°C and 5% CO2. A pre-incubated mixture of antibodies and human TSLP was added to the cells, with final concentrations of QX008N ranging from 0 to 50 ng / ml, tezepelumab ranging from 0 to 400 ng / ml, and TSLP at a final concentration of 0.5 ng / ml. The cells were then incubated at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was discarded, and 120 μl of ONE-Glo-Luciferase Reagent was added to each well for 30 minutes. 100 μl of the sample was then transferred to a white 96-well plate. Luminescence fluorescence signals were measured, and a dose-effect curve was plotted to analyze the antagonistic activity of the antibodies. The dose-effect curve is shown in Figure 4.

[0098] The results shown in Figure 4 show that QX008N can inhibit the phosphorylation of STAT5 in SW756-STAT5-Luciferase reporter gene cells induced by human TSLP. The IC value of QX008N for inhibiting the phosphorylation of STAT5 in SW756-STAT5-Luciferase reporter gene cells induced by human TSLP is 50 The IC value of Tezepelumab for inhibiting STAT5 phosphorylation activity induced by human TSLP in SW756-STAT5-Luciferase reporter gene cells was 0.837 ng / ml. 50 It is 3.8ng / ml.

[0099] Example 4 QX008N and Tezepelumab Neutralize Natural TSLP-Induced STAT5 Phosphorylation Activity in SW756-STAT5-Luciferase Reporter Cells

[0100] The SW756-STAT5-Luciferase reporter gene cell line was used to determine the activity of QX008N in antagonizing the phosphorylation of the intracellular signaling molecule STAT5 mediated by natural TSLP through TSLPR-IL-7R: cells in the culture medium were plated at 4×10 4 Cells were added to 96-well plates and incubated overnight at 37°C and 5% CO2. A pre-incubated mixture of antibodies and native TSLP was added to the cells. The final concentrations of QX008N ranged from 0 to 50 ng / ml, and the final concentration of tezepelumab ranged from 0 to 400 ng / ml. The final concentration of native TSLP was a 62.5-fold dilution of the stock solution. The cells were then incubated at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was discarded, and 120 μl of ONE-Glo-Luciferase Reagent was added to each well for 30 minutes. 100 μl of the sample was then transferred to a white 96-well plate. Luminescence fluorescence signals were measured, and a dose-effect curve was plotted to analyze the antagonistic activity of the antibodies. The dose-effect curve is shown in Figure 5.

[0101] The results shown in Figure 5 show that QX008N can inhibit the natural TSLP-induced STAT5 phosphorylation in SW756-STAT5-Luciferase reporter gene cells. The IC value of QX008N for inhibiting the natural TSLP-induced STAT5 phosphorylation activity in SW756-STAT5-Luciferase reporter gene cells is 50 The IC value of Tezepelumab for inhibiting the STAT5 phosphorylation activity induced by natural TSLP in SW756-STAT5-Luciferase reporter gene cells was 0.462 ng / ml. 50 It is 1.45ng / ml.

[0102] Example 5 QX008N and Tezepelumab Neutralize TSLP-Induced STAT5 Phosphorylation Activity in Cynomolgus Monkey SW756-STAT5-Luciferase Reporter Cells

[0103] The SW756-STAT5-Luciferase reporter gene cell line was used to determine the activity of QX008N in antagonizing the phosphorylation of the intracellular signaling molecule STAT5 mediated by TSLP in cynomolgus monkeys through TSLPR-IL-7R: 4×10 cells were cultured in each well. 4Cells were added to 96-well plates and incubated overnight at 37°C and 5% CO₂. A pre-incubated mixture of antibodies and cynomolgus monkey TSLP was added to the cells, with final concentrations of QX008N ranging from 0 to 50 ng / ml, tezepelumab ranging from 0 to 400 ng / ml, and cynomolgus monkey TSLP at a final concentration of 0.5 ng / ml. The cells were then incubated at 37°C and 5% CO₂ for 24 hours. The cell culture supernatant was discarded, and 120 μl of ONE-Glo-Luciferase Reagent was added to each well for 30 minutes. 100 μl of the solution was then transferred to a white 96-well plate, and the luminescence fluorescence signal was measured. A dose-effect curve was then plotted to analyze the antagonistic activity of the antibody. The dose-effect curve is shown in Figure 6.

[0104] The results shown in Figure 6 show that QX008N can inhibit the phosphorylation of STAT5 in SW756-STAT5-Luciferase reporter gene cells induced by TSLP in cynomolgus monkeys. The IC value of QX008N in inhibiting the phosphorylation activity of STAT5 in SW756-STAT5-Luciferase reporter gene cells induced by TSLP in cynomolgus monkeys is 50 The IC value of Tezepelumab for inhibiting STAT5 phosphorylation activity induced by TSLP in SW756-STAT5-Luciferase reporter gene cells of cynomolgus monkeys was 0.889 ng / ml. 50 It is 1.88ng / ml.

[0105] Example 6 QX008N and Tezepelumab neutralize human TSLP-induced TARC (CCL17) release activity in human whole blood

[0106] The activity of QX008N in antagonizing human TSLP-induced TARC (CCL17) release via the TSLPR-IL-7R was determined using human whole blood: 100 μl of whole blood was added to a 96-well plate and stored at 37°C and 5% CO₂. A pre-incubated mixture of antibody and human TSLP was added to the whole blood at final concentrations ranging from 0 to 10 μg / ml, and a final concentration of 0.5 ng / ml of human TSLP. IL-33 was also added at a final concentration of 0.5 ng / ml. Cells were then cultured at 37°C and 5% CO₂ for 48 hours. The cell culture supernatants were collected and TARC (CCL17) expression in the supernatants was assayed by sandwich ELISA. A dose-response curve was then constructed to analyze the antibody's antagonistic activity. The dose-response curve is shown in Figure 7.

[0107] The results shown in Figure 7 show that QX008N can inhibit the release of TARC (CCL17) from whole blood induced by human TSLP. The IC value of QX008N for inhibiting the release of TARC (CCL17) from whole blood induced by human TSLP is 50 The IC value of Tezepelumab for inhibiting the activity of TARC (CCL17) released from whole blood induced by human TSLP was 0.839 ng / ml. 50 It is 23.9ng / ml.

[0108] Example 7 QX008N and Tezepelumab neutralize human TSLP to induce TARC (CCL17) release from human PBMC cells

[0109] The activity of QX008N in antagonizing human TSLP-induced TARC (CCL17) release via the TSLPR-IL-7R was determined using human PBMCs: PBMCs were isolated by density gradient centrifugation and plated at 300,000 cells / well in 96-well plates. The plates were then stored at 37°C and 5% CO₂. A pre-incubated mixture of antibody and human TSLP was added to the PBMCs at final concentrations ranging from 0 to 10 μg / ml, and a final concentration of 0.5 ng / ml of human TSLP was added. IL-33 was also added at a final concentration of 0.5 ng / ml. The cells were then cultured at 37°C and 5% CO₂ for 48 hours. The cell culture supernatants were collected and TARC (CCL17) expression in the supernatants was assessed by sandwich ELISA. A dose-response curve was constructed to analyze the antagonistic activity of the antibody, as shown in Figure 8.

[0110] The results shown in Figure 8 show that QX008N can inhibit the release of TARC (CCL17) from PBMC cells induced by human TSLP. The IC value of QX008N inhibiting the release of TARC (CCL17) from PBMC cells induced by human TSLP is 50 The IC value of Tezepelumab for inhibiting the release of TARC (CCL17) from PBMC cells induced by human TSLP was 77.1 ng / ml. 50 It is 216ng / ml.

[0111] Although the embodiments of the present application are described above, the present application is not limited to the above-mentioned specific embodiments and application fields. The above-mentioned specific embodiments are merely illustrative and instructive, and not restrictive. A person of ordinary skill in the art, guided by this specification and without departing from the scope of protection of the claims of this application, may also devise various forms, all of which fall within the scope of protection of this application.

Claims

1. A monoclonal antibody against human thymic stromal lymphopoietin (TSLP), characterized in that, it 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: the amino acid sequence of CDR-H1 is as shown in SEQ ID NO: 1; the amino acid sequence of CDR-H2 is as shown in SEQ ID NO: 2; the amino acid sequence of CDR-H3 is as shown in SEQ ID NO: 3; the amino acid sequence of CDR-L1 is as shown in SEQ ID NO: 4; the amino acid sequence of CDR-L2 is as shown in SEQ ID NO: 5; the amino acid sequence of CDR-L3 is as shown in SEQ ID NO:

6.

2. The monoclonal antibody according to claim 1, characterized in that, it comprises a heavy chain variable region and a light chain variable region, wherein, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

8.

3. An isolated nucleic acid, characterized in that, it encodes the monoclonal antibody according to claim 1 or 2.

4. A host cell, characterized in that, it comprises the nucleic acid according to claim 3.

5. A method for producing a monoclonal antibody, characterized in that, the method comprises culturing the host cell according to claim 4 to produce the monoclonal antibody according to claim 1 or 2.

6. A pharmaceutical composition, characterized in that, it comprises the monoclonal antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, it is used for treating diseases related to TSLP-mediated signal transduction.

8. The pharmaceutical composition according to claim 7, characterized in that, the diseases related to TSLP-mediated signal transduction are allergic asthma, atopic dermatitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis fibrosis and inflammatory bowel disease, etc.

9. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a drug for treating diseases related to TSLP-mediated signal transduction.

10. The use according to claim 9, characterized in that, the diseases related to TSLP-mediated signal transduction are allergic asthma, atopic dermatitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis fibrosis and inflammatory bowel disease, etc.