Anti-IL-33 single-domain antibody and derivative thereof

Abstract

The invention provides an anti-IL-33 single domain antibody and a derivative thereof. The anti-IL-33 single-domain antibody disclosed by the invention can block the combination of IL-33 and a receptor thereof, so that a downstream signal channel is blocked.

Description

[0001] Reference to relevant applications

[0002] This application claims priority to the invention patent application filed on September 30, 2024, with application number 202411392487.2 and entitled "Anti-IL-33 Single-Domain Antibody and Its Derivatives", the entire contents of which are incorporated herein by reference. Technical Field

[0003] Generally, this invention relates to the field of antibodies. Specifically, this invention relates to anti-human IL-33 single-domain antibodies and their derivatives. Background Technology

[0004] IL-33, known as an "alarm" cytokine, is released from epithelial and endothelial cells after stimulation by airborne allergens, viruses, cigarette smoke, and air pollutants. Many inflammatory and immune cells are target cells of IL-33, such as mast cells, type 2 innate lymphoid cells (ILC2s), macrophages, dendritic cells, eosinophils, basophils, and CD4+ T cells. IL-33 participates extensively in both innate and adaptive immunity through these cells.

[0005] Full-length IL-33 possesses biological activity, binding to its major receptor ST2 and its minor receptor IL-1 receptor accessory protein (IL-1RAcP), activating the NF-κB signaling pathway and causing target cells to release cytokines. Elastase, protease 3, and cathepsin G produced by neutrophils, as well as chymotrypsin, trypsin, and granzyme B released by mast cells, can all enzymatically cleave IL-33, hydrolyzing it into its mature form, which exhibits 10–30 times greater activity than full-length IL-33.

[0006] ILC2 is a newly discovered group of cells capable of producing Th2-type cytokines such as IL-5 and IL-13. Research indicates that ILC2 plays a crucial role in the development and progression of allergic diseases, even surpassing Th2 in importance. ILC2 is abundant in mucous membranes. ILC2 cells consistently express ST2 at high levels. IL-33 stimulation not only induces ILC2 to produce large amounts of IL-5 and IL-13 but also leads to local tissue expansion of ILC2. ILC2 possesses immune memory. ILC2 cells stimulated by allergens acquire gene expression patterns similar to memory T cells; upon re-stimulation with IL-33, this group of ILC2 cells shows a greater expression than the original ILC2 cells. ILC2 produces more Th2-type cytokines. IL-33 plays a regulatory role in innate immunity through interactions with ILC2, mast cells, eosinophils, basophils and macrophages, and participates in adaptive immunity through interactions with Th2, Th17 and Treg cells.

[0007] Nanobodies, also known as single-domain antibodies, are heavy-chain-only antibodies derived from camels that lack the light chain and are naturally occurring in animals. Compared to traditional IgG antibodies, they possess advantages such as high stability, good water solubility, simple humanization, and strong penetration, making them an emerging force in next-generation therapeutic biopharmaceuticals and clinical diagnostic reagents. Currently, no nanobodies targeting IL-33 have entered the clinical stage. Compared to traditional IgG antibodies, nanobodies offer superior tissue permeability and penetration due to their smaller size, as well as better stability. Therefore, developing nanobody drugs with high blocking activity, excellent clinical efficacy, and low production costs has extremely high pharmaceutical value. Summary of the Invention

[0008] This invention provides an anti-IL-33 single-domain antibody and its derivatives.

[0009] In one embodiment, the present invention provides an antibody that specifically binds to IL-33.

[0010] The term “antibody” is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0011] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document to refer to antibodies that have a structure substantially similar to that of natural antibodies.

[0012] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant domains. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant domain.

[0013] The heavy chains of antibodies can be assigned to one of five classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2).

[0014] Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called Kappa (κ) and Lambda (λ).

[0015] An "antibody fragment" refers to a molecule that is distinct from the intact antibody and contains the portion of the intact antibody that binds to the antigen it binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies, triantibodies, tetraantibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and single-domain antibodies.

[0016] Single-domain antibodies are antibody fragments composed of a single monomeric variable antibody domain. The first type of single-domain antibody was derived from the variable domain of an antibody heavy chain from camelids (nanobodies or V). H H fragment).

[0017] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antibody to bind to a specific antigen can be measured via enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree to which the antibody binds to irrelevant proteins is less than about 10% of the antibody binding to the antigen, as measured by, for example, SPR.

[0018] “Affinity” or “binding affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, “binding affinity” refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be expressed using the dissociation constant (K). D It is represented by ) which is the dissociation and recombination rate constant (k, respectively). off and k on The ratio of the rate constants to the rate constants is given. Thus, equivalent affinities can contain different rate constants, as long as the ratio of the rate constants remains the same. Affinities can be measured using methods commonly known in the art, including those described herein. Specific methods used for measuring affinity include surface plasmon resonance (SPR) and biofilm interferometry (BLI).

[0019] In one implementation, the antibody has K D The value is 5.0 x 10 -8 M or lower, 1.0x 10 -8 M or lower, 5.0x10 -9 M or lower, 1.0x 10-9 M or lower, 5.0x 10 -10 M or lower, 1.0x 10 -10 M or lower, 5.0x 10 -11 M or lower, 1.0x 10 -11 M or lower, 5.0x 10 -8 M to 1.0x 10 -13 Binding affinity of M.

[0020] In one embodiment, the antibody of the present invention is a monoclonal antibody.

[0021] The term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, meaning that the individual antibodies constituting the group are identical and / or bind to the same epitope, except for variant antibodies that may exist in very small amounts, for example, containing naturally occurring mutations or those that may occur during the production of the monoclonal antibody preparation. Unlike polyclonal antibody preparations, which typically contain different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody preparation targets a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the characteristic that the antibody is obtained from a group of substantially homogeneous antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies to be used according to the present invention can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing whole or part of the human immunoglobulin loci.

[0022] In one embodiment, the antibody of the present invention is a chimeric antibody. In one embodiment, the variable region of the chimeric antibody is derived from alpaca, and the constant region of the chimeric antibody is derived from human.

[0023] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from a different source or species.

[0024] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs) (see, for example, Kindt, T.J. et al., Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), page 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to specific antigens can be isolated by screening libraries of complementary VL or VH domains, respectively, using the VH or VL domains of the antibody binding the antigen. See, for example, Portolano, S. et al., J. Immunol. 150:880-887 (1993); Clarkson, T. et al., Nature 352:624-628 (1991)).

[0025] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. While the boundaries of the Fc region in the IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies generated by host cells may undergo post-translational cleavage, removing one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, antibodies generated by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain can include the full-length heavy chain, or it can include cleaved variants of the full-length heavy chain (also called "cleaved variant heavy chains"). This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447), or C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described 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 the Fc region refers to one of the two polypeptides that form the dimer Fc domain, i.e., the polypeptide containing the C-terminal constant region of the immunoglobulin heavy chain that stabilizes its own binding. For example, the subunit of the IgG Fc domain contains the IgG CH2 and IgG CH3 constant domains.

[0026] In one embodiment, the antibody of the present invention is a humanized antibody.

[0027] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody may contain at least one, typically two, variable domains substantially entirely of the following: all or substantially all HVRs (e.g., CDRs) correspond to those of the non-human antibody, and all or substantially all FRs correspond to those of the human antibody. Optionally, a humanized antibody may contain at least a portion of an antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0028] The term "hypervariant region" or "HVR" refers to each region in the antibody variable domain that is sequence-hypervariant ("complementarity-determining region" or "CDR") and / or forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact"). Generally, an antibody contains six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs in this document include:

[0029] (a) Hypervariable rings, present at amino acid residues 26-32 (L1), 50-52 (L2), and 91-96 (L3),

[0030] 26-32(H1), 53-55(H2), and 96-101(H3) (Chothia and Lesk, J. Mol.)

[0031] Biol. 196:901-917 (1987));

[0032] (b) CDR, which is present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 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));

[0033] (c) Antigen contact, which is present at amino acid residues 27c-36 (L1), 46-55 (L2), and 89-96 (L3).

[0034] 30-35b(H1), 47-58(H2), and 93-101(H3) (MacCallum et al. J. Mol. Biol).

[0035] 262:732-745(1996)); and

[0036] Combinations of (d)(a), (b), and / or (c), including HVR amino acid residues 24-34 (L1), 50-56 (L2),

[0037] 89-97(L3), 31-35(H1), 50-63(H2), and 95-102(H3).

[0038] Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domain are designated in this paper according to Kabat et al., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0039] "Frame" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. Generally, the variable domain FR consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences in VH (or VL) generally appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0040] "Human common framework" refers to the framework representing the most frequently occurring amino acid residues in the human immunoglobulin VL or VH framework sequence selection. Typically, the human immunoglobulin VL or VH sequence selection is derived from a variable domain sequence subgroup. Typically, these sequence subgroups are those listed 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 VL, the subgroup is as Kabat et al., see Subgroup Kappa I above. In one embodiment, for VH, the subgroup is as Kabat et al., see Subgroup III above.

[0041] In one embodiment, the antibody of the present invention comprises a VHH domain that specifically binds to IL-33. In one embodiment, the VHH domain comprises CDR1, CDR2, and CDR3 of any one of VHH domains L01 to L03 (see Table 1). In one embodiment, the 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 L01 to L03 (see Table 3). In one embodiment, the VHH domain comprises an amino acid sequence of any one of VHH domains L01 to L03 (see Table 3).

[0042] In one embodiment, the antibody of the present invention is a heavy-chain-only antibody, i.e., composed only of heavy chains and containing no light chains, such as 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 to the same antigen. In one embodiment, the heavy-chain heterodimer binds to the same epitope of the same antigen. In one embodiment, the heavy-chain heterodimer binds to different epitopes of the same antigen. In one embodiment, the heavy-chain heterodimer binds to different antigens.

[0043] In one embodiment, the antibody of the present invention is a single-domain antibody, i.e., it consists only of the VHH domain.

[0044] In one embodiment, the antibody of the present invention is a monospecific antibody that has binding specificity only against IL-33.

[0045] In one embodiment, the antibody of the present invention is a monovalent antibody, i.e., containing a single binding site, for example, consisting of a VHH domain.

[0046] In one embodiment, the antibody of the present invention is a multivalent antibody (e.g., a bivalent antibody, a trivalent antibody, a tetravalent antibody, etc.), that is, it contains multiple binding sites, for example, each binding site is composed of a VHH domain, and the two sites can be fused together via peptide linkers. In one embodiment, the multivalent antibody of the present invention is a monospecific antibody, that is, it has binding specificity only against IL-33. In one embodiment, the multivalent antibody of the present invention is a single epitope antibody. In one embodiment, the multivalent antibody of the present invention is a multi-epitope antibody (e.g., a bi-epitope antibody, a tri-epitope antibody, a tetra-epitope antibody, etc.). In embodiments of multi-epitope antibodies (e.g., bi-epitope antibodies, tri-epitope antibodies, tetra-epitope antibodies, etc.), the binding valence of the antibody of the present invention to each epitope is independent of each other, or the same or different.

[0047] In one embodiment, the antibody of the present invention comprises an Fc region. In one embodiment, the Fc region is the Fc region of IgG. In one embodiment, the Fc region is the Fc region of IgG1, IgG2, or IgG4. 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 that of SEQ ID NO: 10.

[0048] In one embodiment, the antibody of the present invention is a multispecific antibody (e.g., a bispecific antibody, a trispecific antibody, a tetraspecific antibody, etc.). In one embodiment, the multispecific antibody has binding specificity against IL-33 and different antigens. In one embodiment, binding to different antigens prolongs the half-life of the antibody. In one embodiment, the different antigens are serum albumin.

[0049] In one embodiment, the binding specificity to serum albumin is a VHH that specifically binds to serum albumin. In one embodiment, the 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: 9.

[0050] In one embodiment, the VHH that specifically binds to serum albumin is located at the N-terminus of the VHH that specifically binds to IL-33. In another embodiment, the VHH that specifically binds to serum albumin is located at the C-terminus of the VHH that specifically binds to IL-33.

[0051] In one embodiment, VHH that specifically binds to serum albumin is linked via a linker to VHH that specifically binds to IL-33.

[0052] In one embodiment, the antibody of the present invention comprises a module for extending half-life. In one embodiment, the module for extending half-life is an Fc region or a VHH that specifically binds to serum albumin, such as those described above.

[0053] The term "peptide linker" refers to a peptide containing one or more amino acids, typically about 2-20 amino acids, usually glycine (G) and / or serine (S). A suitable linker peptide is, for example, (G4S). n (SG4) n (SG4) n S or G4 (SG4) n Peptide linkers, where "n" is generally an integer from 1 to 10, typically an integer from 1 to 6, and especially 4.

[0054] In one embodiment, the antibody of the present invention is a membrane-integrated antibody (e.g., a chimeric antigen receptor, CAR). In one embodiment, the membrane-integrated antibody includes a hinge region. In one embodiment, the hinge region is the hinge region of IgG1, IgG4, CD8α, CD28, Siglecs, NGFR, or CD34. In one embodiment, the membrane-integrated antibody includes a transmembrane region. In one embodiment, the transmembrane region is the transmembrane region of CD3zeta, CD4, CD8α, CD28, ICOS, 4-1BB, or KIR2DS2. In one embodiment, the membrane-integrated antibody includes an intracellular signaling domain. In one embodiment, the intracellular signaling domain is the signaling domain of CD3ζ or FcγR. In one embodiment, the membrane-integrated antibody includes a co-stimulatory domain. In one embodiment, the co-stimulatory domain is the co-stimulatory domain of CD28, ICOS, 4-1BB, OX40, CD27, CD40, HVEM, GITR, MYD88-CD40, TLR2, or Dectin-1. In one embodiment, the membrane-integrated antibody includes a signal peptide. In one embodiment, the signal peptide is the signal peptide of CD8α.

[0055] The present invention also provides a nucleic acid that encodes the antibody of the present invention.

[0056] The present invention also provides a vector comprising the nucleic acid of the present invention. In one embodiment, the vector is a cloning vector or an expression vector. In one embodiment, the vector is a plasmid, virus, or plasmid.

[0057] The present invention also provides a host cell comprising the nucleic acid of the present invention or the vector of the present invention. In one embodiment, the host cell is a prokaryotic cell or a eukaryotic cell. In one embodiment, the host cell is an immune cell, such as a T cell or an NK cell. In one embodiment, the host cell displays the antibody of the present invention (particularly a membrane-integrated antibody, CAR) on its cell surface (e.g., CAR-T cells or CAR-NK cells).

[0058] The present invention also provides a method for generating antibodies, comprising culturing the host cells of the present invention to express the antibodies.

[0059] The present invention also provides a composition comprising the antibody, nucleic acid, vector, or host cell of the present invention.

[0060] The anti-IL-33 antibody (e.g., a single-domain antibody) of the present invention has one or more of the following characteristics:

[0061] (1) It can bind to human IL-33 antigen with high specificity;

[0062] (2) It can block the binding of IL-33 to its receptor; and / or

[0063] (3) It can block the downstream signaling pathways of IL-33 binding to its receptor. Attached Figure Description

[0064] Figure 1 The blockade effect of antibody L01 on human IL-33 and its receptor-mediated signaling pathways was demonstrated using the human IL-33 reporter cell line as described in Example 3. Detailed Implementation

[0065] The present invention provides the following sequence.

[0066] Table 1: CDR sequences of exemplary antibodies (VHH domain) of the present invention

[0067]

[0068] Table 2: FR sequences of exemplary antibodies (VHH domain) of the present invention

[0069]

[0070] Table 3: Sequences of exemplary antibodies (VHH domains) of the present invention

[0071]

[0072] The sequence of VHH that specifically binds to serum albumin (SEQ ID NO: 9)

[0073] EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTT

[0074] The sequence of LYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Fc(IgG4) (SEQ ID NO: 10)

[0075] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF

[0076] NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY

[0077] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0078] Example 1: Anti-IL-33 single-domain antibodies were obtained by screening alpaca immune libraries.

[0079] Alpacas were immunized five times with a mixture of recombinant human IL-33 protein (purchased from Peptase Biotechnology Co., Ltd., catalog number: IL3-H52H7) 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 (peripheral blood mononuclear cells) were isolated. RNA was isolated and cDNA was reverse transcribed. The VHH coding sequence was amplified by PCR, and the PCR product was recovered to construct a phage display library with a volume of 8.3 × 10⁻⁶. 8 PFU (Positive Cloning Rate) was 90%. Ten differentially expressed sequences were obtained using a phage fishing method developed in-house 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 antibody was purified. Purification was performed using a protein A affinity chromatography column. The purity of the purified antibodies was assessed using SDS-PAGE and SEC-HPLC, with all antibodies achieving a purity of over 95%. One candidate antibody with high IL-33 binding activity was selected through biomembrane interference technology.

[0080] Example 2: Analysis of the affinity of anti-IL-33 single-domain antibody for human IL-33 using Octect

[0081] use The R2 instrument (Sartorius, Germany) utilizes biomembrane interferometry (BLI) to determine the affinity of the VHH+Fc (IgG4) fusion protein for human IL-33. In short, the Fc-carrying antibody is captured using a protein A sensor, and the recombinant IL-33 protein is serially diluted with PBST buffer, resulting in five dilutions (6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM). The instrument detects the binding curves of the antibody with different concentrations of IL-33. The R2 software was used to evaluate the sensor records and obtain dynamic data.

[0082] Table 4: Binding affinity of the single-domain antibody of the present invention to human IL-33 antigen

[0083] Antibody number <![CDATA[K D (M)]]> <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> L01 9.50E-09 6.14E+05 5.84E-03

[0084] Example 3: Determining the blocking activity of anti-IL-33 single-domain antibody against the signaling pathway stimulated by the binding of human IL-33 to its receptor on the cell line using a human IL-33 reporter cell line.

[0085] The human IL-33 reporter cell line (purchased from Jimon Biotechnology) is a luciferase reporter gene cell line constructed based on the IL-33 signaling pathway. IL-33 binds to the heterodimeric receptor complex composed of the ST2 and IL1RAcP subunits, thereby activating downstream signaling pathways and activating luciferase expression. The luciferase reading represents the activation effect of the signaling pathway and can therefore be used to evaluate the in vitro efficacy of IL-33-related drugs. The specific steps are as follows: cells are seeded in 96-well plates at a density of 5 × 10⁶ cells / well. 5 Cells / well, 100 μL per well; IL-33 recombinant protein was added to activate the cell line, bringing the final IL-33 concentration in each well to 2.5 ng / mL; after co-incubation for 1 hour, the anti-IL-33 antibody to be tested was added at an initial concentration of 75 μM, serially diluted 5-fold for a total of 10 dilutions; 33 μL of the corresponding concentration of antibody or blank control was added to each well of cells, and the cells were incubated in a 37℃ CO2 incubator for 23 hours. The samples were then collected and the luminescence signal was detected, and the EC50 value was calculated.

[0086] Table 5: Blocking activity of the single-domain antibody of the present invention against human IL-33 and its receptor pathway

[0087] Antibody number EC50(nM) L01 61.78

[0088] Example 4: Screening for anti-IL-33 single-domain antibodies using a natural (non-immunized) alpaca library

[0089] Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of 40 alpacas. RNA was extracted and cDNA was reverse transcribed. The VHH coding sequence was amplified by PCR, and the PCR product was recovered to construct a phage display library with a volume of 2.5 × 10⁻⁶. 10 PFU (plasma collection of fumes). Anti-IL-33 nanobodies were screened using phage display technology. Fifteen sequences 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 in a shaker, the supernatant was collected, and two candidate antibodies with high IL-33 binding activity were screened using biolayer interference technology. Further, amino acid mutations in the CDR region were performed using internal AI software, and then a solid mutation library was established for screening, identifying two optimal sequences, L02 and L03.

[0090] Example 5: Analysis of the affinity of anti-IL-33 single-domain antibody for human IL-33 using BIAcore

[0091] The affinity of the VHH+Fc (IgG4) fusion protein with sequences L02 and L03 to human IL-33 was determined using a BIAcore 8000 Biosensor instrument (BIAcore AB) and 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 IL-33 protein was serially diluted with HBS-EP+ buffer, resulting in five dilutions (50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM). The instrument detected binding curves between the antibody and different concentrations of IL-33. The kinetic data obtained from the sensor recordings were evaluated using BIAcore evaluation software.

[0092] Table 6: Binding affinity of the single-domain antibody of the present invention to human IL-33 antigen

[0093] Antibody number <![CDATA[K D (M)]]> <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> L02 9.60E-10 1.91E+05 1.83E-04 L03 8.78E-10 1.56E+05 1.37E-04

Claims

1. An antibody that specifically binds to IL-33, comprising a VHH domain that specifically binds to IL-33, said 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: 11, a CDR2 as set forth in SEQ ID NO: 12, and a CDR3 as set forth in SEQ ID NO: 13; or (3) 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.

2. The antibody of claim 1, wherein the 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 SEQ ID NO: 8, 18, or 26.

3. The antibody of claim 1 or 2, 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:

10.

4. The antibody of claim 1 or 2, comprising a half-life prolonging moiety, optionally, a moiety that binds to serum albumin, further optionally, a VHH domain that specifically binds to serum albumin, still further optionally, a VHH domain 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: 9, optionally, located at the N- or C-terminus of the VHH domain that specifically binds to IL-33.

5. The antibody of any one of claims 1 or 2, comprising a hinge region (optionally, a hinge region of an IgG1, 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).

6. A nucleic acid encoding the antibody of any one of claims 1-5.

7. A vector comprising the nucleic acid of claim 6.

8. A host cell comprising the nucleic acid of claim 6 or the vector of claim 7.

9. A method of producing an antibody comprising culturing the host cell of claim 8 so that the antibody is expressed.

10. A composition comprising the antibody of any one of claims 1-5, the nucleic acid of claim 6, the vector of claim 7, or the host cell of claim 8.

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