Anti-TSLP single-domain antibody and derivative thereof

Abstract

Provided in the present invention are an anti-TSLP single-domain antibody and a derivative thereof.

Description

Anti-tslp single-domain antibodies and derivatives thereof

[0001]

[0002] This application claims priority to the application with the application date of September 30, 2024, the application number of PCT / CN2024 / 122841, and the invention name of "Anti-TSLP single-domain antibodies and derivatives thereof", the complete content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] Generally, the present application relates to the field of antibodies. Specifically, the present application relates to anti-human TSLP single-domain antibodies and derivatives thereof. BACKGROUND

[0004] Thymic stromal lymphopoietin (TSLP) is a short-chain four alpha-helix bundle type I IL-2 family cytokine member, homologous to IL-7, mainly released by epithelial cells to respond to various environmental injuries of the human body, and can also be produced by airway smooth muscle cells, keratinocytes, fibroblasts, mast cells, monocyte-macrophages, granulocytes and dendritic cells. Various triggers such as allergens, cigarette smoke extract, cytokines, viruses, bacterial and fungal products, and tryptase activate lung epithelial cells, intestinal epithelial cells, and keratinocytes to release TSLP. TSLP binds to its receptor TSLPR, and then recruits the IL-7Rα subunit to form a TSLP / TSLPR / IL-7Rα ternary complex, activates the JAK1 and JAK2, STAT5A and STAT5B, and STAT1 and STAT3 signaling pathways, and initiates pro-inflammatory signaling. As an alarm signal for epithelial integrity damage, TSLP is located in the early upstream of the inflammatory cascade, and can not only drive the release of downstream Th2 factors IL-4, IL-5, and IL-13, but also participate in non-Th2-driven inflammatory responses.

[0005] Nanobodies, also known as single-domain antibodies, are derived from heavy chain-only antibodies naturally existing in camels, and have the characteristics of high stability, good water solubility, simple humanization, strong penetration, etc. compared with traditional IgG antibodies, and have become a new force in the new generation of therapeutic biological medicines and clinical diagnostic reagents. Currently, there is no nanobody targeting TSLP double epitopes. Compared with single epitope nanobodies, double epitope nanobodies have higher blocking activity. Therefore, developing nanobody drugs with high blocking activity, high clinical efficacy, and low production cost has very high medical value. SUMMARY

[0006] The present application provides a group of anti-TSLP single-domain antibodies and derivatives thereof.

[0007] ​In one embodiment, the present application provides an antibody that specifically binds to TSLP.

[0008] The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0009] The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.

[0010] "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG class antibodies are

[0011] The heavy chains of antibodies can be assigned to one of five types, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), and mu (IgM), some of which can be further divided into subclasses or isotypes, e.g., gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), gamma 4 (IgG4), alpha 1 (IgA1), and alpha 2 (IgA2).

[0012] The light chains of antibodies can be assigned to one of two types, called kappa (K) and lambda (l), based on their amino acid sequences.

[0013] "Antibody fragments" refer to molecules other than intact antibodies that comprise a portion of an intact antibody that is sufficient to bind to an antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and single-domain antibodies.

[0014] Single-domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single-domain antibody was derived from the variable domain of an antibody heavy chain from camelids (nanobodies or VHHs). H H fragments).

[0015] "Specifically binds" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind a particular antigen can be measured via enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one skilled in the art, such as surface plasmon resonance (SPR) technology (analysis on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antibody to an unrelated protein is less than about 10% of the binding of the antibody to the antigen, as measured, e.g., by SPR.

[0016] "Affinity" or "binding affinity" 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 the intrinsic binding affinity reflecting 1 : 1 interactions between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ), which is the ratio of the dissociation and association rate constants (k off and k on , respectively). As such, equivalent affinities can comprise different rate constants, so long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. One particular method for measuring affinity is surface plasmon resonance (SPR).

[0017] In one embodiment, the antibody has a K D value of 5.0 x 10 -8 M or less, 1.0 x 10 -8 M or less, 5.0 x 10 -9 M or less, 1.0 x 10 -9 M or less, 5.0 x 10 -10 M or less, 1.0 x 10 - 10 M or less, 5.0 x 10 -11 M or less, 1.0 x 10 -11 M or less, 5.0 x 10 -8 M to 1.0 x 10 - 13 M.

[0018] In one embodiment, the antibody of the application is a monoclonal antibody.

[0019] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that can arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0020] In one embodiment, the antibody of the application is a chimeric antibody. In one embodiment, the variable region of the chimeric antibody is from a llama and the constant region of the chimeric antibody is from a human.

[0021] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portion of the heavy and / or light chain is derived from a different source or species.

[0022] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T.J. et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind the particular antigen can be isolated using a VH or VL domain from an antibody that binds the antigen by screening libraries of complementary VL or VH domains, respectively. See, e.g., Portolano, S. et al., J. Immunol. 150:880-887 (1993); Clarkson, T. et al., Nature 352:624-628 (1991)).

[0023] "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue Cys226, or Pro230, to the carboxy-terminus of the heavy chain. However, antibodies generated by host cells can undergo post-translational cleavage, with one or more, and particularly one or two, amino acids being excised from the C-terminus of the heavy chain. Thus, an antibody generated by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain can include a full-length heavy chain, or it can include a cleaved variant of the full-length heavy chain (also referred to as a "cleaved variant heavy chain"). This can be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447) of an Fc region can or can not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. A "subunit" of an Fc region refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable association with itself. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0024] In one embodiment, an antibody of the application is a humanized antibody.

[0025] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues both from a non-human HVR and from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, a humanized antibody can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0026] The term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain which are highly divergent in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain the antigen-contacting residues ("antigen contacting"). Generally, antibodies comprise six HVRs: three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). Exemplary HVRs herein include:

[0027] (a) the hypervariable loops which occur at amino acid residues 26-32 (LI), 50- 52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. MoI. Biol. 196:901-917 (1987));

[0028] (b) the CDRs which occur at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (HI), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0029] (c) the antigen contacts which occur at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (HI), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. MoI. Biol. 262:732-745 (1996)); and

[0030] (d) a combination of (a), (b), and / or (c), including HVR amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35 (HI), 50-63 (H2), and 95-102 (H3).

[0031] Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in a variable domain are numbered in accordance with Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) numbering.

[0032] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. Generally, the FRs of a variable domain consist of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0033] "Human consensus framework" refers to a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup is a subgroup as in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

[0034] In one embodiment, the antibody of the application comprises a first VHH domain that specifically binds to TSLP. In one embodiment, the first VHH domain comprises the CDR1, CDR2 and CDR3 of any one of VHH domains T01 to T34, T44-T54, T71 and T72 (see Table 1). In one embodiment, the first VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of VHH domains T01 to T34, T44-T54, T71 and T72 (see Table 3). In one embodiment, the first VHH domain comprises the amino acid sequence of any one of VHH domains T01 to T34, T44-T54, T71 and T72 (see Table 3).

[0035] In one embodiment, the antibody of the application is a heavy chain-only antibody, i.e. composed of heavy chains only, devoid of light chains, e.g. a heavy chain dimer. In one embodiment, the heavy chain dimer is a homodimer. In one embodiment, the heavy chain dimer is a heterodimer. In one embodiment, the heavy chain heterodimer binds the same antigen. In one embodiment, the heavy chain heterodimer binds the same epitope of the same antigen. In one embodiment, the heavy chain heterodimer binds different epitopes of the same antigen. In one embodiment, the heavy chain heterodimer binds different antigens.

[0036] In one embodiment, the antibody of the application is a single domain antibody, i.e. composed of a VHH domain only.

[0037] In one embodiment, the antibody of the application is a monospecific antibody, having binding specificity for TSLP only.

[0038] In one embodiment, the antibody of the application is a monovalent antibody, i.e. comprising one binding site, e.g. composed of one VHH domain.

[0039] In one embodiment, the antibody of the application comprises a first VHH domain that specifically binds to a first epitope of TSLP and a second VHH domain that specifically binds to a second epitope of TSLP, said first epitope of TSLP being different from said second epitope of TSLP. In one embodiment, said first VHH domain binds to the same TSLP epitope as any one of VHH domains T01 to T06, and said second VHH domain binds to the same TSLP epitope as any one of VHH domains T07 to T34. In one embodiment, said first VHH domain binds to the same TSLP epitope as any one of VHH domains T07 to T34, and said second VHH domain binds to the same TSLP epitope as any one of VHH domains T01 to T06. In one embodiment, said first VHH domain comprises the CDR1, CDR2 and CDR3 of any one of VHH domains T01 to T06, and said second VHH domain comprises the CDR1, CDR2 and CDR3 of any one of VHH domains T07 to T34. In one embodiment, said first VHH domain comprises the CDR1, CDR2 and CDR3 of any one of VHH domains T07 to T34, and said second VHH domain comprises the CDR1, CDR2 and CDR3 of any one of VHH domains T01 to T06. In one embodiment, said first VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of VHH domains T01 to T06, and said second VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of VHH domains T07 to T34. In one embodiment, said first VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of VHH domains T07 to T34, and said second VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of VHH domains T01 to T06. In one embodiment, said first VHH domain comprises the amino acid sequence of any one of VHH domains T01 to T06, and said second VHH domain comprises the amino acid sequence of any one of VHH domains T07 to T34. In one embodiment, said first VHH domain comprises the amino acid sequence of any one of VHH domains T07 to T34, and said second VHH domain comprises the amino acid sequence of any one of VHH domains T01 to T06.

[0040] In one embodiment, the first VHH domain binds to the same TSLP epitope as any one of VHH domains T01-T06 and T44-T53, and the second VHH domain binds to the same TSLP epitope as any one of VHH domains T07-T34 and T54. In one embodiment, the first VHH domain binds to the same TSLP epitope as any one of VHH domains T07-T34 and T54, and the second VHH domain binds to the same TSLP epitope as any one of VHH domains T01-T06 and T44-T53. In one embodiment, the first VHH domain comprises the CDR1, CDR2, and CDR3 of any one of VHH domains T01-T06 and T44-T53, and the second VHH domain comprises the CDR1, CDR2, and CDR3 of any one of VHH domains T07-T34 and T54. In one embodiment, the first VHH domain comprises the CDR1, CDR2, and CDR3 of any one of VHH domains T07-T34 and T54, and the second VHH domain comprises the CDR1, CDR2, and CDR3 of any one of VHH domains T01-T06 and T44-T53. In one embodiment, the first VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of VHH domains T01-T06 and T44-T53, and the second VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of VHH domains T07-T34 and T54. In one embodiment, the first VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of VHH domains T07-T34 and T54, and the second VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of VHH domains T01-T06 and T44-T53. In one embodiment, the first VHH domain comprises the amino acid sequence of any one of VHH domains T01-T06 and T44-T53, and the second VHH domain comprises the amino acid sequence of any one of VHH domains T07-T34 and T54. In one embodiment, the first VHH domain comprises the amino acid sequence of any one of VHH domains T07-T34 and T54, and the second VHH domain comprises the amino acid sequence of any one of VHH domains T01-T06 and T44-T53.

[0041] In one embodiment, the first VHH domain binds to the same TSLP epitope as any one of VHH domains T01-T54, and the second VHH domain binds to the same TSLP epitope as any one of VHH domains T71 and T72. In one embodiment, the first VHH domain binds to the same TSLP epitope as any one of VHH domains T71 and T72, and the second VHH domain binds to the same TSLP epitope as any one of VHH domains T01-T54. In one embodiment, the first VHH domain comprises the CDR1, CDR2, and CDR3 of any one of VHH domains T01-T54, and the second VHH domain comprises the CDR1, CDR2, and CDR3 of any one of VHH domains T71 and T72. In one embodiment, the first VHH domain comprises the CDR1, CDR2, and CDR3 of any one of VHH domains T71 and T72, and the second VHH domain comprises the CDR1, CDR2, and CDR3 of any one of VHH domains T01-T54. In one embodiment, the first VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of VHH domains T01-T54, and the second VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of VHH domains T71 and T72. In one embodiment, the first VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of VHH domains T71 and T72, and the second VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of VHH domains T01-T54. In one embodiment, the first VHH domain comprises the amino acid sequence of any one of VHH domains T01-T54, and the second VHH domain comprises the amino acid sequence of any one of VHH domains T71 and T72. In one embodiment, the first VHH domain comprises the amino acid sequence of any one of VHH domains T71 and T72, and the second VHH domain comprises the amino acid sequence of any one of VHH domains T01-T54.

[0042] In one embodiment, the first VHH domain binds the same TSLP epitope as VHH domain T71, the second VHH domain binds the same TSLP epitope as VHH domain T72. In one embodiment, the first VHH domain binds the same TSLP epitope as VHH domain T72, the second VHH domain binds the same TSLP epitope as VHH domain T71. In one embodiment, the first VHH domain comprises the CDR1, CDR2 and CDR3 of VHH domain T71, the second VHH domain comprises the CDR1, CDR2 and CDR3 of VHH domain T72. In one embodiment, the first VHH domain comprises the CDR1, CDR2 and CDR3 of VHH domain T72, the second VHH domain comprises the CDR1, CDR2 and CDR3 of VHH domain T71. In one embodiment, the first VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VHH domain T71, the second VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VHH domain T72. In one embodiment, the first VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VHH domain T72, the second VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VHH domain T71. In one embodiment, the first VHH domain comprises the amino acid sequence of VHH domain T71, the second VHH domain comprises the amino acid sequence of VHH domain T72. In one embodiment, the first VHH domain comprises the amino acid sequence of VHH domain T72, the second VHH domain comprises the amino acid sequence of VHH domain T71.

[0043] In one embodiment, the first VHH domain and the second VHH domain are fused via a peptide linker.

[0044] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids, often glycine (G) and / or serine (S). Suitable linker peptides are, for example, (G4S) n , (SG4) n , (SG4) nS or G4 (SG4) n Peptide linker, wherein "n" is typically an integer from 1 to 10, typically an integer from 1 to 6, in particular 4.

[0045] In one embodiment, the antibody of the application comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of T35 to T40 and T55 to T69. In one embodiment, the antibody of the application comprises the amino acid sequence of any one of T35 to T40 and T55 to T69.

[0046] In one embodiment, the antibody of the present application is a multivalent antibody (e.g., bivalent antibody, trivalent antibody, tetravalent antibody, etc.), i.e., comprises multiple binding sites, e.g., each binding site is constituted by one VHH domain, which can be fused to each other via the peptide linker as described above. In one embodiment, the multivalent antibody of the present application is monospecific antibody, i.e., only has binding specificity for TSLP. In one embodiment, the multivalent antibody of the present application is a monoepitope antibody, e.g., the epitope bound by any one of T01-T06 and T44 to T53 of the present application, the epitope bound by any one of T07-T34 and T54 of the present application, the epitope bound by T71 of the present application, or the epitope bound by T72 of the present application. In one embodiment, the multivalent antibody of the present application is a multi-epitope antibody (e.g., bi-epitope antibody, tri-epitope antibody, tetra-epitope antibody, etc.), e.g., comprises the epitope bound by any one of T01-T06 of the present application and / or the epitope bound by any one of T07-T34 of the present application, and further e.g., comprises the epitope bound by any one of T01-T06 and T44 to T53 of the present application and / or the epitope bound by any one of T07-T34 and T54 of the present application and / or the epitope bound by T71 of the present application and / or the epitope bound by T72 of the present application. In one embodiment, the multivalent antibody of the present application is a bi-epitope antibody, e.g., the epitope bound by any one of T01-T06 of the present application and the epitope bound by any one of T07-T34 of the present application, and further e.g., the epitope bound by any one of T01-T06 and T44 to T53 of the present application and the epitope bound by any one of T07-T34 and T54 of the present application. In the embodiment of multi-epitope antibody (e.g., bi-epitope antibody, tri-epitope antibody, tetra-epitope antibody, etc.), the binding valency of the antibody of the present application for each epitope is independent of each other, or is the same or different. For example, the binding valency of the antibody of the present application for the epitope bound by any one of T01-T06 of the present application is independently 1, 2, 3, 4 or more, and the binding valency of the antibody of the present application for the epitope bound by any one of T07-T34 of the present application is independently 1, 2, 3, 4 or more. For another example, the binding valency of the antibody of the present application for the epitope bound by any one of T01-T06 and T44 to T53 of the present application is independently 1, 2, 3, 4 or more, and the binding valency of the antibody of the present application for the epitope bound by any one of T07-T34 and T54 of the present application is independently 1, 2, 3, 4 or more. For example, the binding valency of the antibody of the present application for the epitope bound by any one of T01-T06 of the present application and the binding valency of the antibody of the present application for the epitope bound by any one of T07-T34 of the present application are the same, both are 1, 2, 3, 4 or more. For another example, the binding valency of the antibody of the present application for the epitope bound by any one of T01-T06 and T44 to T53 of the present application and the binding valency of the antibody of the present application for the epitope bound by any one of T07-T34 and T54 of the present application are the same, both are 1, 2, 3, 4 or more.For example, the binding valence of an antibody of the application to an epitope bound by any of T01-T06 of the application and the binding valence of an antibody of the application to an epitope bound by any of T07-T34 of the application are different, 1+2, 2+1, 1+4, 4+1, 2+4, 4+2, etc. For another example, the binding valence of an antibody of the application to an epitope bound by any of T01-T06 and T44 to T53 of the application and the binding valence of an antibody of the application to an epitope bound by any of T07-T34 and T54 of the application are different, 1+2, 2+1, 1+4, 4+1, 2+4, 4+2, etc.

[0047] In one embodiment, an antibody of the application comprises a first VHH domain that specifically binds to TSLP (e.g., T05) and a second VHH domain that specifically binds to TSLP (e.g., T34). In one embodiment, an antibody of the application comprises, in order from N- to C-terminus, a first VHH domain (e.g., T05) and a second VHH domain (e.g., T34).

[0048] In one embodiment, an antibody of the application comprises an Fc region. In one embodiment, the Fc region is an Fc region of IgG. In one embodiment, the Fc region is an Fc region of IgG1, IgG2, or IgG4, including wild-type and variants, particularly variants that prolong half-life. In one embodiment, the Fc region of IgG4 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 283, 404, or 405.

[0049] In one embodiment, an antibody of the application is a multispecific antibody (e.g., bispecific antibody, trispecific antibody, tetraspecific antibody, etc.). In one embodiment, the multispecific antibody has binding specificities for TSLP and a different antigen. In one embodiment, the binding to the different antigen prolongs the half-life of the antibody. In one embodiment, the different antigen is serum albumin.

[0050] In one embodiment, a multispecific antibody (e.g., bispecific antibody) of the application has binding specificities for TSLP and serum albumin. In one embodiment, a multispecific antibody (e.g., trispecific antibody) of the application has binding specificities for two different TSLP epitopes and serum albumin.

[0051] In one embodiment, a VHH that specifically binds to serum albumin comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 282.

[0052] In one embodiment, the VHH that specifically binds to serum albumin is located N-terminal to the first VHH that specifically binds to TSLP and / or the second VHH that specifically binds to TSLP. In one embodiment, the VHH that specifically binds to serum albumin is located C-terminal to the first VHH that specifically binds to TSLP and / or the second VHH that specifically binds to TSLP.

[0053] In one embodiment, the VHH that specifically binds to serum albumin is located N-terminal, C-terminal or between the first VHH that specifically binds to TSLP and the second VHH that specifically binds to TSLP.

[0054] In one embodiment, the VHH that specifically binds to serum albumin is linked to the first VHH that specifically binds to TSLP and / or the second VHH that specifically binds to TSLP via a linker. In one embodiment, the VHH that specifically binds to serum albumin is linked to the first VHH that specifically binds to TSLP and the second VHH that specifically binds to TSLP via independently selected linkers.

[0055] In one embodiment, the antibody of the application comprises one first VHH domain that specifically binds to TSLP (e.g. T05), one second VHH domain that specifically binds to TSLP (e.g. T34) and one third VHH domain that specifically binds to serum albumin. In one embodiment, the antibody of the application comprises, in order from N- to C-terminus, the third VHH domain, the first VHH domain (e.g. T05) and the second VHH domain (e.g. T34). In one embodiment, the antibody of the application comprises, in order from N- to C-terminus, the first VHH domain (e.g. T05), the third VHH domain and the second VHH domain (e.g. T34). In one embodiment, the antibody of the application comprises, in order from N- to C-terminus, the first VHH domain (e.g. T05), the second VHH domain (e.g. T34) and the third VHH domain.

[0056] In one embodiment, the antibody of the application comprises a half-life extending module. In one embodiment, the half-life extending module is an Fc region or a VHH that specifically binds to serum albumin, e.g. as described hereinabove.

[0057] In one embodiment, the antibody of the application is a membrane-integrated antibody (e.g. chimeric antigen receptor, CAR). In one embodiment, the membrane-integrated antibody comprises a hinge region. In one embodiment, the hinge region is a hinge region of IgGl, IgG4, CD8a, CD28, Siglecs, NGFR, or CD34. In one embodiment, the membrane-integrated antibody comprises a transmembrane region. In one embodiment, the transmembrane region is a transmembrane region of CD3zeta, CD4, CD8a, CD28, ICOS, 4-1BB, or KIR2DS2. In one embodiment, the membrane-integrated antibody comprises an intracellular signaling domain. In one embodiment, the intracellular signaling domain is a signaling domain of CD3zeta or FcyR. In one embodiment, the membrane-integrated antibody comprises a costimulatory domain. In one embodiment, the costimulatory domain is a costimulatory domain of CD28, ICOS, 4-1BB, OX40, CD27, CD40, HVEM, GITR, MYD88-CD40, TLR2, or Dectin-1. In one embodiment, the membrane-integrated antibody comprises a signal peptide. In one embodiment, the signal peptide is a signal peptide of CD8a.

[0058] The application also provides a nucleic acid encoding the antibody of the application.

[0059] The application also provides a vector comprising the nucleic acid of the application. In one embodiment, the vector is a cloning vector or an expression vector. In one embodiment, the vector is a plasmid, a virus, or a cosmid.

[0060] The application also provides a host cell comprising the nucleic acid of the application or the vector of the application. In one embodiment, the host cell is a prokaryotic cell or a eukaryotic cell. In one embodiment, the host cell is an immune cell, e.g. a T cell or an NK cell. In one embodiment, the host cell displays the antibody of the application (in particular a membrane-integrated antibody, CAR) on the cell surface (e.g. a CAR-T cell or a CAR-NK cell).

[0061] The application also provides a method of producing an antibody, comprising culturing the host cell of the application so that the antibody is expressed.

[0062] The application also provides a composition comprising the antibody, the nucleic acid, the vector, or the host cell of the application. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 shows the affinity of T01 (panel A) and T07 (panel B) antibodies to human TLSP as determined using BIAcore as described in Example 2, 6 curves representing 6 different dilution concentrations of the antigen.

[0064] Figure 2 shows the blocking effect of T01 (panel A) and T07 (panel B) antibodies on human TSLP and its receptor-mediated signaling pathway using a human TSLP reporter cell line as described in Example 3.

[0065] Figure 3 shows the blocking effect of bispecific antibodies T35 to T40 and Tezepelumab antibody on the signaling pathway of a human TSLP reporter cell line as described in Example 5.

[0066] Figure 4 shows the binding conformation map of bispecific antibody No. T38 to TSLP antigen (panel A) and the amino acid sites of interaction on TSLP antigen (panel B) as described in Example 6.

[0067] Figure 5 shows the blocking effect of trispecific antibody No. T42 and bispecific antibody No. T38 and Tezepelumab antibody on the signaling pathway of a human TSLP reporter cell line as described in Example 7. DETAILED DESCRIPTION

[0068] The present application provides the following sequences.

[0069] Table 1: CDR sequences of exemplary antibodies (VHH domains) of the present application

[0070] Table 2: FR sequences of exemplary antibodies (VHH domains) of the present application

[0071] Table 3: Sequences of exemplary antibodies (VHH domains) of the present application

[0072] Table 4: Sequences of exemplary bispecific antibodies (VHH domain heterodimers) of the present application

[0073] Table 5: Sequences of exemplary trispecific antibodies of the present application

[0074] Sequence of VHH that specifically binds to serum albumin (SEQ ID NO: 282)

[0075] Sequence of Fc (IgG4) (SEQ ID NO: 283)

[0076] Sequence of Fc (IgG4) variant 1 (SEQ ID NO: 404)

[0077] The sequence of Fc(IgG4) variant 2 (SEQ ID NO: 405)

[0078] Example 1: Anti-TSLP single-domain antibodies were obtained by screening an alpaca immune library.

[0079] Alpacas were immunized five times with a mixture of recombinant human TSLP protein (purchased from ACRO Biosystems, catalog number TSP-H52Ha) and Freund's adjuvant. Peripheral blood was collected after each immunization to determine the titer. After the fifth immunization, peripheral blood was collected, and PBMCs were isolated. RNA was isolated and reverse transcribed into cDNA. The VHH coding sequence was amplified by PCR, and the PCR product was recovered to construct a phage display library. The library size was 3.2 × 10⁻⁶. 8 The positive cloning rate was 96% (PFU). Anti-TSLP nanobodies were screened using phage display technology. After three rounds of "adsorption-washing-enrichment" screening, single-clone ELISA verification was performed, yielding 104 differentially expressed sequences. Fifteen sequences with high OD450 were selected and cloned into pcDNA3.4 (Invitrogen). The plasmid was transfected into CHO-S cells via electroporation, causing the transformed CHO-S cells to express the VHH+Fc(IgG4) fusion protein. After one week of incubation at 37°C, the supernatant was collected, and the antibodies were purified. Purification was performed using a protein A affinity chromatography column, and the purity was detected by SDS-PAGE and SEC-HPLC. All antibodies achieved a purity of over 95%. Two candidate antibodies, T01 and T07, with high TSLP binding activity were obtained through surface plasmon resonance (SPR) screening.

[0080] Example 2: Analysis of the affinity of anti-TSLP single-domain antibody for human TSLP using BIAcore

[0081] The affinity of the VHH+Fc(IgG4) fusion protein for human TSLP was determined using a BIAcore 8000 Biosensor instrument (BIAcore AB) via surface plasmon resonance (SPR) technology. In short, antibodies carrying the Fc group were captured using a protein A chip with a surface density of approximately 500 RU. The recombinant TSLP protein was serially diluted with HBS-EP+ buffer, resulting in six dilutions (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM). The instrument detected binding curves between the antibody and different concentrations of TSLP. The kinetic data obtained from the sensor recordings were evaluated using BIAcore evaluation software.

[0082] Table 6: Binding affinity of single-domain antibodies of the present application to human TSLP antigen

[0083] Example 3: Determination of blocking activity of anti-TSLP single-domain antibodies on the signal pathway triggered by human TSLP binding to its receptor on the cell line using a human TSLP reporter cell line

[0084] The human TSLP reporter cell line (purchased from GenScript) is a luciferase reporter cell line based on the Jak2-Stat signal pathway. When TSLP binds to TSLPR, the binary complex enhances the recruitment of IL-7Ra, forming an extracellular ternary complex, and Jak2 is activated. Jak2 further mediates the phosphorylation of Stats, which is transported into the nucleus, thereby activating the expression of luciferase. The luminescence reading represents the activation effect of the signal pathway. The specific steps are as follows: inoculate the cells in a 96-well plate at a density of 5 x 10 5 Cells / well, 100 μL per well; add TSLP recombinant protein to activate the cell line, and the final concentration of TSLP per well is 15 ng / mL; after 1 hour of co-incubation, add the antibodies T01 and T07 to be tested at an initial concentration of 70 μM, 5-fold gradient dilution, and a total of 10 gradients; add 33 μL of the antibodies or blank control at the corresponding concentration to each well of cells, and continue to incubate in a 37°C CO2 incubator for 23 hours before sampling and detecting the luminescence signal.

[0085] Table 7: Blocking activity of single-domain antibodies of the present application on human TSLP and its receptor pathway

[0086] Example 4: Affinity and cell level blocking activity of anti-TSLP single-domain antibodies after humanization

[0087] The T01 and T07 antibodies were subjected to humanization and drugability modification. The humanization was performed by the CDR grafting method. The basic method is as follows: replace the camel-derived framework region with a selected human germline framework region (FR), and only retain the camel-derived CDR; then perform back mutation on the key amino acid sites in the FR region. In addition, perform amino acid substitution on the DG sites in the CDR and the adjacent region. The T01 humanization modification obtained T02 to T06 and T44-T53 antibodies, and the T07 humanization modification obtained T08 to T34 and T54 antibodies. The affinity of 43 humanized antibodies was determined using the method described in Example 2 above (Table 8). The cell level blocking activity of T08 to T16 humanized antibodies was determined using the method described in Example 3 above (Table 9).

[0088] Table 8: Binding affinity of humanized single-domain antibodies to human TSLP antigen

[0089] Table 9: Cell level blocking activity of humanized single domain antibodies

[0090] Example 5: Bispecific antibody construction and its affinity to human TSLP and cell level blocking activity

[0091] The above single domain antibodies were subjected to structure prediction using in-house developed software, and single domain antibodies with different epitopes were selected for combination to obtain 21 bispecific antibodies, Nos. T35 to T40 and T55 to T69. After expression and purification, cell level blocking activity was determined. The cell level blocking activity of bispecific antibodies was significantly improved compared to single domain antibodies. The structure of bispecific antibodies was VHH-(G4S)4-VHH. Then the affinity value of No. T38 bispecific antibody with better cell level blocking activity was determined using Biacore. The affinity of bispecific antibodies was significantly improved compared to the affinity of single domain antibodies.

[0092] Table 10: Cell level blocking activity of bispecific antibodies

[0093] Table 11: Binding affinity of bispecific antibodies to human TSLP antigen

[0094] Example 6: Epitope structure prediction of bispecific antibodies

[0095] The antigen epitope of No. T38 bispecific antibody was subjected to structure prediction using in-house developed software. No. T38 was connected by T05 and T34, both of which were directed against different antigen epitopes. The epitope bound by T05 single antibody was located at the binding interface of TSLP and TSLPR, i.e. C-terminal Leu156 and Arg153 and Arg72, Ser70, Ser68, Thr66, Asn65, Phe63 and Glu62 of TSLP protein. The epitope bound by T34 single antibody was partially located at the binding interface of TSLP and TSLPR, such as Leu155, Pro154 and Asn152 and Glu62 and Lys59 of TSLP protein, and partially located at the binding interface of TSLP and IL7R, such as Thr53, Ile52, Lys49, Ser45, Leu44, Ala42, Ala41 and Glu37 of TSLP protein. Therefore, the bispecific antibody composed of T05 and T34 can better block the binding of TSLP to ligand-receptor binary complex. The EC50 mass concentration of this bispecific antibody was only 1 / 6 of the reference Tezepelumab.

[0096] Example 7: Trispecific antibody construction and its cell level blocking activity

[0097] The VHH domain that specifically binds to serum albumin was connected to different positions of T38 bispecific antibody to obtain three trispecific antibodies, T41 to T43. The VHH domain that specifically binds to serum albumin was connected to T45 and T54 to obtain T70 trispecific antibody. The structure of the trispecific antibody is VHH-(G4S)4-VHH-(G4S)4-VHH. After expression and purification, the blocking activity at the cell level was determined. Compared with T38 bispecific antibody, T42 trispecific antibody can better block the binding of TSLP to the ligand-receptor binary complex, and its EC50 mass concentration is only 1 / 10 of the reference Tezepelumab.

[0098] Table 12: Cell level blocking activity of trispecific antibodies

[0099] Example 8: Screening of anti-TSLP single-domain antibody using llama natural (non-immune) library

[0100] PBMC (peripheral blood mononuclear cells) were isolated from the peripheral blood of 40 llamas, RNA was extracted and cDNA was reverse transcribed, VHH encoding sequences were amplified by PCR and the PCR products were recovered to construct a phage display library with a library capacity of 6.3 x 10 10 pfu. Anti-TSLP nanobodies were screened using phage display technology, 15 sequences were picked and cloned into pcDNA3.4 (Invitrogen), the plasmid was transfected into CHO-S cells by electroporation method, the transformed CHO-S cells expressed VHH+Fc (IgG4) fusion protein, after incubation at 37°C for one week, the supernatant was collected, and two candidate antibodies with high TSLP binding activity were screened by bio-layer interference technology. Further mutation of amino acids in the CDR region was performed using internal AI software, and then a solid mutation library was established for screening to determine the two optimal sequences T71 and T72.

[0101] Example 9: Analysis of the affinity of anti-TSLP single-domain antibody to human TSLP by BIAcore

[0102] The affinity of the VHH+Fc (IgG4) fusion proteins of sequences T70 and T71 for human TSLP was determined using the surface plasmon resonance technique with a BIAcore 8000 Biosensor instrument (BIAcore AB). Briefly, the Fc-bearing antibodies were captured with a Protein A chip at a surface density of about 500 RU, and the TSLP recombinant protein was diluted in the HBS-EP+ buffer series, so that the resulting series of dilutions (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, for a total of 5 gradients) passed through the sensor chip surface. The instrument detected the binding curves of the antibodies with TSLP at different concentrations. The sensorgrams obtained were evaluated with the BIAcore evaluation software, obtaining the kinetic data.

[0103] Table 13: Binding affinity of the single-domain antibodies of the application to the human TSLP antigen

Claims

1. An antibody that specifically binds to TSLP, comprising a first VHH domain that specifically binds to TSLP, the first VHH domain comprising: (1) a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3; (2) a CDR1 as set forth in SEQ ID NO: 4, a CDR2 as set forth in SEQ ID NO: 5, and a CDR3 as set forth in SEQ ID NO: 6; (3) a CDR1 as set forth in SEQ ID NO: 7, a CDR2 as set forth in SEQ ID NO: 8, and a CDR3 as set forth in SEQ ID NO: 9; (4) a CDR1 as set forth in SEQ ID NO: 10, a CDR2 as set forth in SEQ ID NO: 11, and a CDR3 as set forth in SEQ ID NO: 12; (5) a CDR1 as set forth in SEQ ID NO: 13, a CDR2 as set forth in SEQ ID NO: 14, and a CDR3 as set forth in SEQ ID NO: 15; (6) a CDR1 as set forth in SEQ ID NO: 16, a CDR2 as set forth in SEQ ID NO: 17, and a CDR3 as set forth in SEQ ID NO: 18; (7) a CDR1 as set forth in SEQ ID NO: 19, a CDR2 as set forth in SEQ ID NO: 20, and a CDR3 as set forth in SEQ ID NO: 21; (8) a CDR1 as set forth in SEQ ID NO: 22, a CDR2 as set forth in SEQ ID NO: 23, and a CDR3 as set forth in SEQ ID NO: 24; (9) a CDR1 as set forth in SEQ ID NO: 25, a CDR2 as set forth in SEQ ID NO: 26, and a CDR3 as set forth in SEQ ID NO: 27; (10) a CDR1 as set forth in SEQ ID NO: 28, a CDR2 as set forth in SEQ ID NO: 29, and a CDR3 as set forth in SEQ ID NO: 30; (11) a CDR1 as set forth in SEQ ID NO: 31, a CDR2 as set forth in SEQ ID NO: 32, and a CDR3 as set forth in SEQ ID NO: 33; (12) a CDR1 as set forth in SEQ ID NO: 34, a CDR2 as set forth in SEQ ID NO: 35, and a CDR3 as set forth in SEQ ID NO: 36; (13) a CDR1 as set forth in SEQ ID NO: 37, a CDR2 as set forth in SEQ ID NO: 38, and a CDR3 as set forth in SEQ ID NO: 39; (14) a CDR1 as set forth in SEQ ID NO: 40, a CDR2 as set forth in SEQ ID NO: 41, and a CDR3 as set forth in SEQ ID NO: 42; (15) a CDR1 as depicted in SEQ ID NO: 43, a CDR2 as depicted in SEQ ID NO: 44 and a CDR3 as depicted in SEQ ID NO: 45; (16) a CDR1 as depicted in SEQ ID NO: 46, a CDR2 as depicted in SEQ ID NO: 47 and a CDR3 as depicted in SEQ ID NO: 48; (17) a CDR1 as depicted in SEQ ID NO: 49, a CDR2 as depicted in SEQ ID NO: 50 and a CDR3 as depicted in SEQ ID NO: 51 ; (18) a CDR1 as depicted in SEQ ID NO: 52, a CDR2 as depicted in SEQ ID NO: 53 and a CDR3 as depicted in SEQ ID NO: 54; (19) a CDR1 as depicted in SEQ ID NO: 55, a CDR2 as depicted in SEQ ID NO: 56 and a CDR3 as depicted in SEQ ID NO: 57; (20) a CDR1 as depicted in SEQ ID NO: 58, a CDR2 as depicted in SEQ ID NO: 59 and a CDR3 as depicted in SEQ ID NO: 60; (21) a CDR1 as depicted in SEQ ID NO: 61, a CDR2 as depicted in SEQ ID NO: 62 and a CDR3 as depicted in SEQ ID NO: 63; (22) a CDR1 as depicted in SEQ ID NO: 64, a CDR2 as depicted in SEQ ID NO: 65 and a CDR3 as depicted in SEQ ID NO: 66; (23) a CDR1 as depicted in SEQ ID NO: 67, a CDR2 as depicted in SEQ ID NO: 68 and a CDR3 as depicted in SEQ ID NO: 69; (24) a CDR1 as depicted in SEQ ID NO: 70, a CDR2 as depicted in SEQ ID NO: 71 and a CDR3 as depicted in SEQ ID NO: 72; (25) a CDR1 as depicted in SEQ ID NO: 73, a CDR2 as depicted in SEQ ID NO: 74 and a CDR3 as depicted in SEQ ID NO: 75; (26) a CDR1 as depicted in SEQ ID NO: 76, a CDR2 as depicted in SEQ ID NO: 77 and a CDR3 as depicted in SEQ ID NO: 78; (27) a CDR1 as depicted in SEQ ID NO: 79, a CDR2 as depicted in SEQ ID NO: 80 and a CDR3 as depicted in SEQ ID NO: 81 ; (28) a CDR1 as depicted in SEQ ID NO: 82, a CDR2 as depicted in SEQ ID NO: 83 and a CDR3 as depicted in SEQ ID NO: 84; (29) CDR1 as shown in SEQ ID NO: 85, CDR2 as shown in SEQ ID NO: 86, and CDR3 as shown in SEQ ID NO: 87; (30) CDR1 as shown in SEQ ID NO: 88, CDR2 as shown in SEQ ID NO: 89, and CDR3 as shown in SEQ ID NO: 90; (31) CDR1 as shown in SEQ ID NO: 91, CDR2 as shown in SEQ ID NO: 92, and CDR3 as shown in SEQ ID NO: 93; (32) CDR1 as shown in SEQ ID NO: 94, CDR2 as shown in SEQ ID NO: 95, and CDR3 as shown in SEQ ID NO: 96; (33) CDR1 as shown in SEQ ID NO: 97, CDR2 as shown in SEQ ID NO: 98, and CDR3 as shown in SEQ ID NO: 99; (34) CDR1 as shown in SEQ ID NO: 100, CDR2 as shown in SEQ ID NO: 101, and CDR3 as shown in SEQ ID NO: 102; (35) CDR1 as shown in SEQ ID NO: 284, CDR2 as shown in SEQ ID NO: 285, and CDR3 as shown in SEQ ID NO: 286; (36) CDR1 as shown in SEQ ID NO: 287, CDR2 as shown in SEQ ID NO: 288, and CDR3 as shown in SEQ ID NO: 289; (37) CDR1 as shown in SEQ ID NO: 290, CDR2 as shown in SEQ ID NO: 291, and CDR3 as shown in SEQ ID NO: 292; (38) CDR1 as shown in SEQ ID NO: 293, CDR2 as shown in SEQ ID NO: 294, and CDR3 as shown in SEQ ID NO: 295; (39) CDR1 as shown in SEQ ID NO: 296, CDR2 as shown in SEQ ID NO: 297, and CDR3 as shown in SEQ ID NO: 298; (40) CDR1 as shown in SEQ ID NO: 299, CDR2 as shown in SEQ ID NO: 300, and CDR3 as shown in SEQ ID NO: 301; (41) CDR1 as shown in SEQ ID NO: 302, CDR2 as shown in SEQ ID NO: 303, and CDR3 as shown in SEQ ID NO: 304; (42) CDR1 as shown in SEQ ID NO: 305, CDR2 as shown in SEQ ID NO: 306, and CDR3 as shown in SEQ ID NO: 307; (43) a CDR1 as set forth in SEQ ID NO: 308, a CDR2 as set forth in SEQ ID NO: 309, and a CDR3 as set forth in SEQ ID NO: 310; (44) a CDR1 as set forth in SEQ ID NO: 311, a CDR2 as set forth in SEQ ID NO: 312, and a CDR3 as set forth in SEQ ID NO: 313; (45) a CDR1 as set forth in SEQ ID NO: 314, a CDR2 as set forth in SEQ ID NO: 315, and a CDR3 as set forth in SEQ ID NO: 316; (46) a CDR1 as set forth in SEQ ID NO: 317, a CDR2 as set forth in SEQ ID NO: 318, and a CDR3 as set forth in SEQ ID NO: 319; or (47) a CDR1 as set forth in SEQ ID NO: 320, a CDR2 as set forth in SEQ ID NO: 321, and a CDR3 as set forth in SEQ ID NO:

322.

2. The antibody of claim 1, wherein the first VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS: 239-272 and 375-387.

3. The antibody of claim 1, comprising a second VHH domain that specifically binds to TSLP, the second VHH domain comprising: (1) a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3; (2) a CDR1 as set forth in SEQ ID NO: 4, a CDR2 as set forth in SEQ ID NO: 5, and a CDR3 as set forth in SEQ ID NO: 6; (3) a CDR1 as set forth in SEQ ID NO: 7, a CDR2 as set forth in SEQ ID NO: 8, and a CDR3 as set forth in SEQ ID NO: 9; (4) a CDR1 as set forth in SEQ ID NO: 10, a CDR2 as set forth in SEQ ID NO: 11, and a CDR3 as set forth in SEQ ID NO: 12; (5) a CDR1 as set forth in SEQ ID NO: 13, a CDR2 as set forth in SEQ ID NO: 14, and a CDR3 as set forth in SEQ ID NO: 15; (6) a CDR1 as set forth in SEQ ID NO: 16, a CDR2 as set forth in SEQ ID NO: 17, and a CDR3 as set forth in SEQ ID NO: 18; (7) a CDR1 as set forth in SEQ ID NO: 19, a CDR2 as set forth in SEQ ID NO: 20, and a CDR3 as set forth in SEQ ID NO: 21; (8) a CDR1 as depicted in SEQ ID NO: 22, a CDR2 as depicted in SEQ ID NO: 23 and a CDR3 as depicted in SEQ ID NO: 24; (9) a CDR1 as depicted in SEQ ID NO: 25, a CDR2 as depicted in SEQ ID NO: 26 and a CDR3 as depicted in SEQ ID NO: 27; (10) a CDR1 as depicted in SEQ ID NO: 28, a CDR2 as depicted in SEQ ID NO: 29 and a CDR3 as depicted in SEQ ID NO: 30; (11) a CDR1 as depicted in SEQ ID NO: 31, a CDR2 as depicted in SEQ ID NO: 32 and a CDR3 as depicted in SEQ ID NO: 33; (12) a CDR1 as depicted in SEQ ID NO: 34, a CDR2 as depicted in SEQ ID NO: 35 and a CDR3 as depicted in SEQ ID NO: 36; (13) a CDR1 as depicted in SEQ ID NO: 37, a CDR2 as depicted in SEQ ID NO: 38 and a CDR3 as depicted in SEQ ID NO: 39; (14) a CDR1 as depicted in SEQ ID NO: 40, a CDR2 as depicted in SEQ ID NO: 41 and a CDR3 as depicted in SEQ ID NO: 42; (15) a CDR1 as depicted in SEQ ID NO: 43, a CDR2 as depicted in SEQ ID NO: 44 and a CDR3 as depicted in SEQ ID NO: 45; (16) a CDR1 as depicted in SEQ ID NO: 46, a CDR2 as depicted in SEQ ID NO: 47 and a CDR3 as depicted in SEQ ID NO: 48; (17) a CDR1 as depicted in SEQ ID NO: 49, a CDR2 as depicted in SEQ ID NO: 50 and a CDR3 as depicted in SEQ ID NO: 51; (18) a CDR1 as depicted in SEQ ID NO: 52, a CDR2 as depicted in SEQ ID NO: 53 and a CDR3 as depicted in SEQ ID NO: 54; (19) a CDR1 as depicted in SEQ ID NO: 55, a CDR2 as depicted in SEQ ID NO: 56 and a CDR3 as depicted in SEQ ID NO: 57; (20) a CDR1 as depicted in SEQ ID NO: 58, a CDR2 as depicted in SEQ ID NO: 59 and a CDR3 as depicted in SEQ ID NO: 60; (21) a CDR1 as depicted in SEQ ID NO: 61, a CDR2 as depicted in SEQ ID NO: 62 and a CDR3 as depicted in SEQ ID NO: 63; (22) a CDR1 as depicted in SEQ ID NO: 64, a CDR2 as depicted in SEQ ID NO: 65 and a CDR3 as depicted in SEQ ID NO: 66; (23) a CDR1 as depicted in SEQ ID NO: 67, a CDR2 as depicted in SEQ ID NO: 68 and a CDR3 as depicted in SEQ ID NO: 69; (24) a CDR1 as depicted in SEQ ID NO: 70, a CDR2 as depicted in SEQ ID NO: 71 and a CDR3 as depicted in SEQ ID NO: 72; (25) a CDR1 as depicted in SEQ ID NO: 73, a CDR2 as depicted in SEQ ID NO: 74 and a CDR3 as depicted in SEQ ID NO: 75; (26) a CDR1 as depicted in SEQ ID NO: 76, a CDR2 as depicted in SEQ ID NO: 77 and a CDR3 as depicted in SEQ ID NO: 78; (27) a CDR1 as depicted in SEQ ID NO: 79, a CDR2 as depicted in SEQ ID NO: 80 and a CDR3 as depicted in SEQ ID NO: 81 ; (28) a CDR1 as depicted in SEQ ID NO: 82, a CDR2 as depicted in SEQ ID NO: 83 and a CDR3 as depicted in SEQ ID NO: 84; (29) a CDR1 as depicted in SEQ ID NO: 85, a CDR2 as depicted in SEQ ID NO: 86 and a CDR3 as depicted in SEQ ID NO: 87; (30) a CDR1 as depicted in SEQ ID NO: 88, a CDR2 as depicted in SEQ ID NO: 89 and a CDR3 as depicted in SEQ ID NO: 90; (31) a CDR1 as depicted in SEQ ID NO: 91, a CDR2 as depicted in SEQ ID NO: 92 and a CDR3 as depicted in SEQ ID NO: 93; (32) a CDR1 as depicted in SEQ ID NO: 94, a CDR2 as depicted in SEQ ID NO: 95 and a CDR3 as depicted in SEQ ID NO: 96; (33) a CDR1 as depicted in SEQ ID NO: 97, a CDR2 as depicted in SEQ ID NO: 98 and a CDR3 as depicted in SEQ ID NO: 99; (34) a CDR1 as depicted in SEQ ID NO: 100, a CDR2 as depicted in SEQ ID NO: 101 and a CDR3 as depicted in SEQ ID NO: 102; (35) a CDR1 as depicted in SEQ ID NO: 284, a CDR2 as depicted in SEQ ID NO: 285 and a CDR3 as depicted in SEQ ID NO: 286; (36) CDR1 as set forth in SEQ ID NO: 287, CDR2 as set forth in SEQ ID NO: 288, and CDR3 as set forth in SEQ ID NO: 289; (37) CDR1 as set forth in SEQ ID NO: 290, CDR2 as set forth in SEQ ID NO: 291, and CDR3 as set forth in SEQ ID NO: 292; (38) CDR1 as set forth in SEQ ID NO: 293, CDR2 as set forth in SEQ ID NO: 294, and CDR3 as set forth in SEQ ID NO: 295; (39) CDR1 as set forth in SEQ ID NO: 296, CDR2 as set forth in SEQ ID NO: 297, and CDR3 as set forth in SEQ ID NO: 298; (40) CDR1 as set forth in SEQ ID NO: 299, CDR2 as set forth in SEQ ID NO: 300, and CDR3 as set forth in SEQ ID NO: 301; (41) CDR1 as set forth in SEQ ID NO: 302, CDR2 as set forth in SEQ ID NO: 303, and CDR3 as set forth in SEQ ID NO: 304; (42) CDR1 as set forth in SEQ ID NO: 305, CDR2 as set forth in SEQ ID NO: 306, and CDR3 as set forth in SEQ ID NO: 307; (43) CDR1 as set forth in SEQ ID NO: 308, CDR2 as set forth in SEQ ID NO: 309, and CDR3 as set forth in SEQ ID NO: 310; (44) CDR1 as set forth in SEQ ID NO: 311, CDR2 as set forth in SEQ ID NO: 312, and CDR3 as set forth in SEQ ID NO: 313; (45) CDR1 as set forth in SEQ ID NO: 314, CDR2 as set forth in SEQ ID NO: 315, and CDR3 as set forth in SEQ ID NO: 316; (46) CDR1 as set forth in SEQ ID NO: 317, CDR2 as set forth in SEQ ID NO: 318, and CDR3 as set forth in SEQ ID NO: 319; or (47) CDR1 as set forth in SEQ ID NO: 320, CDR2 as set forth in SEQ ID NO: 321, and CDR3 as set forth in SEQ ID NO:

322.

4. The antibody of claim 3, wherein the second VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 239-272 and 375-387.

5. The antibody of claim 3, wherein, the first VHH domain comprises: (1) CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3; (2) CDR1 as shown in SEQ ID NO: 4, CDR2 as shown in SEQ ID NO: 5, and CDR3 as shown in SEQ ID NO: 6; (3) CDR1 as shown in SEQ ID NO: 7, CDR2 as shown in SEQ ID NO: 8, and CDR3 as shown in SEQ ID NO: 9; (4) CDR1 as shown in SEQ ID NO: 10, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12; (5) CDR1 as shown in SEQ ID NO: 13, CDR2 as shown in SEQ ID NO: 14, and CDR3 as shown in SEQ ID NO: 15; (6) CDR1 as shown in SEQ ID NO: 16, CDR2 as shown in SEQ ID NO: 17, and CDR3 as shown in SEQ ID NO: 18; (35) CDR1 as shown in SEQ ID NO: 284, CDR2 as shown in SEQ ID NO: 285, and CDR3 as shown in SEQ ID NO: 286; (36) CDR1 as shown in SEQ ID NO: 287, CDR2 as shown in SEQ ID NO: 288, and CDR3 as shown in SEQ ID NO: 289; (37) CDR1 as shown in SEQ ID NO: 290, CDR2 as shown in SEQ ID NO: 291, and CDR3 as shown in SEQ ID NO: 292; (38) CDR1 as shown in SEQ ID NO: 293, CDR2 as shown in SEQ ID NO: 294, and CDR3 as shown in SEQ ID NO: 295; (39) CDR1 as shown in SEQ ID NO: 296, CDR2 as shown in SEQ ID NO: 297, and CDR3 as shown in SEQ ID NO: 298; (40) CDR1 as shown in SEQ ID NO: 299, CDR2 as shown in SEQ ID NO: 300, and CDR3 as shown in SEQ ID NO: 301; (41) CDR1 as shown in SEQ ID NO: 302, CDR2 as shown in SEQ ID NO: 303, and CDR3 as shown in SEQ ID NO: 304; (42) CDR1 as shown in SEQ ID NO: 305, CDR2 as shown in SEQ ID NO: 306, and CDR3 as shown in SEQ ID NO: 307; (43) CDR1 as shown in SEQ ID NO: 308, CDR2 as shown in SEQ ID NO: 309, and CDR3 as shown in SEQ ID NO: 310; or (44) CDR1 as shown in SEQ ID NO: 311, CDR2 as shown in SEQ ID NO: 312, and CDR3 as shown in SEQ ID NO: 313, and, the second VHH domain comprises: (7) CDR1 as shown in SEQ ID NO: 19, CDR2 as shown in SEQ ID NO: 20, and CDR3 as shown in SEQ ID NO: 21 ; (8) CDR1 as shown in SEQ ID NO: 22, CDR2 as shown in SEQ ID NO: 23, and CDR3 as shown in SEQ ID NO: 24; (9) CDR1 as shown in SEQ ID NO: 25, CDR2 as shown in SEQ ID NO: 26, and CDR3 as shown in SEQ ID NO: 27; (10) CDR1 as shown in SEQ ID NO: 28, CDR2 as shown in SEQ ID NO: 29, and CDR3 as shown in SEQ ID NO: 30; (11) CDR1 as shown in SEQ ID NO: 31, CDR2 as shown in SEQ ID NO: 32, and CDR3 as shown in SEQ ID NO: 33; (12) CDR1 as shown in SEQ ID NO: 34, CDR2 as shown in SEQ ID NO: 35, and CDR3 as shown in SEQ ID NO: 36; (13) CDR1 as shown in SEQ ID NO: 37, CDR2 as shown in SEQ ID NO: 38, and CDR3 as shown in SEQ ID NO: 39; (14) CDR1 as shown in SEQ ID NO: 40, CDR2 as shown in SEQ ID NO: 41, and CDR3 as shown in SEQ ID NO: 42; (15) CDR1 as shown in SEQ ID NO: 43, CDR2 as shown in SEQ ID NO: 44, and CDR3 as shown in SEQ ID NO: 45; (16) CDR1 as shown in SEQ ID NO: 46, CDR2 as shown in SEQ ID NO: 47, and CDR3 as shown in SEQ ID NO: 48; (17) CDR1 as shown in SEQ ID NO: 49, CDR2 as shown in SEQ ID NO: 50, and CDR3 as shown in SEQ ID NO: 51 ; (18) CDR1 as shown in SEQ ID NO: 52, CDR2 as shown in SEQ ID NO: 53, and CDR3 as shown in SEQ ID NO: 54; (19) a CDR1 as depicted in SEQ ID NO: 55, a CDR2 as depicted in SEQ ID NO: 56 and a CDR3 as depicted in SEQ ID NO: 57; (20) a CDR1 as depicted in SEQ ID NO: 58, a CDR2 as depicted in SEQ ID NO: 59 and a CDR3 as depicted in SEQ ID NO: 60; (21) a CDR1 as depicted in SEQ ID NO: 61, a CDR2 as depicted in SEQ ID NO: 62 and a CDR3 as depicted in SEQ ID NO: 63; (22) a CDR1 as depicted in SEQ ID NO: 64, a CDR2 as depicted in SEQ ID NO: 65 and a CDR3 as depicted in SEQ ID NO: 66; (23) a CDR1 as depicted in SEQ ID NO: 67, a CDR2 as depicted in SEQ ID NO: 68 and a CDR3 as depicted in SEQ ID NO: 69; (24) a CDR1 as depicted in SEQ ID NO: 70, a CDR2 as depicted in SEQ ID NO: 71 and a CDR3 as depicted in SEQ ID NO: 72; (25) a CDR1 as depicted in SEQ ID NO: 73, a CDR2 as depicted in SEQ ID NO: 74 and a CDR3 as depicted in SEQ ID NO: 75; (26) a CDR1 as depicted in SEQ ID NO: 76, a CDR2 as depicted in SEQ ID NO: 77 and a CDR3 as depicted in SEQ ID NO: 78; (27) a CDR1 as depicted in SEQ ID NO: 79, a CDR2 as depicted in SEQ ID NO: 80 and a CDR3 as depicted in SEQ ID NO: 81; (28) a CDR1 as depicted in SEQ ID NO: 82, a CDR2 as depicted in SEQ ID NO: 83 and a CDR3 as depicted in SEQ ID NO: 84; (29) a CDR1 as depicted in SEQ ID NO: 85, a CDR2 as depicted in SEQ ID NO: 86 and a CDR3 as depicted in SEQ ID NO: 87; (30) a CDR1 as depicted in SEQ ID NO: 88, a CDR2 as depicted in SEQ ID NO: 89 and a CDR3 as depicted in SEQ ID NO: 90; (31) a CDR1 as depicted in SEQ ID NO: 91, a CDR2 as depicted in SEQ ID NO: 92 and a CDR3 as depicted in SEQ ID NO: 93; (32) a CDR1 as depicted in SEQ ID NO: 94, a CDR2 as depicted in SEQ ID NO: 95 and a CDR3 as depicted in SEQ ID NO: 96; (33) a CDR1 as depicted in SEQ ID NO: 97, a CDR2 as depicted in SEQ ID NO: 98 and a CDR3 as depicted in SEQ ID NO: 99; (34) a CDR1 as depicted in SEQ ID NO: 100, a CDR2 as depicted in SEQ ID NO: 101 and a CDR3 as depicted in SEQ ID NO: 102; or (45) a CDR1 as depicted in SEQ ID NO: 314, a CDR2 as depicted in SEQ ID NO: 315 and a CDR3 as depicted in SEQ ID NO:

316.

6. The antibody of claim 4, wherein, the first VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 239-244 and 375-384, and, the second VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 245-272 and 385.

7. The antibody of claim 6, comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 273-278 and 388-402.

8. The antibody of any one of claims 1-7, comprising an Fc region, optionally, an Fc region of an IgG, further optionally, an Fc region of an IgG1, IgG2, or IgG4, still further optionally, an Fc region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 283, 404, or 405.

9. The antibody of any one of claims 1-7, comprising a half-life extending moiety, optionally, a moiety that binds to serum albumin, further optionally, a VHH domain that specifically binds to serum albumin, still further optionally, comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 282, and / or is located N-terminal to the first VHH domain and / or the second VHH domain, is located C-terminal to the first VHH domain and / or the second VHH domain, or is located between the first VHH domain and the second VHH domain, still further optionally, the antibody comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 279-281 and 403.

10. The antibody of any one of claims 1-7, comprising a hinge region (optionally, a hinge region of IgGl, IgG4, CD8a, CD28, Siglecs, NGFR, or CD34), a transmembrane region (optionally, a transmembrane region of CD3zeta, CD4, CD8a, CD28, ICOS, 4-1BB, or KIR2DS2), an intracellular signaling domain (optionally, a signaling domain of CD3zeta or FcyR), and optionally, a costimulatory domain (optionally, a costimulatory domain of CD28, ICOS, 4-1BB, OX40, CD27, CD40, HVEM, GITR, MYD88-CD40, TLR2, or Dectin-1), and optionally, a signal peptide (optionally, a signal peptide of CD8a).

11. A nucleic acid encoding the antibody of any one of claims 1-10.

12. A vector comprising the nucleic acid of claim 11.

13. A host cell comprising the nucleic acid of claim 11 or the vector of claim 12.

14. A method of producing an antibody, comprising culturing the host cell of claim 13 such that the antibody is expressed.

15. A composition comprising the antibody of any one of claims 1-10, the nucleic acid of claim 11, the vector of claim 12, or the host cell of claim 13.

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