Anti-IL-1beta antibodies and uses thereof

Abstract

The invention relates to an interleukin-1 beta (IL-1 beta) binding molecule and a multispecific antigen binding molecule based on the IL-1 beta binding molecule, in particular to an IL-1 beta single domain antibody and a bispecific antibody based on the IL-1 beta single domain antibody. The invention also relates to nucleic acid for coding the IL-1beta binding molecule and the multispecific antigen binding molecule based on the IL-1beta binding molecule, an expression vector for expressing the IL-1beta binding molecule and the multispecific antigen binding molecule based on the IL-1beta binding molecule, and a host cell. The invention also relates to a method for preparing the IL-1beta binding molecule and the multispecific antigen binding molecule based on the IL-1beta binding molecule and application of the IL-1beta binding molecule and the multispecific antigen binding molecule based on the IL-1beta binding molecule.

Description

Technical Field

[0001] The present application relates to antibodies. More specifically, the present application relates to single-domain antibodies that specifically bind to IL-1β and bispecific antibodies based thereon, as well as methods for preparing the same and uses thereof. Background Art

[0002] The interleukin 1 (IL-1) family consists of two proinflammatory cytokines (IL-1α and IL-1β) and an IL-1 receptor antagonist (IL-1Ra). IL-1α and IL-1β can effectively stimulate the IL-1 receptor, while IL-1Ra can bind to the IL-1 receptor surface and block signal transduction, making it an endogenous competitive inhibitor of IL-1 (IL-1α or IL-1β). IL-1β is the major circulating form of IL-1. Its precursor is primarily synthesized by monocytes and macrophages. In response to inflammatory stimuli, it is cleaved into its active form by caspase-1, which is activated by the NLRP3 inflammasome. It is a key mediator of inflammatory responses.

[0003] IL-1β regulates the recruitment and activation of effector cells involved in innate and adaptive immunity, and is therefore involved in a wide range of inflammatory diseases, including gouty arthritis, various autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, periodic fever syndromes, and autoinflammatory diseases such as systemic juvenile idiopathic arthritis, as well as the occurrence of cold pyrin-related periodic syndromes in children and adults. The IL-1β pathway has recently been shown to be involved in the occurrence of tumors such as acute myeloid leukemia, liver cancer, lung cancer, and cardiovascular and cerebrovascular diseases. Drugs targeting IL-1β have shown good therapeutic and preventive effects (Cozzolino F et al. Proc Natl Acad Soci USA. 86: 2369 (1989); Nakazaki H et al. Cancer. 70 (3): 709 (1992); (Ridker PM et al. Lancet. 390 (10105): 1833-1842 (2017)).

[0004] There is still a need to provide therapeutic antibodies that bind to human IL-1 β. In particular, there is still a need to provide anti-IL-1 β antibodies with favorable clinical properties. Summary of the Invention

[0005] After in-depth research and creative work, the inventors used human IL-1β protein as an antigen to immunize alpaca (Alpaca) multiple times. Through a large number of screening and modifications, they obtained a single-domain antibody that can specifically bind to human IL-1β, which can effectively block the binding of IL-1β to IL-1R1 and inhibit the release of downstream IL-6 induced by IL-1β. Furthermore, the inventors also constructed a bispecific antibody based on the anti-IL-1β single-domain antibody, which has a more effective anti-tumor effect than using antibodies alone or in combination. Therefore, the single-domain antibodies provided by the present invention and the bispecific antibodies based thereon have the potential to be used to prepare drugs for the prevention or treatment of diseases such as inflammatory diseases, cardiovascular diseases or cancer.

[0006] Thus, in one aspect, the present invention provides an interleukin-1β (IL-1β) binding molecule, comprising a heavy chain variable region (VH), wherein the VH comprises:

[0007] 1) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively;

[0008] 2) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively;

[0009] 3) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 8, respectively;

[0010] 4) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 13, respectively;

[0011] 5) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO: 16, respectively;

[0012] 6) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively;

[0013] 7) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 16, respectively; or

[0014] 8) HCDR1, HCDR2, and HCDR3: They comprise the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 25, and SEQ ID NO: 8, respectively.

[0015] In some embodiments, the IL-1β binding molecule comprises a heavy chain variable region (VH), the VH comprising:

[0016] 1) the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1;

[0017] 2) the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:5;

[0018] 3) the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9;

[0019] 4) the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11;

[0020] 5) the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14;

[0021] 6) the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17;

[0022] 7) the amino acid sequence of SEQ ID NO:21, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:21;

[0023] 8) the amino acid sequence of SEQ ID NO:24, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:24;

[0024] 9) the amino acid sequence of SEQ ID NO:26, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26;

[0025] 10) the amino acid sequence of SEQ ID NO:27, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:27; or

[0026] 11) the amino acid sequence of SEQ ID NO:28, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28.

[0027] In some embodiments, the IL-1β binding molecule is a single domain antibody (VHH), the VHH comprising:

[0028] 1) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively;

[0029] 2) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively;

[0030] 3) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 8, respectively;

[0031] 4) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 13, respectively;

[0032] 5) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO: 16, respectively;

[0033] 6) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively;

[0034] 7) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 16, respectively; or

[0035] 8) HCDR1, HCDR2, and HCDR3: They comprise the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 25, and SEQ ID NO: 8, respectively.

[0036] In some embodiments, the IL-1β binding molecule is a single domain antibody (VHH), the VHH comprising:

[0037] 1) the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1;

[0038] 2) the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:5;

[0039] 3) the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9;

[0040] 4) the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11;

[0041] 5) the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14;

[0042] 6) the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17;

[0043] 7) the amino acid sequence of SEQ ID NO:21, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:21;

[0044] 8) the amino acid sequence of SEQ ID NO:24, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:24;

[0045] 9) the amino acid sequence of SEQ ID NO:26, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26;

[0046] 10) the amino acid sequence of SEQ ID NO:27, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:27; or

[0047] 11) the amino acid sequence of SEQ ID NO:28, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28.

[0048] In some embodiments, the IL-1β binding molecule further comprises one or more amino acid residue mutations but still maintains specific binding to IL-1β.

[0049] In some embodiments, at least one of the mutations is in one or more VH sequences but not in any CDR sequence.

[0050] In some embodiments, the IL-1β binding molecule is an IL-1β antagonist, preferably an anti-IL-1β antibody.

[0051] In some embodiments, the IL-1β binding molecule is a chimeric antibody.

[0052] In some embodiments, the IL-1β binding molecule is a humanized antibody.

[0053] In some embodiments, the IL-1β binding molecule is a heavy chain antibody or a single domain antibody (VHH).

[0054] In some embodiments, the VHH is derived from a camelid species, including an alpaca or a llama.

[0055] In some embodiments, the VH is fused to the Fc domain of IgG.

[0056] In some embodiments, the VH is fused to the Fc domain of human IgG.

[0057] In some embodiments, the IL-1β binding molecule is a heavy chain antibody comprising an IL-1β binding molecule as defined above linked to an Fc domain derived from human IgG.

[0058] In some specific embodiments, the IL-1β binding molecule is a heavy chain antibody comprising an IL-1β binding molecule as defined above linked to an Fc domain derived from human IgG1 or IgG4.

[0059] In some embodiments, the Fc domain further comprises one or more amino acid mutations that reduce or eliminate effector function, including but not limited to complement dependent cytotoxicity (CDC) or antibody dependent cell-mediated cytotoxicity (ADCC).

[0060] In some specific embodiments, the Fc domain is the amino acid sequence of SEQ ID NO:30, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:30.

[0061] In some embodiments, the IL-1β binding molecule comprises:

[0062] 1) the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:31;

[0063] 2) the amino acid sequence of SEQ ID NO:32, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:32;

[0064] 3) the amino acid sequence of SEQ ID NO:33, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:33;

[0065] 4) the amino acid sequence of SEQ ID NO:34, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:34;

[0066] 5) the amino acid sequence of SEQ ID NO:35, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:35;

[0067] 6) the amino acid sequence of SEQ ID NO:36, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:36;

[0068] 7) the amino acid sequence of SEQ ID NO:37, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:37;

[0069] 8) the amino acid sequence of SEQ ID NO:38, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:38;

[0070] 9) the amino acid sequence of SEQ ID NO:39, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:39;

[0071] 10) the amino acid sequence of SEQ ID NO:40, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:40; or

[0072] 11) the amino acid sequence of SEQ ID NO:41, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:41.

[0073] In one aspect, the present invention provides a multispecific antigen-binding molecule comprising a plurality of antigen-binding domains, wherein at least one antigen-binding domain specifically binds to IL-1β, and comprising an IL-1β-binding molecule described herein.

[0074] In some embodiments, the multispecific antigen-binding molecule is a bispecific antigen-binding molecule and comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds IL-1β and comprises an IL-1β binding molecule described herein.

[0075] In some embodiments, the second antigen binding domain is a full-length antibody or an antigen binding fragment, including ScFv, Fab, Fab', F(ab')2, a single domain antibody, or a heavy chain antibody.

[0076] In some embodiments, the first antigen-binding domain in the bispecific antigen-binding molecule is a single-domain antibody and the second antigen-binding domain is a full-length antibody.

[0077] In some embodiments, the constant region of the full-length antibody includes a light chain constant region and a heavy chain constant region, the light chain constant region is a κ chain or λ chain constant region, and the heavy chain constant region is from human IgG1, IgG2, IgG3, IgG4 or a mutant thereof.

[0078] In some embodiments, the first antigen-binding domain in the bispecific antigen-binding molecule is linked directly or via a linker to the C-terminus of the Fc domain of the second antigen-binding domain.

[0079] In some embodiments, the linker fragment is selected from (GS) n 、(GGS) n 、(GGGS) n and (GGGGS) n , n is 1, 2, 3, 4, or 5.

[0080] In some embodiments, the second antigen binding domain specifically binds to an immune checkpoint molecule. Non-limiting examples of some immune checkpoints include PD-1\PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H3, OX40, 4-1BB, etc., preferably specifically binds to PD-L1.

[0081] In some specific embodiments, the second antigen-binding domain in the bispecific antigen-binding molecule is an anti-PD-L1 antibody or an antigen-binding fragment thereof, non-limiting examples of which include but are not limited to atezolizumab, durvalumab, or avelumab, preferably atezolizumab, and the antigen-binding fragment comprises a single-chain Fv, Fab, Fab', F(ab')2, single-domain antibody, or heavy chain antibody of the anti-PD-L1 antibody.

[0082] In some embodiments, the multiple antigen-binding domains in the multispecific antigen-binding molecule are each independently 1, 2, or more.

[0083] In some specific embodiments, the bispecific antigen binding molecule comprises:

[0084] 1) heavy chain: amino acid sequence of SEQ ID NO:46, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:46; and

[0085] 2) Light chain: an amino acid sequence of SEQ ID NO:43, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:43.

[0086] In one aspect, the invention provides an immunoconjugate comprising an IL-1 β binding molecule or a multispecific antigen-binding molecule described herein linked to one or more conjugate moieties.

[0087] In one aspect, the present invention provides a nucleic acid encoding an anti-IL-1β binding molecule or a multispecific antigen-binding molecule described herein.

[0088] In some embodiments, the nucleic acid comprises:

[0089] 1) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 1;

[0090] 2) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 5;

[0091] 3) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 9;

[0092] 4) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 11;

[0093] 5) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 14;

[0094] 6) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 17;

[0095] 7) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 21;

[0096] 8) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 24;

[0097] 9) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 26;

[0098] 10) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 27;

[0099] 11) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 28; or

[0100] 12) The nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO:43 and the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO:46.

[0101] In some preferred embodiments, the nucleic acid 1)-11) further comprises a nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO:30.

[0102] In one aspect, the invention provides an expression vector comprising a nucleic acid described herein.

[0103] In one aspect, the present invention provides a host cell comprising the nucleic acid described herein or the expression vector described herein.

[0104] In one aspect, the present invention provides a method for preparing an IL-1β binding molecule described herein, or a multispecific antigen-binding molecule described herein, comprising 1) culturing a host cell described herein under conditions suitable for expression of the IL-1β binding molecule or the multispecific antigen-binding molecule, 2) recovering the IL-1β binding molecule or the multispecific antigen-binding molecule, and (iii) optionally purifying the IL-1β binding molecule or the multispecific antigen-binding molecule.

[0105] In one aspect, the present invention provides a pharmaceutical composition comprising one or more of an IL-1β binding molecule described herein, or a multispecific antigen-binding molecule described herein, or an immunoconjugate described herein, or a nucleic acid described herein, or an expression vector described herein, and a pharmaceutically acceptable carrier.

[0106] In one aspect, the present invention provides the use of an IL-1β binding molecule as described herein, or a multispecific antigen binding molecule as described herein, or an immunoconjugate as described herein, or a pharmaceutical composition as described herein in the preparation of a medicament for treating or preventing a disease associated with excessive IL-1β in a subject, or for inhibiting or blocking the binding of IL-1β to IL-1R1 in a subject, or downregulating human IL-1β activity or level, or inhibiting activation of downstream signaling pathways mediated by IL-1β and IL-1R1.

[0107] In some embodiments, the drug is administered orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.

[0108] In some embodiments, the disease is an inflammatory disease, a cardiovascular disease, or cancer.

[0109] In some embodiments, the inflammatory disease includes, but is not limited to, the following list: Muir-Wei syndrome (MWS), cryopyrin-associated periodic syndrome (CAPS), neonatal-onset multisystem inflammatory syndrome (NOMIS), rheumatoid arthritis, systemic-onset juvenile idiopathic arthritis (soJIA), gouty arthritis, multiple sclerosis, periodic fever syndromes, chronic obstructive pulmonary disease (COPD), type 1 diabetes, type 2 diabetes, familial cold autoinflammatory syndrome (FCAS), and eye diseases such as age-related macular degeneration.

[0110] In some embodiments, the cardiovascular disease includes, but is not limited to, the following list: atherosclerotic cardiovascular disease (ASCVD), arterial thrombosis, stroke, myocardial infarction, and heart failure.

[0111] In some embodiments, the type of cancer includes, but is not limited to, the following list: lung cancer such as non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), metastatic prostate cancer, low or intermediate risk myelodysplastic leukemia, hepatocellular carcinoma, colon cancer, and localized renal cancer.

[0112] In some embodiments, the drug can also be used in combination with an immune checkpoint inhibitor. In some embodiments, non-limiting examples of the immune checkpoint inhibitor include aptamers and antibodies that block PD-1\PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H3, OX40, and 4-1BB.

[0113] In one aspect, the present invention provides a method for treating or preventing a disease associated with excessive IL-1β in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an IL-1β binding molecule described herein, or a multispecific antigen-binding molecule described herein, or an immunoconjugate described herein, or a pharmaceutical composition described herein.

[0114] In some embodiments, the drug is administered orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.

[0115] In some embodiments, the disease is an inflammatory disease, a cardiovascular disease, or cancer.

[0116] In some embodiments, the inflammatory disease is selected from the list including, but not limited to, Muir-Weid syndrome (MWS), cryopyrin-associated periodic syndrome (CAPS), neonatal-onset multisystem inflammatory syndrome (NOMIS), rheumatoid arthritis, systemic-onset juvenile idiopathic arthritis (soJIA), gouty arthritis, multiple sclerosis, periodic fever syndromes, chronic obstructive pulmonary disease (COPD), type 1 diabetes, type 2 diabetes, familial cold autoinflammatory syndrome (FCAS), and eye diseases such as age-related macular degeneration.

[0117] In some embodiments, the cardiovascular disease is selected from the list including, but not limited to, atherosclerotic cardiovascular disease (ASCVD), arterial thrombosis, stroke, myocardial infarction, and heart failure.

[0118] In some embodiments, the type of cancer is selected from the list including, but not limited to, lung cancer such as non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), metastatic prostate cancer, low or intermediate risk myelodysplastic leukemia, hepatocellular carcinoma, colon cancer, and localized renal cancer.

[0119] In some embodiments, the IL-1β binding molecule, the multispecific antigen-binding molecule, the immunoconjugate, or the pharmaceutical composition may also be used in combination with an immune checkpoint inhibitor.

[0120] In some embodiments, non-limiting examples of the immune checkpoint inhibitors include aptamers and antibodies that block PD-1\PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H3, OX40, and 4-1BB.

[0121] In one aspect, the present invention provides an IL-1β binding molecule as described herein, or a multispecific antigen-binding molecule as described herein, or an immunoconjugate as described herein, or a pharmaceutical composition as described herein, and its use in the preparation of a reagent for diagnosing a disease associated with excessive IL-1β, or detecting IL-1β in a sample or a cell or tissue expressing IL-1β.

[0122] In some embodiments, the disease is an inflammatory disease, a cardiovascular disease, or cancer.

[0123] In some embodiments, the inflammatory disease is selected from the list including, but not limited to, Muir-Weid syndrome (MWS), cryopyrin-associated periodic syndrome (CAPS), neonatal-onset multisystem inflammatory syndrome (NOMIS), rheumatoid arthritis, systemic-onset juvenile idiopathic arthritis (soJIA), gouty arthritis, multiple sclerosis, periodic fever syndromes, chronic obstructive pulmonary disease (COPD), type 1 diabetes, type 2 diabetes, familial cold autoinflammatory syndrome (FCAS), and eye diseases such as age-related macular degeneration.

[0124] In some embodiments, the cardiovascular disease is selected from the list including, but not limited to, atherosclerotic cardiovascular disease (ASCVD), arterial thrombosis, stroke, myocardial infarction, and heart failure.

[0125] In some embodiments, the type of cancer is selected from the list including, but not limited to, lung cancer such as non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), metastatic prostate cancer, low or intermediate risk myelodysplastic leukemia, hepatocellular carcinoma, colon cancer, and localized renal cancer.

[0126] In some embodiments, in the diagnosis or detection, the IL-1β binding molecule, the multispecific antigen-binding molecule, the immunoconjugate, or the pharmaceutical composition further carries a detectable label.

[0127] In one aspect, the present invention provides a kit comprising a pharmaceutical composition, prescribing information, and a container, wherein the pharmaceutical composition comprises one or more of an IL-1β binding molecule described herein, or a multispecific antigen-binding molecule described herein, or an immunoconjugate described herein, or a nucleic acid described herein, or an expression vector described herein and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] Figure 1 : Single domain antibody crude extracts were tested for blocking effect on human IL-1RI-Fc and IL-1β-Fc-biotin. Among them, 7 samples 5, 22, 32, 34, 35, 40, and 41 showed significant blocking efficacy;

[0129] Figure 2 : IgG antibodies were tested for their blocking efficacy against human IL-1RI-Fc and IL-1β-Fc-biotin. Each molecule had a visible blocking effect, with HYB0902 having the best blocking efficacy.

[0130] Figure 3 : IgG antibodies inhibited IL-6 release from MRC-5 cells induced by IL-1β. HYB0902 showed the best inhibitory effect.

[0131] Figure 4 :The results of the engineered IgG antibody inhibiting IL-1β-induced IL-6 release from MRC-5 cells;

[0132] Figure 5 : The results of humanized IgG antibodies inhibiting IL-6 release induced by IL-1β in MRC-5 cells, among which HYB0902-2-hz3 showed the best inhibitory effect;

[0133] Figure 6 : The test results of the preferred molecule HYB009 and the control molecule in the IL-1β-induced IL-6 release inhibition experiment of MRC-5 cells showed that HYB009 had a better inhibitory effect than the control molecule;

[0134] Figure 7 : The test results of the preferred molecule HYB009 and the control molecule in the inhibition experiment of IL-1β-induced IFN-γ release in NK-92 cells showed that HYB009 had a better inhibitory effect than the control molecule;

[0135] Figure 8 : The results of in vivo efficacy testing of the preferred molecule in a human IL-1β-induced IL-6 secretion model in mice showed that HYB009 had efficacy that was non-inferior to that of the control molecule;

[0136] Figure 9: Study results on the synergistic anti-tumor effect of HYB009 and anti-PD-L1 antibody. DETAILED DESCRIPTION

[0137] Although the present invention can be implemented in many different forms, what is disclosed here is its specific illustrative embodiment that proves the principle of the present invention. It should be emphasized that the present invention is not limited to the specific embodiment illustrated. In addition, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0138] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, the terms in the singular should include the plural, and the terms in the plural should include the singular. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular "a", "an" and "the" include plural indicators. In this application, unless otherwise stated, "or" is used to mean "and / or". In this application, "first", "second" have no practical meaning and are only used to distinguish identical terms. In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the range provided in the specification and the appended claims includes all values ​​between endpoints and endpoints.

[0139] Generally, terms relating to, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g., Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Abbas et al., Cellular and Molecular Immunology, 6th ed., WB Saunders Company (2010); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Additionally, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0140] definition

[0141] In order to better understand the present invention, the definitions and explanations of relevant terms are provided below:

[0142] As used herein, the term "IL-1β" (also known as interleukin-1β) (GenBank ID: NP_000567.1) refers to the major circulating form of IL-1. It is produced in the form of a precursor (pro-IL-1β or IL-1β proprotein), which is activated by the NLRP3 inflammasome in many inflammatory diseases and is an important mediator of the inflammatory response.

[0143] As used herein, the term "antibody" or "Ab" generally refers to any form of antibody that exhibits the desired biological or binding activity. It includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, heavy chain antibodies, and single domain antibodies. Antibodies can comprise heavy and light chains. Light chains are classified as κ or λ and are characterized by specific constant regions known in the art. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains can be divided into μ, δ, γ, α, and ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). The VH and VL regions can be further divided into hypervariable regions (called complementarity determining regions (CDRs)) separated by relatively conserved regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus. The CDRs contain most of the residues that form specific interactions with the antigen. Unless otherwise indicated, the numbering and positioning of the CDR amino acid residues in all VL and VH regions described herein are based on the well-known AbM numbering scheme. Antibodies can have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0144] As used herein, the term "anti-IL-1β antibody" refers to an antibody that specifically binds to IL-1β (e.g., human IL-1β). Advantageously, the anti-IL-1β antibody specifically binds to IL-1β with an affinity sufficient to provide diagnostic and / or therapeutic use.

[0145] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to an IL-1β protein is substantially free of antigens other than the IL-1β protein). However, an isolated antibody that specifically binds to a human IL-1β protein may have cross-reactivity with other antigens, such as IL-1β proteins from other species. In addition, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0146] As used herein, the term "heavy chain antibody" or "HCAb" refers to a functional antibody that comprises heavy chains but lacks the light chains typically present in four-chain antibodies. Camelids (such as camels, llamas or alpacas) are known to produce HCAbs.

[0147] As used herein, the term "single domain antibody" or "sdAb" refers to a single antigen-binding polypeptide with three complementary determining regions (CDRs). Only sdAb can bind to the antigen without pairing with the corresponding polypeptide containing CDRs. In some cases, single domain antibodies are obtained from Camelidae HCAb engineering transformations, and their heavy chain variable domains are referred to as "VHH" (the variable domains of the heavy chain of heavy chain antibodies) in this article. Some VHHs are also referred to as nanobodies. Camelidae sdAb is one of the smallest known antigen-binding antibody fragments and is therefore also referred to as "nanoantibodies." A basic VHH has the following structure from N-terminal to C-terminal: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementary determining regions 1 to 3.

[0148] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequences are from one species and the constant region sequences are from another species, for example, wherein the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0149] As used herein, the term "humanized antibody" refers to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0150] As used herein, the term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in mice, in mouse cells, or in hybridomas derived from mouse cells, fully human antibodies may contain murine carbohydrate chains.

[0151] As used herein, the term "full-length antibody" refers to an immunoglobulin molecule that, when naturally present, comprises at least four peptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conserved regions called framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

[0152] As used herein, the term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as scFv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity to the antigen is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as "conservative variants" or "function-conservative variants" of the antibody). The term "binding molecule" refers to both antibodies and their binding fragments.

[0153] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).

[0154] As used herein, the term "multispecific antibody" refers to antibodies that simultaneously target multiple antigens. These antibodies can be produced through immunoprecipitation and purification. Alternatively, they can be obtained through genetic engineering, which offers advantages due to its flexibility in optimizing binding sites, considering synthetic formats, and achieving optimal yields.

[0155] As used herein, the term "specifically binds" or "specifically binds to" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen.

[0156] As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by the polynucleotide inserted therein, the vector is referred to as an expression vector. The vector can be transformed, transduced, or transfected into a host cell to express the genetic material elements carried in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). The vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain an origin of replication.

[0157] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including but not limited to prokaryotic cells such as Escherichia coli (E.coli) or Bacillus subtilis (Bacillus subtilis), fungal cells such as yeast cells or Aspergillus (Aspergillus), insect cells such as S2 Drosophila cells or Sf9, and animal cells such as fibroblasts, COS cells, NSO cells, HeLa cells, BHK cells, CHO cells, HEK293 cells or human cells. CHO cells refer to Chinese hamster ovary cells, which include a variety of commercially available subclones, such as CHO-K1, CHO-S, CHO-DXB11, CHO-DG44, etc. Wherein CHO-K1 is generally used as an expression platform. CHO-K1 is commercially available from ATCC, ECACC, DSMZ and a number of other companies.

[0158] As used herein, the terms "identity" and "similarity" refer to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by alignment and comparison of the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecules being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0159] As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response of antibodies or sensitized T lymphocytes that can be formed in the immune system of an organism after stimulating the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host, and the means of immunity.

[0160] As used herein, the term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0161] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are absolutely required for this killing. NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) page 464. In order to assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337, can be performed. Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model disclosed in PNAS (USA) 95: 652-656 (1998) such as Clynes.

[0162] As used herein, the term "complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed.

[0163] As used herein, the term "EC 50 ”, also known as “half-maximal effective concentration”, is the concentration of a drug, antibody or toxic agent that induces a response that is 50% between baseline and maximum after a specified exposure time. In the context of this application, EC 50 The unit is "nM".

[0164] As used herein, the term "disease associated with excessive IL-1β" refers to any condition caused by, exacerbated by, or otherwise associated with increased expression or activity of IL-1β (eg, human IL-1β).

[0165] As used herein, "inflammatory" includes inflammatory and autoinflammatory diseases. The term "inflammatory disease" includes, but is not limited to, rare genetic diseases that cause excessive production of IL-1β and other diseases that can benefit from regulation, such as antagonizing IL-1β signaling. "Inflammatory disease" may include, but is not limited to, Muir-Wei syndrome (MWS), cryopyrin-associated periodic syndrome (CAPS), neonatal onset multisystem inflammatory syndrome (NOMIS), rheumatoid arthritis, systemic juvenile idiopathic arthritis (soJIA), gouty arthritis, multiple sclerosis, periodic fever syndrome, chronic obstructive pulmonary disease (COPD), type 1 diabetes, type 2 diabetes, familial cold autoinflammatory syndrome (FCAS) and eye diseases such as age-related macular degeneration.

[0166] As used herein, "cardiovascular disease" refers to a class of diseases involving the heart or blood vessels. A non-exhaustive list of CVD manifestations includes, but is not limited to, atherosclerotic cardiovascular disease (ASCVD), arterial thrombosis, stroke and heart failure, angina pectoris, myocardial infarction (MI, commonly known as heart attack), and arrhythmia.

[0167] As used herein, "cancer" refers to a group of diseases that involve abnormal cell growth and may invade or spread to other parts of the body. A non-exhaustive list of cancer types includes, but is not limited to, lung cancer such as non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), metastatic prostate cancer, low or intermediate risk myelodysplastic leukemia, hepatocellular carcinoma, colon cancer, and localized renal cancer. Thus, cancer can involve cells from solid tissues or organs, such as brain cancer, breast cancer, colon cancer, rectal cancer, skin cancer, liver cancer, kidney cancer, lung cancer, pancreatic cancer, prostate cancer, head and neck cancer, ovarian cancer, uterine cancer, bladder cancer, stomach cancer (including esophageal cancer); connective tissue cancer, such as sarcoma or bone cancer; or blood cancer, such as lymphoma, leukemia, and myeloma. Cancers can also be described by their cell of origin, such as cancers that originate from epithelial cells in various parts of the body, or adenomas that originate from glands.

[0168] As used herein, the term "subject" includes any human or non-human animal, preferably a human.

[0169] As used herein, the ability to "block binding" refers to the ability of an antibody to inhibit the binding of two molecules (e.g., human IL-1β and anti-IL-1R1 antibody) to any detectable extent. In some embodiments, an antibody that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%. In some embodiments, the inhibition can be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0170] The term "treatment" as used herein in the context of treating a disease generally refers to treatment and therapy of a human or animal in which some desired therapeutic effect is achieved, for example, inhibition of disease progression, including a decrease in the rate of progression, stagnation of the rate of progression, regression of the disease, improvement of the disease, and cure of the disease. Treatment as a preventative measure (i.e., prevention, prevention) is also included. For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, or some combination thereof. For a tumor, "treatment" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0171] As used herein, the term "therapeutically effective amount" refers to an amount of an active compound, or a material, composition, or dosage form comprising an active compound, which, when administered according to the desired treatment regimen, is effective for producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. Specifically, "therapeutically effective amount" refers to an amount or concentration of an antibody that is effective for treating a disease or condition associated with excessive human IL-1β.

[0172] As used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0173] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th edition, Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0174] As used herein, the term "adjuvant" refers to a nonspecific immunopotentiator that, when delivered to an organism together with an antigen or delivered to an organism in advance, can enhance the immune response to the antigen in the organism or change the type of immune response. There are a variety of adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

[0175] IL-1β binding molecules

[0176] In some aspects, the invention comprises IL-1 β binding molecules.

[0177] Generally speaking, an IL-1β binding molecule may include any molecule that specifically binds to IL-1β. In some cases, an "IL-1β binding molecule" may include an "IL-1β antagonist." An IL-1β binding molecule or IL-1β antagonist may be a polypeptide or protein, such as an antibody, more specifically an anti-IL-1β antibody.

[0178] Antibodies include, but are not limited to, chimeric antibodies, humanized antibodies, or single-domain antibodies. In specific embodiments, the IL-1β binding molecule is a single-domain antibody, which generally refers to an antibody composed of a single monomeric variable antibody domain. Like whole antibodies, single-domain antibodies are capable of selectively binding to a specific antigen.

[0179] More specifically, the IL-1β binding molecule is a heavy chain antibody or a single domain antibody, preferably a single domain antibody, which can be used interchangeably with the terms "VHH", "VHH antibody", "VHH domain", "VHH antibody fragment", "VHH" or "nanobody". VHH molecules from camelid antibodies are one of the smallest known complete antigen-binding domains (approximately 15 kDa, or 1 / 10 the size of conventional IgG), and are therefore well suited for delivery into dense tissues and into the confined spaces between large molecules.

[0180] The single domain antibodies of the present invention disclosed herein can be prepared by a person skilled in the art according to methods known in the art or any future methods. For example, VHHs can be obtained using methods known in the art, such as by immunizing camels and obtaining hybridomas therefrom, or by cloning a library of VHHs of the present invention using molecular biology techniques known in the art, followed by selection using phage display.

[0181] For example, single domain antibodies can be obtained by immunizing llama or alpaca with the desired antigen and then isolating the mRNA encoding heavy chain antibodies. By reverse transcription and polymerase chain reaction, a gene library containing millions of clones of single domain antibodies is produced. Screening techniques such as phage display and ribosome display help to identify clones that bind to antigens. A kind of technology is phage display, in which (preferably human) antibody libraries are synthesized on phage, the libraries are screened with the antigen of interest or its antibody binding portion, and the phage that binds to the antigen is separated, from which immunoreactive fragments can be obtained. The method for preparing and screening such libraries is well known in the art, and the kit for producing phage display libraries is commercially available (e.g., Pharmacia recombinant phage antibody system, catalog number 27-9400-01; and Stratagene SurfZAP™ phage display kit, catalog number 240612). Still other methods and reagents are available for generating and screening antibody display libraries (see, eg, Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).

[0182] When the most potent clones are identified, their DNA sequences are optimized, for example, through affinity maturation or humanization, which prevents the body from mounting an immune response against the antibody.

[0183] Thus, single domain antibodies may be obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of a naturally occurring VHH domain (as described below) or by expressing a nucleic acid encoding such a humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, in particular a mammalian species, such as a human, or by expressing a nucleic acid encoding such a camelized VH domain; (5) by "camelization" of a "domain antibody" or "Dab" as described by Ward et al. (supra), or by expressing a nucleic acid encoding such a camelized VH domain; (6) preparing a protein, polypeptide or other amino acid sequence using synthetic or semisynthetic techniques; (7) by preparing a nucleic acid encoding a VHH using techniques for nucleic acid synthesis and then expressing the nucleic acid thus obtained; and / or (8) by any combination of the foregoing. Suitable methods and techniques for performing the foregoing will be clear to the skilled person based on the disclosure herein and include, for example, the methods and techniques described in more detail below.

[0184] Single domain antibodies are usually produced by cloning variable domain libraries from blood, lymph nodes or spleen cDNA PCR obtained from immune animals into phage display vectors. Antigen-specific single domain antibodies are usually selected by panning libraries on immobilized antigens (e.g., antigens coated on test tube plastic surfaces, biotinylated antigens fixed on streptavidin beads or membrane proteins expressed on cell surfaces). The affinity of adAb can be improved by simulating this strategy in vitro, such as by site-directed mutagenesis in CDR regions and under increased stringency conditions (higher temperature, high or low salt concentration, high or low pH and low antigen concentration), immobilized antigens are further panned (Wesolowski et al., Singledomain antibodies:promisingexperimental and therapeutic tools in infection and immunity.Med Microbiol Immunol (2009) 198:157-174).

[0185] Methods for preparing VHHs that specifically bind to an antigen or epitope are described in the literature, see for example: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645, 17 June, 2009 and WO94 / 04678.

[0186] In some embodiments, the VHH in the IL-1β binding molecule is fused to the Fc domain of an antibody, such as the Fc domain of an IgG (e.g., IgG4 or IgG1). In a specific embodiment, the Fc domain is the Fc domain of human IgG1. By fusing the VHH to the Fc domain, effector functions can be more effectively recruited. Furthermore, the fusion of the VHH to the Fc domain can help the IL-1β binding molecule form dimers and can also help extend the in vivo half-life of the IL-1β binding molecule.

[0187] In some embodiments, the Fc domain fused to the IL-1β binding molecule (eg, IL-1β VHH) may further comprise one or more amino acid mutations that reduce or eliminate the effector function of the Fc domain.

[0188] For ease of description, the IL-1β binding molecules are described as anti-IL-1β antibodies in the following sections.

[0189] In some embodiments, the antibody provided herein further includes one or more conjugate parts. The conjugate part is a part that can be connected to the antibody. For example, a detectable label (such as a luminescent label, a fluorescent label, an enzyme-substrate label), a regulator (such as a polymer, such as PEG that extends half-life) or other therapeutic agents. It is considered that a variety of conjugate parts can be connected to the antibody or its antigen-binding fragment provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York (New York), (1989)). These conjugate parts can be connected to the antibody or its antigen-binding fragment by methods such as covalent bonding, affinity bonding, embedding, coordination bonding, compounding, bonding (association), blending (blending) or adding.

[0190] In some embodiments, the antibodies of the present invention block the binding of human IL-1β to IL-1R1, thereby providing biological activity, including, for example, inhibiting the production of cytokines, thereby alleviating inflammatory responses. Exemplary cytokines include IL-6. The term "IL-6" refers to interleukin-6, which is one of the important cytokines in inflammatory immune responses and can promote B cell secretion of antibodies, promote T cell growth and IL-2 production, etc. Cytokine production can be determined using methods known in the art, such as by ELISA.

[0191] Anti-IL-1β antibody containing CDR

[0192] In some embodiments, the interleukin-1β (IL-1β) binding molecule of the present invention, wherein the IL-1β binding molecule comprises a heavy chain variable region (VH), wherein the VH comprises:

[0193] 1) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively;

[0194] 2) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively;

[0195] 3) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 8, respectively;

[0196] 4) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 13, respectively;

[0197] 5) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO: 16, respectively;

[0198] 6) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively;

[0199] 7) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 16, respectively; or

[0200] 8) HCDR1, HCDR2, and HCDR3: They comprise the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 25, and SEQ ID NO: 8, respectively.

[0201] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using a number of well-known schemes, including Chothia numbering based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat numbering based on antibody sequence variability (Kabat et al, Sequences of Proteins of Immunological Interest, 4 th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM numbering (University of Bath), Contact numbering (University College London), international ImMunoGeneTics database (IMGT numbering) (1999 Nucleic Acids Research, 27, 209-212), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures. The CDRs of the antibodies of the present invention can be defined by those skilled in the art according to any scheme in the art (e.g., different numbering schemes or combinations thereof).

[0202] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different numbering schemes may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different numbering schemes may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different numbering schemes or combinations).

[0203] The variable region and CDR in the antibody sequence can be identified according to the general rule (as mentioned above, for example Kabat numbering system) that this area has developed or by comparing sequence with the database of known variable region.Kontermann and Dubel compile, Antibody Engineering, Springer, New York, NY, 2001 and Dinarello etc., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, described the method for identifying these zones in 2000.The exemplary database of antibody sequence is described in and can be obtained from " Abysis " website (maintained by the ACMartin of the biochemistry and molecular biology department (Department of Biochemistry & Molecular Biology University College London, London, England) of University College London, England) and VBASE2 website www.vbase2.org on www.bioinf.org.uk / abs, as Retter etc., Nucl.Acids Res., 33 (Database issue): described in D671-D674 (2005). Unless otherwise indicated, all CDRs described herein are obtained according to AbM numbering.

[0204] In some specific embodiments, the IL-1β binding molecule is a single domain antibody (VHH), the VHH comprising:

[0205] 1) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively;

[0206] 2) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively;

[0207] 3) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 8, respectively;

[0208] 4) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 13, respectively;

[0209] 5) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO: 16, respectively;

[0210] 6) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively;

[0211] 7) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 16, respectively; or

[0212] 8) HCDR1, HCDR2, and HCDR3: They comprise the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 25, and SEQ ID NO: 8, respectively.

[0213] Anti-IL-1β antibodies defined by VHH sequences

[0214] In some embodiments, the IL-1 β binding molecule comprises at least one immunoglobulin single variable domain (e.g., VHH), wherein the VHH comprises:

[0215] 1) the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1;

[0216] 2) the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:5;

[0217] 3) the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9;

[0218] 4) the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11;

[0219] 5) the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14;

[0220] 6) the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17;

[0221] 7) the amino acid sequence of SEQ ID NO:21, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:21;

[0222] 8) the amino acid sequence of SEQ ID NO:24, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:24;

[0223] 9) the amino acid sequence of SEQ ID NO:26, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26;

[0224] 10) the amino acid sequence of SEQ ID NO:27, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:27; or

[0225] 11) the amino acid sequence of SEQ ID NO:28, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28.

[0226] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available from http: / / www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0227] Multispecific antigen-binding molecules

[0228] In some aspects, the present invention also provides multispecific antigen-binding molecules comprising IL-1βVHH or heavy chain antibodies or fragments thereof as described herein, such as multispecific antibodies. Multispecific antibodies are monoclonal antibodies with binding specificity to at least two different sites (ie, different epitopes on different antigens or different epitopes on the same antigen). In some embodiments, the multispecific antibody comprises a first antigen-binding domain for IL-1β and a second or more antigen-binding domains for one or more non-IL-1β molecules. In some embodiments, the multiple antigen-binding domains in the multispecific antigen-binding molecule are each independently 1, 2 or more.

[0229] In some embodiments, the multispecific antibodies disclosed herein are bispecific antibodies comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds IL-1β and comprises an IL-1β binding molecule described herein.

[0230] In some embodiments, the first antigen-binding domain comprises an IL-1β binding molecule described herein and any IL-1β binding molecule derived therefrom, particularly an IL-1β single-domain antibody. Preferably, the first antigen-binding region comprises or consists of an anti-IL-1β single-domain antibody of the present invention, more preferably, the single-domain antibody is a humanized single-domain antibody.

[0231] As used herein, the antibody portion in a bispecific antibody is "derived from" a parent antibody means that the parent antibody is used as a starting material for making a bispecific antibody as known in the art. The antibody portion may comprise a heavy chain and / or light chain CDR identical to the heavy chain and / or light chain CDRs of the parent antibody. Two antibodies having identical VH and / or VL CDRs means that their CDRs are identical when determined by the same method (e.g., Kabat definition, Chothia definition, AbM definition, and / or contact definition known in the art).

[0232] Alternatively, the antibody portion can comprise a heavy chain and / or light chain CDR that is substantially similar to the heavy chain and / or light chain CDR of the parent antibody (e.g., comprising no more than 5, 4, 3, 2, or 1 amino acid residue changes compared to the parent antibody). In some cases, the antibody portion in the bispecific antibody can have the same heavy chain variable region and / or the same light chain variable region as the parent antibody. For example, the antibody portion in the bispecific antibody can have the same heavy chain and / or the same light chain as the parent antibody.

[0233] In specific examples, HYB0901, HYB0902, HYB0903, HYB0904, HYB0905, HYB0906, HYB0907, or mutants and humanized antibodies derived therefrom disclosed herein can be used as starting materials for making any bispecific antibody disclosed herein.

[0234] The second antigen-binding domain of the bispecific antibodies suitable for use in the present invention may comprise or consist of a full-length antibody or an antigen-binding fragment thereof, as long as it is capable of specifically binding to its targeted antigen, including but not limited to, for example, a full-length antibody, ScFv, Fab, Fab', F(ab')2, single-domain antibody, or heavy-chain antibody that specifically binds to the target antigen. In some embodiments, the heavy chain of the second antigen-binding domain may comprise a mutated Fc region that alters the binding affinity and / or binding specificity of the region to an Fc receptor. In some embodiments, the Fc region suitable for use in the heavy chain antibodies of the present invention is also suitable for use in the bispecific antibodies of the present invention.

[0235] In some embodiments, the heavy chain constant region of the second antigen-binding domain in the bispecific antibodies of the present invention is derived from IgG, such as IgG1, IgG2, IgG3, or IgG4. Preferably, the heavy chain constant region is derived from IgG1 or IgG4. In some embodiments, the light chain constant region of the second antigen-binding domain in the bispecific antibodies of the present invention is a kappa chain or lambda chain constant region, preferably a kappa light chain constant region.

[0236] In some embodiments, the bispecific antibodies disclosed herein may comprise a first antigen-binding domain in the form of a VHH and a second antigen-binding domain in the form of a full-length antibody. The first antigen-binding domain in the form of a VHH may be derived from any anti-IL-1β antibody disclosed herein, for example, HYB0902-2-hz3. For example, the bispecific antibody may comprise a first chain comprising a first antigen-binding domain fused to a heavy chain of a second antigen-binding domain, and a second chain being a light chain of the second antigen-binding domain. In some cases, the heavy chain of the second antigen-binding domain may comprise a mutated Fc domain that has altered binding affinity and / or binding specificity for an Fc receptor.

[0237] In some embodiments, in the bispecific antibodies disclosed herein, the first antigen-binding domain disclosed herein can be connected to the C-terminus of the second antigen-binding domain, for example, connected to the C-terminus of the Fc domain of the second antigen-binding domain. In some embodiments, the first and second antigen-binding domains are connected by a linker. In one embodiment, the linker is a peptide of about 3 to about 20 amino acids in length. For example, the linker comprises (GS) n 、(GGS) n 、(GGGS) nand (GGGGS) n , where n is an integer between 1 and 5.

[0238] In some embodiments, in the bispecific antibodies described herein, the second antigen binding domain specifically binds to immune checkpoint molecules (e.g., those that negatively or positively regulate immune responses), some non-limiting examples of which include PD-1\PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H3, OX40, 4-1BB, etc.

[0239] In some embodiments, the second antigen binding domain in the bispecific antibodies disclosed herein specifically binds to PD-L1, for example, human PD-L1. Any antibody or antigen-binding fragment thereof that can bind to PD-L1 can be used to construct the bispecific antibodies disclosed herein, for example, including but not limited to atezolizumab, durvalumab or avelumab. The anti-PD-L1 antibody portion may comprise the same heavy chain and / or light chain CDR as the parent antibody (e.g., atezolizumab). Alternatively, the antibody portion may comprise a heavy chain and / or light chain CDR that is substantially similar to the heavy chain and / or light chain CDR of the parent antibody (e.g., comprising no more than 5, 4, 3, 2 or 1 amino acid residue changes compared to the parent antibody). In some cases, the anti-PD-L1 antibody portion in the bispecific antibody may have the same heavy chain variable region and / or the same light chain variable region as the parent antibody. For example, the antibody portion of the bispecific antibody can have the same heavy chain and / or the same light chain as the parent antibody.

[0240] The anti-PD-L1 antibody suitable for use in the bispecific antibodies of the present invention may comprise or consist of a full-length antibody or an antigen-binding fragment thereof, as long as it can specifically bind to PD-L1, including but not limited to, for example, a full-length antibody, ScFv, Fab, Fab', F(ab')2, single-domain antibody, or heavy chain antibody that specifically binds to PD-L1. In some embodiments, the heavy chain of the anti-PD-L1 antibody may comprise a mutated Fc region that alters the binding affinity and / or binding specificity of the region to an Fc receptor. In some embodiments, the Fc region suitable for use in the heavy chain antibodies of the present invention is also suitable for use in the bispecific antibodies of the present invention.

[0241] In some embodiments, in the bispecific antibodies disclosed herein, the anti-IL-1β single domain antibody disclosed herein as the first antigen binding domain can be connected to the C-terminus of the anti-PD-L1 antibody, for example, connected to the C-terminus of the Fc domain of the anti-PD-L1 antibody. In some embodiments, the C-termini of the anti-IL-1β single domain antibody and the anti-PD-L1 antibody Fc domain are connected by a connecting fragment (for example, when the anti-IL-1β single domain antibody is connected to the C-terminus of the Fc domain of the anti-PD-L1 antibody). In one embodiment, the linker is a peptide of about 3 to about 20 amino acids in length. For example, the connecting fragment comprises (GS) n 、(GGS) n 、(GGGS) n and (GGGGS) n , wherein n is an integer between 1 and 5, preferably, the linker fragment is (GGGGS) n Amino acid sequence, n is an integer between 1-5, preferably n=3.

[0242] In some embodiments, the bispecific antibodies of the invention have two heavy chains and two light chains, preferably two identical heavy chains and two identical light chains.

[0243] In one embodiment of the present invention, the heavy chain variable region and / or light chain variable region of the anti-PD-L1 antibody in the bispecific antibody of the present invention is derived from atezolizumab.

[0244] In some embodiments, the complementarity determining regions VHCDR1, VHCDR2, and VHCDR3 of the three heavy chain variable regions of the anti-PD-L1 antibody in the bispecific antibody of the present invention are derived from the CDRs in the amino acid sequence shown in SEQ ID NO: 44. Preferably, the CDRs are defined by Kabat.

[0245] In some embodiments, the complementarity determining regions VLCDR1, VLCDR2, and VLCDR3 of the three light chain variable regions of the anti-PD-L1 antibody in the bispecific antibody of the present invention are derived from the CDRs comprising the amino acid sequence shown in SEQ ID NO: 43, preferably, the CDRs are defined by Kabat.

[0246] In some embodiments, the bispecific antibodies of the invention comprise:

[0247] Heavy chain: from N-terminus to C-terminus, heavy chain variable region VH of the second antigen antibody - heavy chain constant region CH1 - heavy chain constant region Fc - connecting fragment - anti-IL-1β VHH; light chain: from N-terminus to C-terminus, light chain variable region - light chain constant region CL of the second antigen antibody, wherein the second antigen is PD-L1; wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 46; and the light chain comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: The sequence shown in NO:43 has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

[0248] In some embodiments, the bispecific antibodies of the invention comprise:

[0249] Heavy chain: from N-terminus to C-terminus, heavy chain variable region VH of the second antigen antibody - heavy chain constant region CH1 - heavy chain constant region Fc - connecting fragment - anti-IL-1β VHH; light chain: from N-terminus to C-terminus, light chain variable region - light chain constant region CL of the second antigen antibody, wherein the second antigen is PD-L1; wherein the heavy chain is the amino acid sequence shown in SEQ ID NO: 46; and the light chain is the amino acid sequence shown in SEQ ID NO: 43.

[0250] In some embodiments, the bispecific antibody of the present invention that specifically binds to IL-1β and PD-L1 has a better tumor inhibition effect than using anti-IL-1β antibody or anti-PD-L1 antibody alone or in combination with anti-IL-1β antibody or anti-PD-L1 antibody.

[0251] Antibody variants

[0252] In certain embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Suitable modifications may be introduced into the nucleotide sequence encoding the antibody, or the amino acid sequence variants of the antibody may be prepared by peptide synthesis. Such modifications include, for example, deletion of residues in the amino acid sequence of the antibody, and / or insertion and / or substitution. Any combination of deletion, insertion, and substitution may be performed to obtain the final construct, as long as the final construct possesses desired characteristics, for example, antigen-specific binding.

[0253] a) Substitution, insertion, and deletion variants

[0254] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Interested sites for substitution mutagenesis include HVRs (Hyper-Variable Regions) and FRs. Conservative substitutions are shown in Table A under the heading "Preferred Substitutions." More substantial variations are provided in Table A under the heading "Exemplary Substitutions," and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0255] Table A: Examples of Conservative Substitutions

[0256]

[0257]

[0258] According to common side chain properties, amino acids can be grouped as follows:

[0259] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, IIe;

[0260] (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0261] (3) Acidic: Asp, Glu;

[0262] (4) Basic: His, Lys, Arg;

[0263] (5) Residues that affect chain orientation: Gly, Pro;

[0264] (6) Aromatic: Trp, Tyr, Phe.

[0265] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0266] A class of substitution variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have changes (e.g., improvements) in certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display, such as those described herein. In short, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0267] HVR can be changed (e.g., replaced), for example to improve antibody affinity. Such changes can be made to HVR "hot spots", i.e., residues encoded by codons that undergo mutations at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigen, wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and reselection of secondary libraries has been described in, e.g., Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-guided mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves an HVR-guided approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0268] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to HVRs that do not substantially reduce binding affinity. For example, such changes may be outside the antigen contact residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.

[0269] In some further embodiments, the antibodies disclosed herein (eg, anti-IL-1β antibodies and multispecific antibodies based thereon) may comprise conservative substitutions or modifications of amino acids in the heavy chain variable region. It is understood in the art that certain conservative sequence modifications can be made without abolishing antigen binding (see, e.g., Brummell et al. (1993) Biochem 32: 1180-8; de Wildt et al. (1997) Prot. Eng. 10: 835-41; Komissarov et al. (1997) J. Biol. Chem. 272: 26864-26870; Hall et al. (1992) J. Immunol. 149: 1605-12; Kelley and O'Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10: 341-6 and Beers et al. (2000) Clin. Can. Res. 6: 2835-43).

[0270] The term "conservative substitution" as used herein refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is substituted by another amino acid residue with a similar side chain, such as a physical or functionally similar residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.) to a corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0271] In some specific embodiments, the IL-1β VHH consists of 1) the amino acid sequence shown in SEQ ID NO: 1; 2) the amino acid sequence shown in SEQ ID NO: 5; 3) the amino acid sequence shown in SEQ ID NO: 9; 4) the amino acid sequence shown in SEQ ID NO: 11; 5) the amino acid sequence shown in SEQ ID NO: 14; 6) the amino acid sequence shown in SEQ ID NO: 17; 7) the amino acid sequence shown in SEQ ID NO: 21; 8) the amino acid sequence shown in SEQ ID NO: 24; 9) the amino acid sequence shown in SEQ ID NO: 26; 10) the amino acid sequence shown in SEQ ID NO: 27; 11) the amino acid sequence shown in SEQ ID NO: 28.

[0272] In some specific embodiments, the IL-1β / PD-L1 bispecific antibody consists of 1) a heavy chain, such as the amino acid sequence shown in SEQ ID NO: 46; and 2) a light chain, such as the amino acid sequence shown in SEQ ID NO: 43.

[0273] b) Glycosylation variants

[0274] In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence to create or eliminate one or more glycosylation sites.

[0275] In the case where antibody comprises Fc district, it is possible to change the carbohydrate of its attachment. The natural antibody produced by mammalian cells generally comprises branched, biantennary oligosaccharide, which is generally attached to the Asn297 of the CH2 domain in the Fc district by N connection. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharide can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and the fucose of the GlcNAc in the biantennary oligosaccharide structure " backbone ". In some embodiments, the oligosaccharide in the antibody of the present invention can be modified to create antibody variants with some improved characteristics.

[0276] c) Fc region variants

[0277] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to thereby generate an Fc region variant. The Fc region variant can be comprised in a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0278] In certain embodiments, the present invention encompasses antibody variants that possess some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important and certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / reduction of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I et al., Proc. Nat'l Acad. Sci USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, e.g., the ACT I™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and the CytoTox96 non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., Proc Nat'l Acad Sci USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and, therefore, lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402.To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS. et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0279] In certain embodiments, where effector function is not desired, the antibodies disclosed herein can be further engineered to introduce at least one mutation in the Fc domain of the antibody that reduces binding of the antibody to an activating Fcγ receptor (FcγR) and / or reduces Fc effector function (such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP)).

[0280] Fc domain positions that can be mutated to reduce antibody binding to activating FcγRs and subsequently reduce effector function are, for example, those described in the following literature: (Xu, Alegre et al. 2000) (Vafa, Gilliland et al. 2014) (Bolt, Routledge et al. 1993) (Chu, Vostiar et al. 2008) (Shields, Namenuk et al. 2001). Fc mutations with minimal ADCC, ADCP, CDC, Fc-mediated cell activation have also been described as sigma mutations of IgG1, IgG2, and IgG4 (Tam, McCarthy et al. 2017).

[0281] Exemplary mutations that can be made, alone or in combination, are K214T, E233P, L234V, L234A, G236 deletion, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S, and P331S mutations on IgG1, IgG2, IgG3, or IgG4.

[0282] Exemplary combination mutations that can be made to reduce ADCC are L234A / L235A on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on IgG1, IgG2, IgG3 or IgG4, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358M on IgG1, H268Q / V309L / A330S / P331S on IgG2, S267E / L328F on G1, L234F / L235E / D265A on IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S on IgG4. Hybrid IgG2 / 4 Fc domains, such as an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4, can also be used.

[0283] In some embodiments, the Fc region is an Fc region from human IgG1 containing the mutation N297A (according to EU numbering).

[0284] In some specific embodiments, the Fc domain is the amino acid sequence of SEQ ID NO:30, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:30.

[0285] d) Cysteine-engineered antibody variants

[0286] In certain embodiments, it may be desirable to create cysteine-engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In specific embodiments, the replaced residues are present at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby localized at accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. Cysteine-engineered antibodies can be generated as described, for example, in U.S. Patent No. 7,521,541.

[0287] e) Antibody derivatives

[0288] In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous modules known in the art and readily available. Modules suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Due to its stability in water, polyethylene glycol propionaldehyde may have advantages in production. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically for a given condition, etc.

[0289] In some specific embodiments, the anti-IL-1β antibody comprises a VHH, and the VHH consists of 1) the amino acid sequence of SEQ ID NO: 1; or 2) the amino acid sequence of SEQ ID NO: 5; or 3) the amino acid sequence of SEQ ID NO: 9; or 4) the amino acid sequence of SEQ ID NO: 11; or 5) the amino acid sequence of SEQ ID NO: 14; or 6) the amino acid sequence of SEQ ID NO: 17; or 7) the amino acid sequence of SEQ ID NO: 21; or 8) the amino acid sequence of SEQ ID NO: 24; or 9) the amino acid sequence of SEQ ID NO: 26; or 10) the amino acid sequence of SEQ ID NO: 27; or 11) the amino acid sequence of SEQ ID NO: 28. In one embodiment, the anti-IL-1β antibody is composed of a VHH consisting of 1) the amino acid sequence of SEQ ID NO: 1; or 2) the amino acid sequence of SEQ ID NO: 5; or 3) the amino acid sequence of SEQ ID NO: 9; or 4) the amino acid sequence of SEQ ID NO: 11; or 5) the amino acid sequence of SEQ ID NO: 14; or 6) the amino acid sequence of SEQ ID NO: 17; or 7) the amino acid sequence of SEQ ID NO: 21; or 8) the amino acid sequence of SEQ ID NO: 24; or 9) the amino acid sequence of SEQ ID NO: 26; or 10) the amino acid sequence of SEQ ID NO: 27; or 11) the amino acid sequence of SEQ ID NO: 28.

[0290] In other specific embodiments, the anti-IL-1β antibody is a heavy chain antibody comprising a VHH fused to the Fc domain of human IgG1 or IgG4. In one embodiment, the anti-IL-1β antibody is a heavy chain antibody consisting of a VHH and the Fc domain of human IgG1, and the antibody comprises:

[0291] 1) the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:31;

[0292] 2) the amino acid sequence of SEQ ID NO:32, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:32;

[0293] 3) the amino acid sequence of SEQ ID NO:33, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:33;

[0294] 4) the amino acid sequence of SEQ ID NO:34, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:34;

[0295] 5) the amino acid sequence of SEQ ID NO:35, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:35;

[0296] 6) the amino acid sequence of SEQ ID NO:36, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:36;

[0297] 7) the amino acid sequence of SEQ ID NO:37, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:37;

[0298] 8) the amino acid sequence of SEQ ID NO:38, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:38;

[0299] 9) the amino acid sequence of SEQ ID NO:39, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:39;

[0300] 10) the amino acid sequence of SEQ ID NO:40, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:40; or

[0301] 11) the amino acid sequence of SEQ ID NO:41, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:41.

[0302] In one embodiment, the anti-IL-1β antibody is a heavy chain antibody composed of a VHH and the Fc domain of human IgG1, and the antibody consists of 1) the amino acid sequence shown in SEQ ID NO:31; or 2) the amino acid sequence shown in SEQ ID NO:32; or 3) the amino acid sequence shown in SEQ ID NO:33; or 4) the amino acid sequence shown in SEQ ID NO:34; or 5) the amino acid sequence shown in SEQ ID NO:35; or 6) the amino acid sequence shown in SEQ ID NO:36; or 7) the amino acid sequence shown in SEQ ID NO:37; or 8) the amino acid sequence shown in SEQ ID NO:38; or 9) the amino acid sequence shown in SEQ ID NO:39; or 10) the amino acid sequence shown in SEQ ID NO:40; or 11) the amino acid sequence shown in SEQ ID NO:41.

[0303] In some specific embodiments, the IL-1β / PD-L1 bispecific antibody consists of 1) a heavy chain, such as the amino acid sequence shown in SEQ ID NO: 46; and 2) a light chain, such as the amino acid sequence shown in SEQ ID NO: 43.

[0304] Nucleic acid molecules encoding the antibodies of the present invention

[0305] In some aspects, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a VHH as disclosed herein.

[0306] The nucleic acids of the present invention can be obtained using standard molecular biology techniques. For antibodies obtained from an immunoglobulin gene library (eg, using phage display technology), nucleic acids encoding such antibodies can be recovered from the gene library.

[0307] Exemplary nucleic acid molecules of the present invention are: 1) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 1; 2) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 5; 3) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 9; 4) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 11; 5) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 14; 6) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 17; 7) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 21; 8) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 24; 9) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 26; 10) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 27; 11) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 28; 12) a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 43 and a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 46; or preferably, the nucleic acids 1)-11) further comprise a nucleotide coding sequence of the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the nucleic acid has at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleotide coding sequence of the above amino acid sequence. In some embodiments, the percent identity is due to the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0308] carrier

[0309] Recombinant techniques known in the art can be used to insert nucleic acid molecules encoding anti-IL-1β antibodies into vectors for further cloning (amplification of DNA) or for expression. In another embodiment, antibodies can be produced by homologous recombination known in the art. DNA encoding monoclonal antibodies is easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the gene encoding the heavy chain of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically, selectable marker genes confer resistance to drugs (e.g., G418, hygromycin, or methotrexate) to host cells into which the vector has been introduced. Selectable marker genes can include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0310] In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc., and includes plasmids, such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., and other laboratory and commercially available vectors. Suitable vectors may include plasmids or viral vectors (such as replication-defective retroviruses, adenoviruses, and adeno-associated viruses). In one embodiment of the invention, the vector may be pET, such as pETbac containing a hexahistidine tag and a c-Myc-tag gene.

[0311] host cells

[0312] The vector comprising the nucleic acid sequence encoding the IL-1 β binding molecules can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vector herein are prokaryotes, yeast or higher eukaryotic cells. Suitable prokaryotes for this purpose include true bacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae such as Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella such as Salmonella typhimurium, Serratia such as Serratia marcescens, and Shigella, and Bacillus such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas such as Pseudomonas aeruginosa and Streptomyces.

[0313] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-IL-1β antibody encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used lower eukaryotic host microorganism. However, many other genera, species, and strains are generally available and can be used in the present invention, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (ATCC 1447); pastoris) (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[0314] Other suitable host cells for expressing the anti-IL-1β antibodies provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A large number of baculovirus strains and variants and corresponding permissive insect host cells have been identified from the following hosts: Spodoptera Frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and according to the present invention, these viruses can be used as viruses herein, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0315] Host cells are transformed with the above-described expression or cloning vectors for anti-IL-1β antibody production and cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify genes encoding the desired sequences.

[0316] Host cells for producing the anti-IL-1β antibodies provided herein can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any culture medium described in Ham et al., Meth.Enz.58:44 (1979); Barnes et al., Anal.Biochem.102:255 (1980); U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or US Pat. Re. 30,985 can be used as a culture medium for host cells. If necessary, any of these culture media may be supplemented with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin (GENTAMYCIN™) drug), trace elements (defined as inorganic compounds, typically present at a final concentration in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those used with the host cell previously selected for expression and will be apparent to those of ordinary skill in the art.

[0317] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris (host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describe a method for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, a cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) over approximately 30 minutes. Cell debris can be removed by centrifugation. In the case where the antibody is secreted into the culture medium, the supernatant from such an expression system is typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, may be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0318] Antibodies produced from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.

[0319] Following any one or more preliminary purification steps, the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5-4.5, preferably at low salt concentration (e.g., about 0-0.25 M salt).

[0320] Pharmaceutical composition

[0321] In some aspects, the present invention relates to a pharmaceutical composition comprising at least one anti-IL-1β antibody as disclosed herein and a pharmaceutically acceptable carrier.

[0322] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-IL-1β antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, combinations of various components known in the art, or more.

[0323] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene, and / or propyl arsenate. As disclosed herein, the compositions comprising antibodies of the present invention include one or more antioxidants, such as methionine, to reduce oxidation of the antibody. Oxidation reduction can prevent or reduce the reduction in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the present invention provides compositions comprising one or more antibodies and one or more antioxidants, such as methionine. The present invention further provides various methods, wherein antibodies are mixed with one or more antioxidants, such as methionine, to prevent the antibodies from being oxidized, to extend their shelf life and / or increase activity.

[0324] To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous carriers such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous carriers such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, bacteriostatic or antimicrobial agents at fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. The antimicrobial agent used as a carrier can be added to a pharmaceutical composition in a multidose container containing phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0325] Administration, formulation and dosage

[0326] The pharmaceutical compositions of the present invention can be administered to a subject in need thereof in vivo via various routes, including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardial, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or by implantation or inhalation. The compositions of the present invention can be formulated into solid, semisolid, liquid, or gaseous formulations, including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. Suitable formulations and routes of administration can be selected based on the intended application and treatment regimen.

[0327] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalants and controlled release forms thereof.

[0328] Preparations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended therein, or otherwise provided (e.g., in liposomes or other microparticles). These liquids can additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the preparation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohol, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such preparations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosage regimens (i.e., dosage, time, and repetition) will depend on specific individual and individual medical history and empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0329] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferating or tumorigenic cells, maintaining the reduction of such tumor cells, reducing the proliferation of tumor cells or delaying the development of metastases. In some embodiments, the dosage administered can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a sustained continuous release formulation of the therapeutic composition of the present invention may be suitable.

[0330] Those skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dosage generally involves a balance between the level of therapeutic benefit and any risks or adverse side effects. The dosage level selected will depend on a variety of factors, including but not limited to the activity of the specific compound, the route of administration, the time of administration, the rate of compound clearance, the duration of treatment, other drugs, compounds and / or materials used in combination, the severity of the disease, and the species, sex, age, weight, disease, general health and previous medical history of the patient. The amount of the compound and the route of administration are ultimately determined by the physician, veterinarian or clinician, but the dosage is generally selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects.

[0331] Typically, IL-1β binding molecules can be administered in various ranges. In some embodiments, the IL-1β binding molecules provided herein can be administered in a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In some of these embodiments, the antibody is administered at a dose of about 50 mg / kg or less, and in some of these embodiments, the dose is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In some embodiments, the dosage can be changed during treatment. For example, in some embodiments, the initial dosage can be higher than the subsequent dosage. In some embodiments, the dosage can be changed during treatment, depending on the subject's response.

[0332] In any case, the antibodies of the present invention are preferably administered to subjects in need thereof as needed. The frequency of administration can be determined by one skilled in the art, such as the attending physician based on the disease being treated, the age of the subject being treated, the severity of the disease being treated, the general health of the subject being treated, and the like.

[0333] In certain preferred embodiments, a course of treatment involving an antibody of the invention will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibody of the invention may be administered daily, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks, or every three months. In this regard, it will be appreciated that the dosage may be varied or the time intervals adjusted based on patient response and clinical practice.

[0334] The dosage and regimen of the disclosed therapeutic compositions may also be determined empirically in individuals given one or more administrations. For example, an individual may be given incremental doses of a therapeutic composition produced as described herein. In selected embodiments, the dosage may be gradually increased or decreased or mitigated, respectively, based on empirically determined or observed side effects or toxicities. In order to assess the efficacy of the selected composition, markers of a specific disease, condition, or disease may be tracked as described above. For cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; improvement assessed by direct tumor biopsy and microscopic examination of tumor specimens; measurement of indirect tumor markers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein, relief of pain or paralysis; improvement in speech, vision, breathing, or other disabilities associated with the tumor; increased appetite; or improvement in quality of life or prolonged survival as measured by accepted tests. One skilled in the art will appreciate that the dosage will vary depending on the individual, the type of tumor disease, the stage of the tumor disease, whether the tumor disease has begun to metastasize to other locations in the individual, and past and concurrent treatments.

[0335] Compatible formulations for parenteral administration (e.g., intravenous injection) may include an IL-1β binding molecule as provided herein at a concentration of about 10 μg / ml to about 100 mg / ml. In some embodiments, the concentration of the IL-1β binding molecule may include 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, or 1 mg / ml. In other preferred embodiments, the concentration of the IL-1 β binding molecule will comprise 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml or 100 mg / ml.

[0336] Application of the present invention

[0337] The IL-1β binding molecules of the present invention have many in vitro and in vivo uses. For example, these molecules can be administered to cultured cells in vitro or ex vivo, or, for example, to human subjects in vivo, to treat or prevent diseases associated with excessive IL-1β in the subject, or to inhibit or block the binding of IL-1β to IL-1R1 in the subject, or to downregulate human IL-1β activity or levels, or to inhibit the activation of downstream signaling pathways mediated by IL-1β and IL-1R1.

[0338] Preferred subjects include human patients suffering from diseases associated with excessive IL-1β. The method is particularly suitable for treating human patients suffering from diseases associated with excessive IL-1β by blocking the binding of IL-1β to IL-1R1, thereby inhibiting the activation of downstream signaling pathways mediated by IL-1β and IL-1R1. In a specific embodiment, the method is particularly suitable for the in vivo treatment of inflammatory diseases, cardiovascular diseases, or cancer. When the anti-IL-1β antibody is administered with another agent, the two agents can be administered in any order or simultaneously.

[0339] The present invention further provides a method for detecting the presence of an IL-1β antigen in a sample or measuring the amount of a human IL-1β antigen, comprising contacting a sample and a control sample with an IL-1β binding molecule under conditions that allow formation of a complex between the IL-1β binding molecule and the IL-1β. Complex formation is then detected, wherein differential complex formation between the sample and the control sample indicates the presence of the IL-1β antigen in the sample. Furthermore, the IL-1β binding molecules of the present invention can be used to purify human IL-1β by immunoaffinity purification.

[0340] Treating inflammatory diseases

[0341] Diseases associated with excessive IL-1β can be inflammatory diseases. IL-1β regulates the recruitment and activation of effector cells involved in innate and adaptive immunity and is therefore involved in a wide range of inflammatory diseases.

[0342] In some embodiments, inflammatory diseases associated with IL-1β include, but are not limited to, Muir-Wei syndrome (MWS), cryopyrin-associated periodic syndrome (CAPS), neonatal-onset multisystem inflammatory syndrome (NOMIS), rheumatoid arthritis, systemic-onset juvenile idiopathic arthritis (soJIA), gouty arthritis, multiple sclerosis, periodic fever syndromes, chronic obstructive pulmonary disease (COPD), type 1 diabetes, type 2 diabetes, familial cold autoinflammatory syndrome (FCAS), and eye diseases such as age-related macular degeneration.

[0343] Treating cardiovascular disease

[0344] Diseases associated with excessive IL-1β can be cardiovascular diseases. Research over the past two decades has emphasized that inflammatory processes are a key component of the pathogenesis of CVD, particularly atherosclerotic cardiovascular disease (ASCVD). Epidemiological data from the mid-1990s showed that inflammation, as determined by high-sensitivity C-reactive protein (hsCRP) or interleukin-6 (IL-6), was strongly associated with future major adverse cardiovascular events (MACE) in both primary and secondary prevention settings, independent of traditional risk factors (Ridker et al. (2018) J. Am. Coll. Cardiol. 72: 3320-3331). Recently, it has also been discovered that multiple factors known to be associated with atherosclerosis can activate the NLRP3 inflammasome, and that activated NLRP3 inflammasomes can mediate IL-1β activation, thereby promoting inflammation. These factors include cholesterol crystals, atherogenic oscillatory flow, hypoxia, and neutrophil extracellular traps, which support a key role for the NLRP3 inflammasome-IL1β pathway in atherogenesis (Ridker (2016) Circ. Res. 118:145-156).

[0345] In some embodiments, cardiovascular diseases associated with IL-1β include, but are not limited to, atherosclerotic cardiovascular disease (ASCVD), arterial thrombosis, stroke and heart failure, angina pectoris, myocardial infarction (MI, commonly known as heart attack), and arrhythmia.

[0346] Treating cancer

[0347] Diseases associated with excessive IL-1β can be cancer, particularly those with an inflammatory basis. Many malignancies arise in areas of chronic inflammation, and inadequate resolution of inflammation can play a major role in tumor invasion, progression, and metastasis (Grivennikov et al. (2010) Cell 140:883-899). IL-1 has been shown to promote tumor growth and metastasis in transplanted mice and human tumors, including sarcomas, melanomas, pancreatic ductal adenocarcinomas, myelomas, and breast cancer. Most importantly, IL-1β and its receptors have been shown to be important drivers of primary carcinogenesis and metastasis in mesenchymal and epithelial cells. In the TME, IL-1 contributes to the establishment of a proinflammatory environment by inducing proinflammatory cytokines and chemokines. Myeloid cells are generally considered to be the primary source of IL-1β within tumors. In pancreatic cancer models, IL-1β was found to be an important driver of the desmoplastic response. Blocking IL-1β can enhance the antitumor activity of anti-PD-1 immunotherapy and is associated with CD8 T cell infiltration. IL-1Ra and anti-IL-1 mAbs have been shown to inhibit primary tumor growth and metastasis. The potential for synergy between IL-1 blockade and immune checkpoint inhibitors has been investigated in various models. For example, in triple-negative breast cancer, treatment with an anti-IL-1β mAb promoted the development of adaptive anti-tumor cell immunity and significantly enhanced the anti-tumor effect of anti-PD-1. Emerging evidence provides a rationale for combining anti-IL-1 strategies with checkpoint blockade immunotherapy.

[0348] In some embodiments, cancers associated with IL-1β include, but are not limited to, lung cancer, such as non-small cell lung cancer (NSCLC); breast cancer, such as triple-negative breast cancer (TNBC); prostate cancer, such as metastatic prostate cancer; blood cancers, such as leukemias, lymphomas, myelomas, such as low- or intermediate-risk myelodysplastic leukemias; gastric cancer, including esophageal cancer; ovarian cancer, kidney cancer, liver cancer, such as hepatocellular carcinoma (HCC); skin cancer, such as melanoma; head and neck cancer; brain cancer; colorectal cancer; bladder cancer; pancreatic cancer; and kidney cancer, such as localized renal cancer.

[0349] Combination with immune checkpoint inhibitors

[0350] The antibodies can be used in combination with immune checkpoint inhibitors. Immune checkpoint inhibition broadly refers to the inhibition of cancer cells to produce checkpoints that hinder or downregulate the immune response. Two known immune checkpoint pathways involve signaling via cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of common signaling molecules that play an important role in all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273) are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the dominant ligand, while PD-L2 has a much more restricted expression pattern. When the ligand binds to PD-1, an inhibitory signal is transmitted to the T cell, which reduces cytokine production and curbs T cell proliferation. Immune checkpoint inhibitors include but are not limited to aptamers and antibodies that block PD-1\PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H3, OX40, and 4-1BB.

[0351] Used in combination with chemotherapy

[0352] The antibodies can be used in combination with chemotherapy or radiotherapy.Antibodies can be used in combination with anticancer agents, cytotoxic agents or chemotherapeutic agents.

[0353] The term "anticancer agent" or "antiproliferative agent" means any agent that can be used to treat cellular acute leukemias such as cancer, and includes, but is not limited to: cytotoxic agents, cytostatics, anti-angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents and immunotherapeutic agents. It should be understood that in selected embodiments as described above, such anticancer agents may comprise conjugates and may be combined with the disclosed site-specific antibodies prior to administration. More specifically, in some embodiments, the selected anticancer agent is linked to the unpaired cysteine ​​of the engineered antibody to provide an immunoconjugate as described herein. Therefore, such immunoconjugates are explicitly included within the scope of the present invention. In other embodiments, the disclosed anticancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.

[0354] As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell destruction. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxins and exotoxins, Staphylococcal enterotoxin A), fungi (e.g., α-sarcin, restrictocin), plants (abrin, ricin, modeccin, viscumin, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, hordeotoxin, Aleurites fordii proteins, dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotonin, phytoncidin inhibitor, gelonin, mitegellin, restrictocin, phenomycin, neomycin, and trichothecenes), or animals (e.g., cytotoxic RNases, such as extracellular pancreatic RNase; DNase I, including fragments and / or variants thereof).

[0355] For purposes of the present invention, "chemotherapeutic agents" include chemical compounds (e.g., cytotoxic agents or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. These chemicals are typically directed against the intracellular processes required for cell growth or division and are therefore particularly effective for cancer cells that typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. Typically, chemotherapeutic agents can include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become cancerous or produce tumorigenic offspring (e.g., TICs). These agents are typically used in combination and are typically the most effective, for example, in regimens such as CHOP or FOLFIRI.

[0356] Examples of anticancer agents that can be used in combination with the site-specific antibodies of the present invention (either as site-specific conjugates or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethyleneimine and methylmelamine, acetogenins, camptothecins, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, hyoscyamine, sarcodictyin, spongistatin, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicins, chromophores of enediyne antibiotics, aclacinomycins, actinomycetes, oxazolidinone, anthramycin, azaserine, bleomycin, actinomycin C, carabicin, carminomycin, carmomycin, chromomycins, dactinomycin, daunorubicin, detopicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycin, mycophenolic acid, nogamycin, olivomycin, peplomycin, potfiromycin, puromycin, triferon-adriamycin, rhodorubicin, streptozocin, streptozotocin, tuberculin, ubenimex, zoloft, daunorubicin; anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, anti-adrenergics, folic acid supplements such as furinic acid acid), aceglucuronolactone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatrexate, defofamine, colcemid, diacrazone, elfornithine, elliptonium acetate, apocillon, etoglucagon, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids, mitoguanidine, mitoxantrone, mopidanmol, nitraerine, pentostatin, methamidine, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazine, procarbazine, polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane; rhizoxin; sizolan; spirogermanamine; tenuzolic acid; triimidoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, baculosporin A, and serpentin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman;Gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine, vinorelbine; norsulfuron; teniposide; edatrexate; daunorubicin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; inhibitors of PKC-α, Raf, H-Ras, EGFR and IL-1β (which reduce cell proliferation), and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included within this definition are anti-hormonal agents used to modulate or inhibit hormonal effects on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the enzyme aromatase that regulates estrogen production in the adrenal glands, and anti-androgens; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as IL-1β expression inhibitors and HER2 expression inhibitors; vaccines, rIL-2; topoisomerase 1 inhibitors; rmRH; vinorelbine and esperamicin, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0357] Used in combination with radiation therapy

[0358] The present invention also provides a combination of antibodies and radiotherapy (i.e., any mechanism for locally inducing DNA damage in tumor cells, such as gamma irradiation, X-rays, UV-irradiation, microwaves, electron emission, etc.). Combination therapies using directed delivery of radioisotopes to tumor cells are also contemplated, and the disclosed immunoconjugates can be used in combination with targeted anticancer agents or other targeted means. Typically, radiotherapy is administered in a pulsed manner over a period of about 1 week to about 2 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple sequential doses.

[0359] diagnosis

[0360] The present invention provides in vitro and in vivo methods for detecting, diagnosing, or monitoring diseases associated with excessive IL-1β. Such methods include identifying individuals with diseases associated with excessive IL-1β for treatment or monitoring the progression of diseases associated with excessive IL-1β, comprising contacting a patient or a sample obtained from a patient (in vivo or in vitro) with an IL-1β antibody as described herein, and detecting the presence or absence or level of binding of the antibody to a bound or free target molecule in the sample. In some embodiments, the antibody will comprise a detectable label or reporter molecule as described herein.

[0361] Samples can be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblotting assays, Western blot analysis, and flow cytometry assays. Compatible in vivo diagnostics or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scans), positron emission tomography (e.g., PET scans), radiography, ultrasound, and the like, known to those skilled in the art.

[0362] Reagent test kit

[0363] The present invention also provides a kit for treating or diagnosing a disease associated with excessive IL-1β, or detecting IL-1β or IL-1β-expressing cells or tissues in a sample. The kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed from a variety of materials, such as glass or plastic, and contains a pharmaceutically effective amount of the disclosed antibodies and variants or derivatives thereof. In other preferred embodiments, the container includes a sterile access port (for example, the container can be an intravenous fluid bag or a vial with a stopper pierceable by a hypodermic needle). Such a kit typically contains a pharmaceutically acceptable formulation of the disclosed antibody in a suitable container and, optionally, one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnosis or combination therapy. For example, in addition to the antibodies of the present invention, such a kit may contain any one or more anticancer agents, such as chemotherapeutic agents or radiotherapeutic agents; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents.

[0364] More specifically, the kit can have a single container containing the disclosed antibodies, which may or may not contain additional components, or they may have different containers for each required reagent. In the case of providing a combined therapeutic agent for combination, a single solution can be premixed in a molar equivalent combination or in a manner such that one component is more than another. Alternatively, the antibody and any optional anticancer agent of the kit can be stored separately in different containers before being administered to a patient. The kit can also include a second / third container device for holding a sterile, pharmaceutically acceptable buffer or other diluent such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0365] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit may be provided as a dry powder. When the reagents or components are provided in dry powder form, the powder may be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in a separate container.

[0366] As briefly described above, the kit may also contain a means for administering the antibody and any optional components to a patient, such as one or more needles, intravenous (IV) bags or syringes, or even eye drops, pipettes or other similar devices, by which the formulation can be injected or introduced into an animal or applied to an affected area of ​​the body. The kits of the invention will typically also include a device for containing vials or the like and other tightly closed components for commercial sale, such as an injection-molded or blow-molded plastic container in which the desired vials and other devices are placed and retained.

[0367] Sequence Listing Overview

[0368] This application is accompanied by a sequence listing comprising a number of nucleic acid and amino acid sequences. Table B below provides an overview of the sequences included.

[0369] Table B: Sequence overview

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376] Example

[0377] The invention described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. These examples are not intended to represent that the experiments below are all or the only experiments performed.

[0378] Example 1 Preparation and screening of single-domain antibodies targeting IL-1β

[0379] 1.1 Alpaca Immunity

[0380] Two alpacas (Alpaca) were immunized subcutaneously with human IL-1β protein (purchased from Sino Biological, 10139-HNAE) at a dose of 0.5 mg per animal. Complete adjuvant was used for the initial immunization, followed by incomplete adjuvant. Each immunization was repeated 2 weeks apart for a total of eight immunizations.

[0381] 1.2 Serum immune titer determination

[0382] Starting from the second immunization, 5 mL of blood was collected one week after each immunization for serum titer determination. The IL-1β antigen protein within 1.1 was coated onto an ELISA plate, 200 ng per well. After blocking and washing, serum samples were serially diluted starting at a 1:2000 ratio. After incubation and washing, color was developed using a secondary antibody against camel single-domain antibody (GenScript, A01861).

[0383] 1.3 Phage library construction

[0384] 10 mL of blood was collected from each camel after the sixth and eighth immunizations, and the blood was combined to construct a phage display library for single-domain antibodies: RNA extraction was first performed, and lymphocytes were isolated using lymphocyte separation solution (Solerbo, P8610). The lymphocytes were treated and lysed using RNA extraction reagent (TaKaRa, 9109), impurities were removed using chloroform, and RNA was precipitated using isopropanol. The precipitate was washed with 75% ethanol, dried at room temperature, and dissolved in water for injection; the RNA was then reverse transcribed using a reverse transcription kit (ThermoScientific, K1622) to prepare cDNA; the DNA containing the single-domain antibody variable region gene was then amplified using the nested PCR method, digested with SfiI, and then ligated with the display vector digested with SfiI using T4 DNA ligase; the ligation product was purified and electroporated into TG1 competent cells, and M13KO7 was used to help phage infection to prepare the phage display library. The effective storage capacity of the final constructed libraries (HYB1-6,8 and HYB2-6,8) were 1.3E+09 and 2.3E+09 cfu, respectively.

[0385] 1.4 Panning of phage libraries

[0386] The antigen (IL-1β-huFc, expressed and purified in the laboratory) was coated on a high-adsorption 96-well ELISA plate at 500 ng per well. 5E+11 and 1E+12 cfu of phage from the library described in 1.3 above were added, respectively. The plates were incubated with shaking at room temperature for binding. The plates were washed eight times with PBST containing 0.1% Tween-20 and then eluted. The titer of the eluted phage was determined, and wells coated with unrelated target proteins were used as parallel controls.

[0387] The results are shown in Table 1. After one round of panning, the eluted phages from the IL-1β-Fc wells were enriched compared to the control wells, with enrichment factors of 11.8 times and 19.8 times, respectively.

[0388] Table 1: Phage library panning data

[0389]

[0390] 1.5 Phage-based screening of single clones

[0391] Use the enriched phage library eluted from 1.4 to infect logarithmic phase TG1 bacteria, spread on plates after dilution, and then pick a single clone and inoculate it into 2YT medium containing Carb and M13KO7 helper phage for overnight culture. On the second day, the culture plates were centrifuged at 4200 rpm, and the supernatant was collected for phage ELISA. Wells coated with IL-1β protein (Beijing Sino Biological, 10139-HNAE) served as assay wells, and wells coated with an unrelated target protein served as control wells. HRP-conjugated anti-M13 antibody (Sino Biological, 11973-MM05T-H) was used as a secondary antibody. Clones with an OD450 nm reading (OD450 nm) above 1.0 in the assay wells and below 0.1 in the control wells were selected for plasmid extraction and gene sequencing. The sequences obtained were translated into amino acid sequences and aligned. Duplicate sequences and CDR3 (CDR region demarcation using the AbM method) sequences with a difference of less than 2 amino acids were removed to identify unique sequences. A total of 62 unique sequences were obtained.

[0392] Example 2 Blocking function assay based on periplasmic extracts of single-domain antibodies

[0393] The unique sequence plasmid was transformed into BL21 Rosetta (DE3) competent bacteria and inoculated into 5 mL of 2×YT medium containing Carb antibiotics. Culture was continued until the OD600 reached 0.4. Then, 2×YT medium containing IPTG and Carb was added to a final IPTG concentration of 0.1 mM. The cells were incubated overnight at 28°C. The next day, the cells were centrifuged at 4200 rpm and 4°C for 20 minutes, resuspended in 1 mL of PBS, and subjected to three freeze-thaw cycles from -80°C to 37°C to release the periplasmic single-domain antibody. The supernatant was then centrifuged at 10,000 rpm and 4°C for 15 minutes to prepare a crude extract of the periplasmic single-domain antibody.

[0394] Then, a single-domain antibody crude extract was used to perform blocking ELISA tests on human IL-1RI and IL-1β. Human IL-1RI-Fc (Biopsy, ILI-H5253) was coated on an ELISA plate at 4°C overnight, 300 ng per well. The next day, the plate was blocked with 1% BSA (bovine serum albumin) at room temperature for 2 hours. The crude extract was mixed with equal volumes of 0.018 μg / mL IL-1β-Fc-biotin (expressed in the laboratory and chemically labeled with biotin). The crude extract was diluted 1:2 starting from the original solution. The crude extract was incubated with IL-1β-Fc-biotin at room temperature for 30 minutes at a 1× stock solution and a 1:2 ratio. The extract was then transferred to a blocked plate coated with IL-1RI-Fc and incubated at room temperature. The plate was then washed and developed with streptavidin-HRP (Shanghai Bioengineering, D111054) at a 1:3000 ratio. The canakinumab biosimilar was used as a positive control (initial concentration of 100 nM), and a crude extract of a single-domain antibody targeting an unrelated target was used as a negative control. Absorbance was read at 450 nm using a microplate reader (Biotek, Synergy H1MF).

[0395] The results are as follows Figure 1 As shown, among the 62 clones, 7 clones (5, 22, 32, 34, 35, 40, and 41) showed significant blocking activity against IL-1β. The crude extracts of the 62 clones were also tested for binding to coat-bound IL-1β on the plate, and it was found that most of them showed significant binding, while control wells containing coat-independent target proteins showed no binding (data not shown).

[0396] Example 3 Expression of IgG Antibodies and Determination of Blocking Activity

[0397] The sequences corresponding to clones 5, 22, 32, 34, 35, 40, and 41 were constructed onto human IgG1 Fc (containing the N297A mutation) and transiently transfected and expressed in HEK293 cells. Purification was performed, and the expressed molecules were named HYB0901, HYB0902, HYB0903, HYB0904, HYB0905, HYB0906, and HYB0907 (SEQ ID NO: 31 to SEQ ID NO: 37). These molecules were then used to perform blocking assays for IL-1RI and IL-1β according to the method in Example 2, using canakinumab biosimilars as a positive control and an isotype single-domain antibody targeting an unrelated target as a negative control.

[0398] The results are as follows Figure 2 As shown in the figure, each molecule has a clear blocking effect, among which HYB0902 has the best blocking effect, and its EC 50 The value was 0.063 μg / mL, which is similar to the 0.067 μg / mL of the reference molecule canakinumab.

[0399] Example 4 Inhibitory effect of antibodies on IL-1β-induced IL-6 release from MRC-5 cells

[0400] MRC-5 cells (human embryonic lung fibroblasts) were plated at 3000 cells / well in a 96-well cell culture plate and incubated in a 37°C incubator overnight. IgG samples such as HYB0901 to HYB0907 and IL-1β purified from human HEK293 cells were added at a final concentration of 4 ng / mL. The cells were cultured in a 37°C, 5% CO2 incubator for 18 h, and the IL-6 secretion level in the supernatant culture medium was then detected using an IL-6 quantification kit (Biosharp, BSEH-009-96T).

[0401] The results are as follows Figure 3 As shown in the figure, it can be seen that HYB0901 to HYB0907 have a certain blocking effect on IL-1β-induced IL-6 release in MRC-5 cells, among which HYB0902 has the best effect. 50 It is 0.188μg / mL, which is similar to the 0.224μg / mL of the control molecule canakinumab.

[0402] Example 5: Modification of post-translational modification sites of antibodies

[0403] Sequence analysis of the antibody HYB0902 revealed a potential isomerization site "DG" within its CDR2. This site was mutated to "EG" and the newly designed IgG molecule was named HYB0902-2 (SEQ ID NO: 38). Similarly, the IgG was transiently transfected into HEK293 cells and expressed and purified. The purified IgG was tested for its inhibitory activity against IL-1β-induced IL-6 release from MRC-5 cells as described in Example 4.

[0404] The results are as follows Figure 4 As shown, it can be seen that the engineered molecule HYB0902-2 has the same cellular blocking efficacy as the parent molecule HYB0902.

[0405] Example 6 Humanization and activity determination of antibodies

[0406] The antibody HYB0902-2 was structurally simulated using SWISS-MODEL. Based on the obtained structure, the camel-derived amino acid sites in the framework region were evaluated for humanization mutations. The human germline gene V 3-23 *01 and J1*01 were selected as target sequences for humanization mutations. Mutations were prioritized at amino acid sites located on the surface of the antibody structure and not adjacent to the CDRs, with subsequent mutations located internally within the antibody structure and adjacent to the CDRs. Based on these principles, several humanized antibody sequences were designed: HYB0902-2-hz1 to HYB0902-2-hz4 (sequences shown in SEQ ID NOs:39 to 42). These sequences were then transiently expressed in HEK293 cells. Following expression, the blocking efficacy of each variant was tested at the cellular level using the methods described in Example 4.

[0407] The results are as follows Figure 5 As shown, the humanized variant molecule HYB0902-2-hz3 has the best blocking effect, and its EC 50 The value was 0.277 μg / mL, which was superior to the parent molecule HYB0902-2 (EC 50 =0.516 μg / mL). HYB0902-2-hz4 lost its blocking effect, indicating that some camel-derived amino acid sites cannot be mutated.

[0408] Example 7 Activity Verification of Preferred Antibody Molecule

[0409] To further verify the activity level of the preferred molecule HYB0902-2-hz3, the blocking activity of IL-1β was tested using MRC-5 cells and NK-92 cells with canakinumab monoclonal antibody as a positive control.

[0410] The activity detection method based on MRC-5 cells is described in Example 4. The results are as follows Figure 6 As shown in the figure, it can be seen that HYB0902-2-hz3 (or HYB009) has a better blocking effect than canakinumab monoclonal antibody. Their EC 50 The values ​​were 0.019 and 0.036 μg / mL, respectively.

[0411] The activity detection method based on NK-92 cells is as follows:

[0412] NK-92 cells were seeded into 96-well cell culture plates at 10,000 cells / well, and gradiently diluted IgG samples, IL-1β expressed and purified by human HEK293 cells at a final concentration of 4 pM, and IL-2 (R&D, BT-002-AFL) at a final concentration of 100 U / mL were added. The cells were cultured in a 37°C, 5% CO2 incubator for 48 h, and the culture plates were centrifuged to obtain the supernatant. The IFN-γ secretion level in the supernatant was detected using a human IFN-γ quantification kit (R&D, SIF50C).

[0413] The results are as follows Figure 7 As shown in the figure, it can be seen that HYB0902-2-hz3 (or HYB009) has a blocking effect that is no less than that of canakinumab monoclonal antibody. Their EC 50 The values ​​were 0.072 and 0.076 μg / mL, respectively.

[0414] Example 8 In vivo pharmacodynamic study of HYB009

[0415] C57BL / 6 mice were intraperitoneally injected with an isotype control antibody (10 μg / kg), various concentrations of HYB009, or canakinumab (1 or 3 μg / kg), with three animals per group. Four hours later, each mouse was subcutaneously injected with 1 μg / kg of human IL-1β (purified from human HEK293 cells). Two hours later, blood was collected for serum preparation, and serum mIL-6 levels were measured using a mouse IL-6 ELISA kit (R&D, SM6000B). The serum mIL-6 levels in the isotype control antibody group were set as 100%, and the inhibition rate (%) of mIL-6 expression by the test antibodies was calculated.

[0416] The results are as follows Figure 8 As shown in the results, it can be seen that HYB009 has an in vivo inhibitory effect on IL-1β comparable to that of canakinumab.

[0417] Example 9 Study on the synergistic anti-tumor effect of HYB009 and anti-PD-L1 antibodies

[0418] To construct a bispecific antibody targeting PD-L1 and IL-1β to investigate the synergistic effects of targeting PD-L1 and IL-1β in anti-tumor responses, the HYB0902-2-hz3 VHH construct was inserted into the C-terminus of the Fc region of the marketed anti-PD-L1 antibody atezolizumab. The light and heavy chain sequences of the constructed bispecific antibody are shown in SEQ ID NO:43 and SEQ ID NO:46, respectively. The antibody was transiently transfected into HEK293 cells and expressed and purified.

[0419] B-hIL1b mice were inoculated with CT-26 colon cancer cells until the tumors grew to an average of approximately 60 mm. 3 At around 40 days, mice were randomly divided into 5 groups of 8 according to body weight and tumor size. They were given 10 mg / kg of isotype control antibody, 10 mg / kg of anti-PD-L1 (atezolizumab), 10 mg / kg of HYB009, 10 mg / kg of anti-PD-L1 and 10 mg / kg of HYB009 combined, and 12 mg / kg of anti-PD-L1×IL-1β bispecific antibody twice a week, and the mouse body weight and tumor volume were continuously monitored.

[0420] The results are as follows Figure 9 As shown in the figure, it can be seen that the anti-PD-L1×IL-1β bispecific antibody has the obvious best tumor inhibition effect, and the combination of anti-PD-L1 and anti-IL-1β also has a better tumor inhibition trend than anti-PD-L1 or anti-IL-1β alone. Considering the wide distribution of IL-1β in the body, the better tumor inhibition effect of the anti-PD-L1×IL-1β bispecific antibody may be derived from the targeting effect of the anti-PD-L1 part on the tumor, thereby effectively increasing the drug distribution in the tumor.

[0421] It should be understood that although the present invention has been described illustratively based on its preferred embodiments, it should not be limited to the above embodiments. For those skilled in the art, various modifications and variations of the present invention are possible. The selection and application of specific antibodies can be adjusted and modified accordingly according to specific needs. Therefore, for those skilled in the art, several simple substitutions can be made without departing from the concept and principles of the present invention, and these should all be included in the scope of protection of the present invention.

Claims

1. An interleukin-1β (IL-1β) binding molecule, wherein the IL-1β binding molecule comprises a heavy chain variable region (VH), wherein the VH comprises: 1) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; 2) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; 3) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; 4) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; 5) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; 6) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; 7) HCDR1, HCDR2, and HCDR3: comprising the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 16, respectively; or 8) HCDR1, HCDR2, and HCDR3: They comprise the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 25, and SEQ ID NO: 8, respectively.

2. The IL-1β binding molecule of claim 1, wherein the VH comprises: 1) the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1; 2) the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:5; 3) the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9; 4) the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11; 5) the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14; 6) the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17; 7) the amino acid sequence of SEQ ID NO:21, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:21; 8) the amino acid sequence of SEQ ID NO:24, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:24; 9) the amino acid sequence of SEQ ID NO:26, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26; 10) the amino acid sequence of SEQ ID NO:27, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:27; or 11) the amino acid sequence of SEQ ID NO:28, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

28. 3 . The IL-1β binding molecule according to claim 1 , further comprising one or more amino acid residue mutations while still retaining specific binding to IL-1β. The IL-1β binding molecule of claim 3 , wherein at least one of the mutations is in the VH sequence but not in any of the CDR sequences. The IL-1β binding molecule according to any one of claims 1 to 4, wherein the IL-1β binding molecule is an IL-1β antagonist, preferably an anti-IL-1β antibody. The IL-1β binding molecule according to any one of claims 1 to 5 , wherein the IL-1β binding molecule is a chimeric antibody. 7 . The IL-1β binding molecule according to claim 1 , wherein the IL-1β binding molecule is a humanized antibody. 8 . The IL-1β binding molecule according to claim 1 , wherein the IL-1β binding molecule is a heavy chain antibody or a single domain antibody (VHH). 9 . The IL-1β binding molecule according to claim 8 , wherein the VHH is derived from a camelid species comprising an alpaca or a llama.

10. The IL-1β binding molecule according to any one of claims 1 to 9, wherein the VH is fused to the Fc domain of IgG. The IL-1β binding molecule according to any one of claims 1 to 10 , wherein the VH is fused to the Fc domain of human IgG. The IL-1β binding molecule according to claim 8 , wherein the IL-1β binding molecule is a heavy chain antibody comprising the IL-1β binding molecule defined in claims 1 to 7 linked to an Fc domain derived from human IgG. The IL-1β binding molecule according to claim 12 , wherein the IL-1β binding molecule is a heavy chain antibody comprising the IL-1β binding molecule defined in claims 1 to 7 linked to an Fc domain derived from human IgG1 or IgG4. 14 . The IL-1β binding molecule according to claim 10 , wherein the Fc domain further comprises one or more amino acid mutations that reduce or eliminate effector function.

15. The IL-1β binding molecule of any one of claims 1 to 14, wherein the Fc domain is the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

30.

16. The IL-1β binding molecule according to any one of claims 1 to 15, comprising: 1) the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:31; 2) the amino acid sequence of SEQ ID NO:32, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:32; 3) the amino acid sequence of SEQ ID NO:33, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:33; 4) the amino acid sequence of SEQ ID NO:34, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:34; 5) the amino acid sequence of SEQ ID NO:35, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:35; 6) the amino acid sequence of SEQ ID NO:36, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:36; 7) the amino acid sequence of SEQ ID NO:37, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:37; 8) the amino acid sequence of SEQ ID NO:38, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:38; 9) the amino acid sequence of SEQ ID NO:39, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:39; 10) the amino acid sequence of SEQ ID NO:40, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:40; or 11) the amino acid sequence of SEQ ID NO:41, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

41. 17 . A multispecific antigen-binding molecule comprising a plurality of antigen-binding domains, wherein at least one antigen-binding domain specifically binds to IL-1β, comprising the IL-1β-binding molecule of any one of claims 1 to 16. The multispecific antigen-binding molecule of claim 17 , which is a bispecific antigen-binding molecule and comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to IL-1β, and comprises the IL-1β-binding molecule of any one of claims 1 to 16.

19. The multispecific antigen-binding molecule of claim 18, wherein the second antigen-binding domain is a full-length antibody or an antigen-binding fragment, wherein the antigen-binding fragment comprises ScFv, Fab, Fab', F(ab')2, a single domain antibody, or a heavy chain antibody.

20. The multispecific antigen-binding molecule of claim 18 or 19, wherein the first antigen-binding domain is a single-domain antibody and the second antigen-binding domain is a full-length antibody.

21. The multispecific antigen-binding molecule of claim 19 or 20, wherein the constant region of the full-length antibody comprises a light chain constant region and a heavy chain constant region, the light chain constant region is a κ chain or a λ chain constant region, and the heavy chain constant region is derived from human IgG1, IgG2, IgG3, IgG4, or a mutant thereof.

22. The multispecific antigen-binding molecule of claim 20 or 21, wherein the first antigen-binding domain is linked to the C-terminus of the Fc domain of the second antigen-binding domain directly or through a linker.

23. The multispecific antigen-binding molecule of claim 22, wherein the linker fragment is selected from (GS) n 、(GGS) n 、(GGGS) n and (GGGGS) n , n is 1, 2, 3, 4, or 5.

24. The multispecific antigen-binding molecule of any one of claims 18-23, wherein the second antigen-binding domain specifically binds to an immune checkpoint molecule, preferably specifically binds to PD-L1.

25. The multispecific antigen-binding molecule of any one of claims 18-24, wherein the second antigen-binding domain is an anti-PD-L1 antibody or an antigen-binding fragment thereof, the anti-PD-L1 antibody comprises atezolizumab, and the antigen-binding fragment comprises a single-chain Fv, Fab, Fab', F(ab')2, single-domain antibody, or heavy chain antibody of the anti-PD-L1 antibody.

26. The multispecific antigen-binding molecule of any one of claims 17-25, wherein the plurality of antigen-binding domains are each independently one, two or more.

27. The multispecific antigen-binding molecule of any one of claims 17 to 26, comprising: 1) heavy chain: amino acid sequence of SEQ ID NO:46, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:46; and 2) Light chain: an amino acid sequence of SEQ ID NO:43, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

43.

28. An immunoconjugate comprising the IL-1β binding molecule of any one of claims 1-16 or the multispecific antigen-binding molecule of any one of claims 17-27 linked to one or more conjugate moieties.

29. A nucleic acid encoding the IL-1β binding molecule of any one of claims 1-16 or the multispecific antigen-binding molecule of any one of claims 17-27.

30. The nucleic acid of claim 29, comprising: 1) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 1; 2) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 5; 3) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 9; 4) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 11; 5) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 14; 6) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 17; 7) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 21; 8) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 24; 9) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 26; 10) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 27; 11) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 28; or 12) the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 43 and the nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO: 46; Preferably, the nucleic acid 1)-11) further comprises a nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO:

30.

31. An expression vector comprising the nucleic acid of claim 29 or 30.

32. A host cell comprising the nucleic acid of claim 29 or 30 or the expression vector of claim 31.

33. A method for preparing the IL-1β binding molecule of any one of claims 1-16, or the multispecific antigen-binding molecule of any one of claims 17-27, the method comprising: 1) culturing the host cell of claim 32 under conditions suitable for expression of the IL-1β binding molecule or the multispecific antigen-binding molecule, 2) recovering the IL-1β binding molecule or the multispecific antigen-binding molecule, and (iii) optionally purifying the IL-1β binding molecule or the multispecific antigen-binding molecule.

34. A pharmaceutical composition comprising one or more of the IL-1β binding molecule of any one of claims 1-16, or the multispecific antigen-binding molecule of any one of claims 17-27, or the immunoconjugate of claim 28, or the nucleic acid of claim 29 or 30, or the expression vector of claim 31, and a pharmaceutically acceptable carrier.

35. Use of the IL-1β binding molecule of any one of claims 1-16, or the multispecific antigen binding molecule of any one of claims 17-27, or the immunoconjugate of claim 28, or the pharmaceutical composition of claim 34 in the preparation of a medicament for treating or preventing a disease associated with excessive IL-1β in a subject, or for inhibiting or blocking the binding of IL-1β to IL-1R1 in a subject, or downregulating the activity or level of IL-1β in a subject, or inhibiting the activation of downstream signaling pathways mediated by IL-1β and IL-1R1.

36. The use according to claim 35, wherein the drug is administered orally, nasally, intravenously, subcutaneously, sublingually or intramuscularly.

37. The use according to claim 35, wherein the disease is an inflammatory disease, a cardiovascular disease or cancer.

38. The use according to claim 35, wherein the drug is used in combination with an immune checkpoint inhibitor.

39. A method for treating or preventing a disease associated with excessive IL-1β in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the IL-1β binding molecule of any one of claims 1-16, or the multispecific antigen-binding molecule of any one of claims 17-27, or the immunoconjugate of claim 28, or the pharmaceutical composition of claim 34.

40. The method of claim 39, wherein the drug is administered orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.

41. The method of claim 39, wherein the disease is an inflammatory disease, a cardiovascular disease, or cancer.

42. The method of claim 39, further comprising using the IL-1β binding molecule, the multispecific antigen binding molecule, the immunoconjugate, or the pharmaceutical composition in combination with an immune checkpoint inhibitor.

43. Use of the IL-1β binding molecule of any one of claims 1-16, or the multispecific antigen-binding molecule of any one of claims 17-27, or the immunoconjugate of claim 28, or the pharmaceutical composition of claim 34 in the preparation of a reagent for diagnosing a disease associated with excessive IL-1β, or detecting IL-1β or cells or tissues expressing IL-1β in a sample.

44. The method of claim 43, wherein the disease is an inflammatory disease, a cardiovascular disease, or cancer.

45. The method of claim 43, wherein in said diagnosis or detection, said IL-1β binding molecule, or said multispecific antigen-binding molecule, or said immunoconjugate, or said pharmaceutical composition further carries a detectable label.

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