Thymus stromal lymphopoietin (TSLP) binding molecules and uses thereof

Abstract

The present disclosure provides a thymic stromal lymphopoietin (TSLP) binding molecule and uses thereof. The antibody disclosed by the invention is combined with different epitopes of Tezepelumab, and antibacterial peptide with antibacterial activity at the C terminal is not blocked, so that the capability of resisting external bacterial infection of TSLP is expected to be reserved. The antibody disclosed by the invention can be used for effectively inhibiting the activation effect of TSLP on STATs and JAK2 signal channels of an HTSLP Report Cell Line cell, and the inhibition activity of the antibody disclosed by the invention is superior to that of a reference antibody such as Tezepelumab. The antibody disclosed by the invention can effectively inhibit the proliferation promotion effect of TSLP on BaF3-TSLPR / IL7R alpha cells, and the inhibitory activity of the antibody is superior to that of a reference antibody such as Tezepelumab.

Description

[0001] Reference to the Sequence Listing

[0002] This application includes a sequence listing in computer-readable form, which is incorporated herein by reference. Technical Field

[0003] The present disclosure belongs to the technical field of antibodies, and particularly relates to a thymic stromal lymphopoietin binding molecule and its uses. Background Art

[0004] Thymic stromal lymphopoietin (TSLP) was initially discovered in the culture supernatant of mouse thymic stromal cells. It is a B lymphocyte growth factor and is mainly expressed by epithelial cells. TSLP belongs to the IL-2 family of cytokines and is most similar to IL-7. The IL-2 family of cytokines includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. They are all globular proteins containing 4 short-chain α helices and all play important roles in promoting and maintaining the T lymphocyte population (Expert Rev Clin Immunol. 2014, 10(11):1463–1474. Nature Communications, 2017, 8). The latest research results show that TSLP is an "alarm protein" of the human immune barrier and regulates immune functions in surface barriers such as skin keratinocytes, lungs, and intestinal epithelial cells. TSLP responds to pathogen stimulation and activates immature dendritic cells, mast cells, basophils, eosinophils, and lymphocytes, causing them to exhibit a type 2 polarized phenotype. Abnormal TSLP signaling can lead to various serious health problems in humans. It has currently been proven that TSLP is associated with diseases such as asthma, atopic dermatitis, allergic rhinitis, psoriasis, chronic obstructive pulmonary disease (COPD), and eosinophilic esophagitis (Nat Immunol. 2010, 11(4):289–293. Expert Rev Clin Immunol. 2014, 10(11):1463–1474. Drugs (2020), 80, 449–458. Adv Immunol. 2009; 101:1–25).

[0005] IL-2 family cytokines bind to their respective specific receptor subunits and share the common receptor subunit γC. However, TSLP binds to two receptors, TSLPR (CRLF2) and IL-7Rα, and does not bind to the γC receptor (Expert Rev Clin Immunol. 2014, 10(11):1463–1474. Adv Immunol. 2009, 101:1–25). The binding of TSLP to TSLPR and IL-7Rα is cooperative. TSLP binds to TSLPR with an affinity of approximately 32 nM. TSLP does not bind directly to IL-7Rα (>100 nM). TSLP binds to IL-7Rα only after forming the TSLP-TSLPR complex, with an affinity of approximately 29 nM (Nature Communications, 2017, 8).

[0006] There is a variable promoter in the TSLP gene region, which can express two proteins, long form TSLP (lfTSLP) and short form TSLP (sfTSLP) (including signal peptide). Compared with lfTSLP, the N-terminus of sfTSLP is truncated, retaining only "one and a half" α-helical structure. Studies have shown that the C-termini of lfTSLP and sfTSLP contain MKK34 antimicrobial peptides with antibacterial activity, and sfTSLP has stronger antibacterial activity. sfTSLP is the main form of TSLP expressed in normal human tissues. It is only constitutively expressed in normal oral mucosa, skin epidermis, salivary glands, and intestinal epithelial cells, exerting antibacterial activity; under inflammatory conditions, the expression of sfTSLP decreases (detected in pathological tissues of atopic dermatitis and segmental ileitis). However, lfTSLP is not expressed in normal tissues, but only in atopic dermatitis, asthma, ulcerative colitis, and oral mucosa of smoking (Pharmaceuticals 2016, 9, 41). When allergens stimulate epithelial cells, TSLP expression in the cells is upregulated, thereby creating a Th2-prone microenvironment, secreting Th2-type cytokines, generating inflammatory Th2 cells, and triggering allergic inflammatory responses. Studies have shown that when TSLP molecules bind to heterodimers composed of TSLPR and IL-7R-α, they activate JAK1 and JAK2, thereby activating signal transducers and activators of transcription 5 and 3 (STAT5, 3) and initiating the expression of downstream genes. TSLP is widely found in a variety of allergic diseases, such as allergic rhinitis, allergic asthma, eczema, etc. In addition, multiple TSLP SNP mutations have been detected in patients with atopic dermatitis, asthma, and eosinophilic esophagitis, which lead to high expression of lfTSLP. And these mutations are familial (Expert Rev Clin Immunol. 2014, 10(11): 1463–1474.). Therefore, it is of great significance to develop antibodies against lfTSLP to block diseases such as atopic dermatitis, asthma, eosinophilic esophagitis, and ulcerative colitis caused by abnormal expression of lfTSLP. At the same time, screening for MKK34 antimicrobial peptides with antibacterial activity without blocking the C-terminus is expected to retain the ability of TSLP to fight against external bacterial infections.

[0007] The currently marketed antibody Tezepelumab binds to the C-terminus of TSLP, which may affect the antimicrobial peptide activity of TSLP and thus cause side effects. In addition, studies have shown that the TSLP-trap molecule that simultaneously blocks TSLP from binding to TSLPR and IL-7Ralpha is 20-30 times more potent than Tezepelumab in inhibiting TSLP-induced STAT5 (Nature Communications, 2017, 8). Therefore, developing TSLP antibodies with different epitopes and stronger biological activities is expected to enhance the drug efficacy and achieve better clinical treatment effects.

[0008] Traditional antibody molecules (mAbs) are structurally composed of two identical heavy chains and two identical light chains, and this structure is very conserved in mammals. In 1993, Hamers-Casterman et al. from the Free University of Brussels discovered and reported that in addition to traditional antibodies, there is another type of antibody in camel blood. Different from the structure of traditional mammalian antibody molecules, this antibody lacks both the light chain of traditional antibodies and the CH1 region of the heavy chain constant region, and is called heavy chain antibody (HcAb). Single-domain antibody (sdAb) is an antibody variable region with only the heavy chain extracted from the serum of camelids. VHH antibody, also known as Nanobody (NB), retains the complete antigen-binding ability of heavy chain antibodies.

[0009] Compared with traditional antibodies, nanobodies have the following advantages: (1) High antigen affinity and the ability to recognize hidden epitopes. Nanobodies are composed of 4 conserved sequences and 3 Complementarity-determining regions (CDRs). The CDR3 of nanobodies contains 16 - 18 amino acid residues, which is longer than the VH of humans and mice (12 and 9 amino acid residues respectively), and can form a convex loop structure, making it easier for nanobodies to bind to antigens. Moreover, due to their small size, they can bind to some epitopes that are difficult to bind. (2) Small molecular weight and strong tissue penetration ability. The molecular weight of nanobodies is only 15 kDa, which is one-tenth of that of ordinary antibodies. Therefore, it is suitable for various administration routes and has a wider range of applicable populations. Additionally, nanobodies have good tissue penetration ability and can quickly and evenly diffuse throughout the body, thus providing new treatment methods for solid tumors and diseases that require drug administration through the blood-brain barrier. (3) Low immunogenicity. The nanobody gene sequence has a high homology with the human VH gene family 3 sequence (VH3) and lacks the Fc segment that is prone to stimulating immune responses, so its immunogenicity in the human body is relatively low. (4) High water solubility, high tolerance, easy refolding, and high expression. In nanobody VHH, the hydrophobic residues in its FR2 are replaced by hydrophilic residues, making the water solubility of nanobodies higher than that of ordinary monoclonal antibodies and reducing polymerization. Therefore, it has more advantages in making multivalent antibodies. Nanobodies have unique physical and chemical properties and can maintain their activity under extreme conditions such as high pressure or acidity, and have reversible unfolding ability. In addition, nanobodies have a simple structure and are encoded by a single gene, and can be mass-produced in bacteria or yeasts, with low production costs. Therefore, they have more industrialization advantages in production, transportation, storage, etc.

[0010] The applications of nanobodies are very extensive, and research and development layouts have been carried out in the fields of autoimmune diseases, tumors, blood diseases, viral infections, orthopedic diseases, neurodegenerative diseases, inflammatory diseases, etc. Pharmaceutical companies such as Sanofi, Merck, Legend Biotech, Boehringer Ingelheim, and Novartis have all laid out in the field of nanobody research and development, and developed antibody drugs for subcutaneous injection and inhaled administration by leveraging the advantages of nanobodies. Autoimmune diseases such as asthma, atopic dermatitis, chronic rhinosinusitis, nasal polyps, urticaria, chronic obstructive pulmonary disease, and eosinophilic esophagitis all require long-term medication. By leveraging the characteristics of small molecular size and high stability of nanobodies, nanobodies can be developed into an inhaled administration method, with better patient compliance. Summary of the Invention

[0011] The currently marketed antibody Tezepelumab binds to the C-terminus of TSLP, which may affect the antimicrobial peptide activity of TSLP and thus cause side effects. In addition, studies have shown that the TSLP-trap molecule that simultaneously blocks TSLP from binding to TSLPR and IL-7Ralpha is 20-30 times more potent than Tezepelumab in inhibiting TSLP-induced STAT5 (Nature Communications, 2017, 8.). Therefore, developing TSLP antibodies with different epitopes and stronger biological activities is expected to enhance the drug efficacy and achieve better clinical treatment effects.

[0012] The inventors immunized alpacas with human TSLP, extracted the peripheral blood of the immunized alpacas, isolated human peripheral blood mononuclear cells (PBMCs), amplified the variable region fragments of the alpaca heavy chain antibodies from PBMC cDNA, inserted them into a phage display vector using conventional molecular biology techniques, constructed a phage display library, and performed liquid phase panning based on streptavidin plus biotin-labeled target antigen and solid phase panning or combined panning based on the ELISA method. After panning, monoclonal clones were randomly selected, cultured, and positive clones were screened. The positive clones were sequenced, the sequences were compared, and candidate antibodies were selected for expression and purification. Then, the candidate antibodies were detected.

[0013] On the one hand, the present invention provides a TSLP-binding molecule, which is characterized by comprising at least one TSLP-binding domain, and the TSLP-binding domain comprises CDR1, CDR2, and CDR3, wherein,

[0014] (i) CDR1, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 3 or consists of SEQ ID NO: 3;

[0015] (ii) CDR2, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 4 and 7 or consists of one of SEQ ID NO: 4 and 7; and

[0016] (iii) CDR3, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 5 or consists of SEQ ID NO: 5.

[0017] In the above-mentioned binding molecule, the TSLP binding domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 6 and 8, or consists of one of SEQ ID NO: 6 and 8.

[0018] In some embodiments, the TSLP binding molecule, wherein the TSLP binding domain is capable of specifically binding to human or monkey TSLP.

[0019] In some embodiments, the TSLP binding molecule, wherein the TSLP binding domain is a nanobody or a functional derivative thereof, preferably, the nanobody or its functional derivative is obtained by screening a nanobody library using TSLP as the bait protein.

[0020] In some embodiments, the TSLP binding molecule comprises two or more TSLP binding domains, and the two or more TSLP binding domains have the same or different amino acid sequences.

[0021] In some embodiments, the TSLP binding molecule further comprises at least one bioactive functional domain or functional fragment, and the bioactive functional domain or functional fragment comprises components such as a carrier protein, an active peptide, a tag peptide, a chaperone protein, etc., which are linked to the TSLP binding domain by covalent bonds, non-covalent bonds or linkers;

[0022] Preferably, the carrier protein includes BSA, OVA, HSA, KLH, Fc, etc.; the active peptide includes a linear peptide or a cyclic peptide and has enzyme activity, cytotoxic activity, antigen-binding activity, etc.; the tag peptide includes a purification tag peptide, a tracer tag peptide, etc.; the chaperone protein includes HSP60, HSP70, HSP90, etc.

[0023] In some embodiments, the TSLP binding molecule, the bioactive functional domain or functional fragment comprises polypeptides, synthetic small molecules, natural active molecules, and the TSLP binding molecule exists in the form of a monomer, a homopolymer or a heteropolymer.

[0024] In some embodiments, the TSLP binding molecule is a nanobody, a heavy chain antibody, a VHH-Fc fusion protein, a monovalent antibody, a bivalent antibody, a multivalent antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, an immunoconjugate.

[0025] In some embodiments, the TSLP binding molecule has one or more properties

[0026] The activity of inhibiting the activation of the STATs and JAK2 signaling pathways in H_TSLP Reporter Cell Line cells by TSLP is at least 1.1-fold, such as 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3-fold that of a reference antibody (such as Tezepelumab); and / or,

[0027] The activity of inhibiting the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells is at least 1.1-fold, such as 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3-fold that of a reference antibody (such as Tezepelumab).

[0028] On the one hand, the present disclosure provides a method for preparing the aforementioned TSLP-binding molecule, comprising:

[0029] (1) Immunizing an animal capable of producing heavy-chain antibodies with TSLP protein;

[0030] (2) Isolating PBMCs, amplifying nucleic acids encoding heavy-chain variable regions, and constructing a phage heavy-chain antibody library;

[0031] (3) Optionally, performing liquid-phase panning using streptavidin plus biotin-labeled target antigen and solid-phase panning or combined panning based on the ELISA method to remove unbound phages;

[0032] (4) Eluting the phage sub-library and infecting the host, culturing the eluted sub-library and infecting with helper phages;

[0033] (5) Optionally, repeating steps (3)-(4) two to four times;

[0034] (6) Infecting with helper phages after culturing, packaging monoclonal recombinant phages, and performing phage identification;

[0035] (7) Sequencing the phage clones positive for phage identification to obtain nucleic acids encoding the TSLP-binding domain.

[0036] Furthermore, the method further comprises:

[0037] (1) Performing humanization modification on the TSLP-binding domain;

[0038] (2) Performing sequence optimization on the humanized TSLP-binding domain.

[0039] On the one hand, the present disclosure provides a composition comprising the aforementioned TSLP-binding molecule; and optionally a pharmaceutically acceptable excipient.

[0040] On the one hand, the present disclosure provides a polynucleotide encoding the aforementioned TSLP-binding molecule.

[0041] On the one hand, the present disclosure provides a nucleic acid construct comprising the aforementioned polynucleotide.

[0042] On the one hand, the present disclosure provides a host cell comprising the aforementioned polynucleotide or the aforementioned nucleic acid construct.

[0043] Furthermore, the present disclosure provides a method for preparing a TSLP-binding molecule, which comprises:

[0044] (1) Culturing the host cell according to claim 15 under conditions suitable for expressing a recombinant foreign protein,

[0045] (2) Optionally, isolating and purifying the TSLP-binding molecule from the cell culture.

[0046] On the one hand, the present disclosure provides the TSLP-binding domain in the aforementioned TSLP-binding molecule.

[0047] On the one hand, the present disclosure provides the use of the aforementioned TSLP-binding molecule or the aforementioned TSLP-binding domain in the preparation of a drug and / or the extension of the half-life of a drug.

[0048] On the one hand, the present disclosure provides the use of the aforementioned TSLP-binding molecule or the aforementioned TSLP-binding domain, characterized in that an active molecule with a prophylactic or therapeutic effect is bound to the TSLP-binding molecule or the TSLP-binding domain by a covalent bond, a non-covalent bond, or a linker molecule.

[0049] On the one hand, the aforementioned TSLP-binding molecule or the aforementioned TSLP-binding domain, its use in TSLP expression-related diseases, and the TSLP expression-related diseases are autoimmune diseases, and the autoimmune diseases are selected from the following group: systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, myasthenia gravis, polymyositis, psoriasis, pemphigus, vitiligo, multiple sclerosis, narcolepsy, neuromyelitis optica, type 1 diabetes, hyperthyroidism, hypothyroidism, Crohn's disease, ulcerative colitis, celiac disease, autoimmune gastritis, primary biliary cholangitis, autoimmune hepatitis, and lupus nephritis.

[0050] The present disclosure has achieved the following beneficial technical effects:

[0051] The antibodies of the present disclosure bind to different epitopes of tezepelumab and do not block the antimicrobial peptides with antimicrobial activity at the C-terminus, and are expected to retain the ability of TSLP to resist external bacterial infections.

[0052] The antibodies of the present disclosure can effectively inhibit the activation of the STATs and JAK2 signaling pathways of the H_TSLP Reporter Cell Line cells by TSLP, and the inhibitory activity is superior to that of reference antibodies such as tezepelumab.

[0053] The antibodies of the present disclosure can effectively inhibit the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells, and the inhibitory activity is superior to that of reference antibodies such as tezepelumab (e.g., at least 1.5 times). Description of the Drawings

[0054] The drawings are used to provide a further understanding of the present disclosure, and constitute a part of the specification. Together with the embodiments of the present disclosure, they are used to explain the present disclosure and do not constitute a limitation to the present disclosure. In the drawings:

[0055] Figure 1a Detection results of the binding activity of candidate antibody TA-10 to TSLP;

[0056] Figure 1b Detection results of the binding activity of candidate antibody P37-18 to TSLP;

[0057] Figure 2a Detection results of the blocking activity of candidate antibody TA-10 on the binding of TSLP to TSLPR at the protein level;

[0058] Figure 2b Detection results of the blocking activity of candidate antibody P37-18 on the binding of TSLP to TSLPR at the protein level;

[0059] Figure 3 Detection results of the blocking activity of candidate antibodies TA-10 and P37-18 on the binding of TSLP to IL-7Rα at the protein level;

[0060] Figure 4a Detection results of the inhibitory activity of candidate antibody TA-10 on the activation of the STATs and JAK2 signaling pathways of H_TSLP Reporter Cell Line cells by TSLP;

[0061] Figure 4b Detection results of the inhibitory activity of candidate antibody P37-18 on the activation of the STATs and JAK2 signaling pathways of H_TSLP Reporter Cell Line cells by TSLP;

[0062] Figure 5a Detection results of the inhibitory effect of candidate antibody TA-10 on the proliferation-promoting activity of TSLP on BaF3-TSLPR / IL7Rα cells;

[0063] Figure 5b Detection results of the inhibitory effect of candidate antibody P37-18 on the proliferation-promoting activity of TSLP on BaF3-TSLPR / IL7Rα cells. Detailed implementation manners

[0064] In the following, certain exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present disclosure. Therefore, the drawings and the description are considered to be exemplary in nature and not restrictive.

[0065] The following describes the preferred embodiments of the present disclosure with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only for the purpose of illustrating and explaining the present disclosure and are not used to limit the present disclosure.

[0066] To better understand the present disclosure, some terms are first defined. Other definitions are listed throughout the detailed implementation manners section.

[0067] Unless otherwise indicated, whether referring to heavy-chain antibodies or conventional 4-chain antibodies in this document, the term "antibody" is used as a general term, including full-size antibodies, their individual chains, and all of their parts, domains, or fragments (including but not limited to antigen-binding domains or fragments such as VHH domains or VH / VL domains, respectively). In addition, as used herein, the term "sequence" (e.g., in terms such as "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence", or "protein sequence") should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding it, unless the context requires a more restrictive interpretation.

[0068] An antibody single variable domain can be used as a "binding unit", "binding domain", or "building unit" (these terms are used interchangeably) for preparing polypeptides containing one or more additional antibody single variable domains that can act as binding units (i.e., for the same or different epitopes against the same target and / or for one or more different targets).

[0069] The term "antibody single variable domain" ("ISVD"), which can be used interchangeably with "single variable domain" ("SVD"), defines a molecule in which the antigen-binding site is present on and formed by a single antibody domain. This makes the antibody single variable domain different from "conventional" antibodies or their fragments, in which two antibody domain variable domains, particularly two variable domains, interact to form the antigen-binding site. Typically, in a conventional antibody, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In such cases, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen-binding site, i.e., a total of 6 CDRs will be involved in the formation of the antigen-binding site. In contrast, the binding site of an antibody single variable domain is formed by a single VH or VL domain. Thus, the antigen-binding site of an antibody single variable domain is formed by no more than three CDRs.

[0070] The terms "antibody single variable domain" and "single variable domain" thus do not include conventional antibodies or their fragments that require the interaction of at least two variable domains to form the antigen-binding site. However, these terms include fragments of conventional antibodies in which the antigen-binding site is formed by a single variable domain.

[0071] Typically, a single variable domain will be an amino acid sequence consisting essentially of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively). Such single variable domains and fragments are most preferred such that they contain an antibody fold or are capable of forming an antibody fold under suitable conditions. Thus, a single variable domain can, for example, comprise a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof; provided that it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain such that the single antigen-binding unit does not need to interact with another variable domain to form a functional antigen-binding unit, as is the case for variable domains present in, for example, conventional antibodies and scFv fragments that require interaction with another variable domain, e.g., via VH / VL interaction, to form a functional antigen-binding domain).

[0072] In one embodiment of the present disclosure, the antibody single variable domain is a light chain variable domain sequence (e.g., a VL sequence) or a heavy chain variable domain sequence (e.g., a VH sequence); more specifically, the antibody single variable domain can be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody.

[0073] For example, a single variable domain or an antibody single variable domain (or an amino acid suitable for use as an antibody single variable domain) can be a (single) domain antibody (or an amino acid suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid suitable for use as a dAb) or a nanobody (as defined herein and including, but not limited to, VHH); other single variable domains, or any suitable fragment of any of them.

[0074] For a general description of (single) domain antibodies, also refer to the prior art cited herein and to EP0368684. For the term "dAb", refer, for example, to Ward et al., 1989 (Nature 341:544-546), refer to Holt et al., 2003 (Trends Biotechnol. 21:484-490); and refer to, for example, WO 04 / 068820, WO 06 / 030220, WO06 / 003388, WO 06 / 059108, WO 07 / 049017, WO 07 / 085815 and other published patent applications of Domantis Ltd. It should also be noted that although they are less preferred in the context of the present disclosure because they are not of mammalian origin, single variable domains can be derived from certain species of sharks (e.g., the so-called "IgNAR domains", see, for example, WO 05 / 18629).

[0075] In particular, an antibody single variable domain can be (as defined herein) or a suitable fragment thereof. [Note: is a registered trademark of Ablynx N.V.] For a general description of nanobodies, refer to the further description below, and to the prior art cited herein, such as, for example, as described in WO 08 / 020079 (page 16).

[0076] For further description of VHHs and nanobodies, reference is made to the review article of Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302), and to the following patent applications mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 of Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193 of Unilever; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 of Vlaams Instituut voor Biotechnologie (VIB); WO 03 / 050531 of Algonomics N.V. and Ablynx N.V.; WO 01 / 90190 of National Research Council of Canada; WO 03 / 025020 of Institute of Antibodies; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 of Ablynx N.V. and further published patent applications of Ablynx N.V. Reference is also made to additional prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of international application WO 06 / 040153, which list and references are incorporated herein by reference. As described in these references, the characteristics of nanobodies (especially VHH sequences and partially humanized nanobodies) can in particular lie in the presence of one or more "signature residues" in one or more framework sequences.Further descriptions of nanobodies, including humanization and / or camelization of nanobodies, as well as other modifications, parts or fragments, derivatives or "nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences), and different modifications for increasing the half-life of nanobodies and their preparation, can be found, for example, in WO 08 / 101985 and WO 08 / 142164.

[0077] Thus, in the context of the present disclosure, the term "antibody single variable domain" or "single variable domain" includes polypeptides derived from non-human sources, preferably from camelids, preferably from the heavy chain antibodies of camelids. As described previously, they can be humanized. In addition, the term includes polypeptides that have been "camelized" from non-camelid sources such as mice or humans, for example, as described in Davies and Riechmann 1994 (FEBS 339:285 - 290), 1995 (Biotechnol. 13:475 - 479), 1996 (Prot. Eng. 9:531 - 537) and Riechmann and Muyldermans 1999 (J. Immunol. Methods 231:25 - 38).

[0078] The term "antibody single variable domain" includes antibody sequences from different sources, including mouse, rat, rabbit, donkey, human and camelid antibody sequences. It also includes fully human, humanized or chimeric antibody sequences. For example, it includes camelid antibody sequences and humanized camelid antibody sequences, or camelized antibody single variable domains, such as the camelized dAbs described by Ward et al., 1989 (see, for example, WO 94 / 04678 and Davies and Riechmann 1994, 1995 and 1996) and camelized VH.

[0079] Similarly, such antibody single variable domains can be derived from any suitable source in any suitable manner and can, for example, be naturally occurring VHH sequences (i.e., from a suitable camelid species) or synthetic or semi-synthetic amino acid sequences, including but not limited to partially or fully "humanized" VHHs, "camelized" antibody sequences (and in particular camelized VH), and nanobodies and / or VHHs obtained by techniques such as affinity maturation (e.g., starting from synthetic, random or naturally occurring antibody sequences such as VHH sequences), CDR grafting, veneering, combining fragments from different antibody sequences, PCR assembly using overlapping primers, and similar techniques known to the person skilled in the art for engineering antibody sequences; or any suitable combination of any of the foregoing.

[0080] The amino acid sequence and structure of an antibody single variable domain can be considered - but not limited to - consisting of four framework regions or "FRs", which are referred to in the art and herein as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4", respectively; the framework regions are separated by three complementarity determining regions or "CDRs", which are referred to in the art as "complementarity determining region 1" or "CDR1"; "complementarity determining region 2" or "CDR2"; and "complementarity determining region 3" or "CDR3", respectively.

[0081] As further described in paragraph q) on pages 58 and 59 of WO 08 / 020079 (incorporated herein by reference), the amino acid residues of an antibody single variable domain are numbered according to the general numbering for VH domains given by Kabat et al. ("Kabat numbering") ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, publication number 91), as applied to the VHH domains of camelids in the article by Riechmann and Muyldermans 2000 (J. Immunol. Methods 240:185 - 195; see, for example, Figure 2 of that publication), and accordingly, the FR1 of the antibody single variable domain contains amino acid residues at positions 1 - 30, the CDR1 of the antibody single variable domain contains amino acid residues at positions 31 - 35, the FR2 of the antibody single variable domain contains amino acids at positions 36 - 49, the CDR2 of the antibody single variable domain contains amino acid residues at positions 50 - 65, the FR3 of the antibody single variable domain contains amino acid residues at positions 66 - 94, the CDR3 of the antibody single variable domain contains amino acid residues at positions 95 - 102, and the FR4 of the antibody single variable domain contains amino acid residues at positions 103 - 113.

[0082] Based on the examples of antibody single variable domain sequences given herein, as well as in WO 08 / 020079, in WO 06 / 040153, and in the additional cited references regarding antibody single variable domains, it will be clear that the exact number of amino acid residues will also depend on the length of the specific CDRs present in the antibody single variable domain. With respect to CDRs, as is well known in the art, there are multiple definitions and conventions for describing the CDRs of VH or VHH fragments, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the position of structural loop regions). For example, refer to the website http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and the claims, even though CDRs according to Kabat may also be mentioned, it is most preferred to define the CDRs based on the Abm definition (which is based on the AbM antibody modeling software of Oxford Molecular), as this is considered to be the best compromise between the Kabat and Chothia definitions. Again, refer to the website http: / / www.bioinf.org.uk / abs / ).

[0083] The term "phage display library" refers to a "library" of phages on the surface of which exogenous peptides or proteins are expressed. The foreign peptides or polypeptides are displayed on the outer surface of the phage capsid. The foreign peptide can be displayed as a recombinant fusion protein incorporated as part of a phage coat protein; a recombinant fusion protein that is not normally a phage coat protein but is capable of being incorporated into the outer surface of the capsid; or a protein or peptide that becomes covalently or non-covalently linked to said protein. This is achieved by inserting an exogenous nucleic acid sequence into the nucleic acid that can be packaged into phage particles. The exogenous nucleic acid sequence can, for example, be inserted into the coding sequence of a phage coat protein gene. If the foreign sequence is cloned in-frame, the protein it encodes will be expressed as part of the coat protein. Thus, a library of nucleic acid sequences, such as a library of antibody lineages made from gene segments encoding an entire B cell lineage of one or more individuals, can be inserted into phages in this way to produce a "phage library". When the peptides and proteins representing those peptides and proteins encoded by the nucleic acid library are displayed by the phages, a "peptide display library" is produced. Although a variety of phages are used in the construction of such libraries, filamentous phages are commonly used (Dunn (1996) Curr. Opin. Biotechnol. 7:547 - 553). See, for example, the following descriptions of phage display libraries.

[0084] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of the antibody molecule is derived from two or more species. Generally, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody of a desired specificity, affinity, and capacity derived from a mammalian species (e.g., mouse, rat, rabbit, etc.), while the constant regions are homologous to sequences derived from another species (usually human) in order to avoid eliciting an immune response in that species.

[0085] The term "chimeric antibody" refers to an antibody in which part of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining part of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison SL et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984). For example, the term "chimeric antibody" can include an antibody (e.g., a human-mouse chimeric antibody) in which the variable regions of the heavy and light chains of the antibody are from a first antibody (e.g., a murine antibody), while the constant regions of the heavy and light chains of the antibody are from a second antibody (e.g., a human antibody).

[0086] The term "multispecific antibody", the antibodies of the present disclosure can be monospecific, bispecific or multispecific. Multispecific antibodies can be specific for different epitopes of a target polypeptide or can contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, Journal of Immunology 147: 60-69; Kufer et al., 2004, Trends Biotechnol. 22: 238-244. The antibodies of the present disclosure can be linked to or co-expressed with another functional molecule (such as another peptide or protein). For example, an antibody or a fragment thereof can be functionally linked (such as by chemical coupling, gene fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to prepare a bispecific or multispecific antibody with a second binding specificity. Variations of the bispecific antibody formats described above are encompassed within the scope of the present disclosure. Other exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, for example, scFv- or bispecific antibody-based bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knob-in-hole structures, common light chains (such as common light chains with knob-in-hole structures, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobodies, IgG1 / IgG2, dual action Fab (DAF)-IgG and Mab2 bispecific formats (for a review of the formats see, for example, Klein et al. 2012, mAbs 4:6, 1-11 and the references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid binding, for example, where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, for example, Kazane et al., Journal of the American Chemical Society (J. Am. Chem. Soc.) [e-published: December 4, 2012).

[0087] The term "single-domain antibody" is obtained by genetic engineering methods and mainly has 3 categories. The first category is the heavy-chain variable region obtained from camelid HCAb, which is a single folding unit and retains the complete antigen-binding activity and is the smallest natural antibody fragment. The second category is the heavy-chain variable region obtained from cartilaginous fish such as sharks IgNAR, denoted by VNAR. The third category is the heavy-chain or light-chain variable region obtained from human or murine monoclonal antibodies, which retains the antigen-binding activity but with greatly reduced affinity and solubility.

[0088] The term "Fc region" or "Fc" refers to the C-terminal region of an antibody heavy chain, which contains at least a portion of the hinge region, CH2 domain, and CH3 domain, and mediates the binding of the antibody to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component of the classical complement system (e.g., C1q), including native sequence Fc regions and variant Fc regions. Generally, the human IgG heavy chain Fc region is the segment from the amino acid residue at position Cys226 or Pro230 to the carboxyl terminus, although its boundaries may vary. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering system) may or may not be present. Fc can also refer to this isolated region, or in the case of a protein polypeptide containing Fc, such as a "binding protein containing an Fc region", also referred to as an "Fc fusion protein" (e.g., an antibody or an immunoadhesin). The native sequence Fc regions in the antibodies of the present disclosure include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In the IgG, IgA, and IgD antibody isotypes, the Fc region contains the CH2 and CH3 constant domains of each of the two heavy chains of the antibody; the IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

[0089] The term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. The term "immunological binding" refers to the specific binding reaction that occurs between an antibody molecule and an antigen (for which the antibody is specific). The strength or affinity of an immunological binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. The immunological binding properties between two molecules can be quantified using methods well known in the art. One method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation. The "association rate constant" (Ka or Kon) and the "dissociation rate constant" (Kd or Koff) for a specific antibody-antigen interaction can both be calculated from the concentration and the actual rates of association and dissociation, see Malmqvist M, 1993, Nature, 361:186-187. The ratio of Kd / Ka is equal to the dissociation constant KD, see Davies DR et al., 1990, Annual Rev Biochem., 59:439-473. The KD, Ka, and Kd values can be measured using any valid method. In a preferred embodiment, the dissociation constant is measured by bioluminescence interferometry. In other preferred embodiments, surface plasmon resonance techniques (e.g., Biacore) or KinExa can be used to measure the dissociation constant.

[0090] The terms "antibody-drug conjugate" and "immunoconjugate", which are composed of an antibody, a linker, and a drug, where the linker is a cleavable linker combination or a non-cleavable linker. The antibody is a globular protein containing a series of amino acid sites available for conjugating the drug-linker. Due to its tertiary and quaternary structures, only solvent-accessible amino acids are available for conjugation. In fact, high-yield conjugation usually occurs on the ε-amino group of lysine residues or the sulfhydryl group of cysteine residues. The large number of lysine side chains on the surface of the antibody protein results in a large number of sites available for drug conjugation, leading to the generation of antibody-drug conjugates that are mixtures containing different drug conjugation numbers (drug / antibody ratio, DAR) and conjugation sites. The conjugate products provided by the present disclosure, although still mixtures, have a narrow DAR distribution range compared to antibody-drug conjugates conjugated in the traditional manner. Its average DAR value is close to 4, approaching the average DAR value (2-4) range of the optimal antibody-drug conjugates. In addition, the conjugate products rarely contain naked antibodies (DAR = 0), which do not contribute to cytotoxic killing. At the same time, the conjugate products also do not contain highly conjugated products (DAR = 8), which are cleared rapidly in vivo compared to the low-DAR components. Therefore, the heterogeneity of the antibody-drug conjugate products provided by the present disclosure is greatly improved.

[0091] The antibody that constitutes the antibody-drug conjugate in the present disclosure preferably retains its antigen-binding ability in its original wild state. Therefore, the antibody in the present disclosure can, preferably specifically, bind to the antigen. To develop effective cellular-level targets for cancer diagnosis and treatment, researchers have sought transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells and are expressed little or not at all on the surface of one or more non-cancer cells. Generally, such tumor-associated polypeptides are more highly expressed on the surface of cancer cells compared to the surface of non-cancer cells. Identifying such tumor-associated factors can greatly improve the specific targeting characteristics of antibody-based cancer treatment.

[0092] The terms "conjugation", "linking", "coupling" refer to the association of two or more molecules. Linking can also be genetic (i.e., recombinant fusion). In a specific context, the terms include referring to the linking of a ligand (such as an antibody moiety) to an effector molecule. Such linking can be achieved using a variety of techniques recognized in the art, for example, by chemical or recombinant means. "Chemical means" refers to the reaction between the antibody moiety and the effector molecule to form a covalent bond between the two molecules to form a single molecule.

[0093] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. The term is intended to include radioactive isotopes (e.g., I131, I125, Y90, Re186), chemotherapeutic agents, and toxins (such as enzymatically active toxins of bacterial, fungal, plant, or animal origin), or fragments thereof.

[0094] The terms "vector", "nucleic acid construct" refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell after being introduced into the host cell and thereby replicate along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are effectively ligated. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA techniques are usually in the form of plasmids. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which perform equivalent functions, are also included.

[0095] The term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.

[0096] The term "polypeptide" refers to a chain containing at least two contiguous linked amino acid residues, and there is no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications, such as but not limited to glycosylation, phosphorylation, or disulfide bonds. A "protein" can comprise one or more polypeptides.

[0097] The term "host cell" is a cell in which a vector can propagate and the DNA thereof can be expressed, and the cell can be a prokaryotic cell or a eukaryotic cell. The term also includes any progeny of the test host cell. It should be understood that not all progeny are identical to the parental cell, as mutations may occur during replication, and such progeny are included.

[0098] Examples

[0099] The present invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in the relevant field (for example, referring to "Molecular Cloning: A Laboratory Manual", Third Edition, J. Sambrook et al., translated by Huang Peitang et al., Science Press) or according to the product specifications are followed. Raw materials, reagents or instruments for which the manufacturer is not indicated are all conventional products that can be obtained commercially.

[0100] An overview of some of the sequences contained in the antibodies of the examples is provided below.

[0101] Tezepelumab heavy chain (SEQ ID NO: 1)

[0102] QMQLVESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEWVAVIWYDGSNK

[0103] HYADSVKGRFTITRDNSKNTLNLQMNSLRAEDTAVYYCARAPQWELVHEAFDIWGQGT

[0104] MVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

[0105] VLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAG

[0106] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN

[0107] STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREE

[0108] MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW

[0109] QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0110] Tezepelumab light chain (SEQ ID NO: 2)

[0111] SYVLTQPPSVSVAPGQTARITCGGNNLGSKSVHWYQQKPGQAPVLVVYDDSDRPSWIPER

[0112] FSGSNSGNTATLTISRGEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGQPKAAPSVTLF

[0113] PPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYL

[0114] SLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSTA - 10 and CDR1 of P37 - 18 (SEQ ID NO: 3)

[0115] LFNIG CDR2 of TA - 10 (SEQ ID NO: 4)

[0116] IISTSDGNPYYVDSVKG CDR3 of TA - 10 and P37 - 18 (SEQ ID NO: 5)

[0117] AVGGDQRLTPDAFGA TA - 10 (SEQ ID NO: 6)

[0118] QLQLVESGGGLVQPGGSLRLSCVVSGFTLDLFNIGWFRQAHGKEREVVSIISTSDGNPYYV

[0119] DSVKGRFTISRDNAKNAVYLQMNSLKVEDTAVYSCAAAVGGDQRLTPDAFGAWGQGTQ

[0120] VTVSS

[0121] CDR2 of P37 - 18 (SEQ ID NO: 7)

[0122] IISTSDGNPYYVDSVRG P37 - 18 (SEQ ID NO: 8)

[0123] QLQLVESGGGLVQPGGSLRLSCVVSGFTLDLFNIGWFRQAHGKEREVVSIISTSDGNPYYV

[0124] DSVRGRFTTSRDNAKNAVYLQMNSLKVEDTAVYSCAAAVGGDQRLTPDAFGAWGQGTQ

[0125] VTVSS

[0126] Example 1: Immunizing Alpacas with Thymic Stromal Lymphopoietin

[0127] Alpacas were immunized with the HEK293-expressed thymic stromal lymphopoietin protein (Human TSLP(R127A,R130A)Protein, His Tag, purchased from ACRO BIOSYSTEMS, catalog number TSP-H52Ha)(Accession#Q969D9-1(R127A,R130A)). For the first immunization, complete Freund's adjuvant was mixed and emulsified with the antigen protein (0.5 mg) at a ratio of 1:1 and injected subcutaneously at multiple points. Every 2 weeks, booster immunizations were performed by emulsifying Freund's incomplete adjuvant with the antigen protein (0.5 mg) at a ratio of 1:1. A total of 7 immunizations were carried out. Blood was drawn after the 4th, 5th, 6th, and 7th immunizations to separate antiserum. The ELISA plate was coated with the immunogen protein, and the titer of the antiserum was measured by ELISA. The total IgG titers of the alpaca antisera measured at the 4th, 5th, 6th, and 7th times were all greater than 1.0E5.

[0128] Example 2: Construction of an Immunized Alpaca Nanobody Phage Display Library

[0129] Peripheral blood mononuclear cells (PBMCs) were isolated from the immunized alpaca peripheral blood by an outsourcing immunization company. The PBMC Total RNA was extracted using the HiPure Total RNA Mini Kit (Magen, catalog number R4111-02), and III First-Strand Synthesis System for RT-PCR (Invitrogen, Catalog No. 18080-051) kit was used for reverse transcription into cDNA. A set of forward and reverse primers matching the variable region gene of the heavy chain of the alpaca antibody was used to amplify the variable region fragment of the heavy chain of the alpaca antibody from the PBMC cDNA, and the phage display vector was inserted using conventional molecular biology techniques, and the antibody library was constructed by electrotransferring ER2738 electrotransfer competent cells (BIOSEARCH, Catalog No. 60522-2). The bacterial library was expanded and cultured to the logarithmic growth phase, and the helper phage M13K07 (M13KO7 Helper Phage purchased from NEB, Catalog No. N0315S, amplified by itself after infecting the host) was added for superinfection to amplify and rescue the nanoantibody phage display library. The antibody library capacity obtained in this example is 1.89E+9, which is greater than 1.0E+9, and the titer of the obtained nanobody phage display library is 1.76E+13 cfu / mL, which is greater than 1.0E+12 cfu / mL.

[0130] Example 3: Screening of Nanobody Phage Display Library

[0131] The panning of the phage display library uses liquid panning based on streptavidin plus biotin-labeled target antigen and solid-phase panning or combined panning based on the ELISA method. During liquid panning, the phage library of >1.0E+12 cfu and streptavidin-conjugated magnetic beads BeaverBeads Mag Streptavidin (Beaver Biotechnology, product number 22305-1) were blocked separately using SuperBlock PBS Blocking Buffer (Thermo, product number 37580). After washing, Biotinylated Human TSLP (R127A,R130A) Protein,His,Avi tag (ACRO BIOSYSTEMS, product number TSP-H82E0) was added to the magnetic beads for incubation. After rinsing with PBST (1xPBS, 0.05% Tween20), the blocked phage library was added and mixed evenly at room temperature for reaction. After rinsing with PBST, the magnetic beads were separated, and the phage sub-library was eluted with 1 mg / mL Trypsin and used to infect the host ER2738. The eluted sub-library was cultured and infected with the helper phage M13K07 for rescuing the phage sub-library for the next round of panning. During combined panning, first, the TSLPR-His (Sino Biological, product number 29749-H08H) protein and the Human TSLP (R127A,R130A)-His protein were used to form a complex on the ELISA plate. The blocked phage display library was added for incubation, and the supernatant was taken for liquid panning. After multiple rounds of panning of adsorption-rinsing-elution-host infection, monoclonal clones were randomly picked from the eluted sub-library counting plate. After culturing in deep well plates, they were infected with the helper phage M13K07, and the monoclonal recombinant phages were packaged. The positive clones that could bind to the immunogen were screened by ELISA using the phage-containing supernatant. The positive clones were sequenced, the sequences were aligned, and the candidate antibodies were selected for expression and purification.

[0132] Example 4: Expression and purification of candidate antibodies

[0133] A suitable signal peptide was added to the N-terminus of the candidate antibody sequence, and Human IgG1 (N297G) Fc (Uniprot: P01857-2, Glu99-Lys330, N297G) was fused to the C-terminus. After codon optimization, it was cloned into the expression vector pCDNA3.4 (GENEWIZ) and transfected into ExpiCHO-S cells (Thermo Fisher Scientific) for expression according to the manufacturer's instructions. The supernatant was collected and purified by Protein A (Cytiva) to obtain the candidate antibody protein.

[0134] The amino acid sequence of the variable region of the candidate antibody protein TA-10 is shown in SEQ ID NO:6;

[0135] The amino acid sequence of the variable region of the candidate antibody protein P37-18 is shown in SEQ ID NO:8.

[0136] Example 5: Determination of the level binding activity of the candidate antibody protein

[0137] The level binding activity of the candidate antibody protein was determined by ELISA: The enzyme-linked immunosorbent assay (ELISA) plate (Thermo, cat. no. 442404) was coated with Human TSLP(R127A,R130A)Protein,His Tag (ACRO BIOSYSTEMS, cat. no. TSP-H52Ha) overnight. After washing with PBST (1xPBS, 0.05% Tween 20), 2% BSA was added and blocked at room temperature for 1 h. The candidate antibody was serially diluted 3-fold with 1% BSA and added to the blocked and rinsed ELISA plate (Thermo, cat. no. 442404), and incubated at room temperature for 1 h. After washing with PBST, rabbit anti-human IgG H&L (HRP) (abcam, cat. no. ab6759) diluted with 1% BSA was added and incubated at room temperature for 1 h. After washing with PBST, TMB was added for color development. After termination, the OD450 value was read with an enzyme-linked immunosorbent assay reader and a curve was plotted. The results are as Figure 1a - Figure 1b shown, both TA-10 and P37-18 can bind to Human TSLP(R127A,R130A)Protein,His Tag.

[0138] Example 6: Determination of the level TSLPR blocking activity of the candidate antibody protein

[0139] The enzyme-linked immunosorbent assay (ELISA) plate (Thermo, cat. no. 442404) was coated with CRLF2 / TSLPR Protein,Human,Recombinant(His Tag) (Sino Biological, cat. no. 29749-H08H) overnight. After washing with PBST, 2% BSA was added and blocked at room temperature for 1 h. After washing with PBST, the candidate antibody serially diluted with 1% BSA was added, and then TSLP-mFc diluted to 0.04 μg / mL with 1% BSA was added to the blocked and rinsed ELISA plate, and incubated at room temperature for 1 h. After washing with PBST, rabbit anti-human IgG H&L (HRP) (abcam, cat. no. ab6759) diluted with 1% BSA was added and incubated at room temperature for 1 h. After washing with PBST, TMB was added for color development. After termination, the OD450 value was read with an enzyme-linked immunosorbent assay reader and a curve was plotted. The results are as Figure 2a - Figure 2b shown, the blocking activities of TA-10 and P37-18 on the binding of TSLP to TSLPR at the protein level are very weak.

[0140] Example 7: Determination of the level IL-7Ra blocking activity of the candidate antibody protein

[0141] a) Coat the ELISA plate overnight with 1 μg / mL Human IL-7Ra Fc (purchased from ACRO BIOSYSTEMS, catalog number ILA-H525a), wash the plate with PBST, and then block the ELISA plate;

[0142] b) Incubate TSLPR-His (Sino Biological, catalog number 29749-H08H) and TSLP-mFc (ACROBIOSYSTEMS, catalog number TSP-H5255) with 1% BSA at room temperature for 1 h;

[0143] c) Gradient dilute the antibody to be tested;

[0144] d) After washing the plate with PBST, add the diluted antibody and the TSLPR-His-TSLP-mFc mixture to the ELISA plate successively. Do not add the antibody to the last well in each row as a control, and incubate at room temperature for 1 h;

[0145] e) Add Goat Anti-Mouse IgG, HRP diluted with 1% BSA and incubate at room temperature for 1 h;

[0146] f) After washing the plate with PBST, add the TMB chromogenic reagent and incubate at room temperature for color development, then add the stop solution to terminate the reaction, and read the OD450 value with an ELISA reader.

[0147] Blocking percentage = (OD450 of control well – OD450 of well with antibody) / OD450 of control well.

[0148] The results are as Figure 3 shown that P37-18 and TA-10 have good blocking activities against the binding of TSLP to IL-7Ra at the protein level.

[0149] Example 8: Detection of the affinity of candidate antibodies for human TSLP

[0150] Using the BLI technology, capture biotinylated TSLP (Acro, TSP-H82E0) with an SA probe and determine its binding affinity for TA-10, P37-18, and Tezepelumab. Antigen concentration: 5 μg / ml; antibody concentration: 50, 25, 12.5, 6.25, 3.125, 0 nM; experimental conditions: Loading-Threshold 0.1 nm, Baseline-250 s, Association-120 s, Dissociatioon-600 s.

[0151] The affinity results (Table 1) show that both TA-10 and P37-18 can bind to human TSLP with high affinity.

[0152] Table 1 Detection results of the affinity of candidate antibodies

[0153]

[0154] Example 9: Effect of candidate antibodies on inhibiting the activation of STATs and JAK2 signaling pathways in H_TSLP Reporter Cell Line by TSLP

[0155] The H_TSLP Reporter Cell Line is a Luciferase reporter cell line constructed based on the Jak2-Stat signaling pathway. After TSLP binds to TSLPR, the dimer enhances the recruitment of IL-7Rα to form an extracellular ternary complex, and Jak2 is activated. Jak2 further mediates the phosphorylation of Stats, causing Stats to translocate into the nucleus, thereby activating the expression of luciferase. This method can effectively detect the inhibitory effect of anti-TSLP antibodies on the activation of H_TSLP Reporter Cell Line by TSLP. Specific operation: Wash the H_TSLP Reporter Cell Line cells (GemGen, product number: GM-C15572) in the logarithmic growth phase once with 1640 + 1% FBS + 1% PS and adjust the cell density to 2E6 cells / ml, then inoculate them into a white 96-well cell culture plate (Corning, product number: 3917), 50 μl / well; incubate overnight at 37 °C and 5% CO2. The next day, mix an appropriate amount of TSLP (Acro Biosystems, product number: TSP-H52Ha) with different concentrations of anti-TSLP antibodies (starting concentration 4 * 25 nM, 10-fold serial dilution, 8 concentration points) in equal volume and incubate in the dark at 37 °C for 15 minutes. Add the TSLP and antibody mixture to the 96-well plate inoculated with H_TSLP Reporter Cell Line cells and incubate at 37 °C and 5% CO2 for 6 hours. Add Bright-Lumi TM II Firefly Luciferase Reporter Gene Detection Kit (Beyotime, product number: RG052M), 100 μl / well; incubate in the dark at room temperature for 5 - 10 minutes. Use a multifunctional microplate reader (MD, SpectraMax i3X) to detect the LUM value. Use the formula: Inhibition percentage (%) = (RLU TSLP对照 -RLU 样品 ) / (RLU TSLP control - RLU cell control ) * 100% to calculate the inhibition efficiency of the sample. Where: RLU 样品 represents the well with TSLP, anti-TSLP antibody, and cells added; RLU cell control represents the well with only cells added; RLU TSLP对照Indicates the wells with TSLP and cells added. Using the final concentration of the antibody as the abscissa and the inhibition percentage of the antibody at the corresponding concentration as the ordinate for non-linear fitting to calculate the IC50 value. The results are as follows Figure 4a - Figure 4b Show that TA-10 and P37-18 antibodies can effectively inhibit the activation of the STATs and JAK2 signaling pathways of H_TSLP Reporter Cell Line cells by TSLP, and the inhibitory activities are 1.48 and 1.65 times that of Tezepelumab, respectively.

[0156] Example 10: Inhibitory effect of anti-human TSLP antibody on the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells

[0157] BaF3-TSLPR / IL7Rα cells are a stable cell line with high expression of TSLP receptor and rely on mIL3 for proliferation. When mIL3 is not added, TSLP can effectively promote the proliferation of BaF3-TSLPR / IL7Rα cells. This method can effectively evaluate the inhibitory effect of anti-TSLP antibody on the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells. Specific operation: Prepare a 4*0.625 ng / ml TSLP (Acro Biosystems, catalog number: TSP-H52Ha) solution with 1640 + 10% FBS + 1% PS analysis buffer, add it to a 96-well U-bottom plate, 50 μl / well; Dilute the anti-TSLP antibody to different concentrations (starting concentration 4*166.67 ng / ml, 6-fold serial dilution, 8 concentration points) with 1640 + 10% FBS + 1% PS analysis buffer and add it to the 96-well U-bottom plate with TSLP added, 50 μl / well, incubate at 37 °C for 15 minutes; Resuspend BaF3-TSLPR / IL7Rα with 1640 + 10% FBS + 1% PS analysis buffer and adjust the density to 5E4 / ml, add it to the above 96-well U-bottom plate, 100 μl / well; Incubate at 37 °C for 72 hours; Transfer the incubated cells to a new white 96-well plate, 100 μl / well; Detect with CellCounting-Lite2.0 Luminescent Cell Viability Assay (Novoprotein, catalog number: DD1101-02), 100 μl / well, incubate at room temperature in the dark for 5 - 10 minutes, and read the LUM value on a multi-functional microplate reader. Use the formula: Inhibition percentage (%) = (RLU TSLP对照 -RLU 样品 ) / (RLU TSLP对照 -RLU cell control ) * 100% to calculate the inhibition efficiency of the sample. Where: RLU 样品 Indicates the well with TSLP, anti-TSLP antibody, and cells added; RLUcell control Indicate the wells with only cells added; RLU TSLP对照 Indicate the wells with TSLP and cells added. Using the final concentration of the antibody as the abscissa and the inhibition percentage of the antibody at the corresponding concentration as the ordinate, perform non-linear fitting to calculate the IC50 value. The results ( Figure 5a - Figure 5b ) show that TA-10 and P37-18 antibodies can effectively inhibit the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells, and the inhibitory activities are 2.7 and 2.6 times that of Tezepelumab, respectively.

[0158] Example 11: Epitope competition experiment between candidate antibodies and Tezepelumab

[0159] Experimental principle: Through the BLI technology, use the SA probe to capture the biotinylated first antibody, bind to the antigen, and then bind to the second antibody for epitope grouping.

[0160] Experimental steps: (1) The SA probe binds to the biotinylated first antibody Tezepelumab; (2) Bind to the antigen human TSLP; (3) Then bind to the solvent (blank control) or the non-biotinylated second antibody P37-18 or TA-10 respectively.

[0161] Experimental results: Both P37-18 and TA-10 bind to different epitopes of Tezepelumab binding to human TSLP (Table 2). According to the published literature (Nat Commun. 2017; 8:14937.), Tezepelumab binds in the C-terminal region. The nanobodies P37-18 and TA-10 screened in the present disclosure do not cross-react with the epitope bound by Tezepelumab and will not block the antibacterial peptide MKK34 with antibacterial activity at the C-terminal, and are expected to retain the ability of TSLP to resist external bacterial infections.

[0162] Table 2 Second antibody binding signal values

[0163]

[0164] Finally, it should be noted that the above are only the preferred embodiments of the present disclosure and are not used to limit the present disclosure. Although the present disclosure has been described in detail with reference to the embodiments, for those skilled in the art, they can still modify the technical solutions recorded in each embodiment, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A TSLP-binding molecule comprising at least one TSLP-binding domain, said TSLP-binding domain comprising CDR1, CDR2 and CDR3, wherein, (i) CDR1, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 3 or consists of SEQ ID NO: 3; (ii) CDR2, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 4 and 7 or consists of one of SEQ ID NO: 4 and 7; and (iii) CDR3, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 5 or consists of SEQ ID NO:

5.

2. The TSLP-binding molecule according to claim 1, wherein the TSLP-binding domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 6 and 8, or consists of one of SEQ ID NO: 6 and 8.

3. The TSLP-binding molecule according to any one of the preceding claims, wherein the TSLP-binding domain is a nanobody or a functional derivative thereof, preferably, the nanobody or its functional derivative is obtained by screening a nanobody library using TSLP as the bait protein.

4. The TSLP-binding molecule according to any one of the preceding claims, which comprises two or more TSLP-binding domains, and the two or more TSLP-binding domains have the same or different amino acid sequences.

5. The TSLP-binding molecule according to any one of the preceding claims, which further comprises at least one bioactive functional domain or functional fragment, and the bioactive functional domain or functional fragment comprises components such as a carrier protein, an active peptide, a tag peptide, a chaperone protein, etc., which are linked to the TSLP-binding domain by covalent bonds, non-covalent bonds or linkers; wherein preferably, the carrier protein comprises BSA, OVA, HSA, KLH, Fc, etc.; the active peptide comprises a linear peptide or a cyclic peptide and has enzyme activity, cytotoxic activity, antigen-binding activity, etc.; the tag peptide comprises a purification tag peptide, a tracer tag peptide, etc.; the chaperone protein comprises HSP60, HSP70, HSP90, etc.

6. The TSLP-binding molecule according to any one of the preceding claims, which is a nanobody, a heavy-chain antibody, a VHH-Fc fusion protein, a monovalent antibody, a bivalent antibody, a multivalent antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, an immunoconjugate.

7. A polynucleotide encoding the TSLP-binding molecule according to any one of claims 1-6.

8. Use of the TSLP binding molecule according to any one of claims 1-6 in the preparation of a drug and / or the extension of the half-life of a drug.

9. The TSLP binding molecule according to any one of claims 1-6, characterized in that, An active molecule having a prophylactic or therapeutic effect is bound to the TSLP binding molecule or the TSLP binding domain by a covalent bond, a non-covalent bond, or a linker molecule.

10. The TSLP binding molecule according to any one of claims 1-6, its use in diseases related to TSLP expression, the diseases related to TSLP expression being autoimmune diseases, and the autoimmune diseases being selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, myasthenia gravis, polymyositis, psoriasis, pemphigus, vitiligo, multiple sclerosis, narcolepsy, neuromyelitis optica, type 1 diabetes, hyperthyroidism, hypothyroidism, Crohn's disease, ulcerative colitis, celiac disease, autoimmune gastritis, primary biliary cholangitis, autoimmune hepatitis, and lupus nephritis.

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