CN113444177A

CN113444177A - anti-IL-1 beta antibodies, pharmaceutical compositions thereof, and uses thereof

Info

Publication number: CN113444177A
Application number: CN202110316624.4A
Authority: CN
Inventors: 夏瑜; 李百勇; 王忠民; 陈娜
Original assignee: Akeso Biopharma Inc
Current assignee: Akeso Pharmaceuticals Inc
Priority date: 2020-03-27
Filing date: 2021-03-24
Publication date: 2021-09-28
Also published as: WO2021190553A1

Abstract

The invention relates to an anti-IL-1 beta antibody, a pharmaceutical composition thereof and application thereof. Specifically, the invention relates to an anti-IL-1 β antibody or antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises: the amino acid sequences are respectively shown as SEQ ID NO: 17-SEQ ID NO: HCDR1-HCDR3 shown at 19; and the light chain variable region of the antibody comprises: the amino acid sequences are respectively shown as SEQ ID NO: 20-SEQ ID NO: LCDR1-LCDR3 shown at 22.

Description

anti-IL-1 beta antibodies, pharmaceutical compositions thereof, and uses thereof

Technical Field

The invention belongs to the field of immunology, and relates to an anti-IL-1 beta antibody, a pharmaceutical composition thereof and application thereof. Specifically, the present invention relates to an antibody against human IL-1 β; more specifically, the present invention relates to a monoclonal antibody against human IL-1 β; in particular, the invention relates to a humanized monoclonal antibody against human IL-1 beta.

Background

IL-1 β is a member of the IL-1 family, which plays a key role in innate immunity and inflammation development. IL-1 beta family members contain 7 cytokines (IL-1 alpha, IL-1 beta, IL-18, IL-33, IL-36 alpha, IL-36 beta, IL-36 gamma, IL-37) and three specific receptor antagonists (IL-1Ra, IL-36Ra and IL-38). IL-1. beta. is enzymatically produced from the inactive precursor protein ProIL-1. beta. this process is mediated primarily by the inflammatory corpuscle NLRP3(NOD-like receptor protein 3) (Jesus AA and Goldbach-Manual R. IL-1 Block in autoinflammatory syndrome. Annu Rev Med 2014).

The receptors of IL-1 beta and IL-1 alpha are IL-1R1 and IL-1RAcP heterodimers, wherein IL-1R1 is a receptor subunit specifically bound by IL-1 beta and IL-1 alpha, and IL-1RAcP (interleukin-1-receptor synergrin) is a common constituent of receptors of other multiple members of the IL-1 family, such as IL-33, IL-36 and the like. IL-1R1 is a transmembrane receptor, can be combined with IL-1 beta and IL-1RAcP to form a receptor complex, activate a downstream intracellular related signaling pathway and mediate IL-1 beta related biological effects. IL-1 β has naturally occurring negative regulator molecules including IL-1R2, soluble IL-1R1 and IL-1R2, and IL-1 receptor antagonists (IL-1 Ra). IL-R2 can bind to IL-1 beta, however, since the intracellular segment is short and therefore unable to activate downstream pathways, soluble IL-1R1 and IL-1R2 bind to IL-1 and prevent it from further binding to membrane-localized receptors; IL-1Ra binds to IL-1R1 and thus blocks the binding of IL-1 α and IL-1 β to the corresponding receptors (Boraschi D, ItaliaaniP, Weil S, Martin MU. the family of the interleukin-1 receptors. Immunol Rev.2018; 281 (1): 197-232).

IL-1 β is produced primarily by monocytes and macrophages, and binding of IL-1 β to its receptor activates downstream NF-. kappa.B and MAPK dependent proinflammatory intracellular signaling cascades. IL-1. beta. is an effective inducer of COX-2 expression, producing large amounts of prostaglandin E2(PGE2) to suppress IFN-. gamma.production and thus Th 1-type immune responses against viruses and intracellular parasitic bacteria dominated by Th 1-type T cells (Napoli G, acta-Rodriguez EV, Lanzavecchia A, Sallusto F. prostaglandin E2 enghances Th17 responses via modulation of IL-17 and IFN-gamma production by memory CD4+ T cells. European journal of immunology. 2009; 39 (5): 1301-12.). Another key effect of IL-1 β is to promote the secretion of IL-6. IL-6 is a key factor in inducing the differentiation of naive T cells into Th17 type T cells (Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al. regenerative developmental pathways for the generation of pathological effects TH17 and regulatory T cells. Nature.2006; 441 (7090): 235-8.); IL-6 may also inhibit the immunosuppressive function of regulatory T cells and prevent the conversion of Th17 type T cells into immunosuppressive Treg type T cells (Korn T, Mitsdoerffer M, Cronford AL, Awasthi A, Dardalhon VA, Galileos G, et a1.IL-6 controls Th17 immunity in vivo inhibition of the compliance T cells in Foxp3+ regulatory T cells proceedings of the National Academy of science of the United States of America 2008; 105(47) 18460-5. Pascal C, comfort R. topathology-dependent CD4+ CD25+ 3. benthal. 3. T-6. Regulation of the National Academy of science of culture of America 2008. 3. Pat No. 3. D-5. Pascal culture of No. 3. D-3. No. 3. D. No. 3. A. IL-1 β -mediated Th 17-type immune responses inhibit killing of virus-infected cells by CD8+ cytotoxic T-cells and enhance the survival of virus-infected cells, thereby potentially reducing the body's defense against viruses (Hou W, Kang HS, Kim BS. Thl 7 cell enhancement viral persistence and inhibition T cell cytoxicity in a model of viral infection. the Journal of experimental media.2009; 206 (2): 313-28.). In addition, IL-1 β increases secretion of GM-CSF by inducing proliferation and activation of Th17 cells (Mufazalov IA, Schelmbauer C, Regen T, Kuschmann J, Wanke F, Gabriel LA, Hauptmann J, Muller W, Pinteaux E, Kurschaus FC, Waisman A. IL-1 signaling is clinical for expansion bus generation of autoreactive GM-CSF + Th17 cells EMBO J.20174; 36 (1): 102-115); endothelial Cells, Fibroblasts, chondrocytes, smooth muscle Cells, and the like, can be produced under Induction of IL-1 and TNF- α (GM-CSF S. Matthew Fitzgerald, David S. Chi, H.Kenton Hall, Scott A. Reynolds, Omoloa amide, Steven A. Lee, and guide Krishanswamy. GM-CSF expression Human Lung fibers by IL-1 β, TNF- α, and MacrophaContact. journal of interference & Cytokine research. 23, No.2.A. M. A. Baqui, Timothy F. Meiller, Jennifer J. Chon, bee-Tuo, William A. kernel. Colony. 341. gradient, molecular protein of microorganism F. Maillar, molecular protein of culture of microorganism of 1. particle-culture P1991. microorganism of culture P. Microduct of microorganism of culture of microorganism of 3. III, microorganism of culture of microorganism of cell of microorganism of culture of microorganism of P. 12. C. 12. III, cell of microorganism of FIGS.

There is evidence that IL-1 β plays a critical role in the lethal systemic inflammatory response caused by viral infection. In severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) infection, studies demonstrated that SARS-CoV 3a protein can directly activate NLRP3inflammasome in macrophages, mediated secretion of IL-1 β, which may be the mechanism observed in SARS as IL-1 β, and its downstream IL-6 elevation (Chen IY, Moriyama M, Chang MF, Ichinohe T.Severe acid reactivity Synthesis resonance chromatography virous virogene 3a activities the NLRPRPInflamamame. Front Microbiol. ecollection 2019). A significant reduction in the number of CD4+ and CD8+ T cells in the blood of patients at necropsy of patients with new coronavirus pneumonia (COVID-19) death caused by infection with a virus of the family Conaviridae, but a significant increase in the proportion of Th17 cells, was found (Xu Z, Shi L, Wang YJ, Zhang JY, HuangL, Zhang C et al medical definitions of COVID-19 associated with access restriction syndrome, the Lancet Respiratory medicine.2020). Several studies have reported that IL-6, GM-CSF are significantly elevated in severe and critically ill patients with novel coronary pneumonia (Characteristics of lymphoma patients and cytokines in periheral blods of 123 hospitated tissues with 2019 Novel Coronavir Pneumania (NCP). Wan SX, Yi QJ, Fan SB, Lv JL, Zhang XX, Guo L, Lang CH, Xiao Q, Xiao KH, et a1.medRxiv 2020.02.10.20021832; Chenlei, Liuhui, Liuwei et al.2019 novel coronavirus pneumonia 29 clinical profiles.Zhonghua tuberculosis and respiration, 2020, 43: E005). These suggest that excessive, fatal inflammatory responses are present in patients, and inhibition of the IL-1 β signaling pathway may be useful in treating such diseases. anti-IL-6 antibodies have been currently approved for the treatment of cytokine release syndrome in the development of CAT-T therapy. However, as an upstream molecule of IL-6, inhibition of IL-1 β -related signaling pathways may be more advantageous for the treatment of excessive, fatal inflammatory responses caused by viral infections, tumors, or cellular immunotherapy. Norelli et al report that in a mouse model of lymphoma treated by chimeric antigen receptor T cells (CAR-T), IL-1 receptor antagonist Anakinra, a recombinant human IL-1Ra, can effectively alleviate the clinical symptoms of cytokine release syndrome caused by CAT-T cell treatment, and alleviate the nervous system toxicity of CAT-T therapy, thereby greatly prolonging the disease-free survival period of experimental animals; while anti-IL-6 antibodies failed to protect mice from delayed lethal neurotoxicity characterized by nervous system inflammation (Norelli M, Camisa B, Barbiera G, Falcone L, Purevdorj A, Genua M, Sanvito F, Ponzoni M, Doglioni C, Cristospori P, Traversari C, Bordignon C, Ciceri F, Ostuni R, Bonini C, Casucci M, Bondaza A. monocyte-derived IL-1 and IL-6 area differiential required for cytokinase-release syndrome and neurooxydity CAR T cells 748. Nat.201Jun; 24 (6): 739-).

As mentioned previously, pathogenic microbial (viral, bacterial or fungal or other) infections, burns, pancreatitis, blood transfusions or allogeneic blood cell transplantation, trauma, drugs (antibodies, immune agonists, or cytokines), chimeric antigen cell therapy, surgery, radiation therapy, chemotherapy, etc. may all result in systemic inflammatory responses, manifested by increased secretion of IL-1 β, and even cytokine release syndrome characterized by increased secretion of IL-1 β, manifested by multiple organ injury in the body including acute kidney injury, acute respiratory injury such as Acute Respiratory Distress Syndrome (ARDS), disseminated intravascular coagulation, hyper-ferritin, nervous system inflammation, and the like.

Although IL-1 receptor antagonists such as Anakinra are currently in clinical use, the development of anti-IL-1 β antibody drugs with stable pharmacokinetic profiles has great potential for clinical use due to their rapid in vivo metabolic rates, half-lives of only a few hours, and the need for daily injections to maintain efficacy.

Disclosure of Invention

Through intensive research and creative work, the inventor utilizes a mammalian cell expression system to express recombinant IL-1 beta-His as an antigen to immunize a mouse, and obtains hybridoma cells through fusion of mouse spleen cells and myeloma cells. The inventor obtains the following hybridoma cell strains by screening a large number of samples:

the hybridoma cell line LT010 is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 6 months and 21 days, and the preservation number is CCTCC NO: C2018133.

the inventors have surprisingly found that:

hybridoma cell line LT010 can secrete specific monoclonal antibody (named 3H6) specifically combined with human IL-1 beta, and the monoclonal antibody can effectively block the combination of IL-1 beta and IL-1R 1;

furthermore, the inventor creatively prepares a humanized antibody (respectively named as 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1) of anti-human IL-1 beta, which can effectively bind to human IL-1 beta, block the binding of IL-1 beta and the receptor IL-1R1 thereof, and inhibit the activation of the downstream signal path of IL-1 beta; has the functions of reducing, preventing or treating systemic inflammatory reaction caused by pathogenic microorganism (virus, bacteria or fungi or other) infection, burn, pancreatitis, blood transfusion or allogeneic blood cell transplantation, trauma, medicine (antibody, immune agonist or cytokine), chimeric antigen cell therapy, operation, radiotherapy and chemotherapy.

The following invention is thus provided:

one aspect of the invention relates to an anti-IL-1 beta antibody or antigen-binding fragment thereof, wherein,

the heavy chain variable region of the antibody comprises: the amino acid sequences are respectively shown as SEQ ID NO: 17-SEQ ID NO: HCDR1-HCDR3 shown at 19; and

the light chain variable region of the antibody comprises: the amino acid sequences are respectively shown as SEQ ID NO: 20-SEQ ID NO: LCDR1-LCDR3 shown at 22.

Preferably, the IL-1 β is human IL-1 β.

The heavy chain variable region and the light chain variable region determine the binding of the antigen; the variable region of each chain contains three hypervariable regions, called Complementarity Determining Regions (CDRs) (CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR3, CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR 3; named by Kabat et al, see Sequences of Proteins of Immunological interest.

Antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 of the invention can be found to have the same HCDR1-3 and LCDR1-3 by technical means well known to those skilled in the art, e.g. by analysis of the VBASE2 database:

the amino acid sequences of 3 HCDR regions of the heavy chain variable region are as follows:

HCDR1：GFSLSTSGMG(SEQ ID NO：17)，

HCDR2：IYWDDDK(SEQ ID NO：18)，

HCDR3：ARSAYYSFAY(SEQ ID NO：19)；

the amino acid sequences of the 3 CDR regions of the light chain variable region are as follows:

LCDR1：QDVDTD(SEQ ID NO：20)，

LCDR2：WAS(SEQ ID NO：21)，

LCDR3：QQYSSYPT(SEQ ID NO：22)。

in one or more embodiments of the invention, wherein,

the amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10 and SEQ ID NO: 14 or a sequence identical to SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10 and SEQ ID NO: 14, respectively, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; and

the amino acid sequence of the variable region of the light chain of the antibody is selected from SEQ ID NO: 4 and SEQ ID NO: 8. SEQ ID NO: 12 and SEQ ID NO: 16 or a sequence identical to SEQ ID NO: 4 and SEQ ID NO: 8. SEQ ID NO: 12 and SEQ ID NO: 16, respectively, has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity.

In some embodiments of the invention, the antibody is selected from the group consisting of:

(1) as shown in SEQ ID NO: 2 or a variant of SEQ ID NO: 2 and a VH as set forth in SEQ ID NO: 4 or a sequence identical to SEQ ID NO: 4, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity;

(2) as shown in SEQ ID NO: 6 or a sequence identical to SEQ ID NO: 6 and a VH as set forth in SEQ ID NO: 8 or a variant of SEQ ID NO: 8, a VL represented by a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity;

(3) as shown in SEQ ID NO: 10 or a sequence identical to SEQ ID NO: 10 and a VH as set forth in SEQ ID NO: 12 or a variant of SEQ ID NO: 12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical; and

(4) as shown in SEQ ID NO: 14 or a sequence identical to SEQ ID NO: 14 and a VH as set forth in SEQ ID NO: 16 or a sequence identical to SEQ ID NO: 16, or a VL represented by a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity.

In one or more embodiments of the invention, the antibody or antigen-binding fragment thereof is selected from the group consisting of a Fab, Fab ', F (ab') 2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody (e.g., scFv), humanized antibody, chimeric antibody, or diabody.

In one or more embodiments of the invention, wherein the antibody is less than 10^-5M, e.g. less than 10^- ⁶M, less than 10^-7M, less than 10^-8M, less than 10^-9M or less than 10^-10K of M or less_DBinds to IL-1 β protein; preferably, said K_DMeasured by a Biacore molecular interaction instrument; preferably, said K_DMeasured by Fortebio molecular interaction instrument.

In some embodiments of the invention, wherein the antibody has an EC of less than about 100nM, e.g., less than about 10nM, less than about 1nM, less than about 0.9nM, less than about 0.8nM, less than about 0.7nM, less than about 0.6nM, less than about 0.5nM, less than about 0.4nM, less than about 0.3nM, less than about 0.2nM, less than about 0.1nM or less₅₀Binds to IL-1 beta protein. In particular, the EC₅₀Measured by indirect ELISA method.

In one or more embodiments of the invention, wherein,

the antibody comprises a non-CDR region, and the non-CDR region is from a species other than murine, e.g., from a human antibody.

In some embodiments of the invention, the constant regions of the antibodies are humanized, e.g., the heavy chain constant regions are each humanized with an Ig gamma-1chain C region, e.g., ACCESSION: p01857 or Ig gamma-4chain C region, such as ACCESSION: p01861.1; light chain constant regions were all made using Ig kappa chain C regions, such as access: p01834.

In one or more embodiments of the present invention, the antibody or the antigen binding fragment thereof is a monoclonal antibody produced by a hybridoma cell line LT010, wherein the hybridoma cell line LT010 is preserved in the chinese typical culture collection center (CCTCC) with the preservation number of CCTCC NO: C2018133.

in one or more embodiments of the invention, the antibody is a monoclonal antibody.

Another aspect of the present invention relates to an antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or the antigen-binding fragment thereof is the antibody or the antigen-binding fragment thereof according to any one of the present invention; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a chemotherapeutic drug.

The chemotherapeutic agent may be a conventional tumor chemotherapeutic agent, such as an alkylating agent, an antimetabolite, an antitumor antibiotic, a plant-based anticancer agent, a hormone, an immunosuppressive agent, and the like.

In one or more embodiments of the invention, the antibody or antigen-binding fragment thereof is linked to a small molecule drug via a linker; the linker may be one known to those skilled in the art, for example, a hydrazone bond, a disulfide bond, or a peptide bond.

In one or more embodiments of the invention, the antibody or antigen-binding fragment thereof is linked to the small molecule drug at a molar ratio; for example, the molar ratio is 1: (2-4).

Yet another aspect of the invention relates to a bispecific antibody (also called bifunctional antibody) comprising a first protein functional region and a second protein functional region, wherein:

the first protein functional region targets IL-1 beta,

the second protein functional region targets a target other than IL-1 β, e.g., IL-17A;

wherein the first protein functional region is an antibody or antigen-binding fragment thereof according to any one of the present invention;

preferably, the bispecific antibody is an IgG-scFv format;

preferably, the first and second electrodes are formed of a metal,

(1) the first protein functional region is an antibody or antigen-binding fragment thereof according to any one of the present invention, and the second protein functional region is a single chain antibody;

or,

(2) the first protein functional region is a single-chain antibody, and the heavy chain variable region of the first protein functional region comprises an amino acid sequence shown as SEQ ID NO: 17-SEQ ID NO: 19, HCDR1-HCDR3 wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20-SEQ ID NO: 22, and the second protein functional region is an antibody (e.g., a monoclonal antibody).

In some embodiments of the invention, the bispecific antibody is one wherein the first protein functional region and the second protein functional region are linked directly or via a linker fragment;

preferably, the linker fragment is (GGGGS) m, m being a positive integer, e.g. 1, 2, 3, 4, 5 or 6;

preferably, the linker is ss (ggggs) n, n being a positive integer, e.g. 1, 2, 3, 4, 5 or 6.

In some embodiments of the invention, the bispecific antibody of item (2),

the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10 and SEQ ID NO: 14 or a sequence identical to SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10 and SEQ ID NO: 14, respectively, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; and

the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 4 and SEQ ID NO: 8. SEQ ID NO: 12 and SEQ ID NO: 16 or a sequence identical to SEQ ID NO: 4. SEQ ID NO: 8. SEQ ID NO: 12 and SEQ ID NO: 16, respectively, has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity.

In some embodiments of the invention, the bispecific antibody of item (2),

the amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO: 2 or a variant of SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single chain antibody is SEQ ID NO: 4 or a sequence identical to SEQ ID NO: 4, a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; or

The amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO: 6 or a sequence identical to SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single chain antibody is SEQ ID NO: 8 or a sequence corresponding to SEQ ID NO: 8, a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; or

The amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO: 10 or a sequence identical to SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single chain antibody is SEQ ID NO: 12 or a variant of SEQ ID NO: 12, a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; or

The amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO: 14 or a sequence identical to SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single chain antibody is SEQ ID NO: 16 or a sequence identical to SEQ ID NO: 16, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity.

In some embodiments of the invention, the first protein functional region and the second protein functional region of the bispecific antibody are independently 1, 2, or 2 or more.

In some embodiments of the invention, in item (2) of the bispecific antibody, the constant region of the monoclonal antibody is selected from the constant regions of human IgG1, IgG2, IgG3 or IgG 4.

In some embodiments of the invention, the single chain antibody of the bispecific antibody is linked to the C-terminus of the heavy chain of the antibody or monoclonal antibody.

Yet another aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody heavy chain variable region and a nucleic acid sequence encoding an antibody light chain variable region, wherein,

the heavy chain variable region of the antibody comprises amino acid sequences respectively shown as SEQ ID NO: 17-SEQ ID NO: 19, and the light chain variable region of the antibody comprises amino acid sequences as set forth in SEQ ID NOs: 20-SEQ ID NO: LCDR1-LCDR3 not found in 22;

preferably, the amino acid sequence of the heavy chain variable region of the antibody is selected from the group consisting of SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10 and SEQ ID NO: 14 or a sequence identical to SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10 and SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of said antibody is selected from the group consisting of SEQ ID NOs: 4 and SEQ ID NO: 8. SEQ ID NO: 12 and SEQ ID NO: 16 or a sequence identical to SEQ ID NO: 4 and SEQ ID NO: 8. SEQ JD NO: 12 and SEQ ID NO: 16, a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity;

more preferably, the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 2 or a variant of SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 4 or a sequence identical to SEQ ID NO: 4, a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; or the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 6 or a sequence identical to SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 8 or a variant of SEQ ID NO: 8, a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; or the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 10 or a sequence identical to SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 12 or a sequence corresponding to SEQ ID NO: 12, a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; or the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 14 or a sequence identical to SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 16 or a sequence corresponding to SEQ ID NO: 16, and further preferably, the isolated nucleic acid molecule comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity:

SEQ ID NO: 1 and SEQ ID NO: 3, or a nucleic acid sequence as set forth in (3),

SEQ ID NO: 5 and SEQ ID NO: 7, or a nucleic acid sequence as set forth in SEQ ID NO,

SEQ ID NO: 9 and SEQ ID NO: 11, or

SEQ ID NO: 13 and SEQ ID NO: 15, or a nucleic acid sequence as set forth in seq id no.

The isolated nucleic acid molecule may be a single nucleic acid molecule or may be multiple nucleic acid molecules, e.g., two nucleic acid molecules. In the case of a single nucleic acid molecule, the heavy chain variable region and the light chain variable region of the antibody may be expressed by the same nucleic acid molecule, e.g., by the same or different expression cassettes located on the same nucleic acid molecule. When a plurality of nucleic acid molecules, for example, two nucleic acid molecules, the heavy chain variable region and the light chain variable region of the antibody may be expressed by different nucleic acid molecules, respectively.

Yet another aspect of the invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the invention. The recombinant vector may be one or more. When the nucleic acid molecule is a plurality of (e.g., two), a plurality of (e.g., two) nucleic acid molecules may be expressed by the same recombinant vector or may be expressed by different recombinant vectors.

Yet another aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention, or comprising a recombinant vector of the invention.

Yet another aspect of the invention relates to a method of making an antibody or antigen-binding fragment thereof according to any one of the invention, comprising the steps of culturing a host cell of the invention under suitable conditions, and recovering the antibody or antigen-binding fragment thereof from the cell culture.

The invention also relates to a hybridoma cell strain LT010 which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2018133.

yet another aspect of the invention relates to a pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of the present invention, an antibody drug conjugate according to the invention, or a bispecific antibody according to the invention; optionally, it further comprises a pharmaceutically acceptable carrier and/or excipient.

Yet another aspect of the invention relates to the use of an effective amount of an antibody or antigen-binding fragment thereof according to any one of the present invention, an antibody drug conjugate according to the invention or a bispecific antibody according to the invention for the preparation of a medicament for the treatment and/or prevention of a systemic inflammation characterized by abnormal increased secretion of IL-1 β;

preferably, the systemic inflammation includes systemic inflammation syndrome, cytokine release syndrome, Multiple Organ Dysfunction Syndrome (MODS), acute respiratory distress syndrome;

preferably, the systemic inflammation is caused by infection with a pathogenic microorganism;

preferably, the pathogenic microorganism is a virus, bacterium, fungus, rickettsia, chlamydia, mycoplasma, parasite, prion;

preferably, the virus includes RNA viruses and DNA viruses;

preferably, the RNA virus comprises a virus of the family coronaviridae;

preferably, the viruses of the family Coronaviridae include 2019 novel coronaviruses (2019-nCoV or SARS-CoV-2, causing novel coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing middle east respiratory syndrome).

In yet another aspect of the present invention, the use of an antibody or antigen-binding fragment thereof according to any one of the present invention, an antibody drug conjugate according to the present invention, or a bispecific antibody according to the present invention for the preparation of a medicament comprising:

a drug which blocks the binding of human IL-1 beta to human IL-1R1 and/or human IL-1R2,

a drug which down-regulates the activity or level of human IL-1 beta, or

An agent that inhibits the activation of a downstream signaling pathway mediated by the binding of human IL-1 β to human IL-1R1 and/or human IL-1R 2.

In one embodiment of the invention, the human IL-1R1 and/or human IL-1R2 is cell surface human IL-1R1 and/or human IL-1R 2.

In one embodiment of the invention, the use is non-therapeutic and/or non-diagnostic.

In one or more embodiments of the invention, the antibody or antigen-binding fragment thereof, the antibody drug conjugate of the invention, or the bispecific antibody of the invention, for use in the manufacture of a medicament for the treatment and/or prevention of systemic inflammation (characterized by abnormal increased secretion of IL-1 β);

preferably, the virus includes RNA viruses and DNA viruses;

preferably, the RNA virus comprises a virus of the family coronaviridae;

preferably, the viruses of the family Coronaviridae include 2019 novel coronaviruses (2019-nCoV or SARS-CoV-2, causing novel coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing middle east respiratory syndrome),

preferably, the antibody or antigen binding fragment thereof is used alone or in combination with one or more antiviral drugs;

preferably, the antiviral drug is an antiretroviral drug or an interferon;

preferably, the antiretroviral drug is one wherein: antiretroviral drugs are RNA-dependent RNA polymerase (RdRp) inhibitors, or nucleoside analogs, or peptoids, or strand transfer inhibitors of human immunodeficiency virus type 1(HIV-1) integrase (INSTI), or HIV protease inhibitors.

Preferably, the antiretroviral drug is faviravir (favipiravir), Reidesvir (Remdesivir), Becravir, Saquinavir (Saquinavir), Bilatavir (Bictegravir), Lopinavir (Lopinavir), Dorteravir (Dolutogravir);

more preferably, the drug is favipiravir.

In one or more embodiments of the invention, the antibody or antigen-binding fragment thereof, the antibody drug conjugate of the invention, or the bispecific antibody of the invention is used for:

blocking the binding of human IL-1 beta to human IL-1R1 and/or human IL-1R2,

down-regulating human IL-1 beta activity or level, or

Inhibit the activation of downstream signaling pathways mediated by the binding of human IL-1 β to human IL-1R1 and/or human IL-1R 2.

Yet another aspect of the invention relates to an in vivo or in vitro method comprising the step of applying cells with an effective amount of an antibody or antigen-binding fragment thereof according to any one of the invention, an antibody drug conjugate according to the invention or a bispecific antibody according to the invention, said method being selected from the group consisting of:

blocking the binding of human IL-1 beta to human IL-1R1 and/or human IL-1R2,

a method of down-regulating the activity or level of human IL-1 beta, or

Methods of inhibiting activation of downstream signaling pathways mediated by binding of human IL-1 β to human IL-1R1 and/or human IL-1R 2.

In one embodiment of the invention, the in vitro method is of non-therapeutic and/or non-diagnostic purpose.

Yet another aspect of the present invention relates to a method of treating and/or preventing a systemic inflammation characterized by abnormal increased secretion of IL-1 β, comprising the step of administering to a subject or patient in need thereof an effective amount of an antibody or antigen-binding fragment thereof according to any one of the present invention, an antibody drug conjugate according to the present invention, or a bispecific antibody according to the present invention;

preferably, the virus includes RNA viruses and DNA viruses;

preferably, the RNA virus comprises a virus of the family coronaviridae;

In any of the embodiments of the invention, the antibody or antigen binding fragment thereof, the antibody drug conjugate, or the bispecific antibody can be used alone or in combination with one or more antiviral drugs.

In one aspect of the invention, a kit is provided comprising an effective amount (e.g., 0.001mg to 1000mg) of the antibody or antigen-binding fragment thereof, the antibody drug conjugate, or the bispecific antibody, and optionally, an effective amount of one or more antiviral drugs (e.g., 100-2400mg), wherein the antiviral drugs are as described above.

In another aspect of the invention, a single pharmaceutical dosage unit is contemplated comprising 0.001mg to 1000mg of the antibody or antigen-binding fragment thereof of the invention, preferably 0.001mg to 900mg, 0.001mg to 800mg, 0.001mg to 700mg, 0.001mg to 600mg, 0.001mg to 500mg, 0.001mg to 400mg, 0.001mg to 300mg, 0.001mg to 200mg, 0.001mg to 100mg, more preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, or 1000mg of the antibody or antigen-binding fragment thereof of the invention.

In any of the above embodiments of the invention, the effective amount of the antibody or antigen-binding fragment thereof, the antibody drug conjugate or the bispecific antibody of the invention is 0.001mg to 1000mg, preferably 0.001mg to 900mg, 0.001mg to 800mg, 0.001mg to 700mg, 0.001mg to 600mg, 0.001mg to 500mg, 0.001mg to 400mg, 0.001mg to 300mg, 0.001mg to 200mg, 0.001mg to 100mg, more preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000 mg. Alternatively, in any of the above embodiments of the invention, the effective amount of the antibody or antigen-binding fragment thereof, the antibody drug conjugate or the bispecific antibody of the invention is 0.1-100mg/kg, preferably 1-90mg/kg, 1-80mg/kg, 1-70mg/kg, 1-60mg/kg, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1-20mg/kg or 1-10mg/kg, based on the weight of the subject.

In any of the above embodiments of the invention, the effective amount of one or more antiviral drugs (e.g., Favipiravir) is 100,2400 mg, preferably 100mg-2300mg, 100mg-2200mg, 100mg-2100mg, 100mg-2000mg, 100mg-1900mg, 100mg-1800mg, 100mg-1700mg, 100mg-1600mg, 100mg-1800mg, more preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000 mg. Alternatively, in any of the above embodiments of the invention, the effective amount of the antiviral drug is 0.1-100mg/kg, preferably 1-90mg/kg, 1-80mg/kg, 1-70mg/kg, 1-60mg/kg, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1-20mg/kg or 1-10mg/kg, based on the weight of the subject or patient.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in the present invention are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

In the present invention, the term "single pharmaceutical dosage unit" denotes a single pharmaceutical dosage form, e.g. in units of one ampoule, of an antibody or antigen-binding fragment thereof, of said antibody drug conjugate or of said bispecific antibody (or a pharmaceutical composition comprising the same) of the present invention to be administered to a subject or patient at the time of a dosing regimen.

As used herein, reference to the amino acid sequence of IL-1 β includes the full length of the IL-1 β protein (GenBank ID: NP-000567.1), as well as fusion proteins of IL-1 β, such as those fused to the Fc protein fragment (mFc or hFc) of a mouse or human IgG, or multiple His. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced into the amino acid sequence of IL-1 β without affecting its biological function. Thus, in the present invention, the term "IL-1. beta. shall include all such sequences, as well as natural or artificial variants thereof. Also, when a sequence fragment of an IL-1 β protein is described, it includes not only the sequence fragment but also the corresponding sequence fragment in its natural or artificial variant.

As used herein, reference to the amino acid sequence of IL-1R1 includes the full length of IL-1R1 protein (GenBank ID: NP-000868), as well as fusion proteins of IL-1R1, such as fragments fused to the Fc protein fragment (mFc or hFc) of mouse or human IgG, or multiple His. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced into the amino acid sequence of the IL-1R1 protein without affecting its biological function. Thus, in the present invention, the term "IL-1R 1" is intended to include all such sequences as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the IL-1R1 protein, it includes a fragment of the IL-1R1 sequence, and also includes a fragment of the corresponding sequence in natural or artificial variants thereof.

As used herein, reference to the amino acid sequence of IL-1R2 includes the full length of IL-1R2 protein (GenBank ID: CAA42441.1), as well as fusion proteins of IL-1R2, such as fragments fused to the Fc protein fragment (mFc or hFc) of mouse or human IgG, or multiple His. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced into the amino acid sequence of the IL-1R2 protein without affecting its biological function. Thus, in the present invention, the term "IL-1R 2" is intended to include all such sequences as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the IL-1R2 protein, it includes a fragment of the IL-1R2 sequence, and also includes a fragment of the corresponding sequence in natural or artificial variants thereof.

As used herein, surgeryLanguage EC₅₀Refers to the concentration of the half maximal effect (concentration for 50% of the maximum effect), and refers to the concentration that causes 50% of the maximal effect.

The term "antibody" as used herein refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. 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 3 domains (CH1, CH2, and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation (called complementarity determining regions or CDRs) interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to the various regions or domains follows either Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J.mol.biol.196: 901-; chothia et al (1989) Nature 342: 878-883. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, ch.7(Paul, w., ed., 2 nd edition, Raven Press, n.y. (1989), which is incorporated herein by reference in its entirety for all purposes.

In some cases, the antigen-binding fragment of an antibody is a single chain antibody (e.g., an scFv), in which the VL and VH domains are paired by a linker that enables them to be produced as a single polypeptide chain to form a monovalent molecule (see, e.g., Bird et al, Science 242: 423426(1988) and Huston et al, proc.natl.acad.sci.usa 85: 58795883 (1988)). Such scFv molecules can have the general structure: NH (NH)₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used (Holliger et al (1993), Proc. Natl. Acad. Sci. USA 90: 6444-. Other linkers useful in the present invention are prepared by Alfthan et al (1995), Protein Eng.8: 725-731, Choi et al (2001), Eur.J.Immunol.31: 94-106, Hu et al (1996), Cancer Res.56: 3055-3061, Kipriyanov et al (1999), J.mol.biol.293: 41-56 and Rovers et al (2001), Cancer Immunol.

In some cases, the antigen-binding fragment of the antibody is a diabody, i.e., a diabody in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and create two antigen-binding sites (see, e.g., Holliger P. et al, Proc. Natl. Acad. Sci. USA 90: 6444-.

In other cases, the antigen-binding fragment of an antibody is a "bifunctional antibody". Bifunctional antibodies, also known as Bispecific antibodies (Bispecific antibodies), are specific drugs that target two different antigens simultaneously, and can be produced by immunoselection purification. In addition, the method can also be obtained through genetic engineering, and the genetic engineering has corresponding flexibility in the aspects of combination site optimization, consideration of synthetic forms, yield and the like, so that the method has certain advantages. Currently, more than 45 forms have been demonstrated (Muller D, Kontermann RE Bispecific antibodies for cancer immunology: Current perspectives.BioDrugs 2010; 24: 89-98). A number of bispecific antibodies have been developed in the form of IgG-ScFv, i.e., Morrison model (1997Coloma MJ, Morrison SL. design and production of novel bispecific antibodies. Nature Biotechnology, 1997; 15: 159. 163. due to the advantages of this form, similar to naturally occurring IgG, in antibody engineering, expression and purification, it has proven to be one of the ideal forms of bifunctional antibodies (Miller BR, Demarest SJ, et. Stablity engineering of scfvs. for the expression of bispecific and variant antibodies. protein Eng Sel 2010; 549: 549-57; Fitzgerald J, Lungovsky A. rational engineering of variants. 2011. variants. 299. multiple variants. 3. multiple of proteins. 299).

Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., monoclonal antibody 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3, or 3H6H4L1 provided by the present invention) using conventional techniques (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods) known to those skilled in the art, and the antigen-binding fragments of antibodies are specifically screened for specificity in the same manner as for intact antibodies.

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using hybridoma technology first reported by Kohler et al (Nature, 256: 495, 1975), but can also be obtained using recombinant DNA technology (see, e.g., U.S. P4, 816, 567).

As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or a portion of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody of the desired specificity, affinity, or reactivity. In addition, some amino acid residues of the Framework Region (FR) of the acceptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further refine or optimize the performance of the antibody. For more details on humanized antibodies, see, e.g., Jones et al, Nature, 321: 522525 (1986); reichmann et al, Nature, 332: 323329 (1988); presta, curr, op.struct.biol., 2: 593596 (1992); and Clark, immunol. today 21: 397402(2000).

As used herein, the term "isolated" or "isolated" refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" or "isolated" does not exclude the presence of substances mixed artificially or synthetically or other impurities which do not affect the activity of the substance.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

As used herein, the term "bispecific", "dual specificity" or "bifunctional" antigen binding proteins or antibodies are hybrid antigen binding proteins or antibodies, respectively, having two different antigen binding sites. A bispecific antibody is a multispecific antigen-binding protein or multispecific antibody and may be produced by a variety of methods, including, but not limited to, fusion of hybridomas or attachment of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, clin. exp. immunol.79: 315- > 321; kostelny et al 1992, J.Immunol.148: 1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind two different epitopes that are present on the same or different protein targets.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody is present in an amount less than about 10^-5M, e.g. less than about 10^-6M, less than about 10^-7M, less than about 10^-8M, less than about 10^-9M or less than about 10^-10M or less affinity (K)_D) Binding the antigen.

As used herein, the term "K_D"refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, the antibody (e.g., monoclonal antibody 3H6, 3H6H1L1, 3H6H2L2, or 3H6H3L3 of the invention) is present at less than about 10^-5M, e.g. less than about 10^-6M, less than about 10^-7M, less than about 10^-8M, less than about 10^-9M or less than about 10^-10Dissociation equilibrium constant (K) of M or less_D) Binding antigen (e.g., IL-1. beta. protein). K can be determined using methods known to those skilled in the art_DFor example, using a Biacore molecular interaction instrument.

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the terms "hybridoma" and "hybridoma cell line" are used interchangeably, and when referring to the terms "hybridoma" and "hybridoma cell line," it also includes subclones and progeny cells of the hybridoma. For example, when referring to hybridoma cell line LT010, it also refers to subclones and progeny cells of hybridoma cell line LT 010.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject or patient and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR, 19th ed. pennsylvania: mach Publishing Company, 1995) and include, but are not limited to: pH regulator, surfactant, adjuvant, and ionic strength enhancer. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a disease (e.g., RA) preventing effective amount refers to an amount sufficient to prevent, or delay the onset of disease (e.g., RA); a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.

The invention has the beneficial effects that:

the anti-IL-1 beta antibodies of the invention, in particular humanized anti-IL-1 beta antibodies, have one or more of the following technical effects:

(1) effectively bind to human IL-1 beta, block the binding of IL-1 beta to its receptor IL-1R 1;

(2) inhibit activation of the IL-1 β downstream signaling pathway;

(3) can specifically inhibit the activity of IL-1 beta for inducing MRC-5 cells to secrete IL-6;

(4) can effectively block the activation of NF-kB by IL-1 beta;

(5) has the potential for preparing medicines for inhibiting IL-1 beta;

(6) has the potential of being used for preparing medicines for preventing and/or treating systemic inflammation caused by pathogenic microorganism infection, including systemic inflammation syndrome, cytokine release syndrome, Multiple Organ Dysfunction Syndrome (MODS) and acute respiratory distress syndrome.

Drawings

FIG. 1: results of the binding activity assay of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 to human IL-1. beta. -His-Bio.

FIG. 2: the results of the binding activity of 3H6H4L1 to human IL-1. beta. -His-Bio were examined.

FIG. 3: 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 competed with human IL-1R1(1-332) -his for binding to human IL-1. beta. -hFc.

FIG. 4: the activity of 3H6H4L1 competing with human IL-1R1(1-332) -his for binding to human IL-1. beta. -hFc was examined.

FIG. 5: 3H6H4L1 and human IL-1 beta affinity constant detection results. Note: curves 1-5 show the analyte concentrations at 25nM, 12.5nM, 6.25nM, 3.13nM, and 1.56nM, respectively.

FIG. 6: and (3) detecting the affinity constant of Canakinumab and human IL-1 beta. Note: curves 1-5 show the analyte concentrations at 25nM, 12.5nM, 6.25nM, 3.13nM, and 1.56nM, respectively.

FIG. 7: 3H6H4L1 has the effect of inhibiting IL-1 beta induced MRC-5 to secrete IL-6.

FIG. 8: neutralizing biological activity of 3H6H4L1 on IL-1 beta at different doses.

FIG. 9: 3H6H4L1 blocks the dose-response curve for IL-1 β.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature of the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, but are conventional products available on the market.

In the following examples of the present invention, BALB/c mice used were purchased from the center for medical laboratory animals in Guangdong province.

In the following examples of the invention, the homotarget marketed drug antibody Canakinumab (trade name) was used

) As a control antibody, purchased from Novartis (Novartis).

Preparation example 1: fusion proteins human IL-1. beta. -His, IL-1R1(1-332) -His, IL-1. beta. -hFc and human IL-1. beta. - Preparation of His-Bio

The protein sequences of human IL-1. beta. (Genbank ID: NP-000567.1) and IL-1R1(Genbank ID: NP-000868, amino acids 1-332) were searched from the NCBI GenBank protein database. The amino acid sequences of human IL-1 beta and IL-1R1 are respectively fused with His tag sequence (6 His) and human IgG Fc purification tag sequence (Ig gamma-1chain C region, GenbankID: P01857 position 114-330); the above fusion proteins are abbreviated as human IL-1. beta. -His, IL-1R1(1-332) -His, IL-1. beta. -hFc, respectively.

The protein sample quality was qualified by SDS-PAGE.

Use of

Preparing a biotinylated coupled human IL-1 beta-His protein sample (called human IL-1 beta-His-Bio for short) by using Sulfo-NHS-LC-Biotinylation Kit (Thermo scientific); the specific preparation method is carried out by referring to the instruction of the kit.

The above-mentioned fusion protein was prepared and used in the following examples.

Example 1: preparation of anti-IL-1 beta murine antibody 3H6

1. Preparation of hybridoma cell line LT010

A BALB/c mouse (purchased from Guangdong medical experimental animal center) is immunized by taking human IL-1 beta-his as an antigen, and splenocytes of the immunized mouse are taken to be fused with myeloma cells of the mouse to prepare hybridoma cells. The hybridoma cells were screened by ELISA using IL-1. beta. -His-Bio as an antigen, and hybridoma cells capable of secreting an antibody specifically binding to IL-1. beta. -His-Bio were obtained. And (3) screening the hybridoma obtained by ELISA by competitive ELISA to obtain a hybridoma capable of secreting an antibody which is in competitive combination with the receptor IL-1R1(1-332) -His to IL-1 beta-hFc, and performing a limiting dilution method to obtain a stable hybridoma cell line. Methods for hybridoma cell preparation reference is made to currently established Methods (e.g., Stewart, S.J., "Monoclonal Antibody Production", in Basic Methods in Antibody Production and Characterization, Eds.G.C.Howard and D.R.Bethell, Boca Raton: CRC Press, 2000).

The inventors named the hybridoma cell line LT010 (IL-1. beta. -3H6) and named the monoclonal antibody secreted therefrom 3H 6.

The hybridoma cell line LT010(IL-1 beta-3H 6) is preserved in China Center for Type Culture Collection (CCTCC) in 6 and 21 months in 2018, and the preservation number is CCTCC NO: c2018133, the preservation address is China, Wuhan university, postcode: 430072.

2. preparation of anti-IL-1 beta antibody 3H6

The LT010 cell line prepared above was cultured in a hybridoma-containing serum-free medium (hybridoma-containing serum-free medium containing 1% streptomycin and 4% Glutamax in 5% CO)₂Cultured in a cell culture chamber at 37 ℃) and after 7 days, cell culture supernatant was collected and purified by high-speed centrifugation, microfiltration membrane vacuum filtration and HiTrap protein A HP column to obtain antibody 3H 6. The purified 3H6 sample is qualified by SDS-PAGE electrophoresis.

Example 2: sequence analysis of anti-IL-1. beta. antibody 3H6

mRNA was extracted from the LT010 cell line cultured in example 1 according to the method of the Total RNA extraction kit (Tiangen, cat # DP 430). According to Invitrogen

III First-Sthe trand Synthesis System for RT-PCR kit instructions Synthesis of cDNA, and PCR amplification. The PCR amplification product was directly subjected to TA Cloning, and the specific operation was carried out with reference to the pEASY-T1 Cloning Kit (Transgen CT101) Kit instruction.

The products of the TA clones were sequenced directly, with the following results:

nucleic acid sequence encoding the heavy chain variable region of antibody 3H 6: (354bp)

The amino acid sequence of the heavy chain variable region of antibody 3H6 is as follows: (118aa, in which the underlined amino acid sequence is a CDR region)

Nucleic acid sequence encoding the light chain variable region of antibody 3H 6: (318bp)

The amino acid sequence of the light chain variable region of antibody 3H6 is as follows: (106aa wherein the amino acid sequence of the underlined markers is a CDR region)

Example 3: design and production of humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 against IL-1. beta Prepare for

1. Design of light and heavy chain sequences of anti-IL-1 beta humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1

Based on the three-dimensional crystal structure of the IL-1 β protein (van Oosteoprum J, Priester JP, Gr ü tter MG, Schmitz A. the structure of human interleukin-1beta at 2.8A resolution. J Structure biol.1991, 107 (2): 189-95.) and the sequence information obtained in example 2, the sequences of the heavy chain variable regions of the humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 and the sequences of the light chain variable regions (3H6H1L1, 3H6H2L2 and 3H6H3L3 antibody constant region sequences from the NCBI database, the heavy chain constant region is Ig gamma-1C region, AChSION: P01857, the light chain constant region is CEsCA 834, the light chain constant region is Ig cesSigan, the light chain constant region is CEsSicesH 3H 3L3, the light chain constant region is Ig constant region H3H 8H 7, the light chain constant region is from the constant region Ig sSigan C834, the light chain constant region is from the constant region Sigan P01861.1, the constant region is from the constant region Sigan, the constant region from the constant region of Ig sH 7, the constant region is from the constant region of the AChSIgan, the constant region of the AChSIgan, the constant region of the CEsH 4, the constant region of the AChC, the constant region of the same, and the same, and the same, such as the same, and the same, respectively, and the same, and the same, were designed by the same, respectively, and the same, respectively, and the same, respectively, were designed as the same, and.

The heavy chain variable region sequence and the light chain variable region sequence of humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 are as follows:

(1) humanized monoclonal antibody 3H6H1L1

Nucleic acid sequence encoding the heavy chain variable region of antibody 3H6H1L 1: (354bp)

The amino acid sequence of the heavy chain variable region of antibody 3H6H1L1 is as follows: (118aa, in which the underlined amino acid sequence is a CDR region)

Nucleic acid sequence encoding the light chain variable region of antibody 3H6H1L 1: (318bp)

The amino acid sequence of the light chain variable region of antibody 3H6H1L1 is as follows: (106aa wherein the amino acid sequence of the underlined markers is a CDR region)

(2) Humanized monoclonal antibody 3H6H2L2

Nucleic acid sequence encoding the heavy chain variable region of antibody 3H6H2L 2: (354bp)

The amino acid sequence of the heavy chain variable region of antibody 3H6H2L2 is as follows: (118aa, in which the underlined amino acid sequence is a CDR region)

Nucleic acid sequence encoding the light chain variable region of antibody 3H6H2L 2: (318bp)

The amino acid sequence of the light chain variable region of antibody 3H6H2L2 is as follows: (106aa wherein the amino acid sequence of the underlined markers is a CDR region)

(3) Humanized monoclonal antibody 3H6H3L3

Nucleic acid sequence encoding the heavy chain variable region of antibody 3H6H3L 3: (354bp)

The amino acid sequence of the heavy chain variable region of antibody 3H6H3L3 is as follows: (118aa, in which the underlined amino acid sequence is a CDR region)

Nucleic acid sequence encoding the light chain variable region of antibody 3H6H3L 3: (318bp)

The amino acid sequence of the light chain variable region of antibody 3H6H3L3 is as follows: (106aa wherein the amino acid sequence of the underlined markers is a CDR region)

(4) Humanized monoclonal antibody 3H6H4L1

The nucleic acid sequence encoding the heavy chain variable region of antibody 3H6H4L1 is set forth in SEQ ID NO: 5, respectively.

The amino acid sequence of the heavy chain variable region of antibody 3H6H4L1 is set forth in SEQ ID NO: and 6.

The nucleic acid sequence encoding the light chain variable region of antibody 3H6H4L1 is set forth in SEQ ID NO: shown at 7.

The amino acid sequence of the light chain variable region of antibody 3H6H4L1 is set forth in SEQ ID NO: shown in fig. 8.

2. Preparation of humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1

The constant regions of the heavy chains of 3H6H1L1, 3H6H2L2 and 3H6H3L3 all adopt Ig gamma-1chain C region, ACCESSION: p01857; light chain constant regions were all replaced with Ig kappa chain C region, access: p01834;

the heavy chain constant region of 3H6H4L1 is Ig gamma-4chain C region, ACCESSION: p01861.1; the light chain constant region is Ig kappa chain C region, ACCESSION: p01834.

Cloning cDNA of a 3H6H1L1 heavy chain and a light chain, cDNA of a 3H6H2L2 heavy chain and a light chain, cDNA of a 3H6H3L3 heavy chain and a light chain, and cDNA of a 3H6H4L1 heavy chain and a light chain into a pUC57simple vector (provided by Kinsley company) respectively to obtain 8 recombinant plasmids, namely pUC57simple-3H6H1 and pUC57simple-3H6L 1; pUC57simple3H6H2 and pUC57simple-3H6L 2; pUC57simple-3H6H3 and pUC57simple-3H6L 3; pUC57simple-3H6H4 and pUC57simple-3H6L 1. And subcloned into pcDNA3.1 vector, respectively. Co-transfecting 293F cells with a plasmid containing a heavy chain and a recombinant plasmid containing a light chain, collecting a culture solution, and purifying to obtain humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L 1; and the result is correct by SDS-PAGE detection.

Example 4: antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 bind to human IL-1 β -his-bio Activity study (ELISA method)

The plate was coated with 2. mu.g/mL SA (streptavidin), 50. mu.L per well and incubated overnight at 4 ℃. Plates were washed once and residual liquid removed, blocked with 300 μ L of 1% BSA solution (dissolved in PBS) per well, and incubated for 2 hours at 37 ℃. The plate was washed three times and the residual liquid was removed. Human IL-1. beta. -His-Bio was diluted to 0.2. mu.g/mL, 50. mu.L/well with PBST, incubated at 37 ℃ for 30 minutes, washed three times and the residual liquid removed. The antibody was diluted to 1. mu.g/mL as in Table 1 or 0.333. mu.g/mL as in Table 2, respectively, as the starting concentration, and a gradient dilution of 1: 3 was performed for 7 concentrations, and blank controls were provided, each in 2 duplicate wells, each in a volume of 100. mu.L, and incubated at 37 ℃ for 30 minutes. After washing the plate three times, the coated ELISA plate was patted to remove the residual liquid, 50. mu.L of a horseradish peroxidase-labeled secondary goat anti-human IgG (H + L) antibody (cat # 109-. Among them, 50. mu.L of horseradish peroxidase-labeled secondary goat anti-human IgG (H + L) antibody working solution (corresponding to the wells in which 3H6H1L1, 3H6H2L2, 3H6H3L3, 3H6H4L1 and Canakiumab are located) and 50. mu.L of horseradish peroxidase-labeled secondary goat anti-mouse IgG (H + L) antibody working solution (corresponding to the wells in which 3H6 is located). The plate was washed four times and the residual liquid was removed, 50. mu.L of TMB developing solution was added to each well, and after development for 5 minutes in the dark at room temperature, 50. mu.L of stop solution was added to each well to terminate the developing reaction. Immediately putting the ELISA plate into an ELISA reader, and reading the OD value of each hole of the ELISA plate by selecting the wavelength of 450 nm.

The data were analyzed using SoftMax Pro 6.2.1 software. The results of plotting the 4-parameter fit curves using the antibody concentration as the abscissa and the absorbance value as the ordinate are shown in FIG. 1 and FIG. 2, respectively. The results of the detection of the binding activity of 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 to human IL-1. beta. -His-Bio are shown in tables 1 and 2, respectively.

Table 1: results of examining the binding activity of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 to human IL-1. beta. -His-Bio

Table 2: results of detection of binding Activity between 3H6H4L1 and human IL-1. beta. -His-Bio

The results show that:

3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 are all capable of effectively binding to human IL-1 beta-His-Bio, and the binding efficiency is dose-dependent;

under the same detection conditions, the binding efficiency of 3H6H1L1, 3H6H2L2 and 3H6H4L1 and the antigen human IL-1 beta-His-Bio presents a dose-dependent relationship, and the binding activity is superior to that of a medicine Canakinsub on the market at the same target point; 3H6H3L3 binding activity and Canakinumab is comparable.

Example 5: antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 with human IL-1R1(1-332) -his Activity study (ELISA method) of competitive binding to human IL-1. beta. -hFc

The plate was coated with 4. mu.g/mL human IL-1. beta. -hFc, 50. mu.L per well, and incubated overnight at 4 ℃. The plate was washed once and the residual liquid removed, blocked by adding 300. mu.L of 1% BSA solution (dissolved in PBS) per well, incubated at 37 ℃ for 2 hours, washed three times and the residual liquid removed. Antibody dilution to 2 μ g/mL (final concentration 1 μ g/mL) as the starting concentration, 1: 3, and 2 duplicate wells with 50 μ L of each well are prepared by gradient dilution and blank control, and incubated for 10 minutes at room temperature. Human IL-1R1(1-332) -his at 0.08. mu.g/mL (final concentration 0.04. mu.g/mL) or 0.1. mu.g/mL (final concentration 0.05. mu.g/mL) was added to the microplate, 50. mu.L per well volume and antibody volume 1: 1 mix gently, 100. mu.L of final volume per well, incubate for 30min at 37 ℃. The plate was washed three times and the residual liquid was removed, and 50. mu.L of anti-His-tagged murine monoclonal antibody (HRP-labeled) (cat # CW0285M, manufacturer: Kangji century) working solution was added to each well and incubated at 37 ℃ for 30 minutes. The plate was washed four times and the residual liquid was removed, 50. mu.l of TMB developing solution was added to each well, and after 10 minutes or 5 minutes of development in the dark at room temperature, 50. mu.L of stop solution was added to each well to terminate the development reaction. Immediately putting the ELISA plate into an ELISA reader, and reading the OD value of each hole of the ELISA plate by selecting the wavelength of 450 nm.

The data were analyzed using SoftMax Pro 6.2.1 software and plotted as a 4-parameter fit curve with antibody concentration as the abscissa and absorbance values as the ordinate, the results being shown in FIGS. 3 and 4. The results of the activity assays of 3H6, 3H6H1L1, 3H6H2L2, and 3H6H3L3 and 3H6H4L1 in competition with human IL-1R1(1-332) -his for binding to human IL-1. beta. -hFc are shown in tables 3 and 4.

Table 3: results of activity assays for competitive binding of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 to human IL-1R1(1-332) -his to human IL-1 β -hFc

Table 4: activity detection result of 3H6H4L1 and human IL-1R1(1-332) -his for competing binding to human IL-1 beta-hFc

The results show that:

3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 can effectively block the combination of the antigen human IL-1 beta-hFc and the receptor human IL-1R1(1-332) -his, the blocking efficiency presents a dose-dependent relation, and the competitive binding activity is superior to that of the Canakinsub which is a medicine on the market at the same target point.

Example 6: determination of the affinity constant of antibody 3H6H4L1 for human IL-1. beta

The affinity constant of the antibody and human IL-1 beta-his was determined using a Biacore molecular interaction instrument. PBST is used as a buffer solution, the antibody is fixed on the surface of a CM5 chip by adopting an amino coupling mode, and the fixed signal value is about 1000 RU. The antibody binds to human IL-1. beta. at a concentration of 1.56-25nM (two-fold gradient dilution), a flow rate of 30. mu.l/min, an association time of 120s, and a dissociation time of 600 s. Chip uses 3M MgCl₂Regeneration was carried out at a flow rate of 30. mu.l/min for a period of 30 s. Data acquisition was performed using Biacore Control 2.0 software and data analysis was performed using Biacore T200 Evaluation 2.0 software. The results are shown in table 5, fig. 5 and fig. 6.

Table 5: 3H6H4L1 and human IL-1 beta affinity constant detection result

Name of antibody

K_D(M)

ka(1/Ms)

SE(ka)

kd(1/s)

SE(kd)

Rmax(RU)

3H6H4L1

8.79E-11

1.44E+06

3.15E+03

1.27E-04

1.75E-07

112.49-122.37

Canakinumab

9.79E-11

5.24E+05

6.84E+02

5.13E-05

1.43E-07

76.61-86.37

The results show that: the affinity constant of 3H6H4L1 and human IL-1 beta is 8.79E-11M, and the affinity constant of Canakinumab and human IL-1 beta is 9.79E-11M, which indicates that 3H6H4L1 has stronger binding capacity with human IL-1 beta.

Example 7: cell biological Activity Studies of antibody 3H6H4L1

1.3H 6H4L1 assay for the cytological Activity of IL-1 beta blockade of IL-1 beta Induction of IL-6 secretion by MRC-5 cells

Human MRC-5 cells (purchased from cell center of Chinese academy of sciences) are digested and counted conventionally, and 7, 500 cells/well are inoculated into a flat-bottom 96-well plate and placed in a cell culture box for culture; after 24h (when cell growth reached 80% confluence), dosing treatments were performed: the antibody is set to 4 concentrations (0.003nM, 0.03nM, 0.3nM, 3nM), IL-1 beta (purchased from Nano Biological, cat # 10139-HNAE) is set to three concentrations (5pM, 50pM, 500pM), the IL-1 beta in the antibody group is set to 50pM (the antibody and IL-1 beta are incubated at 37 ℃ for 30min), and a blank control group and an isotype control group are set at the same time; after adding medicine, placing the mixture in a cell culture box for culturing for 24 hours; cell supernatants were collected and assayed using an IL-6ELISA Kit (purchased from David Biotechnology Co., Ltd., cat # 1110602). The results of the measurements are shown in FIG. 7and Table 6.

Table 6: inhibition of IL-1 beta induced release of IL-6 from MRC-5 cells by 3H6H4L1

The result shows that IL-1 beta can induce MRC-5 cells to secrete IL-6 in a gradient manner, 3H6H4L1 can obviously inhibit the induction effect of IL-1 beta on MRC-5 secretion release IL-6, and the obvious dose dependence relationship is realized. The combination shows that 3H6H4L1 can block IL-1 beta-mediated secretion of immune factor IL-6 by specifically binding IL-1 beta, and the blocking efficiency is equivalent to that of a targeted control antibody Canakinumab.

2.3H 6H4L1 blocks IL-1 beta activation of NF- κ B signaling pathway

The experiment detects the neutralization bioactivity of 3H6H4L1 blocking IL-1 beta to activate NF-kB signal channel by a fluorescent reporter gene method.

(1) Construction of 293T-NF-kB-LUC cells

Digesting 293T cells by pancreatin, and carrying out subculture; 2h before transfection, replacing an opti-DMEM culture medium; adding 500 mu L of opti-DMEM medium into a sterile EP tube, and then adding 3 mu g of plasmid pNF-kB-Luc2P-hygro (Akeso); 293T-NF kappa B-Luc cells were then constructed by infection with the transfection reagent PEI (cat # 23966, Polyscience) and expression vectors; after 8h of transfection, the fresh medium was replaced; 24h after transfection, Hygromycin B (Invitrogen, cat # 10687010) was added to a final concentration of 100. mu.g/mL for selection, with control wells 293T for untransfected plasmid. After 7-10 days, the cells in the control wells are completely dead, and the screened cells are harvested and cultured in an amplification way. Dosing was continued at 100. mu.g/mL. Obtaining the 293T-NF-kB-LUC stable transgenic cell strain.

(2) Neutralization bioactivity assay of 3H6H4L1 blocking IL-1 β activation of NF-. kappa.B signaling pathway 293T-NF-. kappa.B-Luc cells were routinely digested and seeded into 96-well plates at 20,000 cells per well. Cell patchAfter the completion of the wall, IL-1. beta. was added to a final concentration of 1.65ng/mL, and a blank was set. Antibodies, Canakinumab, 3H6H4L1, were added simultaneously, with 8 gradients per antibody, at final concentrations: 1600ng/mL, 800ng/mL, 200ng/mL, 50ng/mL, 12.5ng/mL, 3.125ng/mL, 0.3125ng/mL, 0.03125 ng/mL. After co-culturing for 4 hours, the supernatant was removed, 50. mu.L of PBS was added, and 50. mu.L of Bright-Glo was added^TMSubstrate (Promega, cat # E2620), reaction for 5min, and detection on machine.

The results are shown in FIGS. 8 and 9.

The results show that:

the IL-1 beta is dependent on a reporter gene detection system which takes 293T-NF kB-Luc as a working cell, and the IL-1 beta can activate an NF-kB signal channel of the 293T-NF kB-Luc cell in a dose-dependent manner, so that the expression of the reporter gene is started. The system can be used for evaluating the neutralization bioactivity of 3H6H4L1 on IL-1 beta. The research result shows that after being specifically combined with IL-1 beta, 3H6H4L1 can efficiently inhibit the activation of a signal channel mediated by the IL-1 beta, the neutralizing biological activity of 3H6H4L1 on the IL-1 beta is shown, the neutralizing activity EC50 is 0.0966nM, and the neutralizing activity ECS0 of Canakinguumab on the market at the same target point is 0.0801nM, and the activities of the two are equivalent.

While the preferred embodiments of the present invention have been illustrated and described, it will be understood by those skilled in the art that the present invention is not limited to the details of the embodiments shown and described, but is capable of numerous equivalents and substitutions without departing from the spirit of the invention and its scope is defined by the claims appended hereto.

Claims

1. Use of an anti-IL-1 beta antibody or antigen-binding fragment thereof, an antibody drug conjugate or a bispecific antibody for the treatment or prevention of a systemic inflammatory condition characterized by abnormal increased secretion of IL-1 beta or for the manufacture of a medicament for the treatment and/or prophylactic treatment or prevention of a systemic inflammatory condition characterized by abnormal increased secretion of IL-1 beta,

the light chain variable region of the antibody comprises: the amino acid sequences are respectively shown as SEQ ID NO: 20-SEQ ID NO: LCDR1-LCDR3 shown at 22,

preferably, the virus includes RNA viruses and DNA viruses;

preferably, the RNA virus comprises a virus of the family coronaviridae;

2. The use according to claim 1, wherein,

the amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10 and SEQ ID NO: 14 or a sequence identical to SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10 and SEQ ID NO: 14, a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; and

the amino acid sequence of the variable region of the light chain of the antibody is selected from SEQ ID NO: 4 and SEQ ID NO: 8. SEQ ID NO: 12 and SEQ ID NO: 16 or a sequence identical to SEQ ID NO: 4 and SEQ ID NO: 8. SEQ ID NO: 12 and SEQ ID NO: 16, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein,

the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 2 or a variant of SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 4 or a sequence identical to SEQ ID NO: 4, a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; or

The amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 6 or a sequence identical to SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 8 or a variant of SEQ ID NO: 8, a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; or

The amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 10 or a sequence identical to SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 12 or a variant of SEQ ID NO: 12, a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity; or

The amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 14 or a sequence identical to SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 16 or a sequence identical to SEQ ID NO: 16, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity.

4. The use according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is selected from Fab, Fab ', F (ab') 2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody or diabody.

5. The use according to any one of claims 1 to 3, wherein the antibody is administered at less than 10%^-5M, e.g. less than 10^-6M, less than 10^-7M, less than 10^-8M, less than 10^-9M or less than 10^-10K of M or less_DBinds to IL-1 β protein; preferably, said K_DMeasured by Biacore molecular interaction instrument.

6. The use according to any one of claims 1 to 3, wherein the antibody comprises non-CDR regions from a species other than murine, such as from a human antibody.

7. The use according to any one of claims 1 to 3, wherein the antibody has a constant region derived from a human antibody;

preferably, the constant region of the antibody is selected from the constant regions of human IgG1, IgG2, IgG3 or IgG 4.

8. The use of any one of claims 1 to 3, wherein said antibody has a heavy chain constant region of Ig gamma-1chain C region or Ig gamma-4chain C region and a light chain constant region of Ig kappa chain C region.

9. The use according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody produced by a hybridoma cell line LT010 deposited in China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC NO: C2018133.

10. the use of any one of claims 1 to 3, wherein the antibody drug conjugate comprises the antibody or antigen-binding fragment thereof and a small molecule drug; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug.

11. The use of claim 10, wherein the antibody or antigen-binding fragment thereof is linked to a small molecule drug via a linker; for example, the linker is a hydrazone bond, a disulfide bond, or a peptide bond.

12. The use of claim 10 or 11, wherein the antibody or antigen-binding fragment thereof is linked to a small molecule drug at a molar ratio; for example, the molar ratio is 1: (2-4).

13. The use of any one of claims 1 to 3, wherein the bispecific antibody comprises a first protein functional region and a second protein functional region, wherein:

the first protein functional region targets IL-1 beta,

the second protein functional region targets a target other than IL-1 β (e.g., IL-17A);

wherein the first protein functional region is the antibody or antigen-binding fragment of any one of claims 1-9;

preferably, the bispecific antibody is an IgG-scFv format;

preferably, the first protein functional region is an antibody of any one of claims 1-9 and the second protein functional region is a single chain antibody; or

Preferably, the first protein functional region is a single chain antibody of claim 4 and the second protein functional region is an antibody.

14. The use of claim 13, wherein the first and second protein functional regions are linked directly or via a linker fragment;

15. The use of claim 13, wherein the first and second protein functional regions are independently 1, 2 or more than 2.

16. The use of any one of claims 13 to 15, wherein the single chain antibody is attached to the C-terminus of the heavy chain of the antibody.

17. The use according to any one of claims 1-16, wherein the dose of said antibody or antigen-binding fragment thereof, said antigen-drug conjugate or said bispecific antibody is an effective amount, preferably said effective amount is 0.001mg-1000mg, more preferably 0.001mg-900mg, 0.001mg-800mg, 0.001mg-700mg, 0.001mg-600mg, 0.001mg-500mg, 0.001mg-400mg, 0.001mg-300mg, 0.001mg-200mg, 0.001mg-100mg, most preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg, or said effective amount is 0.1-100mg/kg, preferably 1-90mg/kg, 1-80mg/kg, 1-70mg/kg, 1-60mg/kg, based on the body weight of the subject or patient, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1-20mg/kg or 1-10 mg/kg.

18. A method of treating and/or prophylactically treating and/or preventing systemic inflammation, comprising administering to a subject or patient in need thereof an effective amount of an antibody or antigen-binding fragment thereof, antibody drug conjugate or bispecific antibody as claimed in any one of claims 1 to 16,

preferably, the antibody or antigen-binding fragment thereof, antibody drug conjugate, or bispecific antibody is used alone or in combination with an effective amount of one or more antiviral drugs;

preferably, the antiviral drug is an antiretroviral drug or an interferon, preferably wherein: antiretroviral drugs are RNA-dependent RNA polymerase (RdRp) inhibitors, or nucleoside analogs, or peptoids, or strand transfer inhibitors of human immunodeficiency virus type 1(HIV-1) integrase (INSTI), or HIV protease inhibitors.

Preferably, the antiviral drug is an antiretroviral drug, preferably, the antiretroviral drug is faviravir (favipiravir), redevivir (Remdesivir), Beclabuvir, Saquinavir (Saquinavir), bervalvir (bicegravir), Lopinavir (Lopinavir), dolitegravir (Dolutegravir); more preferably, the chemotherapeutic agent is favipiravir;

more preferably, the effective amount of the antibody or antigen-binding fragment thereof, antibody drug conjugate or bispecific antibody is 0.001mg to 1000mg, more preferably 0.001mg to 900mg, 0.001mg to 800mg, 0.001mg to 700mg, 0.001mg to 600mg, 0.001mg to 500mg, 0.001mg to 400mg, 0.001mg to 300mg, 0.001mg to 200mg, 0.001mg to 100mg, most preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg, or the effective amount of the antibody or antigen-binding fragment thereof, antibody drug conjugate or bispecific antibody is 0.1 to 100mg/kg, preferably 1 to 90mg/kg, 1 to 80mg/kg, 1 to 70mg/kg, 1 to 60mg/kg, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1-20mg/kg or 1-10 mg/kg;

more preferably, the effective amount of the antiviral drug is 100-2400mg, preferably 100-2300 mg, 100-2200 mg, 100-2100 mg, 100-2000 mg, 100-1900 mg, 100-1800 mg, 100-1700 mg, 100-1600 mg, 100-1800 mg, more preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, or the effective amount of the antiviral drug is 0.1-100mg/kg, preferably 1-90mg/kg, 1-80mg/kg, 1-70mg/kg, 1-60mg/kg, 1-50mg/kg, 100mg-1800 mg/mg, 100mg-1800 mg/kg, 100mg/kg, 100 mg/400 mg/kg, 500 mg-1800mg, 100mg/kg, 1-60mg/kg, 1000 mg/kg, 100mg/kg, 1800 mg/kg, 100 mg/g, 100 mg/1800 mg/g, 100 mg/or 1800 mg/g, 100 mg/or 1800 mg/g, 100 mg/or 1800 mg/g, 100 mg/g, or 1800 mg/g, or 1600 mg/g, or 1800 mg/g, or 1000 mg/g, 100 mg/g, or 1800 mg/g, or 1600 mg/g, 100 mg/or 100 mg/g, or 100 mg/or 1000 mg/g, or 100 mg/g, 100 mg/or 100 mg/g, or 100 mg/g, or 1-1800 mg/g, 1-40mg/kg, 1-30mg/kg, 1-20mg/kg or 1-10 mg/kg.