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

Abstract

The present invention provides an anti-IL-33 single-domain antibody and a derivative thereof. The anti-IL-33 single-domain antibody of the present invention can block the binding of IL-33 to a receptor thereof, thereby blocking downstream signaling pathways.

Description

Anti-il-33 single-domain antibodies and derivatives thereof

[0001] Incorporation by Reference of Related Applications

[0002] This application claims priority to the application with the filing date of September 30, 2024, the application number of PCT / CN2024 / 122846, and the invention name of “Anti-il-33 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 IL-33 single-domain antibodies and derivatives thereof. BACKGROUND

[0004] IL-33 is known as an “alarmin” cytokine, which is released after stimulation of epithelial and endothelial cells by airborne allergens, viruses, cigarette smoke, and air pollutants, etc. Many inflammatory and immune cells are the target cells of IL-33, such as mast cells, type 2 innate lymphoid-like cells (ILC2s), macrophages, dendritic cells, eosinophils, basophils, CD4+ T cells, etc. IL-33 is widely involved in the innate immunity and adaptive immunity of the body through these cells.

[0005] Full-length IL-33 has biological activity, can bind to the main receptor ST2 and the secondary receptor IL-1 receptor accessory protein (IL-1RAcP), activate the NF-κB signaling pathway, and make the target cells release cytokines. Elastase, proteinase 3 and cathepsin G produced by neutrophils, and chymase, trypsin and granzyme B released by mast cells can enzymatically cleave IL-33, making it hydrolyzed into mature IL-33, whose activity is 10-30 times higher than that of full-length IL-33.

[0006] ILC2 is a newly discovered group of cells, which has the ability to produce Th2-type cytokines such as IL-5 and IL-13. Research results show that ILC2 plays an important role in the occurrence and development of allergic diseases, and its position has even surpassed Th2. ILC2 is rich in mucosa. ILC2 cells continuously express ST2 at a high level. IL-33 stimulation can not only make ILC2 produce a large amount of IL-5 and IL-13, but also expand ILC2 in the local tissue. ILC2 has immune memory. ILC2 stimulated by allergens will obtain a gene expression pattern similar to that of memory T cells, and this group of ILC2 will express more ST2 than the original ILC2 when stimulated by IL-33 again. ILC2) produce more Th2-type cytokines. IL-33 plays a regulatory role in innate immunity by interacting with ILC2, mast cells, eosinophils, basophils, and macrophages, and participates in the acquired immune process by interacting with Th2, Th17, and Treg cells.

[0007] 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. relative to traditional IgG antibodies, and have become a new force in the new generation of therapeutic biological medicines and clinical diagnostic reagents. At present, there is no nanobody against IL-33 that has entered the clinical stage. Relative to traditional IgG-type antibodies, nanobodies have better tissue penetration and penetration due to their small size, and better stability. Therefore, developing nanobody drugs with high activity, high clinical efficacy, and low production cost has very high medical value. SUMMARY

[0008] The present application provides a group of anti-IL-33 single-domain antibodies and derivatives thereof.

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

[0010] 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.

[0011] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure.

[0012] A "native antibody" refers to a naturally occurring immunoglobulin molecule with varying structures. For example, a native IgG class antibody is a heterotetrameric glycoprotein of about 150,000 Daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region.

[0013] The heavy chains of antibodies can be assigned to one of five types, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), some of which can be further divided into subclasses, e.g., gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), gamma 4 (IgG4), alpha 1 (IgA1) and alpha 2 (IgA2).

[0014] The light chains of antibodies can be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequences of their constant domains.

[0015] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the 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.

[0016] A single-domain antibody is an antibody fragment 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 Camelidae (nanobody or VHH H H fragment).

[0017] "Specifically binds" means binding that 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 by, e.g., SPR.

[0018] "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 interaction between members of a binding pair (e.g., antibody and 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 onThus, equivalent affinity can include different rate constants, as 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) and biolayer interferometry (BLI).

[0019] In one embodiment, the antibody has a Kd D of 5.0 x 10 -8 M or lower, 1.0 x 10 -8 M or lower, 5.0 x 10 -9 M or lower, 1.0 x 10 -9 M or lower, 5.0 x 10 -10 M or lower, 1.0 x 10 - 10 M or lower, 5.0 x 10 -11 M or lower, 1.0 x 10 -11 M or lower, 5.0 x 10 -8 M to 1.0 x 10 - 13 M.

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

[0021] 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.

[0022] 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.

[0023] 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.

[0024] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an 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 a particular antigen can be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library 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)).

[0025] The "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, 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 IgG CH2 and IgG CH3 constant domains.

[0026] In one embodiment, an antibody of the application is a humanized antibody.

[0027] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. 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, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0028] The term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts"). Generally, antibodies comprise six HVRs: three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). Exemplary HVRs herein include:

[0029] (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. Mol. Biol. 196:901-917 (1987));

[0030] (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));

[0031] (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. Mol. Biol. 262:732-745 (1996)); and

[0032] (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).

[0033] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered in accordance with the Kabat et al., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) numbering.

[0034] "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 in a VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0035] "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.

[0036] In one embodiment, the antibody of the application comprises a VHH domain that specifically binds to IL-33. In one embodiment, the VHH domain comprises the CDR1, CDR2 and CDR3 of any one of VHH domains L01 to L37 (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 L37 (see Table 3). In one embodiment, the VHH domain comprises the amino acid sequence of any one of VHH domains L01 to L37 (see Table 3).

[0037] In one embodiment, the antibody of the application is a heavy chain-only antibody, i.e. composed of heavy chains only, without 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 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.

[0038] In one embodiment, the antibody of the application is a single domain antibody, i.e. composed of VHH domains only.

[0039] In one embodiment, the antibody of the application is a monospecific antibody, having binding specificity for IL-33 only.

[0040] 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.

[0041] In one embodiment, the antibody of the application is a multivalent antibody (e.g. bivalent antibody, trivalent antibody, tetravalent antibody, etc.), i.e. comprising multiple binding sites, e.g. each binding site composed of one VHH domain, which can be fused to each other via a peptide linker. In one embodiment, the multivalent antibody of the application is a monospecific antibody, i.e. having binding specificity for IL-33 only. In one embodiment, the multivalent antibody of the application is a monoeptopic antibody. In one embodiment, the multivalent antibody of the application is a polyepitopic antibody (e.g. bi-epitopic antibody, tri-epitopic antibody, tetra-epitopic antibody, etc.). In the embodiment of a polyepitopic antibody (e.g. bi-epitopic antibody, tri-epitopic antibody, tetra-epitopic antibody, etc.), the binding valency of the antibody of the application for each epitope is independent from each other, or the same or different.

[0042] In one embodiment, the 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 IgGl, 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: 202, 299, or 300.

[0043] In one embodiment, the 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 IL-33 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.

[0044] In one embodiment, the binding specificity for 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: 201.

[0045] In one embodiment, the VHH that specifically binds to serum albumin is located N-terminal to the VHH that specifically binds to IL-33. In one embodiment, the VHH that specifically binds to serum albumin is located C-terminal to the VHH that specifically binds to IL-33.

[0046] In one embodiment, the VHH that specifically binds to serum albumin is connected to the VHH that specifically binds to IL-33 via a linker.

[0047] In one embodiment, the antibody of the application comprises a half-life prolonging module. In one embodiment, the half-life prolonging module is an Fc region or a VHH that specifically binds to serum albumin, e.g., as described above.

[0048] 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, e.g., (G4S) n , (SG4) n , (SG4) n S or G4(SG4) nPeptide linker, wherein "n" is generally an integer from 1 to 10, typically an integer from 1 to 6, in particular 4.

[0049] 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.

[0050] The present application also provides a nucleic acid encoding the antibody of the application.

[0051] The present 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.

[0052] The present 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).

[0053] The present application also provides a method of producing an antibody, comprising culturing the host cell of the application so that the antibody is expressed.

[0054] The present application also provides a composition comprising the antibody, the nucleic acid, the vector or the host cell of the application.

[0055] The anti-IL-33 antibody (e.g. single-domain antibody) of the application has one or more of the following properties:

[0056] (1) is capable of binding to human IL-33 antigen with high specificity;

[0057] (2) are capable of blocking the binding of IL-33 to its receptor; and / or

[0058] (3) are capable of blocking the downstream signaling pathway of the binding of IL-33 to its receptor. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 shows the determination of the blocking effect of L01, L14, L15, L18, L21, L23, L24, L25 antibodies on the human IL-33 and its receptor mediated signaling pathway using a human IL-33 reporter cell line as described in Example 4.

[0060] Figure 2 shows the determination of the blocking effect of L32 and L25 antibodies on the human IL-33 and its receptor mediated signaling pathway using a human IL-33 reporter cell line as described in Example 5. DETAILED DESCRIPTION

[0061] The present application provides the following sequences.

[0062] Table 1 : CDR sequences of exemplary antibodies (VHH domains) of the present application

[0063] Table 2: FR sequences of exemplary antibodies (VHH domains) of the present application

[0064] Table 3: Sequences of exemplary antibodies (VHH domains) of the present application

[0065] Sequence of VHH that specifically binds to serum albumin (SEQ ID NO: 201 )

[0066] Sequence of Fc (IgG4) (SEQ ID NO: 202)

[0067] Sequence of Fc (IgG4) variant 1 (SEQ ID NO: 299)

[0068] Sequence of Fc (IgG4) variant 2 (SEQ ID NO: 300)

[0069] Example 1 : Screening of anti-IL-33 single domain antibodies using a llama immunized library

[0070] The llama was immunized five times with a mixture of human IL-33 recombinant protein (purchased from Boster Biological Technology Co., Ltd., Item No.: IL3-H52H7) and Freund's adjuvant. The titer of the llama peripheral blood was determined after each immunization. The peripheral blood was collected after the fifth immunization and PBMC (peripheral blood mononuclear cells) was separated, RNA was isolated and cDNA was reverse transcribed, VHH coding sequence was amplified by PCR and the PCR product was recovered to construct a phage display library with a capacity of 8.3 x 10 8 pfu, and the positive clone rate was 90%. Ten different sequences were obtained using the phage rapid fishing method developed by the team. The best one (No. L01) was selected for CDR single-point position mutation, and 12 sequences (L02-L13) were obtained. The L01-L13 sequences were cloned into pcDNA3.4 (Invitrogen), and the plasmid was transfected into CHO-S cells by electroporation to make the transformed CHO-S cells express VHH+Fc (IgG4) fusion protein. After incubation at 37°C for one week, the supernatant was collected and the antibody was purified. The antibody was purified using a protein A affinity chromatography column, and the purity of the antibody was detected by SDS-PAGE and SEC-HPLC detection methods, respectively. The purity of all antibodies reached more than 95%.

[0071] Example 2: Analysis of the affinity of anti-IL-33 single-domain antibody to human IL-33 by Octect

[0072] Using R2 instrument (Sartorius, Germany) to determine the affinity of VHH+Fc (IgG4) fusion protein to human IL-33 by using biofilm interference technology (BLI). Briefly, the Fc-containing antibody was captured by a protein A sensor, and the IL-33 recombinant protein was diluted with a series of PBST buffers to obtain a series of dilutions (100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM, a total of 5 gradients). The obtained series of dilutions were passed through the surface of the sensor. The instrument detected the binding curve of the antibody to different concentrations of IL-33. The obtained sensor record was evaluated by using R2 software to obtain kinetic data.

[0073] Table 4: Binding affinity of the single-domain antibody of the present application to human IL-33 antigen

[0074] Example 3: Determination of the blocking activity of anti-IL-33 single-domain antibody to the signal pathway triggered by the binding of human IL-33 to its receptor on the cell line by using human IL-33 reporter cell line

[0075] Human IL-33 reporter cell line (purchased from GenScript) is a luciferase reporter cell line based on IL-33 signaling pathway. IL-33 binds to the heterodimeric receptor complex composed of ST2 and IL1RAcP subunits, then activates the downstream signaling pathway, thereby activating the expression of luciferase. The luciferase reading represents the activation effect of the signaling pathway, and therefore can be used for in vitro effect evaluation of IL-33 related drugs. The specific steps are as follows: inoculate cells in a 96-well plate at a density of 5x10 5 cells / well, with a volume of 100 μL per well; add IL-33 recombinant protein to activate the cell line, so that the final concentration of IL-33 in each well is 2.5 ng / mL; after 1 hour of co-incubation, add the anti-IL-33 antibody to be tested at an initial concentration of 75 μM, with 5-fold gradient dilution, a total of 10 gradients; add 33 μL of antibody 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, then collect samples for detection of luminescence signal, and calculate the EC50 value.

[0076] Table 5: Blocking activity of single-domain antibodies of the present application on human IL-33 and its receptor pathway

[0077] Example 4: Affinity and cell level blocking activity of anti-IL-33 single-domain antibodies after humanization modification

[0078] L01 and L02 antibodies were humanized and modified for drugability. Humanization was performed using the CDR grafting method. The basic approach is to replace the camelid framework region with a selected human germline framework region, and only retain the camelid CDR; then perform back mutation on the key amino acid sites in FR. L14 to L25 antibodies were obtained. The affinity of the antibodies was determined using the method described in Example 2 above (Table 6). The cell level blocking activity of the antibodies was determined using the method described in Example 3 above (Table 7).

[0079] Table 6: Affinity of humanized single-domain antibodies to human IL-33 antigen

[0080] Table 7: Cell level blocking activity of humanized single-domain antibodies

[0081] Example 5: Point mutation modification of antibodies, affinity of anti-IL-33 modified single-domain antibodies to human IL-33

[0082] The 29th amino acid of CDR1, the 50th, 56th, 58th and 61st amino acids of CDR2, the 99th and 104th amino acids of CDR3 of the L25 antibody were mutated and modified, and L26, L27, L28, L29, L30, L31, L32, L33, L34, L35, L36 and L37 were screened. The affinity of the antibodies was determined using the method described in Example 2 above (Table 8). The cell level blocking activity of the antibodies was determined using the method described in Example 3 above (Table 9). The affinity and activity of the modified antibodies were greatly improved.

[0083] Table 8: Affinity of modified single domain antibodies to human IL-33 antigen

[0084] Table 9: Cell level blocking activity of modified single domain antibodies

Claims

1. An antibody that specifically binds to IL-33, comprising a VHH domain that specifically binds to IL-33, the 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: 203, a CDR2 as depicted in SEQ ID NO: 204 and a CDR3 as depicted in SEQ ID NO: 205; (27) a CDR1 as depicted in SEQ ID NO: 206, a CDR2 as depicted in SEQ ID NO: 207 and a CDR3 as depicted in SEQ ID NO: 208; (28) a CDR1 as depicted in SEQ ID NO: 209, a CDR2 as depicted in SEQ ID NO: 210 and a CDR3 as depicted in SEQ ID NO: 211; (29) a CDR1 as set forth in SEQ ID NO: 212, a CDR2 as set forth in SEQ ID NO: 213, and a CDR3 as set forth in SEQ ID NO: 214; (30) a CDR1 as set forth in SEQ ID NO: 215, a CDR2 as set forth in SEQ ID NO: 216, and a CDR3 as set forth in SEQ ID NO: 217; (31) a CDR1 as set forth in SEQ ID NO: 218, a CDR2 as set forth in SEQ ID NO: 219, and a CDR3 as set forth in SEQ ID NO: 220; (32) a CDR1 as set forth in SEQ ID NO: 221, a CDR2 as set forth in SEQ ID NO: 222, and a CDR3 as set forth in SEQ ID NO: 223; (33) a CDR1 as set forth in SEQ ID NO: 224, a CDR2 as set forth in SEQ ID NO: 225, and a CDR3 as set forth in SEQ ID NO: 226; (34) a CDR1 as set forth in SEQ ID NO: 227, a CDR2 as set forth in SEQ ID NO: 228, and a CDR3 as set forth in SEQ ID NO: 229; (35) a CDR1 as set forth in SEQ ID NO: 230, a CDR2 as set forth in SEQ ID NO: 231, and a CDR3 as set forth in SEQ ID NO: 232; (36) a CDR1 as set forth in SEQ ID NO: 233, a CDR2 as set forth in SEQ ID NO: 234, and a CDR3 as set forth in SEQ ID NO: 235; or (37) a CDR1 as set forth in SEQ ID NO: 236, a CDR2 as set forth in SEQ ID NO: 237, and a CDR3 as set forth in SEQ ID NO:

238.

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 any one of SEQ ID NOs: 176-200 and 287-298.

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 IgGl, 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: 202, 299, or 300.

4. The antibody of claim 1 or 2, comprising a half-life prolonging moiety, optionally a moiety that binds serum albumin, further optionally a VHH domain that specifically binds 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: 201, optionally located N-terminal or C-terminal to a VHH domain that specifically binds IL-33.

5. The antibody of any one of claims 1 or 2, 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).

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 such 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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