Monoclonal antibody to TSLP and use thereof

Abstract

The present invention relates to the field of biotechnology and medicine, in particular to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to thymic stromal lymphopoietin (TSLP). The invention further relates to nucleic acids encoding said antibody, expression vectors, host cells and methods for producing same, methods for producing the antibodies according to the invention, pharmaceutical compositions comprising the antibody according to the invention, pharmaceutical compositions comprising the antibody according to the invention and other therapeutically active compounds, methods for treating diseases or disorders mediated by thymic stromal lymphopoietin (TSLP), uses of the antibodies or pharmaceutical compositions thereof for treating diseases or disorders mediated by TSLP, and uses of the antibodies and other therapeutically active compounds for treating diseases or disorders mediated by TSLP.

Description

[0001] MONOCLONAL ANTIBODY TO TSLP AND USE THEREOF

[0002] Field of the invention

[0003] The present invention relates to the field of biotechnology and medicine, in particular to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to thymic stromal lymphopoietin (TSLP). The invention further relates to nucleic acids encoding said antibody, expression vectors, host cells and methods for producing same, methods for producing the antibodies according to the invention, pharmaceutical compositions comprising the antibody according to the invention, pharmaceutical compositions comprising the antibody according to the invention and other therapeutically active compounds, methods for treating diseases or disorders mediated by thymic stromal lymphopoietin (TSLP), uses of the antibodies or pharmaceutical compositions thereof for treating diseases or disorders mediated by TSLP, and uses of the antibodies and other therapeutically active compounds for treating diseases or disorders mediated by TSLP.

[0004] Background of the invention

[0005] Monoclonal antibodies in the form of chimeric, humanized or fully human molecules have proven to be useful as effective medicine for treating multiple disorders and diseases.

[0006] Thymic stromal lymphopoietin (TSLP) is a cytokine produced by epithelial cells that plays a crucial role in initiating type 2 inflammation through both innate and adaptive immune responses (Mitchell PD, O'Byrne PM. Epithelial-derived cytokines in asthma. CHEST. 2017; 151(6): 1338— 1344; Corren J, Ziegler SF. TSLP: from allergy to cancer. Nat Immunol. 2019;20(12): 1603-1609). TSLP is primarily found in epithelial cells of barrier surfaces such as the skin, airways, and gastrointestinal tract, where it helps the organism sensitize the external environment (Gauvreau, G. M., Sehmi, R., Ambrose, C. S., & Griffiths, J. M. (2020). Thymic stromal lymphopoietin: its role and potential as a therapeutic target in asthma. Expert Opinion on Therapeutic Targets, 24(8), 777-792). TSLP is also produced by fibroblasts and dendritic cells in response to a variety of external factors.

[0007] The functions of TSLP are diverse (Brusselle G, Bracke K. Targeting immune pathways for therapy in asthma and chronic obstructive pulmonary disease. Ann Am Thorac Soc. 2014; 1 l(Suppl 5):S322-328), and include:

[0008] • facilitation of development of Th2 cells by means of increasing OX40L expression in dendritic cells,

[0009] • promotion of differentiation of CD4+ T cells into Th2 cells,

[0010] • induction of basophils to produce IL-4, activation of ILC2s (group 2 innate lymphoid cell), induction of corticosteroid resistance.

[0011] Patent documents W02009035577, WO2021043221, WO2021104053 disclose antibodies to TSLP.

[0012] In connection with the above, there is a need to create antibodies that specifically bind to TSLP.

[0013] Disclosure of the invention

[0014] The authors of the present group of inventions have developed antibodies that specifically bind to TSLP and have high affinity parameters for binding to TSLP. The antibodies of the invention block TSLP-dependent STAT5 signaling of the HEK293 STAT5-FLuc TSLPR / IL-7Ra cell line and do so with more than 2-fold activity than that of the studied prior-art anti-TSLP antibody. The antibodies of the invention have high stability in both human and mouse serum, in particular compared to that of the studied prior-art TSLP antibody. The antibodies of the invention competitively block the binding of TSLPR-Fc (SEQ ID NO: 34) to TSLP. The antibodies of the invention do not compete with other test antibodies for binding to TSLP, which fact suggests that they interact with different epitopes. The antibodies of the invention downregulate TARC secretion by peripheral blood mononuclear cells, and do so more efficiently than the studied prior-art antibodies to TSLP. The antibodies of the invention reduce leukocyte infiltration in the lung tissue, goblet cell hyperplasia and the concentration of ovalbumin-specific IgE in serum of test animals. The antibodies of the invention reduce the proportion of eosinophils to CD45+cells in bronchoalveolar lavage fluid and the concentration of total IgE in test animals compared to the placebo group as well as compared to the group receiving the prior-art antibody to TSLP.

[0015] Definitions and general methods

[0016] Unless defined otherwise herein, all technical and scientific terms used in connection with the present invention will have the same meaning as is commonly understood by those skilled in the art.

[0017] Furthermore, unless otherwise required by context, singular terms shall include plural terms, and the plural terms shall include the singular terms. Typically, the present classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medical and pharmaceutical chemistry, as well as hybridization and chemistry of protein and nucleic acids described herein are well known by those skilled and widely used in the art. Enzyme reactions and purification methods are performed according to the manufacturer's guidelines, as is common in the art, or as described herein.

[0018] The term "KD" in this description refers to the affinity constant (or equilibrium dissociation constant), which is calculated from the ratio of Kd to Ka (i.e. Kd / Ka), and it is expressed as a molar concentration (M). "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g. an antigen). Unless indicated otherwise, "binding affinity" refers to intrinsic (characteristic, true) binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g. antibody and antigen). The affinity of a molecule X for its binding partner Y can generally be represented by the equilibrium dissociation constant (KD). The preferred Kd value is about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or less. Affinity can be measured by common methods known in the art, including those described in the present description. Low- affinity antibodies typically bind an antigen slowly and tend to dissociate readily, whereas high- affinity antibodies typically bind an antigen faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any one of these methods may be used for the purposes of the present invention.

[0019] The term "Kd", "koff' or "kdis" refers to the off rate constant of a particular interaction between a binding molecule and antigen. The koff dissociation rate constant can be measured using bio-layer interferometry: for example, using the Octet™ system; as well as by surface plasmon resonance: for example, using the Biacore™ system.

[0020] The term "Ka", "kon" or "on-rate" refers to the association rate constant.

[0021] The term "ED50" (EC50) (50% effective dose / concentration, half maximal effective concentration) refers to concentrations of a formulation producing 50% biological effect (which may include cytotoxicity).

[0022] The terms "anti-TSLP antibody", "antibody to TSLP", "antibody that specifically binds to TSLP" are interchangeable in the context of the present application and refer to an antibody that specifically binds to TSLP.

[0023] Antigen — a protein which the antibody (antibodies) in question specifically binds to.

[0024] Genetic construct (plasmid) — an artificially created circular DNA molecule comprising various elements necessary for the expression of target genes and replication within organisms.

[0025] Proliferation — division of cells.

[0026] Reporter cell line — a cell line carrying a reporter gene for evaluation of intracellular signaling.

[0027] The terms "transformation" and "transforming" refer to altering of the genetic characteristics of a cell, and the cell is considered transformed if it has been modified by way of introducing a foreign DNA to the cell. The term "transfection" refers to a method for DNA delivery where foreign DNA molecules are delivered to a cell using chemical or physical methods: as part of liposomal complexes, as a result of electroporation, and the like.

[0028] Chromatography — a method of separation and analysis of mixtures of substances, as well as a study of physico-chemical properties of substances. It is based on distribution of substances between two phases - the stationary phase (solid phase or liquid coated on an inert carrier) and the mobile phase (gas or liquid phase, eluent).

[0029] CHO — Chinese hamster ovary cell line.

[0030] Fc (fragment crystallizable) — constant fragment region of an antibody.

[0031] Luc — luciferase.

[0032] PBMC — peripheral blood mononuclear cells.

[0033] ELISA - enzyme-linked immunosorbent assay.

[0034] As used in the present description and claims that follow, unless otherwise dictated by the context, the words "include" and "comprise", or variations thereof such as "includes", "including", "comprises", or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0035] Antibody

[0036] The present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP.

[0037] The term "monoclonal antibody" or "mAb" refers to an antibody that is synthesized and isolated as an individual clonal population of cells.

[0038] The antibody of the invention is a recombinant antibody.

[0039] The term "recombinant antibody" refers to an antibody that is expressed in a cell or cell line comprising nucleotide sequence(s) encoding an antibody, wherein said nucleotide sequence(s) is (are) not associated with the cell in nature.

[0040] In one aspect, the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP comprising:

[0041] (a) a light chain variable domain comprising:

[0042] (i) CDR1 with the amino acid sequence of SEQ ID NO: 1;

[0043] (ii) CDR2 with an amino acid sequence selected from the group: SEQ ID NO: 2 or SEQ ID NO: 3; and

[0044] (iii) CDR3 with the amino acid sequence of SEQ ID NO: 4; and

[0045] (b) a heavy chain variable domain comprising: (i) CDR1 with the amino acid sequence of SEQ ID NO: 5;

[0046] (ii) CDR2 with an amino acid sequence selected from the group: SEQ ID NO: 6 or SEQ ID

[0047] NO: 7; and iii) CDR3 with the amino acid sequence of SEQ ID NO: 8.

[0048] In one embodiment of the invention, the antibody according to the invention is an isolated antibody.

[0049] The term "isolated" used to describe various antibodies according to the present description refers to an antibody which has been identified and isolated and / or regenerated from a cell or cell culture, in which the antibody is expressed. Impurities (contaminant components) from natural environment are materials which typically interfere with diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. The isolated polypeptide is typically prepared by at least one purification step.

[0050] The term "antibody" or "immunoglobulin" (Ig) as used in the present description includes whole antibodies. The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated referred to in the present description as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated referred to in the present description as VL) and light chain constant region. The light chain constant domain can be CK (kappa light chain constant domain) or CL (lambda light chain constant domain). Preferably the light chain is a lambda (A) light chain, and the light chain constant domain is preferably CL.

[0051] Antibodies according to the invention can be of any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, preferably IgGl).

[0052] VL and VH regions may be further subdivided into hyper-variability regions called complementarity determining regions (CDRs), located between regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains form a binding domain that interacts with an antigen.

[0053] The constant regions of antibodies may mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system. The term "antigen-binding portion" of antibody or "antigen-binding fragment", as used in the present description, refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It was shown that the antigen-binding function of antibody can be performed by fragments of a full-length antibody. Examples of binding fragments which are included within the term "antigen-binding portion" of an antibody include (i) Fab-fragment, monovalent fragment, consisting of VL, VH, CL and CHI domains; (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab-fragments linked by a disulfide bridge at the hinge region; (iii) Fd-fragment consisting of VH and CHI domains; (iv) Fv-fragment consisting of VL and VH domains of a single arm of an antibody; (v) dAb-fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH / VHH domain. In addition, two regions of the Fv-fragment, VL and VH, are encoded by different genes, they can be joined using recombinant methods using a synthetic linker that enables to receive them as a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as a single-chain Fv (scFv); see e.g. Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). It is assumed that such single-stranded molecules are also included within the term "antigen -binding portion" of antibody. Such antibody fragments are produced using conventional techniques known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies are.

[0054] "Kabat numbering scheme" or "numbering according to Kabat" as used in the present application refers to the system for numbering of amino acid residues that are more variable (i.e. hypervariable) than other amino acid residues in variable regions of heavy and light chains of antibody (Kabat et al. Ann. N.Y. Acad. Sci., 190:382-93 (1971); Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0055] The antibody of the present invention "which specifically binds" to a target antigen refers to an antibody that binds an antigen with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting a protein or cell or tissue expressing the antigen.

[0056] The term "specifically binds to" a particular polypeptide or an epitope on a particular target polypeptide may be described by example of a molecule having a Kd for the target of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or at least about 100 pM or less. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on a polypeptide.

[0057] In some embodiments of the invention, the monoclonal antibody or antigen-binding fragment thereof includes:

[0058] (i) a light chain variable domain comprising:

[0059] CDR1 with the amino acid sequence of SEQ ID NO: 1,

[0060] CDR2 with the amino acid sequence of SEQ ID NO: 2 and

[0061] CDR3 with the amino acid sequence of SEQ ID NO: 4; or

[0062] (ii) a light chain variable domain comprising:

[0063] CDR1 with the amino acid sequence of SEQ ID NO: 1,

[0064] CDR2 with the amino acid sequence of SEQ ID NO: 3 and

[0065] CDR3 with the amino acid sequence of SEQ ID NO: 4.

[0066] In some embodiments of the invention, the monoclonal antibody or antigen-binding fragment thereof includes:

[0067] (i) a heavy chain variable domain comprising:

[0068] CDR1 with the amino acid sequence of SEQ ID NO: 5,

[0069] CDR2 with the amino acid sequence of SEQ ID NO: 6 and

[0070] CDR3 with the amino acid sequence of SEQ ID NO: 8; or

[0071] (ii) a heavy chain variable domain comprising:

[0072] CDR1 with the amino acid sequence of SEQ ID NO: 5,

[0073] CDR2 with the amino acid sequence of SEQ ID NO: 7 and

[0074] CDR3 with the amino acid sequence of SEQ ID NO: 8.

[0075] In some embodiments of the invention, the monoclonal antibody or antigen-binding fragment thereof includes:

[0076] (i) (a) a light chain variable domain comprising:

[0077] CDR1 with the amino acid sequence of SEQ ID NO: 1,

[0078] CDR2 with the amino acid sequence of SEQ ID NO: 2 and

[0079] CDR3 with the amino acid sequence of SEQ ID NO: 4; and

[0080] (b) a heavy chain variable domain comprising:

[0081] CDR1 with the amino acid sequence of SEQ ID NO: 5,

[0082] CDR2 with the amino acid sequence of SEQ ID NO: 6 and

[0083] CDR3 with the amino acid sequence of SEQ ID NO: 8; or

[0084] (ii) (a) a light chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1,

[0085] CDR2 with the amino acid sequence of SEQ ID NO: 3 and

[0086] CDR3 with the amino acid sequence of SEQ ID NO: 4; and

[0087] (b) a heavy chain variable domain comprising:

[0088] CDR1 with the amino acid sequence of SEQ ID NO: 5,

[0089] CDR2 with the amino acid sequence of SEQ ID NO: 7 and

[0090] CDR3 with the amino acid sequence of SEQ ID NO: 8; or

[0091] (iii) (a) a light chain variable domain comprising:

[0092] CDR1 with the amino acid sequence of SEQ ID NO: 1,

[0093] CDR2 with the amino acid sequence of SEQ ID NO: 3 and

[0094] CDR3 with the amino acid sequence of SEQ ID NO: 4; and

[0095] (b) a heavy chain variable domain comprising:

[0096] CDR1 with the amino acid sequence of SEQ ID NO: 5,

[0097] CDR2 with the amino acid sequence of SEQ ID NO: 6 and

[0098] CDR3 with the amino acid sequence of SEQ ID NO: 8.

[0099] In some embodiments of the invention, the monoclonal antibody or antigen-binding fragment thereof includes a light chain variable domain that comprises an amino acid sequence that is selected from the group: SEQ ID NO: 9 or SEQ ID NO: 10.

[0100] In some embodiments of the invention, the monoclonal antibody or antigen-binding fragment thereof includes a heavy chain variable domain that comprises an amino acid sequence that is selected from the group: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.

[0101] In some embodiments of the invention, the monoclonal antibody or antigen-binding fragment thereof includes:

[0102] (a) a light chain variable domain which comprises an amino acid sequence selected from the group: SEQ ID NO: 9 or SEQ ID NO: 10; and

[0103] (b) a heavy chain variable domain that comprises an amino acid sequence that is selected from the group: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.

[0104] In some embodiments of the invention, the monoclonal antibody or antigen-binding fragment thereof includes:

[0105] (i) (a) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 9 and

[0106] (b) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:

[0107] 11; or (ii) (a) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:

[0108] 9 and

[0109] (b) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 12; or

[0110] (iii) (a) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:

[0111] 10 and

[0112] (b) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 13; or

[0113] (iv) (a) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 10 and

[0114] (b) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 14; or

[0115] (v) (a) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 10 and

[0116] (b) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 11.

[0117] In some embodiments of the invention, the monoclonal antibody that specifically binds to TSLP is a full-length IgG antibody.

[0118] In some embodiments of the invention, the monoclonal antibody that specifically binds to TSLP is a full-length IgG antibody that is of human IgGl, IgG2, IgG3 or IgG4 isotype.

[0119] In some embodiments of the invention, the monoclonal antibody that specifically binds to TSLP is a full-length IgG antibody that is of human IgGl isotype.

[0120] In some embodiments of the invention, the monoclonal antibody comprises, in the Fc fragment, mutations M252Y, S254T, T256E, according to the EU numbering scheme of amino acids of antibodies, in the CH2 region (Edelman G.M. et al., Proc. Natl. Acad. Sci. USA 63 (1969) pp. 78- 85; Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991).

[0121] In some embodiments of the invention, the monoclonal antibody comprises mutations L234A and L235A, according to the EU numbering scheme of amino acids of antibodies, in the CH2 region.

[0122] In some embodiments of the invention, the monoclonal antibody comprises deletions 446G and 447K, according to the EU numbering scheme of amino acids of antibodies, in the CH3 region.

[0123] In some embodiments of the invention, the monoclonal antibody comprises a light chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 15 or SEQ ID NO: 16. In some embodiments of the invention, the monoclonal antibody includes a heavy chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

[0124] In some embodiments of the invention, the monoclonal antibody includes:

[0125] (i) (a) a light chain comprising an amino acid sequence selected from the group: SEQ ID NO: 15 or SEQ ID NO: 16, and

[0126] (b) a heavy chain comprising an amino acid sequence selected from the group: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

[0127] In some embodiments of the invention, the monoclonal antibody includes:

[0128] (i) (a) a light chain comprising the amino acid sequence of SEQ ID NO: 15, and

[0129] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 17; or

[0130] (ii) (a) a light chain comprising the amino acid sequence of SEQ ID NO: 15, and

[0131] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 18; or

[0132] (iii) (a) a light chain comprising the amino acid sequence of SEQ ID NO: 16, and

[0133] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 19; or

[0134] (iv) (a) a light chain comprising the amino acid sequence of SEQ ID NO: 16, and

[0135] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 20; or

[0136] (v) (a) a light chain comprising the amino acid sequence of SEQ ID NO: 16, and

[0137] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 17.

[0138] In some embodiments, the monoclonal antibody that specifically binds to TSLP is an antibody selected from the group: AB1, AB2, AB3, AB4, or AB5.

[0139] In some embodiments of the invention, the monoclonal antibody that specifically binds to TSLP is antibody AB1.

[0140] Antibody AB1 includes:

[0141] (a) a light chain comprising the amino acid sequence of SEQ ID NO: 15; and

[0142] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 17.

[0143] Antibody AB1 includes:

[0144] (a) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 9;

[0145] (b) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 11.

[0146] Antibody AB1 includes:

[0147] (a) a light chain variable domain comprising:

[0148] (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,

[0149] (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2, (iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 4, and

[0150] (b) a heavy chain variable domain comprising:

[0151] (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 5,

[0152] (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 6,

[0153] (iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 8.

[0154] In some embodiments of the invention, the monoclonal antibody that specifically binds to TSLP is antibody AB2.

[0155] Antibody AB2 includes:

[0156] (a) a light chain comprising the amino acid sequence of SEQ ID NO: 15; and

[0157] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 18.

[0158] Antibody AB2 includes:

[0159] (a) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 9;

[0160] (b) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:

[0161] 12.

[0162] Antibody AB2 includes:

[0163] (a) a light chain variable domain comprising:

[0164] (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,

[0165] (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,

[0166] (iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 4, and

[0167] (b) a heavy chain variable domain comprising:

[0168] (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 5,

[0169] (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 6,

[0170] (iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 8.

[0171] In some embodiments of the invention, the monoclonal antibody that specifically binds to TSLP is antibody AB 3.

[0172] Antibody AB3 includes:

[0173] (a) a light chain comprising the amino acid sequence of SEQ ID NO: 16; and

[0174] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 19.

[0175] Antibody AB3 includes:

[0176] (a) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 10;

[0177] (b) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:

[0178] 13.

[0179] Antibody AB3 includes: (a) a light chain variable domain comprising:

[0180] (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,

[0181] (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 3,

[0182] (iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 4, and

[0183] (b) a heavy chain variable domain comprising:

[0184] (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 5,

[0185] (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 7,

[0186] (iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 8.

[0187] In some embodiments of the invention, the monoclonal antibody that specifically binds to TSLP is antibody AB4.

[0188] Antibody AB4 includes:

[0189] (a) a light chain comprising the amino acid sequence of SEQ ID NO: 16; and

[0190] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 20.

[0191] Antibody AB4 includes:

[0192] (a) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 10;

[0193] (b) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 14.

[0194] Antibody AB4 includes:

[0195] (a) a light chain variable domain comprising:

[0196] (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,

[0197] (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 3,

[0198] (iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 4, and

[0199] (b) a heavy chain variable domain comprising:

[0200] (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 5,

[0201] (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 6,

[0202] (iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 8.

[0203] In some embodiments of the invention, the monoclonal antibody that specifically binds to TSLP is antibody AB 5.

[0204] Antibody AB 5 includes:

[0205] (a) a light chain comprising the amino acid sequence of SEQ ID NO: 16; and

[0206] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 17.

[0207] Antibody AB 5 includes:

[0208] (a) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 10; (b) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:

[0209] 11.

[0210] Antibody AB 5 includes:

[0211] (a) a light chain variable domain comprising:

[0212] (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,

[0213] (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 3,

[0214] (iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 4, and

[0215] (b) a heavy chain variable domain comprising:

[0216] (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 5,

[0217] (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 6,

[0218] (iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 8.

[0219] The hypervariable regions of variable domains of light and heavy chains (LCDR1, 2, 3 and HCDR1, 2, 3) of all the above antibodies are provided in accordance with the Kabat nomenclature. Those skilled will appreciate that the hypervariable regions of variable domains of light and heavy chains (LCDR1, 2, 3 and HCDR1, 2, 3) may also be represented in accordance with other commonly known numbering scheme, for example, IMGT, Chothia or AbM. Thus, all of the above antibodies which are characterized by means of hypervariable regions of variable domains of light and heavy chains (LCDR1, 2, 3 and HCDR1, 2, 3) using the IMGT, Chothia or AbM numbering scheme are also encompassed by the present invention.

[0220] The authors of the present group of inventions have developed antibodies that specifically bind to TSLP and have high affinity parameters for binding to TSLP. The antibodies of the invention block TSLP-dependent STAT5 signaling of the HEK293 STAT5-FLuc TSLPR / IL-7Ra cell line and do so with more than 2-fold activity than that of the studied prior-art anti-TSLP antibody. The antibodies of the invention have high stability in both human and mouse serum, in particular compared to that of the studied prior-art TSLP antibody. The antibodies of the invention competitively block the binding of TSLPR-Fc (SEQ ID NO: 34) to TSLP. The antibodies of the invention do not compete with other test antibodies for binding to TSLP, which fact suggests that they interact with different epitopes. The antibodies of the invention downregulate TARC secretion by peripheral blood mononuclear cells, and do so more efficiently than the studied prior-art antibodies to TSLP. The antibodies of the invention reduce leukocyte infiltration in the lung tissue, goblet cell hyperplasia and the concentration of ovalbumin-specific IgE in serum of test animals. The antibodies of the invention reduce the proportion of eosinophils to CD45+cells in bronchoalveolar lavage fluid and the concentration of total IgE in test animals compared to the placebo group as well as compared to the group receiving the prior-art antibody to TSLP. Nucleic acid molecule

[0221] In one aspect, the present invention relates to a nucleic acid that encodes any one of the above antibody or antigen-binding fragment thereof that specifically binds to TSLP.

[0222] In any one of said embodiments, the nucleic acid molecules may be isolated.

[0223] The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", used interchangeably in the present description, mean a precise sequence of nucleotides, modified or not, determining a fragment or a region of a nucleic acid, containing or not containing unnatural nucleotides, and being either a double-strand DNA or RNA, a single-strand DNA or RNA, or transcription products of said DNAs, or reverse transcription products of said RNAs.

[0224] Unless otherwise indicated, the term nucleotide sequence encompasses its complement. Thus, a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof.

[0225] An "isolated" nucleic acid molecule is one which is identified and separated from at least one nucleic acid molecule-impurity. An isolated nucleic acid molecule is different from the form or set in which it is found under natural conditions. Thus, an isolated nucleic acid molecule is different from a nucleic acid molecule that exists in cells under natural conditions.

[0226] In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NO: 1-20. A nucleic acid molecule may also comprise any combination of said nucleotide sequences.

[0227] As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequence of the light chain or heavy chain of the antibody according to the invention or fragments thereof (VH, VL, CDR, etc.). It is well within the skill of those trained in the art to create these alternative DNA sequences encoding one and the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

[0228] In some embodiments of the invention, the nucleic acid is DNA.

[0229] The nucleic acid molecule of the present invention may be isolated from any source that produces the monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP. In certain embodiments of the invention, the nucleic acid molecule of the invention may be synthesized by way of chemical synthesis, rather than isolated.

[0230] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibodies AB1, AB2, and includes a nucleotide sequence with SEQ ID NO: 21. In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibodies AB3, AB4, AB5, and includes a nucleotide sequence with SEQ ID NO: 22.

[0231] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibodies AB1, AB5, and includes a nucleotide sequence with SEQ ID NO: 23.

[0232] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibody AB2, and includes the nucleotide sequence with SEQ ID NO: 24.

[0233] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibody AB3, and includes the nucleotide sequence with SEQ ID NO: 25.

[0234] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibody AB4, and includes the nucleotide sequence with SEQ ID NO: 26.

[0235] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibodies AB1, AB2, and includes a nucleotide sequence with SEQ ID NO: 27.

[0236] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibodies AB3, AB4, AB5, and includes a nucleotide sequence with SEQ ID NO: 28.

[0237] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibodies AB1, AB5, and includes a nucleotide sequence with SEQ ID NO: 29.

[0238] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody AB2, and includes the nucleotide sequence with SEQ ID NO: 30.

[0239] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody AB3, and includes the nucleotide sequence with SEQ ID NO: 31.

[0240] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody AB4, and includes the nucleotide sequence with SEQ ID NO: 32. The nucleic acid molecules may be used to express the recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP.

[0241] Vector

[0242] In one aspect, the present invention relates to an expression vector comprising any one of the above nucleic acid molecules that encode the corresponding amino acid sequences of the antibody that specifically binds to TSLP, or portions thereof (for example, heavy chain and / or light chain binding domain sequences). The present invention relates to a vector suitable for the expression of any one of nucleotide sequences described herein.

[0243] The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0244] As used in the present description, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence.

[0245] In some embodiments of the invention, the vector is a plasmid, i.e. a circular double stranded piece of DNA into which additional DNA segments may be inserted.

[0246] In some embodiments of the invention, the vector is a viral (expression) vector, wherein additional DNA segments may be inserted into the viral genome.

[0247] In some embodiments of the invention, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial site of replication origin and episomal vectors). In further embodiments of the invention, the vectors (e.g. non-episomal vectors) may be integrated into the genome of a host cell upon introduction into a host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0248] In some embodiments of the invention, expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, and the like. DNA molecules may be inserted into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of DNA. An expression vector and expression control sequences may be chosen to be compatible with the expression host cell used.

[0249] In one embodiment of the invention, DNA molecules encoding partially or fully heavy and light chain sequences can be inserted into distinct vectors.

[0250] In one embodiment, any combination of the above DNA molecules is introduced into the same expression vector. In one embodiment of the invention, DNA molecules may be introduced into an expression vector by standard methods (e.g. ligation of complementary restriction sites on a gene fragment of antibody and vector, or blunt end ligation if no restriction sites are present).

[0251] In some embodiments of the invention, a suitable vector is one that includes restriction sites such that any VH or VL sequence can easily be inserted and expressed, as described above. A recombinant expression vector can also encode a signal peptide that facilitates secretion of an antibody chain from a host cell. An antibody chain sequence may be cloned into a vector such that the signal peptide is linked in-frame to the N-terminus of an immunoglobulin chain. A signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e. a signal peptide from a non-immunoglobulin protein).

[0252] In some embodiments of the invention, the vector may include an expression control sequence. The term "expression control sequence" as used in the present description refers to polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are inserted. It will be understood by those skilled in the art that the design of an expression vector, including the selection of expression control sequences, may depend on such factors as the choice of the type of a host cell to be transformed, the required level of expression of antibody, and so forth. Expression control sequences include appropriate transcription initiation and termination sequences, promoter sequences, enhancer sequences and / or insulator sequences; efficient RNA processing signals such as splicing sites and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such expression control sequences differs depending upon the host organism; in prokaryotes, such expression control sequences typically include a promoter, a ribosome binding site, as well as transcription termination sequences; in eukaryotes, such expression control sequences typically include promoters and transcription termination sequences. Preferred expression control sequences for an expression host cell in a mammal include viral elements that ensure high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from a retroviral LTR (long terminal repeat), cytomegalovirus (CMV) (such as a CMV enhancer / promoter), simian virus 40 (SV40) (such as a SV40 promoter / enhancer), adenovirus, (e.g. the major late promoter adenovirus (AdMLP)), polyomavirus and strong mammalian promoters such as TTR promoter, native immunoglobulin promoter or actin promoter. Expression control sequences encompass at least all components whose presence is important for expression and processing.

[0253] In some embodiments of the invention, in addition to antibody chain genes and expression control sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of a vector in host cells (e.g. origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which a vector has been introduced.

[0254] Host cell

[0255] In one aspect, the present invention relates to a method for producing a host cell to produce any above antibody or antigen-binding fragment thereof that specifically binds to TSLP, and includes transformation of the cell with the above vector.

[0256] In one aspect, the present invention relates to a host cell for producing any above antibody or antigen-binding fragment thereof that specifically binds to TSLP, comprising any one of the above nucleic acids.

[0257] The term "host cell" as used herein refers to a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may include, for example, the above-described vector according to the invention. The present invention further relates to host cells that comprise, for example, a nucleotide sequence encoding a heavy chain or antigen-binding portions thereof, a nucleotide sequence encoding a light chain or antigen-binding portions thereof, or both. It should be understood that "host cell" refers not only to a particular subject cell but to the progeny of such cell as well. Since modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to a parental cell; however, such cells are still included within the scope of the term "host cell" as used herein.

[0258] Nucleic acid molecules encoding the monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP according to the invention and vectors comprising these nucleic acid molecules may be used for transfection of a mammalian cell, plant cell, bacterial cell, or yeast cell. Transfection may be carried out by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, transfection by electroporation, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of the polynucleotides in liposomes, and direct microinjection of DNA into nuclei. In addition, the nucleic acid molecules may be introduced into mammalian cells by viral (expression) vectors.

[0259] Mammalian cell lines used as hosts for transformation are well known in the art and include a plurality of immortalized cell lines. These include, e.g., Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549, SK-HEP1, HUH7, Hep-RG cells and a number of other cell lines. Cell lines are selected by way of determining which cell lines have high expression levels and provide for necessary characteristics of the protein being produced. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells. When recombinant expression vectors encoding the monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP are introduced into mammalian host cells, the antibodies or fragments thereof are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibodies or fragments thereof in host cells or, more preferably, secretion of the antibodies or fragments thereof into the culture medium in which the host cells are grown. The monoclonal antibody or antigen -binding fragment thereof that specifically binds to TSLP may be isolated from culture medium using standard protein purification techniques. Plant host cells include e.g. Nicotiana, Arabidopsis, duckweed, com, wheat, potato, etc. Bacterial host cells include, for example, genera such as Escherichia and Streptomyces. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

[0260] Furthermore, level of production of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP from a production cell line may be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions.

[0261] It is likely that the monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP from various cell lines will have a different glycosylation profile as compared to one another. However, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP encoded by nucleic acid molecules described herein, or comprising amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the binding molecules, and, in general, regardless of the presence or absence of post-translational modifications.

[0262] The above host cell does not relate to a host cell produced using human embryos.

[0263] The above host cell does not relate to a host cell produced by modifying the genetic integrity of human germline cells.

[0264] Method for producing antibody

[0265] In one aspect, the present invention relates to a method for producing the antibody or antigenbinding fragment thereof that specifically binds to TSLP, comprising culturing the above host cell in a growth medium under conditions sufficient to produce said antibody or fragment thereof, followed by isolation and purification of the resulting antibody or fragment thereof.

[0266] Pharmaceutical compositions Another aspect of the invention is a pharmaceutical composition comprising, as an active ingredient (or as the only active ingredient), the monoclonal antibody according to the present invention or antigen-binding fragment thereof that specifically binds to TSLP.

[0267] In one aspect, the present invention relates to a pharmaceutical composition that comprises any above-mentioned antibody or antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients.

[0268] In one aspect, the present invention relates to a pharmaceutical composition used for treating a disease or disorder mediated by TSLP, which comprises any above antibody or antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients.

[0269] In one aspect, the present invention relates to a pharmaceutical composition used for treating a disease or disorder mediated by TSLP, which comprises any above antibody or antigen-binding fragment thereof in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.

[0270] "Pharmaceutical composition" means a composition comprising the antibody according to the invention and at least one of components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible fillers, solvents, diluents, carriers, auxiliary, distributing and sensing agents, delivery agents.

[0271] The term "pharmaceutically acceptable" refers to one or more compatible liquid or solid components that are suitable for administration in a mammal, preferably in a human.

[0272] The term "excipient" is used herein to describe any ingredient other than the antibody according to the present invention. These are substances of inorganic or organic nature which are used in the pharmaceutical production / manufacturing in order to give drug products the necessary physicochemical properties.

[0273] In some embodiments, the compositions are intended to improve the condition or treat diseases or disorders that may be mediated by TSLP.

[0274] The term "disease or disorder mediated by TSLP" refers to any disease or disorder that is either directly, or indirectly associated with TSLP, including pathogenesis, progression, recurrence or chronification of a disease or disorder.

[0275] "Treat", "treatment" and "therapy" refer to a method of alleviating or abrogating a biological disorder and / or at least one of attendant symptoms thereof.

[0276] The term "disorder" means any condition that would benefit from treatment according to the present invention. The definition of the term includes chronic and acute disorders or diseases including those pathological conditions that predispose the mammal to the disorder in question. "Therapeutically effective amount" refers to that amount of the therapeutic agent being administered during treatment which will relieve to some extent one or more of the symptoms of the disease being treated. A therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex and weight of the patient, and whether the monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP is being administered as a stand-alone treatment and / or in combination with one or more additional drugs, treatments.

[0277] In one aspect, the subject of treatment, or patient, is a human subject. Said subject may be either male or female, of any age.

[0278] The pharmaceutical compositions of the present invention and methods of preparation thereof will be undoubtedly apparent to those skilled in the art. The pharmaceutical compositions should preferably be manufactured in compliance with the GMP (Good Manufacturing Practice) requirements.

[0279] In some embodiments of the pharmaceutical composition, it may include a buffer composition, tonicity agents (osmolyte or osmotic agent), stabilizers and / or solubilizers.

[0280] The pharmaceutical composition according to the invention is a stable composition.

[0281] A pharmaceutical composition is "stable" if the active agent retains physical stability and / or chemical stability and / or biological activity thereof during the specified shelf life at a storage temperature, for example, of 2-8 °C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. Storage period is adjusted based on the results of stability test in accelerated or natural aging conditions.

[0282] In some embodiments, the pharmaceutical composition is a dosage form for parenteral administration.

[0283] In some embodiments, the pharmaceutical composition is an injectable dosage form.

[0284] In some embodiments, the dosage form is an infusion solution.

[0285] In some embodiments, the dosage form is a solution for subcutaneous administration.

[0286] The dosage forms may be manufactured without limitation, in unit dosage form, such as in ampoules, vials, plastic containers, pre-filled syringes, autoinjection devices.

[0287] In some embodiments, the pharmaceutical composition is a pharmaceutical composition provided in dry, i.e. powder or granular, form for reconstitution with a suitable solvent (e.g., sterile pyrogen-free water) prior to administration. Such medicinal formulation may be prepared by, for example, lyophilization, i.e. a process, which is known in the art as freeze drying, and which involves freezing a product followed by removal of solvent from frozen material.

[0288] In some embodiments, the pharmaceutical composition is a lyophilizate for preparing a solution for infusion. In some embodiments, the pharmaceutical composition is a lyophilizate for preparing a solution for subcutaneous administration.

[0289] In some embodiments, the pharmaceutical composition is a concentrate for preparing a solution for infusion.

[0290] In some embodiments, the pharmaceutical composition is a concentrate for preparing a solution for subcutaneous administration.

[0291] In some embodiments, the pharmaceutical composition is a dosage form ready for subcutaneous injection.

[0292] In some embodiments, the pharmaceutical composition is a dosage form ready for infusion.

[0293] In one aspect, the present invention relates to a pharmaceutical composition that comprises a monoclonal antibody according to the present invention or antigen-binding fragment thereof that specifically binds to TSLP, and at least one other therapeutically active compound.

[0294] In one aspect, the present invention relates to a pharmaceutical composition for treating a disease or disorder mediated by TSLP, comprising any above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound.

[0295] In one aspect, the present invention relates to a pharmaceutical composition comprising any above antibody or antigen-binding fragment thereof and further at least one other therapeutically active compound.

[0296] In one aspect, the present invention relates to a pharmaceutical composition for treating a disease or disorder mediated by TSLP, comprising any above antibody or antigen-binding fragment thereof and further at least one other therapeutically active compound.

[0297] In one aspect, the present invention relates to a pharmaceutical composition for treating a disease or disorder mediated by TSLP, comprising any above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound, which is an antibody, a small molecule, a hormone therapy agent or any combination thereof.

[0298] In some embodiments of the pharmaceutical composition, the other therapeutically active compound is selected from the group comprising: rituximab, omalizumab, reslizumab, dupilumab, benralizumab, mepolizumab, disease-modifying anti-rheumatic drugs (DMARDs), glucocorticoids, or any combination thereof.

[0299] In some embodiments of the pharmaceutical composition, the disease or disorder mediated by TSLP is selected from the group: bronchial asthma, eosinophilic granulomatosis with polyangiitis, chronic rhinosinusitis with nasal polyps or eosinophilic esophagitis.

[0300] Therapeutic use of monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP In one aspect, the antibody or antigen-binding fragment thereof that specifically binds to TSLP is used in the treatment of diseases or disorders mediated by TSLP.

[0301] In one aspect, the subject of treatment, or patient, is a human subject. Said subject may be either male or female, of any age.

[0302] In one aspect, the present invention relates to a method for treating a disease or disorder mediated by TSLP, comprising administering in a subject in need of such treatment any above antibody or antigen-binding fragment thereof or said pharmaceutical composition, in a therapeutically effective amount.

[0303] In one aspect, the present invention relates to a method for treating a disease or disorder mediated by TSLP, comprising administering in a subject in need of such treatment any above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound in a therapeutically effective amount.

[0304] In some embodiments of the method for treating, the disease or disorder mediated by TSLP is selected from the group: bronchial asthma, eosinophilic granulomatosis with polyangiitis, chronic rhinosinusitis with nasal polyps or eosinophilic esophagitis.

[0305] In some embodiments of the method of treatment, the other therapeutically active compound is an antibody, small molecule, hormone therapy agent, or any combination thereof.

[0306] In one aspect, the present invention relates to the use of the above antibody or antigen-binding fragment thereof or the above pharmaceutical composition for treating in a subject in need of such treatment a disease or disorder mediated by TSLP.

[0307] In one aspect, the present invention relates to the use of the above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound for treating in a subject in need of such treatment a disease or disorder mediated by TSLP.

[0308] In some embodiments of the use, the disease or disorder mediated by TSLP is selected from the group: bronchial asthma, eosinophilic granulomatosis with polyangiitis, chronic rhinosinusitis with nasal polyps or eosinophilic esophagitis.

[0309] In some embodiments of the use, the other therapeutically active compound is an antibody, small molecule, hormone therapy agent, or any combination thereof.

[0310] The uses or methods used herein relating to the antibody or antigen-binding fragment thereof that specifically binds to TSLP with one or more other therapeutic agents are contemplated to mean, refer to and include the following:

[0311] 1) simultaneous administration of such combination of the antibody or antigen-binding fragment thereof that specifically binds to TSLP and therapeutic agent to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said patient,

[0312] 2) simultaneous administration of such combination of the antibody or antigen-binding fragment thereof that specifically binds to TSLP and therapeutic agent to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said patient, whereupon said components are released at substantially the same time to said patient,

[0313] 3) sequential administration of such combination of the antibody or antigen-binding fragment thereof that specifically binds to TSLP and therapeutic agent to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said patient with a significant time interval between each administration, whereupon said components are released at substantially different times to said patient; and

[0314] 4) sequential administration of such combination of the antibody or antigen-binding fragment thereof that specifically binds to TSLP and therapeutic agent to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components in a controlled manner, whereupon they are concurrently, consecutively, or jointly released at the same and / or different times to said patient, where each portion may be administered by either the same or different routes.

[0315] The antibody or antigen-binding fragment thereof that specifically binds to TSLP may be administered without further therapeutic treatment, i.e. as an independent therapy.

[0316] In some embodiments of the method of treatment or the use, the antibody or antigen-binding fragment thereof that specifically binds to TSLP may be administered in combination with other therapeutically active compound that is selected from the group: rituximab, omalizumab, reslizumab, dupilumab, benralizumab, mepolizumab, disease-modifying anti-rheumatic drugs (DMARDs), glucocorticoids, or any combination thereof.

[0317] In some embodiments of the method of treatment or the use, the antibody or antigen-binding fragment thereof that specifically binds to TSLP may be administered in combination with glucocorticoids.

[0318] In some embodiments of the method of treatment or the use, the antibody or antigen-binding fragment thereof that specifically binds to TSLP may be administered in combination with glucocorticoids and other therapeutically active compound that is selected from the group: rituximab, omalizumab, reslizumab, dupilumab, benralizumab, mepolizumab or disease-modifying antirheumatic drugs (DMARDs). The antibody or antigen-binding fragment thereof that specifically binds to TSLP and the pharmaceutical composition according to the present invention are suitable for parenteral administration in the form of sterile medicinal products intended for administration into the body of a subject by breaching the integrity of the skin or mucous membranes, bypassing the gastrointestinal tract by means of injection or infusion. In particular, it is contemplated that parenteral administration includes, inter alia, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial, transdermal injection or infusion; and kidney dialytic infusion techniques.

[0319] In some embodiments of the method of treatment or the use, the antibody or antigen-binding fragment that specifically binds to TSLP or the pharmaceutical composition is administered intravenously.

[0320] In some embodiments, intravenous administration is carried out by using infusion, prolonged infusion, or long-lasting continuous infusion.

[0321] In some embodiments of the method of treatment or the use, the antibody or antigen-binding fragment that specifically binds to TSLP or the pharmaceutical composition is administered subcutaneously.

[0322] In some embodiments, subcutaneous administration is carried out by using subcutaneous injection.

[0323] In some embodiments of the method for treating or the use, a suitable dose of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to TSLP according to the present invention will be in the range of 0.1-200 mg / kg.

[0324] Brief description of drawings

[0325] Figure 1 is a schematic illustration of a plasmid vector for expressing the heavy chain of antibodies.

[0326] Figure 2 is a schematic illustration of a plasmid vector for expressing the light chain of antibodies.

[0327] Figure 3 is a diagram showing STAT5-dependent reporter gene expression induced by human TSLP.

[0328] Expression of the reporter gene encoding firefly luciferase is induced in HEK293 STAT5- FLuc TSLPR / IL-7Ra cells in the presence of TSLP. Addition of antagonistic anti-TSLP antibodies blocks TSLP-dependent activation of the reporter gene, which, after adding a luciferase substrate, is manifested in decreased luminescence intensity. The diagram shows luminescence intensity versus the concentration of anti-TSLP antibodies. The curves are fit to a four-parameter logistic equation.

[0329] Figure 4 is a diagram showing the stability of antibodies in serum.

[0330] The bars correspond to the stability of test anti-TSLP antibody drugs following incubation in mouse or human serum for 14 days at 37°C.

[0331] Figure 5 is a diagram showing the binding of biotinylated molecules to TSLP.

[0332] The ability of biotinylated molecules to bind human TSLP immobilized on the plate surface was confirmed by ELISA. The bars correspond to the optical density of the samples, which correlates with the number of immobilized complexes of TSLP / biotinylated protein formed.

[0333] Figure 6 is a diagram showing the competition of molecules for binding to TSLP.

[0334] Competition of biotinylated molecules and immobilized AB5 or anti-TSLP non-comp. for binding to TSLP was demonstrated by ELISA.

[0335] As a positive control used is a non-competitive anti-TSLP antibody (anti-TSLP non-comp.) immobilized on the plate surface, showing the possibility of binding of TSLPR-Fc to the complex of TSLP and anti-TSLP antibody immobilized on the plate surface. The bars correspond to the optical density of the samples, which correlates with the number of immobilized complexes of anti-TSLP antibody / TSLP / biotinylated protein formed.

[0336] Figure 7 is a diagram showing TSLP-dependent proliferation of the TF-1 TSLPR / IL-7Ra cell line, inhibited by anti-TSLP antibodies.

[0337] Proliferation of the TF-1 TSLPR / IL-7Ra cell line is dependent on the presence of TSLP in the growth medium. Addition of antagonistic anti-TSLP antibodies blocks TSLP-dependent cell proliferation, which is manifested in decreased fluorescence intensity of the added Alamar Blue reagent. The diagram shows fluorescence intensity versus concentration of anti-TSLP antibodies. The curves are fit to a four-parameter logistic equation.

[0338] Figure 8 is a diagram showing blocking, by anti-TSLP antibodies, of TARC secretion by dendritic cells.

[0339] In the presence of TSLP, dendritic cells within PBMCs secrete TARC. Addition of antagonistic anti-TSLP antibodies blocks TSLP-dependent secretion of TARC whose level in the growth fluid is measured by ELISA. The bars correspond to the optical density correlating with the TARC level in the growth fluid of PBMCs cultured in the presence of TSLP and test anti-TSLP antibodies.

[0340] Figure 9 is a diagram showing the individual proportion of eosinophils to CD45+cells in bronchoalveolar lavage fluid. 1 The proportion of eosinophils (CD45+ / CD1 lc7SiglecF+) to CD45+cells in bronchoalveolar lavage fluid was determined by flow cytometry.

[0341] Figure 10 is a diagram showing the individual eosinophil to macrophage ratio in bronchoalveolar lavage fluid.

[0342] The macrophage count does not change significantly compared to healthy animals, which allows the macrophages to be used as a reference cell population for an alternative assessment of the degree of infiltration of eosinophils in the lung tissue. Eosinophils (CD45+ / CD1 lc7SiglecF+) and macrophages (CD45+ / CD1 lc+) in bronchoalveolar lavage fluid were detected by flow cytometry.

[0343] Figure 11 is a diagram showing the individual histological assessment of the degree of leukocyte infiltration in the lung tissue.

[0344] Inflammatory cell infiltration (inflammation) in mouse lungs was scored on a scale from 0 to 4:

[0345] 0 - no inflammation;

[0346] 1 - few cells;

[0347] 2 - a 1 -cell-layer ring;

[0348] 3 - a 2-4-cell-layer ring;

[0349] 4 - a ring more than 4 cell layers deep.

[0350] A lung tissue sample from each animal was assessed in six fields of view, and then the total score was calculated.

[0351] Figure 12 is a diagram showing the individual histological assessment of the degree of goblet cell hyperplasia.

[0352] GC hyperplasia in the lungs was scored on a scale from 0 to 4:

[0353] 0 - up to 0.5% of PAS-positive cells over the area of the bronchial mucosa;

[0354] 1 - up to 25% of PAS-positive cells over the area of the bronchial mucosa;

[0355] 2 - 26 to 50% of PAS-positive cells over the area of bronchial mucosa;

[0356] 3 - 51 to 75% of PAS-positive cells over the area of bronchial mucosa;

[0357] 4 - more than 76% of PAS-positive cells over the area of the bronchial mucosa.

[0358] The assessment was performed for each animal in six fields of view, and then the total score was calculated.

[0359] Figure 13 is a diagram showing the individual histological assessment of the total score of the degree of leukocyte infiltration in the lung tissue and goblet cell hyperplasia.

[0360] The values represent the sum of the total score of the degree of leukocyte infiltration in the lung tissue and the total score of goblet cell hyperplasia for each animal.

[0361] Figure 14 is a diagram showing the individual concentration of total IgE in serum. The concentration of total IgE in the serum of individual mice was determined by ELISA.

[0362] Figure 15 is a diagram showing the individual concentration of ovalbumin-specific IgE in serum.

[0363] The concentration of ovalbumin-specific IgE in the serum of individual mice was determined by ELISA.

[0364] Examples

[0365] The following examples are provided for better understanding of the invention. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

[0366] Materials and general methods

[0367] General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Amino acids of antibody chains are numbered according to EU numbering (Edelman, G.M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991).

[0368] Recombinant DNA techniques

[0369] Standard methods were used to manipulate DNA as described in Sambrook, J. et al, Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer protocols.

[0370] Gene synthesis

[0371] Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The gene segments of 300-1400 bp long, flanked by singular restriction sites, were assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequently cloned via the restriction sites. The DNA sequences of the cloned gene fragments were confirmed by DNA sequencing.

[0372] DNA sequence determination

[0373] DNA sequences were determined by Sanger sequencing.

[0374] DNA and protein sequence analysis and sequence data management

[0375] The Unipro's UGENE suite version 1.29 and SnapGene version 6.1 were used for sequence creation, mapping, analysis, annotation and illustration.

[0376] Expression vectors

[0377] To produce the antibodies described herein, we used variants of genetic constructs intended for the expression of a transgene in eukaryotic cells (for example, in Chinese hamster ovary (CHO) cells), as well as for maintaining plasmid copy number in prokaryotic cells (for example, E. coli). In addition to the transgene encoding the sequence of a given antibody, the vectors contained all the elements necessary for the expression of protein in eukaryotic cells, as well as all the elements necessary for maintaining plasmid copy number in prokaryotic cells.

[0378] The fusion genes comprising the subject antibody chains as described below were generated by PCR and / or gene synthesis and assembled using known recombinant methods and techniques by connection of the according nucleic acid segments, e.g. using unique restriction sites in the corresponding vectors. The resulting nucleic acid sequences were verified by DNA sequencing. For eukaryotic cell transfections, larger quantities of the plasmids were prepared by preparation thereof from transformed E. coli cultures.

[0379] Example 1. Preparation of monoclonal anti-TSLP antibody drugs

[0380] Prepared was a panel of plasmid genetic constructs for producing antibodies, consisting of heavy and light chains, by mammalian cells into growth fluid (Figures 1 and 2). The light chains of antibodies consist of a variable domain (VL) and a constant portion (IGLC). The heavy chains of antibodies consist of a variable domain (VH) and a constant portion (IGHG). The studies, whose results are given below, also used prior art anti-TSLP antibodies, in particular

[0381] - tezepelumab which includes a light chain with the amino acid sequence of SEQ ID NO: 39 and a light chain with the amino acid sequence of SEQ ID NO: 40;

[0382] - anti-TSLP antibody hulC5F12E9-V8 (SEQ ID NO: 41 and SEQ ID NO: 42);

[0383] - anti-TSLP antibody Ab24 (SEQ ID NO: 43 and SEQ ID NO: 44).

[0384] The antibodies are produced in an episomal expression system based on the CHO-K1-S cell line. Cells are cotransfected with plasmid genetic constructs encoding light and heavy chains of antibodies, according to Table 1. Chromatographic purification of antibodies from growth fluid is carried out using immobilized protein A sorbent.

[0385] Table 1. Names of antibodies of the invention and sequences of heavy and light chains.

[0386] Example 2. Blocking of TSLP-dependent signaling

[0387] The activity of monoclonal antibody drugs is assessed using a reporter cell assay. HEK293 STAT5-FLuc TSLPR / IL-7Ra cells are produced by stably transfecting the HEK293 cell line with the following genetic constructs:

[0388] • one comprising a firefly luciferase-encoding gene under the control of a STAT5- dependent promoter

[0389] • one comprising a gene encoding human TSLPR (SEQ ID NO: 35) under the control of the CMV promoter

[0390] • one comprising a gene encoding human IL-7Ra (SEQ ID NO: 36) under the control of the CMV promoter

[0391] To the wells of a 96-well culture plate added are 100 pl of DMEM culture medium comprising 10% fetal bovine serum (FBS), 4*104 / ml HEK293 STAT5-FLuc TSLPR / IL-7Ra cells, 370 ng / ml TSLP-hise (SEQ ID NO: 33), and serial dilutions of test antibodies. The plate is Incubated for 16 hours at 37°C 5% CO2. A luciferase substrate is added, the plate is incubated in the dark at room temperature for 10 minutes, and the luminescence intensity of the samples is measured. For test antibodies, luminescence intensity versus antibody concentration is determined using four-parameter logistic fitting (Figure 3). The relative activity (RA) of antibodies is calculated using the formula:

[0392] The results of the study are shown in Table 2.

[0393] Table 2. TSLP blocking activity of test antibodies compared to tezepelumab.

[0394] Antibodies AB1, AB2, AB3, AB4 and AB5 block TSLP-dependent STAT5 signaling of the HEK293 STAT5-FLuc TSLPR / IL-7Ra cell line more than 2 times more actively compared to tezepelumab.

[0395] Example 3. Serum stability

[0396] The stability of antibodies during storage in human or mouse serum was measured. Antibody drugs are diluted with human or mouse serum to a concentration of 25 mp / ml, and samples are incubated for 14 days at 4 °C or 37 °C. The concentration of anti-TSLP antibodies in the samples was measured by ELISA. The antigen is immobilized on the surface of the wells of 96-well ELISA plates by adding 100 pl of a 1 pg / ml TSLP-hise solution in carbonate buffer to each well, and the plate is incubated at 4°C for 18 hours. After washing 3 times with a water-based saline solution comprising sodium chloride, Tris and HC1 (hereinafter TBS), the plate surface is blocked by adding 200 pl of a 1% dry milk solution to the wells, and the plate is incubated for 30 minutes at 37°C. After washing 3 times with TBS, 100 pl of serum antibody samples or antibody calibration solutions are added to the wells, and the plate is incubated for 60 minutes at 37°C. After washing 3 times with a solution of TBS + 0.1% polysorbate 20 (hereinafter TBS-T), 100 pl of a solution of anti-human IgG antibodies conjugated with horseradish peroxidase are added to each well, and the plate is incubated for 60 minutes at 37°C.

[0397] After washing 3 times with TBS-T, 100 pl of 3,3’,5,5’-tetramethylbenzidine (hereinafter TMB) solution is added to each well, and the plate is incubated in the dark at room temperature until color develops (approximately 10 minutes). The reaction is stopped by adding 50 pl of 5% sulfuric acid to each well. The optical density of solutions is measured at a wavelength of 450 nm. The concentration of antibodies in samples is determined according to respective calibration curves. The stability of the molecules is determined by the formula: concentration 37°C stability = - - - — x 100% concentration (4 C)

[0398] The results of the study are shown in Figure 4.

[0399] Antibodies AB1, AB2, AB3, AB4 and AB5 show greater stability in both human and mouse serum compared to tezepelumab.

[0400] Example 4. Biolayer interferometry

[0401] The equilibrium dissociation constant for AB5 and TSLP is measured by biolayer interferometry. The antigen from a 1.25 pg / ml TSLP-hise solution is immobilized onto the surface of Amine Reactive Second-Generation (AR2G) Biosensors. Binding kinetic parameters are measured upon addition of a solution of AB5 at 150, 75 or 37.5 ng / ml. Table 3 shows the resulting values.

[0402] Table 3. Kinetic constants for AB5 / TSLP-hise antigen interactions

[0403] Thus, AB 5 has a KD in the subpicomolar range and shows a high degree of interaction with the TSLP antigen.

[0404] Example 5. TSLP binding competition Biotinylation of TSLPR-Fc, anti-TSLP non-comp. (SEQ ID NO: 45 and 46), AB5, tezepelumab, hulC5F12E9-V8, and Ab24 was performed in phosphate-buffered saline (hereinafter PBS) supplemented with a 20-fold molar excess of succinimidyl-6-biotinamide hexanoate. The solutions were incubated for 30 minutes at room temperature, followed by dialysis against PBS. Their ability to bind to TSLP is measured by ELISA. The antigen is immobilized on the surface of the wells of a 96-well ELISA plate by adding 50 pl of a solution comprising 1 pg / ml TSLP-hise in PBS to each well, and the plate is incubated at 4°C for 18 hours. After washing 3 times with PBS, the plate surface is blocked by adding 200 pl of 1% dry milk solution to each well, and the plate is incubated for 30 minutes at 37°C. After washing 3 times with PBS, 50 pl of 1 pg / ml biotinylated TSLPR-Fc, anti- TSLP non-comp., AB5, tezepelumab, hulC5F12E9-V8 and Ab24 are added to the wells and the plate is incubated for 60 minutes at 37°C. After washing 3 times with a solution of PBS + 0.1% polysorbate 20 (hereinafter PBS-T), 50 pl of streptavidin solution conjugated with horseradish peroxidase are added to each well and the plate is incubated for 30 minutes at 37°C. After washing 3 times with PBS-T, 50 pl of TMB solution are added to each well, and the plate is incubated in the dark at room temperature until color develops (approximately 10 minutes). The reaction is stopped by adding 30 pl of 10% sulfuric acid to each well. The optical density of solutions is determined at a wavelength of 450 nm (Figure 5). Biotinylation does not block the binding of test molecules to TSLP.

[0405] Competition for binding of AB5 and biotinylated molecules to TSLP was assessed by ELISA. Antibodies are immobilized on the surface of the wells of a 96-well ELISA plate by adding 50 pl of a solution comprising 1 pg / ml AB5 or anti-TSLP non-comp. in carbonate buffer to each well, and the plate is incubated at 4°C for 18 hours. After washing 3 times with PBS, the plate surface is blocked by adding 200 pl of 1% dry milk solution to each well, and the plate is incubated for 30 minutes at 37°C. After washing 3 times with PBS, 50 pl of a solution comprising 10 pg / ml TSLP-hise are added to each well, and the plate is incubated for 60 minutes at 37°C. After washing 3 times with PBS-T, 50 pl of a solution comprising 1 pg / ml biotinylated antibodies are added to each well, and the plate is incubated for 60 minutes at 37°C. After washing 3 times with PBS-T, 50 pl of streptavidin solution conjugated with horseradish peroxidase are added to each well, and the plate is incubated for 30 minutes at 37°C. After washing 3 times with PBS-T, 50 pl of TMB solution are added to each well, and the plate is incubated in the dark at room temperature until color develops (approximately 10 minutes). The reaction is stopped by adding 30 pl of 10% sulfuric acid to each well. The optical density of solutions is determined at a wavelength of 450 nm (Figure 6).

[0406] As a positive control used is a non-competitive anti-TSLP antibody (anti-TSLP non-comp.) immobilized on the plate surface, showing the ability of binding of TSLPR-Fc to the complex of TSLP and anti-TSLP antibody immobilized on the plate surface. AB5 competitively blocks binding of TSLPR-Fc (SEQ ID NO: 34) to TSLP.

[0407] AB5 does not compete with other test antibodies for binding to TSLP, which fact suggests that they interact with different epitopes.

[0408] Example 6. Blocking of TSLP-dependent proliferation of TF-1 TSLPR / LL-7Ra cell line

[0409] TF-1 TSLPR / IL-7Ra cells are produced by stably transfecting the TF-1 cell line with genetic constructs:

[0410] • one comprising a gene encoding human TSLPR (SEQ ID NO: 35) under the control of the CMV promoter

[0411] • one comprising a gene encoding human IL-7Ra (SEQ ID NO: 36) under the control of the CMV promoter

[0412] To each well of a 96-well culture plate added are 200 pl of RPMI1640 culture medium comprising 10% fetal bovine serum (FBS), 104 / ml TF-1 TSLPR / IL-7Ra cells, 8 ng / ml TSLP-hise (SEQ ID NO: 33), serial dilutions of test antibodies. The plates are incubated for 72 hours at 37 °C (5% CO2). 20 pl of Alamar Blue is added to each the wells, and the plate is incubated for 5 hours at 37 °C 5% CO2. The fluorescence intensity of the well contents is determined at an excitation / emission wavelength of 530 / 590 nm. For test antibodies, fluorescence intensity versus antibody concentration is determined using four-parameter logistic fitting (Figure 7). The relative activity (RA) of antibodies is calculated using the formula:

[0413] The results of the study are shown in Table 4.

[0414] Table 4. Activity of blocking of TSLP-dependent proliferation of TF-1 TSLPR / IL-7Ra cell line by test antibodies compared to tezepelumab.

[0415] The antibody AB5 blocks TSLP-dependent proliferation of the TF-1 TSLPR / IL-7Ra cell line with more than 2-fold activity than the reproduced antibodies tezepelumab, hulC5F12E9-V8 and Ab24.

[0416] Example 7. Blocking of secretion of TARC (thymus and activation-regulated chemokine) To the wells of a 24-well culture plate added are 500 pl of the RPMI1640 culture medium comprising 10% fetal bovine serum (FBS), 4x l06 / ml peripheral blood mononuclear cells, 50 ng / ml TSLP-hise, 50 pg / ml of test anti-TSLP antibodies. The plates are incubated for 16 hours at 37 °C 5% CO2. The level of TARC in the growth fluid is measured using the Human CCL17 / TARC DuoSet ELISA kit (R&D Systems). All test antibodies downregulate TARC secretion by peripheral blood mononuclear cells; AB5 shows more efficient downregulation thereof compared to hulC5F12E9-V8, Ab24, and tezepelumab (Figure 8).

[0417] Example 8. Ovalbumin-induced bronchial asthma mouse model

[0418] The anti-inflammatory activity of anti-TSLP antibodies is measured in a mouse model of bronchial asthma using transgenic B-hTSLP / hTSLPR mice plus (Biocytogen). The transgenic B- hTSLP / hTSLPR mice plus include exons 1-4 of human Tslp instead of exons 1-5 of the mouse Tslp gene, which encode the full-length protein. The mice also include a sequence encoding the leader peptide, the extracellular and transmembrane portion of human TSLPR, and the cytoplasmic portion of mouse TSLPR which is inserted into the second exon of the mouse TSLPR gene. During the expression of the TSLPR gene, synthesized will be the chimeric TSLPR protein but not the mouse TSLPR protein.

[0419] The animals are sensitized by intraperitoneal administration of a mixture of ovalbumin solution with an adjuvant based on aluminum hydroxide suspension on days 0, 7 and 14. Bronchial asthma provocation is carried out by intranasal administration of a challenge dose of ovalbumin on days 21, 22, 23, 24 and 25. The animals are euthanized and biological material is collected on day 26. Figures 9-15 show the results of the analysis of the samples. Subcutaneous administration of placebo or of 100 mg / kg anti-TSLP antibody drugs is performed on days 0, 7, 14, and 21.

[0420] The group of animals receiving AB5 exhibited reduced leukocyte infiltration in the lung tissue, goblet cell hyperplasia, and serum ovalbumin-specific IgE concentrations compared to the placebo group. This group also showed a reduced proportion of eosinophils to CD45+cells in bronchoalveolar lavage fluid and reduced total IgE concentrations compared to the placebo group as well as compared to the tezepelumab group.

Claims

Claims1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to thymic stromal lymphopoietin (TSLP), comprising:(a) a light chain variable domain comprising:(i) CDR1 with the amino acid sequence of SEQ ID NO: 1;(ii) CDR2 with an amino acid sequence selected from the group: SEQ ID NO: 2 or SEQ ID NO: 3; and(iii) CDR3 with the amino acid sequence of SEQ ID NO: 4; and(b) a heavy chain variable domain comprising:(i) CDR1 with the amino acid sequence of SEQ ID NO: 5;(ii) CDR2 with an amino acid sequence selected from the group: SEQ ID NO: 6 or SEQ ID NO: 7; and iii) CDR3 with the amino acid sequence of SEQ ID NO: 8.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising: (i) a light chain variable domain comprising:CDR1 with the amino acid sequence of SEQ ID NO: 1,CDR2 with the amino acid sequence of SEQ ID NO: 2 andCDR3 with the amino acid sequence of SEQ ID NO: 4; or(ii) a light chain variable domain comprising:CDR1 with the amino acid sequence of SEQ ID NO: 1,CDR2 with the amino acid sequence of SEQ ID NO: 3 andCDR3 with the amino acid sequence of SEQ ID NO: 4.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising:(i) a heavy chain variable domain comprising:CDR1 with the amino acid sequence of SEQ ID NO: 5,CDR2 with the amino acid sequence of SEQ ID NO: 6 andCDR3 with the amino acid sequence of SEQ ID NO: 8; or(ii) a heavy chain variable domain comprising:CDR1 with the amino acid sequence of SEQ ID NO: 5,CDR2 with the amino acid sequence of SEQ ID NO: 7 andCDR3 with the amino acid sequence of SEQ ID NO: 8.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising:(i) (a) a light chain variable domain comprising:CDR1 with the amino acid sequence of SEQ ID NO: 1,CDR2 with the amino acid sequence of SEQ ID NO: 2 andCDR3 with the amino acid sequence of SEQ ID NO: 4; and(b) a heavy chain variable domain comprising:CDR1 with the amino acid sequence of SEQ ID NO: 5,CDR2 with the amino acid sequence of SEQ ID NO: 6 andCDR3 with the amino acid sequence of SEQ ID NO: 8; or(ii) (a) a light chain variable domain comprising:CDR1 with the amino acid sequence of SEQ ID NO: 1,CDR2 with the amino acid sequence of SEQ ID NO: 3 andCDR3 with the amino acid sequence of SEQ ID NO: 4; and(b) a heavy chain variable domain comprising:CDR1 with the amino acid sequence of SEQ ID NO: 5,CDR2 with the amino acid sequence of SEQ ID NO: 7 andCDR3 with the amino acid sequence of SEQ ID NO: 8; or(iii) (a) a light chain variable domain comprising:CDR1 with the amino acid sequence of SEQ ID NO: 1,CDR2 with the amino acid sequence of SEQ ID NO: 3 andCDR3 with the amino acid sequence of SEQ ID NO: 4; and(b) a heavy chain variable domain comprising:CDR1 with the amino acid sequence of SEQ ID NO: 5,CDR2 with the amino acid sequence of SEQ ID NO: 6 andCDR3 with the amino acid sequence of SEQ ID NO: 8.

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable domain comprises an amino acid sequence selected from the group: SEQ ID NO: 9 or SEQ ID NO: 10.

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain comprises an amino acid sequence selected from the group: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.

7. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein:(a) the light chain variable domain comprises an amino acid sequence selected from the group: SEQ ID NO: 9 or SEQ ID NO: 10; and(b) the heavy chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.

8. The monoclonal antibody or antigen-binding fragment thereof according to claim 7, wherein:(i) (a) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 9 and(b) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 11; or(ii) (a) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 9 and(b) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 12; or(iii) (a) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 10 and(b) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 13; or(iv) (a) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 10 and(b) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 14; or(v) (a) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 10 and(b) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 11.

9. The monoclonal antibody according to any one of claims 1-8, wherein the antibody that specifically binds to TSLP is a full-length IgG antibody.

10. The monoclonal antibody according to claim 9, wherein the full-length IgG antibody is of human IgGl, IgG2, IgG3 or IgG4 isotype.

11. The monoclonal antibody according to claim 10, wherein the full-length IgG antibody is of human IgGl isotype.

12. The monoclonal antibody according to claim 11, wherein the antibody comprises the deletion 446G and 447K according to the EU numbering scheme for amino acids of antibodies in the CH3 region.

13. The monoclonal antibody according to claim 11, wherein the antibody comprises the mutations L234A and L235A according to the EU numbering scheme for amino acids of antibodies.

14. The monoclonal antibody according to claim 11, wherein the antibody comprises the mutations M252Y, S254T, T256E according to the EU numbering scheme for amino acids of antibodies.

15. The monoclonal antibody according to claim 1, comprising a light chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 15 or SEQ ID NO: 16.

16. The monoclonal antibody according to claim 1, comprising a heavy chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

17. The monoclonal antibody according to claim 1, comprising:(i) (a) a light chain comprising an amino acid sequence selected from the group: SEQ ID NO: 15 or SEQ ID NO: 16, and(b) a heavy chain comprising an amino acid sequence selected from the group: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

18. The monoclonal antibody according to claim 17, comprising:(i) (a) a light chain comprising the amino acid sequence of SEQ ID NO: 15, and(b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 17; or(ii) (a) a light chain comprising the amino acid sequence of SEQ ID NO: 15, and(b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 18; or(iii) (a) a light chain comprising the amino acid sequence of SEQ ID NO: 16, and(b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 19; or(iv) (a) a light chain comprising the amino acid sequence of SEQ ID NO: 16, and(b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 20; or(v) (a) a light chain comprising the amino acid sequence of SEQ ID NO: 16, and(b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 17.

19. A nucleic acid that encodes the antibody or antigen-binding fragment thereof according to any one of claims 1-18.

20. The nucleic acid according to claim 19, wherein the nucleic acid is DNA.

21. An expression vector comprising the nucleic acid according to any one of claims 19-20.

22. A method for producing a host cell to produce the antibody or antigen-binding fragment thereof according to any one of claims 1-18, comprising transforming the cell by the vector according to claim 21.

23. A host cell for producing the antibody or antigen-binding fragment thereof according to any one of claims 1-18, comprising the nucleic acid according to any one of claims 19-20.

24. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1-18, comprising culturing the host cell according to claim 23 in a growth medium under conditions sufficient to produce said antibody, followed by isolation and purification of the resulting antibody.

25. A pharmaceutical composition for treating a disease or disorder mediated by TSLP comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-18 in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.

26. The pharmaceutical composition according to claim 25, wherein the disease or disorder mediated by TSLP is selected from the group: bronchial asthma, eosinophilic granulomatosis with polyangiitis, chronic rhinosinusitis with nasal polyps or eosinophilic esophagitis.

27. A pharmaceutical composition for treating a disease or disorder mediated by TSLP, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-18 and at least one other therapeutically active compound.

28. The pharmaceutical composition according to claim 27, wherein the disease or disorder mediated by TSLP is selected from the group: bronchial asthma, eosinophilic granulomatosis with polyangiitis, chronic rhinosinusitis with nasal polyps or eosinophilic esophagitis.

29. The pharmaceutical composition according to any one of claims 27-28, wherein the other therapeutically active compound is an antibody, a small molecule, a hormone therapy agent or any combination thereof.

30. A method for treating a disease or disorder mediated by TSLP, comprising administering to a subject in need of such treatment the antibody or antigen -binding fragment thereof according to any one of claims 1-18 or the pharmaceutical composition according to any one of claims 25-29 in a therapeutically effective amount.

31. The method for treating a disease or disorder according to claim 30, wherein the disease or disorder mediated by TSLP is selected from the group: bronchial asthma, eosinophilic granulomatosis with polyangiitis, chronic rhinosinusitis with nasal polyps or eosinophilic esophagitis.

32. A method for treating a disease or disorder mediated by TSLP, comprising administering to a subject in need of such treatment the antibody or antigen -binding fragment thereof according toany one of claims 1-18 or the pharmaceutical composition according to any one of claims 25-29 and at least one other therapeutically active compound in a therapeutically effective amount.

33. The method for treating a disease or disorder according to claim 32, wherein the disease or disorder mediated by TSLP is selected from the group: bronchial asthma, eosinophilic granulomatosis with polyangiitis, chronic rhinosinusitis with nasal polyps or eosinophilic esophagitis.

34. The method for treating a disease or disorder according to any one of claims 32-33, wherein the other therapeutically active compound is an antibody, a small molecule, a hormone therapy agent or any combination thereof.

35. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-18 or the pharmaceutical composition according to any one of claims 25-29 for treating an disease or disorder mediated by TSLP in a subject in need of such treatment.

36. The use according to claim 35, wherein the disease or disorder mediated by TSLP is selected from the group: bronchial asthma, eosinophilic granulomatosis with polyangiitis, chronic rhinosinusitis with nasal polyps or eosinophilic esophagitis.

37. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-18 or the pharmaceutical composition according to any one of claims 25-29 and at least one other therapeutically active compound for treating a disease or disorder mediated by TSLP in a subject in need of such treatment.

38. The use according to claim 37, wherein the disease or disorder mediated by TSLP is selected from the group: bronchial asthma, eosinophilic granulomatosis with polyangiitis, chronic rhinosinusitis with nasal polyps or eosinophilic esophagitis.

39. The use according to any one of claims 37-38, wherein the other therapeutically active compound is an antibody, a small molecule, a hormone therapy agent or any combination thereof.

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