EA040391B1 - HUMANIZED ANTI-IL-1R3 ANTIBODIES - Google Patents

Description

The field of technology to which the invention belongs

The invention relates to humanized aHTu-IL-1R3 antibodies and their use.

State of the art

Interleukin 1 receptor accessory protein (IL1RAP), also referred to as IL1R3, is a co-receptor for the interleukin-1 type 1 receptor (IL1R1) and is essential for IL-1 signaling. Upon binding to IL-1, IL-1R1 binds to IL-IRAcP to form a functional signaling receptor complex that stimulates NFkB activity.

IL-33, its ST2 receptor and IL-1RAcP also form a complex (IL-33/ST2/IL-1RAcP) with similar NFkB activation activity as the IL-1e/IL-1R1/IL-1RAcP complex. IL-36 (IL36α (IL-1F6), IL-36e (IL-1F8) and IL-36y (IL-1F9)), their receptors IL-36R and IL-1RAcP also form a complex (IL-36/IL-36R /IL-1RAcP) with similar NFkB activation activity as the IL-1e/IL-1R1/IL-1RAcP complex.

Human NF-kB is an important regulator of the expression of several genes responsible for inflammation, immune response, and apoptosis. Therefore, dysfunction of NFkB is one of the causes of pathology in various diseases, including autoimmune diseases, degenerative diseases, inflammation, and malignant tumors. For example, in the treatment of osteoarthritis (OA), the NF-kB pathway is an important target. Thus, agents that regulate the human NFkB pathway through downregulation of the signaling activity of the human IL-1R1/IL-1RAcP complex are potential therapeutic agents useful in the treatment of various human diseases. In particular, high affinity neutralizing antibodies would be excellent therapeutic agents.

For more than 15 years, attempts have been made to develop functional monoclonal antibodies to human IL1RAcP. However, many attempts have failed and existing antibodies still suffer from various shortcomings.

From WO 199623067 an anti-IL-1RAcP antibody is known that specifically binds to a mouse IL-1 receptor accessory protein. Examples 15 and 16 describe attempts to obtain anti-human IL-1RAcP antibodies that neutralize the biological activity of IL-1. However, WO 199623067 does not present such an antibody and Example 16, describing the IL-1 induced assay of IL-6, is only hypothetical. In J. Immunol. 1998; 160:3170-3179, Do-Young Yoon D.-Y. and Charles A. Dinarello CA describe polyclonal antibodies to mouse IL-1RAcP domains II and III that inhibit but do not bind to IL-1e. However, at higher concentrations of IL-1e (1000 pg/ml), this polyclonal antiserum did not block D10S cell proliferation. (D10S is a subclone of the mouse T-helper lineage clone D10.G4.1 that proliferate at subfemtomolar (attomolar) levels of IL-1e or α in the absence of mitogens, see Orencole S.F. and Dinarello C.A.; Cytokine 1 (1989) 14-22) . Jaras M. et al., PNAS 107 (2010) 16280-16285 describe the use of a rabbit polyclonal anti-IL1RAcP antibody KMT-1 to kill CML stem cells. This antibody induces rabbit Fc-induced ADCC in an IL1RAcP-independent manner. Jaras et al. it is believed that potential future therapeutic antibodies targeting IL1RAP will have low toxicity in normal hematopoietic cells. Polyclonal rabbit antibodies to mouse IL-lRAcP are also mentioned in Do-Young Yoon and Charles A. Dinarello, Journal of Biochemistry and Molecular Biology, Vol. 40, no. 4, July 2007, pp. 562-570.

WO 2002064630 also relates to IL-1RAcP and its use, but does not describe anti-IL1RacP antibodies. WO 2004022718 and WO 2009120903 only talk about the theoretical possibility of obtaining antibodies to CSF1R, IL13RA1, IL1RAP, IFNAR1, IL5R, INSR, IL1RL1, LTK and TACSTD1 in accordance with the prior art. However, this document also does not describe antibodies to IL-1RAcP. WO 2011021014 and WO 2012098407 (US 20140017167) relate to KMT-1 antiserum based on polyclonal rabbit anti-human IL-1RAcP (see Jaras et al., 2010) and its use. WO 2014100772 describes an anti-IL-1RAcP antibody that binds to IL-1RAcP. However, this document lacks activity to inhibit any functional signaling receptor complex (like IL-1e/IL-1R1/IL-1RAcP) stimulating NFkB activity. US 6280955 relates to IL-1RAcP and its use, but this document does not disclose anti-IL-1RAcP antibodies. US 7390880 mentions the N-terminal fragment of IL1RAcP but does not describe anti-IL-1RAcP antibodies.

WO 2004100987 relates to the use of an interleukin-1 (IL-1) receptor antagonist for the preparation of a medicament for the treatment of neointimal hyperplasia, and to the use of an IL-1 antagonist to treat neointimal hyperplasia. As such an antagonist, an anti-IL-1RAcP antibody is proposed, which, however, is not described. US 2003026806 relates to antibodies that bind IL-1. WO 2002064630 relates to an IL-1 antagonist and IL-1RAcP protein. While this document mentions the use of IL-1RAcP to screen for IL-1RAcP antagonists, no such method or antagonist is disclosed.

This indicates the extreme difficulty in determining high-affinity and highly specific monoclonal antibodies with strong neutralizing activity against IL-1R3. The present invention encompasses a humanized anti-IL-1R3 antibody that is high affinity and specific for IL-1R3, has strong IL-1R3 neutralizing activity, and has improved stability.

The essence of the invention

The invention relates to a humanized anti-IgG1 _LALA antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, or a polypeptide containing at least a portion of said antibody that is sufficient to provide IL-1R3-binding specificity.

The specified antibody suppresses the activity of NFkB induced by IL-1R3. Thus, it inhibits NFkB activity stimulated by IL-1a, IL-1β, IL-33 and/or IL-36.

The present invention also relates to an antibody of the invention for use in the treatment of a disease mediated by IL-1R3.

The invention also relates to a method for treating a J-Sch3-mediated disease in a patient, comprising administering to the patient a pharmaceutically effective amount of said antibody.

The present invention also relates to a pharmaceutical composition containing a pharmaceutically acceptable carrier and a therapeutically effective amount of said antibody.

Definitions.

The term rabbit according to the invention means an animal belonging to the taxonomic group Lagomorpha (hares), which includes the families (hares and rabbits) and Ochotonidae (pikas, pikas), preferably in the genus Oryctolagus (rabbits).

The term antibody encompasses various structural forms of antibodies, including, without limitation, whole antibodies and antibody fragments, provided that they exhibit the properties in accordance with the invention.

The term rabbit monoclonal antibody according to the invention means a monoclonal antibody obtained by immunization of a rabbit and isolated from a cell of this rabbit that produces an antigen, and also means an antibody that is further modified, preferably a humanized antibody, a chimeric antibody, a fragment thereof, or an antibody further genetically modified and obtained by a recombinant method, provided that it retains its characteristic properties in accordance with the invention. Preferably, the antibody is derived from a B cell or rabbit hybridoma cell of said rabbit.

The term antibody producing cell according to the invention means a rabbit B cell that produces antibodies, preferably a B cell or a rabbit hybridoma cell.

Native antibodies are usually heterotetrameric glycoproteins consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a single covalent disulfide bond, with the heavy chains of different immunoglobulin isotypes having different numbers of disulfide bonds. Thus, each heavy and light chain has regularly spaced interchain disulfide bridges. Each heavy chain contains at one end a variable domain (VH) followed by a number of constant domains. Each light chain contains a variable domain (VL) at one end and a constant domain at the other end. The light chain constant domain is aligned with the first heavy chain constant domain, and the light chain variable domain is aligned with the heavy chain variable domain. It is generally accepted that specific amino acid residues form the boundary between the variable domains of the light and heavy chains.

Percentage (%) amino acid sequence identity relative to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after sequence alignment and, if necessary, the introduction of gaps (gaps) to achieve maximum percent identity sequences, disregarding any conservative substitutions within sequence identity. Alignment to determine amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using the available computer programs BLAST, BLAST-2, ALIGN or Megalign (DNASTAR).

The term VL (or VH) region has the same meaning as the VL (or VH) domain.

The terms Fc receptor or FcR in accordance with the invention refer to a human receptor that binds to the Fc fragment of an antibody. FcRs bind to IgG antibodies and include receptor subclasses FcyRI, FcyRII and FcyRIII, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB (inhibiting receptor), which have similar amino acid sequences that differ mainly in cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains in its cytoplasmic domain an inhibitory immunoreceptor tyrosine-based (ITIM) motif (reviewed by M. Daeron, Annu Rev. Immunol 15:203-234 (1997)). FcRIIIA (CD16a) mediates ADCC. An overview of FcR species can be found in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and Haas et al., J. Lab. CHn. Med. 126:330-41 (1995). These and all other FcRs are covered by the term FcR. The term therefore includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al. J. Immunol. 117:587 (1976) and Kim et al. J. Immunol. 24:249 (1994)) and mediates slower catabolism, thus providing a longer half-life.

As used herein, the term antibody effector function(s) or effector function refers to an IgG Fc effector domain(s) (eg, an immunoglobulin Fc fragment). Such a function may be effective, for example, by binding the Fc effector domain(s) to an Fc receptor on an immune cell with phagocytic or lytic activity, or by binding the Fc effector domain(s) to components of the complement system. Typical effector functions are ADCC, ADCP and CDC.

An antibody fragment refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, without limitation, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabody; linear antibodies;

single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments.

An antibody that binds to the same epitope as a reference antibody refers to an antibody that blocks the reference antibody from binding to its antigen in a competition assay by 50% or more and, conversely, a reference antibody blocks said antibody from binding to its antigen in a competition assay by 50% or more. 50% or more. This application provides an example of a competitive analysis.

The term antibody-dependent cell-mediated cytotoxicity and ADCC refers to a cell-mediated response in which non-specific cytotoxic cells expressing an FcR (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize the bound antibody on the target cell and then cause lysis of the target cell. The main cells mediating ADCC, NK cells, only express FcyRIII, while monocytes express FcyRI, FcyRII, and FcyRIII.

The term antibody-dependent cellular phagocytosis and ADCP refers to the process by which antibody-coated cells are internalized, in whole or in part, by phagocytic immune cells (eg, macrophages, neutrophils, and dendritic cells) that bind to the Fc portion of an immunoglobulin.

C1q is a polypeptide that includes an immunoglobulin Fc binding site. C1q together with two serine proteases, C1r and C1s, form the C1 complex, the first component of the complement-dependent cytotoxic (CDC) pathway. Human C1q is commercially available from, for example, Quidel, San Diego, Calif.

An antibody class refers to the type of constant domain or constant region that a heavy chain has. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, y, and μ, respectively.

An effective amount of an agent, such as a pharmaceutical composition, refers to the effective amount in doses and periods of time necessary to achieve the desired therapeutic or prophylactic result.

The term Fc fragment is used to refer to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc fragments and variant Fc fragments. Unless otherwise indicated herein, the numbering of amino acid residues in an Fc fragment or constant region follows the EC numbering system, also referred to as the EU index, as described by Rabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

A variant Fc fragment contains an amino acid sequence that differs from a native Fc fragment or a wild-type Fc fragment by virtue of at least one amino acid modification, as defined in this application. The term Fc variant as used in this application refers to a polypeptide containing a modification in the Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications in their composition. Thus, for example, P329G is an Fc variant with a proline substitution with glycine at position 329 relative to the parent Fc polypeptide, numbering according to the EU index. The wild-type amino acid identity may not be established, in which case the above variant

- 3 040391 is designated as P329G. For all provisions discussed in the present invention, the numbering corresponds to the EU-index. The EU index or Kabat EU index or EU numbering scheme refers to the EU numbering of antibodies (Edelman, et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85, incorporated herein in its entirety. completeness as a reference). The modification may be an addition, deletion or substitution. Substitutions may include naturally occurring amino acids and non-naturally occurring amino acids. Options may include non-naturally occurring amino acids. Examples can be found in US Pat. No. 6,586,207; W098/48032; WO 03/073238; US 2004/0214988A1; WO 05/35727 A2; WO 05/74524 A2; Chin J.W. et al., Journal of the American Chemical Society 124 (2002) 9026-9027; Chin, J.W. and Schultz P.G., Chem. BioChem. 11 (2002) 1135-1137; Chin J.W. et al., PICAS United States of America 99 (2002) 11020-11024; and Wang L., Schultz P.G., Chem. (2002) 1-10, all of which are incorporated herein in their entirety by reference.

The term polypeptide containing an Fc fragment refers to a polypeptide, such as an antibody or immunoadhesin (see below for definitions), that contains an Fc fragment.

The terms Fc receptor or FcR are used to describe a receptor that binds to the Fc portion of an antibody. An FcR that binds to an IgG antibody (γ receptor) includes the FcyRI, FcyRII, and FcyRIII subclass receptors, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB (inhibiting receptor), which have similar amino acid sequences that differ mainly in cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains in its cytoplasmic domain an inhibitory immunoreceptor tyrosine-based motif (ITAM) (reviewed by Daeron M., Annu Rev. Immunol., 15 (1997) 203-234). An overview of FcR species can be found in Ravetch, and Kinet, Annu. Rev. Immunol. 9 (1991) 457-492; Capel et al. Immunomethods 4 (1994) 25-34; and de Haas et al., J. Lab. Clin. Med. 126 (1995) 330-41. The term FcR encompasses other FcRs, including the receptors defined below. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al. J. Immunol. 117:1976 (587) and Kim et al. J. Immunol. 24:1994 (249)).

An IgG Fc ligand used herein preferably means a polypeptide derived from any organism that binds to an IgG Fc fragment of an antibody to form an Fc/Fc ligand complex. Fc ligands include, without limitation, FcyR, FcyR, FcyR, FcRn, Clq, C3, mannan-binding lectin, mannose receptor, staphylococcal A protein, streptococcal G protein, and viral FcyR. Fc ligands also include Fc receptor (FcRH) homologues, which are a family of Fc receptors that are homologous to FcyR (Davis, et al., Immunological Reviews 190 (2002) 123-136, incorporated herein in its entirety. as reference). Fc ligands may include as yet undiscovered molecules that bind to Fc. Specific IgG Fc ligands are FcRn and Fcγ receptors. By Fc ligand as used herein is meant a molecule, preferably a polypeptide, derived from any organism and which binds to an Fc fragment of an antibody to form an Fc/Fc ligand complex.

The term Fc γ receptor, FcyR or Fc-yR, as used herein, means a member of the family of proteins that bind to the Fc fragment of an IgG antibody and are encoded by the FcyR gene. In humans, this family includes, without limitation, FcyRI (CD64), including the FcyRIA, FcyRIB, and FcyRIC isoforms; FcyRII (CD32), including isoforms FcyRIIA (including allotypes H131 and R131), FcyRIIB (including FcyRIIB-1 and FcyRIIB-2) and FcyRIIc; and FcyRIII (CD16), including the isoforms FcyRIIIA (including allotypes VI58 and F158) and FcyRIIIb (including allotypes FcyRIIB-NA1 and FcyRIIB-NA2) (Jefferis et al., Immunol. Lett. 82 (2002) 57-65 , incorporated herein in its entirety by reference), as well as any undiscovered isoforms or allotypes of human FcyR. The FcyR may be from any organism, including, without limitation, humans, mice, rats, rabbits, and monkeys. Mouse FcyRs include, without limitation, FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and FcyRIII-2 (CD16-2), as well as any undiscovered mouse FcyR isoforms or allotypes.

FcRn or neonatal Fc receptor as used herein means a protein that binds to the Fc fragment of an IgG antibody and is encoded at least in part by the FcRn gene. The FcRn may be from any organism, including, without limitation, humans, mice, rats, rabbits, and monkeys. As is known in the art, a functional FcRn protein contains two polypeptides, often referred to as a heavy chain and a light chain. The light chain is β-2-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise indicated, an FcRn or FcRn protein refers to a FcRn heavy chain complex with β-2-microglobulin.

An immunoconjugate means an antibody conjugated to one or more cytotoxic agents such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin, another antibody, or a radioactive isotope.

Antibody fragments comprise a portion of a full length antibody, preferably its variable regions, or at least its antigen binding site. Examples of antibody fragments include diabodies, Fab fragments, and single chain antibody molecules. ScFv antibodies are described, for example, in Huston J.S., Methods in Enzymol. 203 (1991) 46-88.

The term monoclonal antibody or composition of monoclonal antibodies used in this application refers to the production of antibody molecules of a single amino acid composition.

The term humanized antibody or humanized version of an antibody refers to antibodies in which the human variable region has been modified to contain the CDRs of an antibody of the invention. In a preferred embodiment, the VH CDR and VL CDR are grafted onto a human antibody framework region to produce a humanized antibody. See, for example, Riechmann L. et al., Nature 332 (1988) 323-327; and Neuberger M.S. et al., Nature 314 (1985) 268270. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. Human antibody sequences may be naturally occurring human antibody sequences. Human heavy and light chain variable framework regions are listed, for example, in Lefranc M.-P., Current Protocols in Immunology (2000) - Appendix IP A.1P.1-A.1P.37 and are available through IMGT, the international information system ImMunoGeneTics ® (http://imgt.cines.fr) or at: http://vbase.mrc-cp.cam.ac.uk.

The terms specific binding, against a target (to a target) or antibody against (to) a target, as used in this application, refer to the binding of an antibody to a corresponding antigen (target) measured by an ELISA method, where said ELISA preferably comprises applying the corresponding antigen to a solid support, adding said antibody under conditions allowing formation of an immune complex with the corresponding antigen or protein, detecting said immune complex by measuring an optical density (OD) value with a secondary antibody that binds to an antibody of the invention, and using peroxidase mediated staining.

The term antigen according to the invention refers to an antigen used for immunization or to a protein containing said antigen as part of a sequence. For example, a fragment of the extracellular domain of a protein (eg, the first 20 amino acids) can be used for immunization, and for detection/analysis, etc. the extracellular domain of this protein or the full-length protein can be used.

The term specifically binding or specifically recognizable as used herein means that an antibody exhibits a marked affinity for an antigen and preferably has no significant cross-specificity.

Marked binding affinity includes binding with an affinity of at least 10exp7M ^-1 , in particular at least 10exp8M ^-1 , more specifically at least 10exp9M ^-1 or more specifically at least 10exp10M ^-1 .

An antibody that does not show significant cross-specificity is an antibody whose unwanted binding to another protein is not detectable. An antibody specific for an epitope according to the invention will not, for example, cross-react significantly with other epitopes for IL-1R3. Specific binding can be determined according to any means recognized in the art for determining such binding, for example, using competitive binding assays (eg, ELISA).

All protein terms used in this application refer to human proteins. If we are talking about a protein from another species, this is indicated explicitly.

The term IL-1a as used in this application refers to human IL-1 (UniProtKB P01583). The term IL-1e as used in this application refers to human ΓΕ-1β (UniProtKB P01584). IL-1 stimulates thymocyte proliferation by inducing IL-2 release, B cell maturation and proliferation, and fibroblast growth factor activity. IL-1 proteins are involved in the inflammatory response, referred to as endogenous pyrogens (UniProtKB).

The term IL-33 as used in this application refers to human IL-33 (UniProtKB O95760), a cytokine that binds to and signals through the IL1RL1/ST2 receptor, which in turn activates NF-k-B and MAPK signaling pathways in target cells (UniProtKB).

The term IL-36 as used herein refers to human IL-36a (UniProtKB Q9UHA7), IL-36e (UniProtKB Q9NZH7) and or IL-36y (UniProtKB Q9NZH8). IL-36 are cytokines that bind to the IL1RL2/IL-36R receptor and signal through this receptor, which in turn activates NF-k-B and MAPK signaling pathways in target cells associated with the pro-inflammatory response. IL-36 appears to be involved in the cutaneous inflammatory response by acting on keratinocytes, dendritic cells, and indirectly on T cells to stimulate tissue infiltration, cell maturation, and cell proliferation (UniProtKB).

As used herein, the term NFkB refers to the human nuclear factor NF-k-B, which consists of a p105 subunit (P19838) and a p100 subunit (Q00653).

NFkB inhibition is measured in accordance with the invention as suppression of NFKB-dependent luciferase gene expression in human cells. Such methods are, for example, those described in Windheim M. et al., Mol. cell. Biol. 28 (2008) 1783-1791; Huang J. et al. PNAS USA 94 (1997) 12829-12832; Xiaoxia L. et al., Mol. Cell, Biol. 19 (1999) 4643-4652. When it comes to mice

NFkB, this is explicitly stated.

The variable region (or domain) of an antibody of the invention (light chain variable region (VL), heavy chain variable region (VH)) as used herein means each of a pair of light and heavy chain regions that are directly involved in antibody binding with an antigen. The variable regions of the light and heavy chains have the same general structure, and each region contains four structural (FR) regions, the sequences of which are largely conserved, associated with three complementarity-determining regions, CDRs. An antibody according to the invention comprises a VH region and a VL region, or portions thereof, both of which together are sufficient for specific binding to the respective antigen.

The term antigen-binding portion of an antibody, as used herein, refers to the amino acid residues of an antibody that are responsible for binding the antigen. The antigen-binding portion of an antibody preferably contains amino acid residues from complementarity-determining regions or CDRs. CDR sequences were determined according to Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may include fewer amino acids or additional amino acids corresponding to a truncation or insertion in the FR or CDR of the variable region.

The phrases parenteral administration and parenterally administered as used in this application mean methods of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

The term cancer as used herein can be, for example, lung cancer, non-small cell lung cancer, bronchioloalveolar lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer. , rectal cancer, anal cancer, stomach cancer, gastrointestinal cancer, colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, cancer esophagus, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra cancer, penis cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, carcinoma renal pelvis, mesothelioma, hepatocellular carcinoma, bile duct cancer, malignant tumors of the central nervous system (CNS), spinal tumors, glioma brainstem, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory variants of any of the above cancers, or a combination of one or more of the above cancers.

Detailed description of the invention

Humanization of antibodies derived from immunized mammals is required as a developmental feature when said antibodies are intended for use as therapeutic agents in humans. The goal of humanization is to preserve, as far as possible, the original characteristics (binding specificity, activity) of an antibody, while reducing the immunological side effects that can occur when non-humanized antibodies derived from another organism are administered to humans. The present invention is based on the known humanization strategy by CDR grafting. In the present application, a large number of active humanized antibodies were created in parallel, and the best candidates were selected for further evaluation.

As stated in the introduction of the present application, it is extremely difficult to identify monoclonal antibodies with high affinity, high specificity and strong neutralizing activity against IL-1R3. The present invention encompasses a humanized anti-[L-1R3 antibody with high affinity and specificity for IL-1R3, potent IL-1R3 neutralizing activity and improved stability.

In particular, the present invention relates to a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to provide IL-1R3-binding specificity, comprising:

a) a heavy chain variable region (VH) containing complementarity determining regions containing CDR-H1, CDR-K2 and CDR-H3, where the CDR-H1 region contains an amino acid sequence selected from the group SEQ ID

- 6 040391

NO:69-85, where the CDR-H2 region contains an amino acid sequence selected from the group of SEQ ID NO:86-102, and where the CDR-H3 region contains an amino acid sequence selected from the group of SEQ ID NO:103-119; And

b) a light chain variable region (VL) containing complementarity determining regions containing CDR-L1, CDR-L2 and CDR-L3, where the CDR-L1 region contains an amino acid sequence selected from the group of SEQ ID NO:120-136, where the region The CDR-L2 contains an amino acid sequence selected from the group of SEQ ID NOs: 137-153, and where the CDR-L3 region contains an amino acid sequence selected from the group of SEQ ID NOs: 154-170 and 175.

In one embodiment, an antibody of the invention contains a substitution at position 2 of CDR-L3. Said substitution may be a substitution of cysteine for serine.

Moreover, the present invention includes a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to confer IL-1R3 binding specificity, wherein the antibody contains a heavy chain variable region (VH) that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90% identical to a VH region selected from the group consisting of VH regions shown in SEQ ID NO:1-34 and SEQ ID NO:173.

In one embodiment, a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to confer IL-1R3 binding specificity, contains a heavy chain variable region sequence ( VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group of VH sequences according to the invention.

In some embodiments, a VH sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, when this antibody retains the ability to specifically bind, according to the invention, with the corresponding antigen.

The present invention also relates to a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of the specified antibody that is sufficient to provide IL-1R3-binding specificity, and the antibody contains a light chain variable region ( VL) that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90% identical to a VL region selected from the group consisting of VL regions, shown in SEQ ID NOS: 35-68 and 174.

In another embodiment, a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to provide IL-1R3 binding specificity, contains a light chain variable region (VL), having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the VL sequences of the invention.

In some embodiments, a VL sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, with This antibody retains the ability to specifically bind to the corresponding antigen.

In some embodiments, a total of 1 to 10 amino acids are replaced, inserted, and/or deleted in said VL sequences. In some embodiments, substitutions, insertions, or deletions occur in regions outside of the CDR (ie, in the FR). The present invention also relates to the affinity of mature antibodies, which can be obtained by methods known in this field. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by shuffling the VH and VL domains.

Random mutagenesis of CDR and/or scaffold residues is described by: Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 1 54(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992) and WO2010108127.

In some embodiments, a total of 1 to 10 amino acids are replaced, inserted, and/or deleted in each of said VH or VL sequences. In one embodiment, an antibody of the invention contains a substitution at position 90 of the VH sequence, or

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VL. Preferably, the amino acid at position 90 is replaced by serine. This substitution is preferably at position 90 of the light chain variable region (VL). In a preferred embodiment, the cysteine is at position 90 in SEQ ID. NO: 62 is replaced by serine. However, the antibodies of the present invention are not limited to amino acid substitution at position 90, and may contain any substitution, deletion, or insertion that results in a functional antibody having the properties of an antibody of the invention. Therefore, the VL and VH sequences of the present invention may also contain additional mutations at different positions.

In some embodiments, the substitutions, insertions, or deletions are in regions outside of the CDR (ie, in the FR).

In other embodiments, the substitutions, insertions, or deletions are in the CDRs. In one preferred embodiment, an antibody of the invention contains a substitution at position 2 of CDR-L3. Preferably, this is the replacement of cysteine with serine. In one embodiment, said substitution is in SEQ ID NO:164.

The present invention also includes a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to confer IL-1R3 binding specificity, wherein the antibody contains a heavy chain variable region (VH ) containing an amino acid sequence selected from the group of SEQ ID NOs: 1-34 and 173.

Preferably the heavy chain variable region (VH) sequence is SEQ ID NO: 1, alternatively SEQ ID NO: 2 or SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or alternatively SEQ ID NO: 34 or 173.

Moreover, the present invention relates to a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to provide IL-1R3 binding specificity, and the antibody contains a light variable region chain (VL) containing an amino acid sequence selected from the group of SEQ ID NOs: 35-68 and 174.

Even more preferred is the light chain variable region (VL) sequence which is SEQ ID NO: 35 or SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 , SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or alternatively SEQ ID NO: 68 or 174.

A humanized antibody of the invention that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to provide GE-A3 binding specificity, also contains a VH region and a VL region containing the respective regions CDR1, CDR2 and CDR3 of an antibody selected from the group consisting of: MAB-15-0139, MAB-15-0106MAB-15-0108, MAB-15-0110, MAB-15-0117, 15-0140, MAV-15-0115, MAV-15-0125, MAV-15-0119, MAV-15-0109, MAV-15-0097, MAV-15-0135, MAV-15-0133, MAV-15- 0107, MAV-15-0128, MAV-15-0116, MAV-16-0004, MAV-16-0009, MAV-16-0028, MAV-16-0031, MAV-16-0043, MAV-16-0049, MAV-16-0045, MAV-16-0040, MAV-16-0036, MAV-16-0046, MAV-16-0030, MAV-16-0021, MAV-16-0019, MAV-16-0015, MAV- 16-0027, MAV-16-0048, MAV-16-0041, MAV-16-0149, MAV-16-0150.

In one embodiment, a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to provide IL-1R3 binding specificity, comprises SEQ ID NOs: 1 and 35 or SEQ ID NOs: 2 and 36. An antibody of the invention may comprise SEQ ID NOs: 3 and 37 or SEQ ID NOs: 4 and 38 or SEQ ID NOs: 5 and 39 or SEQ ID NOS: 6 and 40 or SEQ ID NOs: 7 and 41 or SEQ ID NOs: 8 and 42 or SEQ ID NOs: 9 and 43 or SEQ ID NOs: 10 and 44 or SEQ ID NOs: 11 and 45 or SEQ ID NOs: 12 and 46. Alternatively, an antibody of the invention comprises SEQ ID NOs: 13 and 47, or SEQ ID NOs: 14 and 48, or SEQ ID NOs: 15 and 49, or SEQ ID NOs: 16 and 50, or SEQ ID NOs: 17 and 51 , or SEQ ID NOs: 18 and 52, or SEQ ID NOs: 19 and 53, or SEQ ID NOs: 20 and 54, or SEQ ID NOs: 21 and 55, or SEQ ID NOs: 22 and 56, or SEQ ID NOs : 23 and 57 or SEQ ID NOs: 24 and 58 or SEQ ID NOs: 25 and 59 or SEQ ID NOs: 26 and 60 or SEQ ID NOs: 27 and 61.

Alternatively, an antibody of the invention comprises SEQ ID NOS: 28 and 62, or SEQ ID NOS: 29 and 63, or SEQ ID NOS: 30 and 64, or SEQ ID NOS: 31 and 65, or SEQ ID NOS: 32 and 66, or SEQ ID NOs: 33 and 67, or SEQ ID NOs: 34 and 68.

Most preferably, a humanized antibody that specifically binds to IL-8 040391

1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to provide IL-R3 binding specificity, contains the constant region sequences CR-H (SEQ ID NO: 172) and CR-L (SEQ ID NO : 171) and a VH region selected from the group of SEQ ID NOs: 1-34 and 173, and a VL region selected from the group of SEQ ID NOs: 35-68 and 174.

A humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to confer IL-1R3 binding specificity, also contains CR-H constant region sequences (SEQ ID NO. 172) and CR-L (SEQ ID NO. 171) and VH and VL regions containing the respective CDR1, CDR2 and CDR3 regions of an antibody selected from the group consisting of: MAB-15-0139, MAB-15-0106, MAB- 15-0108, MAV-15-0110, MAV-15-0117, MAV-15-0121, MAV-15-0140, MAV-15-0115, MAV-15-0125, MAV-15-0119, MAV-15- 0109, MAV-15-0097, MAV-15-0135, MAV-15-0133, MAV-15-0107, MAV-15-0128, MAV-15-0116, MAV-16-0004, MAV-16-0009, MAV-16-0028, MAV-16-0031, MAV-16-0043, MAV-16-0049, MAV-16-0045, MAV-16-0040, MAV-16-0036, MAV-16-0046, MAV- 16-0030, MAV-16-0021, MAV-16-0019, MAV-16-0015, MAV-16-0027, MAV-16-0048, MAV-16-0041, MAV-16-0149 and MAV-16- 150.

In accordance with the preferred therapeutic use of the antibodies of the invention, the effector functions (such as ADCC) of the antibodies of the invention are reduced or absent. Unlike other prior art antibodies such as C7AN04 (as in WO 2015/132602 A1), the antibodies of the invention do not have increased effector functions and do not induce ADCC.

Preferably, the humanized antibodies of the invention show reduced or absent Fcγ receptor signaling.

Therefore, the invention also relates to a humanized antibody that contains at least amino acid substitutions in L234A and L235A of a human IgG1 Fc fragment or S228P and L235E of a human IgG4 Fc fragment.

In one embodiment, the antibody is a humanized IgG1LALA antibody.

In one embodiment of the present invention, a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to provide IL-1R3 binding specificity, suppresses NFkB activity induced by IL -1R3.

In another embodiment, a humanized antibody of the invention that specifically binds to IL-1R3, or a fragment or derivative thereof containing at least a portion of said antibody that is sufficient to confer IL-1R3 binding specificity, binds to the same epitope, as an antibody selected from the group of antibodies MAB-15-0139, MAB-15-0106, MAB-15-0108, MAB-15-0110, MAB-15-0117, MAB-15-0121, MAB-15-0140, MAV-15-0115, MAV-15-0125, MAV-15-0119, MAV-15-0109, MAV-15-0097, MAV-15-0135, MAV-15-0133, MAV-15-0107, MAV- 15-0128, MAV-15-0116, MAV-16-0004, MAV-16-0009, MAV-16-0028, MAV-16-0031, MAV-16-0043, MAV-16-0049, MAV-16- 0045, MAV-16-0040, MAV-16-0036, MAV-16-0046, MAV-16-0030, MAV-16-0021, MAV-16-0019, MAV-16-0015, MAV-16-0027, MAV-16-0048, MAV-16-0041, MAV-16-0149 and MAV-16-150.

The antibodies of the invention have the advantage of being highly efficient in binding to their target. They have a high binding capacity for their antigen, IL1R3, but not for other receptors. The binding properties of the antibodies were studied by biochemical enzyme-linked immunosorbent assay (ELISA) and cell binding assay (flow cytometry), which are shown in FIG. 2, 6 and 7.

Preferred antibodies of the invention show a half maximum effective concentration (EC ₅₀ ) of less than 30 ng/ml, preferably less than 20 ng/ml. In other embodiments, they showed an EC ₅₀ of less than 15, 10, or less than 5 ng/ml. Preferred antibodies of the invention showed an EC ₅₀ of 16.3 ng/ml in a biochemical ELISA experiment (see FIG. 7).

The antibodies of the invention also showed a very high level of binding to their antigen in experiments in which human IL1R3 was expressed in different cell lines, while the antibodies did not bind to cell lines that do not express IL1R3 (for example, NIH-3T3, see Fig. 5) .

In IL1R3 highly expressed SK-MEL-30 cell lines (see FIG. 6, Example 8), antibody EC ₅₀ is preferably less than 400 ng/ml, more preferably less than 350 ng/ml or less than 310 ng/ml.

In one preferred embodiment, the antibodies of the invention inhibit NFkB activity stimulated by IL-1a and/or IL-Sch. In FIG. 3, 4 and 8 show the strong inhibitory activity of the antibodies of the invention.

In one embodiment, a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to confer IL-1R3 binding specificity, suppresses

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NFkB activity stimulated by IL-1a.

In another embodiment, a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof containing at least a portion of said antibody that is sufficient to confer IL-1R3 binding specificity, inhibits NFkB activity stimulated by IL-1β.

It is preferred that the antibody of the invention inhibits IL-Ιβ-stimulated NFkB activity in HEK293T/17-FR cells with an EC ₅₀ value of less than 100 ng/ml, preferably less than 95, 85, 75, 65, 55, 45, 35, 25, 20 ng/ml, and most preferably less than 15 ng/ml (eg see FIG. 3).

It is also preferred that the antibody of the invention inhibits IL-1a stimulated NFkB activity in A549-NFkB-RE-Luc cells with an EC ₅₀ value of less than 1000 ng/ml, preferably less than 500, 300, 200 ng/ml and most preferably less than 100 ng/ml (e.g. see FIG. 8)

Also preferred is that the antibody of the invention suppresses NFkB activity stimulated by IL-1a in A549-NFkB-RE-Luc cells with an EC ₅₀ value of less than 700 ng/ml, preferably less than 600, 300, 200, 100 ng/ ml and most preferably less than 50 ng/ml (for example, see Fig. 8).

The invention also relates to a humanized antibody that inhibits NFkB activity stimulated by a complex selected from the group consisting of: IL-1e/IL-1R1/IL-1RAcP, IL-1a/IL1R1/IL-1RAcP, IL-33/ST2/ IL-1RACP and/or IL-36/IL-36R/IL-IRAcP.

Moreover, the humanized antibody of the invention suppresses NFkB expression in lysates of A549-NFkB-RE-Luc cells (Steady-Glo™ Luciferase Assay System; Promega, Cat No. E2510) stimulated with 0.1 ng/mL human IL-1 a , IL-1e, human IL-33 and/or IL-36 (molecular weight see UniProtKB/Swiss-Prot), at a concentration of 10 μg/ml (rabbit IgG isotype with a molecular weight of 150 kDa) by 50% or more, preferably 70% or more, preferably 80% or more, or preferably 90% or more, or more preferably 95% or more, relative to the results of the same assay without said antibody of the invention.

In one embodiment, a humanized antibody of the invention inhibits luciferase activity stimulated by IL-1a, IL-1e, IL-33 and/or IL-36, respectively, in HEK 293T/17 cells (HEK 293T/17-FR cells transfected with luciferase under NF-kB reporter gene control), HEK-Blue-IL33™ cells (Invivogen) or HEK-293/17-IF cells.

Preferably, said IL-1a stimulated luciferase activity is inhibited by 50% or more, preferably 70% or more, preferably 80% or more, preferably 90% or more, and more preferably 95% or more. Preferably, said luciferase activity stimulated by IL-1a is inhibited by 95%.

Preferably, said IL-1e stimulated luciferase activity is inhibited by 50% or more, preferably 70% or more, preferably 80% or more, preferably 90% or more, and more preferably 95% or more. Preferably, luciferase activity stimulated by IL-1e is inhibited by 95%.

Preferably, said IL-33 stimulated luciferase activity is inhibited by 50% or more, preferably 70% or more, preferably 80% or more, preferably 90% or more, and more preferably 95% or more. Preferably said luciferase activity stimulated by L-33 is inhibited by 95%.

Preferably, said IL-36 stimulated luciferase activity is inhibited by 50% or more, preferably 70% or more, preferably 80% or more, preferably 90% or more, and more preferably 95% or more. Preferably said L-36 stimulated luciferase activity is inhibited by 95%.

In addition, the antibodies of the invention suppress the release of IL-6 mediated by human IL-1a and IL-1b and are superior to polyclonal antibodies. This strong inhibitory activity is shown and illustrated in FIG. 9. In these experiments, EC ₅₀ values indicate that humanized anti-I-1I3 IgG1-LALA antibodies outperform goat anti-human IL-1R3 polyclonal antibodies AF676 (FIG. R&D Systems). In preferred embodiments, the antibodies inhibit IL-a mediated release of IL-6 with an EC50 of less than 2500 μg/mL, preferably less than 1500 ng/mL, less than 1000 ng/mL, less than 600 ng/mL, less than 400 ng/mL. ml or less than 300 ng/ml. It is also preferred that the antibodies of the invention inhibit IL-1b mediated release of IL-6 with an EC50 of less than 500 ng/ml, preferably less than 400 ng/ml, less than 300 ng/ml, less than 200 ng/ml or less than 150 ng/ml.

In another embodiment, the antibodies suppress IL-33 mediated NfkB signaling. In FIG. 10 shows the inhibitory activity of selected antibodies of the invention in HEK-Blue-IL33™ cells and shows their superiority over polyclonal antibodies. In preferred embodiments, the antibodies suppress IL-33 mediated NfkB signaling with

- 10 040391

EC ₅₀ less than 20,000 ng/ml, preferably less than 18,000 ng/ml, less than 3,000 ng/ml, less than

1000 ng/ml, less than 500 ng/ml or less than 400 ng/ml.

The antibodies of the invention can also suppress IL-36 mediated NfkB signaling (FIG. 11). They preferably inhibit IL-36 mediated NfkB signaling with an EC ₅₀ of less than 100 ng/ml, preferably less than 50 ng/ml, less than 40 ng/ml, less than 30 ng/ml, less than 20 ng/ml or less than 15 ng/ml.

The inventors unexpectedly found that the antibodies of the invention suppress the release of cytokines mediated by various stimuli. For example, antibodies inhibit the release of cytokines mediated by IL-1a, IL-33 and IL-36a. The results for the selected antibody are shown in FIG. 12. For example, the MAB-16-0030 antibody inhibits cytokine release mediated by all three stimuli, while IL-1Ra affects only IL-1a mediated cytokine release.

Diseases associated with acute or chronic inflammation are maintained or developed by exposure to multiple cytokines acting either simultaneously or sequentially. Early alarm signals such as IL-1a and IL-33 can trigger other cytokines, including IL-1b and IL-36, thus creating a strong inflammatory environment. Therefore, the concomitant suppression of signaling mediated by numerous cytokines provides effective control of inflammatory processes. A key aspect of the antibodies of the invention is that they inhibit signaling from a variety of cytokines by blocking the IL1R3 receptor.

Binding of antibodies to immune cells can lead to debilitating and detrimental effects, for example by direct induction of apoptotic signaling pathways, stimulation of excessive cytokine release, or antibody effector function, antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, another aspect of the antibodies of the invention is that they do not induce immune cell depletion mediated by direct induction of apoptotic signaling pathways, stimulation of excessive cytokine release, or antibody effector function, antibody-dependent cell-mediated cytotoxicity.

It is important to note that the antibodies of the invention do not affect the viability of immune cells. For example, they do not affect the viability of human peripheral blood mononuclear cells (PBMC)), and do not induce the release of IL-6 in PBMC (see Fig. 13).

The antibodies of the invention suppress the functional activation of cytokine release not only in various cell lines as described above, but also in PBMC or whole blood cells of donors. They inhibit the release of cytokines mediated by various specific or complex stimuli. For example, they suppress the activation of LPS stimulated PBMCs, heat inactivated Candida albicans, IL-12/IL-33 or anti-CD3/CD28 antibodies (see FIGS. 14 and 15).

In addition, in one embodiment, the humanized anti-IL-1R3 IgG1-LALA antibodies of the present invention are capable of inhibiting the release of IFNy, IL-6, TNF-α, IL-13, IL-17, and IL-10 in mixed lymphocyte culture reactions. (see Fig. 16).

In addition, the antibodies of the invention are particularly useful in the treatment of diseases caused by dysregulation of the target, IL1R3.

Therefore, the present invention relates to a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to confer IL-1R3 binding specificity, for the treatment of IL-1R3 mediated diseases.

The present invention also relates to a method of treating an IL-1R3 mediated disease in a patient, comprising administering to the patient a pharmaceutically effective amount of said antibody, or a derivative or fragment thereof, of the invention.

The invention also relates to a pharmaceutical composition containing a pharmaceutically acceptable carrier and a therapeutically effective amount of a humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to provide IL-1R3-binding specificity, in accordance with the invention.

As used herein, the term pharmaceutical carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion).

The composition of the present invention can be administered in a variety of ways known in the art. As will be appreciated by one of skill in the art, the route and/or mode of administration will vary depending on the desired results. In order to administer a compound of the invention by certain routes of administration, it may be necessary to coat said compound or co-administer said compound with a material to prevent its inactivation. For example, the compound may be administered to the subject in a suitable vehicle, such as liposomes, or

- 11 040391 thinner. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

The phrases parenteral administration and parenterally administered as used in this application mean methods of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersing agents. The presence of microorganisms can be prevented by sterilization procedures, see above, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It is also possible to include isotonic agents such as sugars, sodium chloride, and the like in the composition. In addition, prolonged absorption of the injectable pharmaceutical form can be obtained by including agents that delay absorption, such as aluminum monostearate and gelatin. Regardless of the route of administration chosen, the compounds of the present invention, which may be used in a suitable hydrated form and/or pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known in the art. Actual dosage levels of active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response in a particular patient, for a particular composition and route of administration, while not being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the specific compositions of the present invention used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and/or materials used in combination with the specific used compositions, age, sex, weight, condition, general health and previous medical history of the patient receiving treatment, and similar factors well known in the medical field.

One aspect of the invention is a pharmaceutical composition according to the invention for use in the treatment of cancer, as defined in this application.

Another aspect of the invention is a method of treating an IL-1R3 mediated disease in a patient, comprising administering to the patient a pharmaceutical composition of the invention.

Such IL-1R3 mediated diseases may include cancer. According to the literature, a negative prognosis and progression of cancer is associated with an increase in the levels of IL-1a, IL-1e, IL33, IL-36 and/or increased expression of IL-1R3.

The term cancer as used herein can be, for example, lung cancer, non-small cell lung cancer, bronchioloalveolar lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer. , rectal cancer, anal cancer, stomach cancer, gastrointestinal cancer, colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, cancer esophagus, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra cancer, penis cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, carcinoma renal pelvis, mesothelioma, hepatocellular carcinoma, bile duct cancer, malignant tumors of the central nervous system (CNS), spinal tumors, glioma brainstem, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory variants of any of the above cancers, or a combination of one or more of the above cancers.

Preferably such cancer is leukemia, breast cancer, colon cancer, lung cancer or pancreatic cancer. Most preferably, the cancer is leukemia.

In one embodiment, the IL-1R3 mediated disease is selected from the group consisting of leukemia, breast cancer, colon cancer, lung cancer, pancreatic cancer, liver cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, gastrointestinal cancer. intestinal tract, estrogen receptor positive breast cancer, head and neck squamous cell carcinoma, mesothelioma, gallbladder cancer, ovarian cancer, bladder cancer, prostate cancer, thyroid cancer, Hodgkin's disease, MALT lymphoma, salivary gland cancer, or melanoma.

Some types of tumors can be caused or stimulated by cells in the microenvironment.

- 12 040391 tumors secreting inflammatory cytokines such as IL-Ια, IL-Ιβ, IL-33, IL-36. In some cases, the expression of such cytokines leads to the development of tumor resistance. Concomitant use of cytokine inhibitors and anti-cancer compounds greatly improves the response rate of such treatments or may impair tumor resistance. This is provided by the present invention, since a wide range of suppression of cytokine-induced signaling is achieved. Such cancer-indicated activity is achieved not through direct debilitating activity of cancer cells, but through suppression of cancer-associated inflammation through modulation of IL1R3 signaling pathways. The antibodies of the present invention provide a very advantageous activity profile as they provide effective suppression of cancer-associated chronic inflammation while at the same time not causing undesirable side effects as they do not exhibit antibody effector function and thus do not affect the viability of target cells. expressing IL-1R3.

Therefore, in one embodiment of the present invention, the antibody or pharmaceutical composition is used to treat patients who have a tumor, such as a solid tumor, and are under-responsive or tumor resistant to cytotoxic, cytostatic, or targeted/immunotherapy.

In one aspect of the invention, the humanized antibody and/or pharmaceutical composition of the invention is for use in the treatment of cancer in combination with one or more cytotoxic, cytostatic or anti-cancer target compounds. The use of cytokine inhibitors and cytotoxic, cytostatic, or targeted anticancer compounds greatly improves the response rate of such treatments or may impair tumor resistance.

In such aspects of the invention, the cancer is preferably selected from the group consisting of pancreatic cancer, liver cancer, lung cancer (asbestos-induced inflammation, infections, smoking, silica-associated), non-small cell lung cancer, colorectal/colitis-associated cancer (associated with inflammatory bowel disease), gastric cancer, gastrointestinal cancer, estrogen receptor-positive breast cancer, head and neck squamous cell carcinoma, mesothelioma, gallbladder cancer (associated with chronic cholecystitis associated with gallstones), cancer ovarian cancer, bladder cancer, prostate cancer, E. coli infection-associated prostate cancer, thyroid cancer, Hodgkin's disease, MALT lymphoma, salivary gland cancer, melanoma, endometriosis-associated endometrial carcinoma, esophageal cancer associated with Barrett's esophagitis.

In one aspect of the method of the present invention, the IL1R3 antibody or pharmaceutical composition of the invention is administered simultaneously with one or more cytotoxic, cytostatic, or anticancer targets. In another aspect, the antibody or pharmaceutical composition is administered sequentially with one or more cytotoxic, cytostatic, or anticancer targets.

In the latter case, it is preferred that the antibody be administered after treatment with one or more cytotoxic, cytostatic, or targeted anticancer agents.

The cytotoxic or cytostatic agents of the invention may be taxanes, anthracyclines, alkylating agents, histone deacetylase inhibitors, topoisomerase inhibitors, kinase inhibitors, nucleotide analogs, peptide antibiotics, platinum-based agents, and checkpoint inhibitors.

Preferably the targeted anti-cancer agents are selected from one of the following compounds or combinations thereof: anti-EGFR compounds such as cetuximab, gefitinib, erlotinib, lapatinib, panitumumab, and anti-HER2 compounds such as trastuzumab, ado-trastuzumab emtansine, pertuzumab.

In addition, it is preferred that the targeted anti-cancer agents are checkpoint inhibitors. These can be, without limitation, anti-PD1 compounds such as pembrolizumab and nivolumab, and anti-PDL1 compounds such as atezolizumab, avelumab and durvavumab, and anti-CTBA-4 compounds such as ipilimumab and tremelimumab.

Examples

The following examples are described in combination with the drawings and tables to illustrate the invention.

Example 1 Detection of antibodies specific to P013_03 (human IL-1R3) in B cell supernatants.

The principle of analysis.

NUNC Maxisorp 384-well microtiter plates are coated with P013_03. After blocking, specific antibodies from B-cell supernatants are allowed to bind to the antigen and then detected with a POD-labeled antibody. Samples were evaluated at a 1:2 dilution.

Materials.

Plates: NUNC Maxisorp 384-well plates; catalog number 464718.

Proteins: P013-03 (conc. 1.5 mg/ml, assay concentration 0.5 µg/ml).

Standard Ab: P013-02 (conc. 1 mg/mL, initial assay concentration 2 µg/mL).

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Identifying Ab: anti-rabbit IgG, peroxidase-linked species-specific complete antibody (from donkey) (ECL); G.E.; part number NA9340; dilution for analysis: 1:5000.

PBS: packaged buffers, pre-mixed PBS buffer, 10x; Roche Applied Sciences; catalog number 11666789001.

BSA: fraction V of bovine serum albumin from bovine serum; Roche Applied Sciences; catalog number 10735086001.

Twin 20: Twin 20; Carl Roth; catalog number 9127.2.

TMB: TMB solution; Life Technologies; catalog number SB02.

HCl: 1M hydrochloric acid Titripur; Merck; catalog number 1090571000.

ELISA Buffer: PBS, 0.5% BSA, 0.05% Tween.

Wash Buffer: PBS, 0.1% Tween.

Blocking buffer: PBS, 2% BSA, 0.05% Tween.

Samples: 1:2 dilution in ELISA buffer.

Procedure.

1. Add 12.5 µl of P013-03 (0.5 µg/ml) in PBS to a NUNC Maxisorp 384-well plate and incubate for 1 hour at room temperature.

2. Wash 3 times with 90 µl wash buffer.

3. Add 90 µl of blocking buffer to each well and incubate for 1 hour at room temperature.

4. Wash 3 times with wash buffer.

5. Add 12.5 µl of standard antibody at a 1:2 dilution or sample diluted 1:2 with ELISA buffer and incubate for 1 hour at room temperature.

6. Wash 3 times with wash buffer.

7. Add 12.5 µl 1:5000 POD antibody in ELISA buffer and incubate for 1 hour at room temperature.

8. Wash 6 times with wash buffer.

9. Add 15 µl of TMB.

10. Add 15 µl of HCl after sufficient color development.

11. Measure absorbance at 450 nm/620 nm.

Example 2 Identification of P013-specific antibodies inhibiting the P013 receptor using Luciferase reporter technology.

The principle of analysis.

293T/17-FR cells expressing the firefly NF-kB-RE luciferase reporter were seeded in cell culture plates coated with poly-D-lysine. After stimulation with P013, the 293T/17-FR lysate is tested for the presence of activated NF-kB using the SteadyGlo Lumiferase Assay Kit. Provide the binding of supernatants containing functional antibodies, P013 and suppress the activation of NF-kB, which is manifested as a low signal. Samples were evaluated at a 1:2 dilution in P013 solution.

Materials.

Plates: cell plates: PDL Costar 384-well cell culture plates; catalog number 3844.

Assay plates: Lumitrac 384-well white plates; Corning; catalog number 3572.

Cells: 293T/17-FR; concentration for analysis 250,000 cells/ml.

The higher the cell passage number, the lower the signal!

Proteins: P013 05 (concentration 0.03 mg/ml, assay concentration 115 g/ml, working concentration 230 g/ml).

Standard Ab: P013-06 (concentration 0.2 mg/ml, initial concentration for analysis 6 µg/ml).

Kit: system for luciferase analysis Steady-Glo; Promega; catalog number E2510.

Cell medium: DMEM medium; PAN Biotech; catalog number P04-04510.

FCS: fetal bovine serum, HyClone; Thermo; catalog number St30070.03.

Media for 293T/17-FR: DMEM, 10% FCS, (+ 20 µg/ml hygromycin-B, culture only) conditioned B cell media (identifying MAB).

Samples: 1:2 dilution with P013_05 in DMEM+10% FCS.

Procedure.

1. Cell culture procedure: confluent 293T/17-FR cells are detached from the surface with trypsin/EDTA (incubate only 30 at RT) every Monday (seeding: 5x106 cells/T175 flask) and Friday (seeding: 3x106 cells/T175 flask) ).

2. Seeding cells (0.25x106 cells/ml) in 25 µl DMEM+10% FCS on a 384-well PDL plate (Corning, cat. no. 3844) and incubating overnight at 37° C. and 5% CO ₂ .

3. Aspirate the medium and add 12.5 µl of sample or P013 06 diluted 1:3 in conditioned

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5% CO2 (program: 3. Aspiration and transfer of samples).

4. Add 12.5 µl of P013_05 in DMEM+10% FCS and incubate for 5 hours at 37°C and 5% CO2 (program: 4 Add P013 05).

5. Cultured cells are equilibrated at room temperature for 10 minutes.

6. Add 25 µl Steady-Glo reagent and mix several times with a pipette (program: 6 Steady Glo).

7. Wait 5 minutes to transfer 45 µl of the supernatant to a Lumitrac 384-well white plate (Corning, cat. no. 3572) (program: 7_Transfer 45ul).

8. Luminescence is measured with a Tesap reader: Integration time: 0.5 s.

Example 3 Determination of specific antibodies to huIL1RaP, msIL1RaP, CD134 and CD137 in B cell supernatants.

The principle of analysis.

NUNC Maxisorp 384-well microtiter plates are coated with the target protein. After blocking, specific antibodies from the B-cell supernatants are allowed to bind to the targets and then detected with a POD-labeled antibody.

Materials.

Plates: NUNC Maxisorp 384-well plates; catalog number 464718.

Proteins: digested huIL1RaP (P026_12; concentration 0.96 mg/ml; assay concentration 0.25 μg/ml);

cleaved muIL1RaP (P026 13; concentration 0.93 mg/ml, assay concentration 0.25 μg/ml);

cleaved CD134 (P026 14; concentration 0.51 mg/ml, assay concentration 0.25 µg/ml);

cleaved CD137 (P026 15; lot 2; concentration 1.1 mg/ml, assay concentration 1 μg/ml).

Standard Ab: human anti-IL-1RAcP/IL-1R3 antibody (P013_6/P026_08, concentration 0.2 or 0.399 mg/ml, assay starting concentration 2 μg/ml, used for huIL1RaP and msIL1RaP);

MAB-14-0283 (concentration 0.6 mg/ml, starting concentration for assay 2 µg/ml, used for CD134);

MAB-14-0285 (concentration 1 mg/ml, assay starting concentration 2 µg/ml used for CD137).

Identifier Ab: Samples: anti-rabbit IgG, peroxidase-linked species-specific Fab fragment ₂ (from donkey) (ECL); G.E.; part number NA9340; assay dilution: 1:5000 in ELISA buffer.

For MAB-14-0283 and MAB-0285: peroxidase-associated species-specific anti-human IgG Fab ₂ fragment (from goat) (HRP); AbD Serotec; part number STAR126P; assay dilution: 1:5000 in ELISA buffer.

For huIL1RaP and msIL1RaP: AffiniPure peroxidase-conjugated donkey anti-goat IgG (H+L); Jackson Immuno Research; catalog number 705-035-003; assay dilution: 1:5000 in ELISA buffer.

PBS: packaged buffers, pre-mixed PBS buffer, 10x; Roche Applied Sciences; catalog number 11666789001.

BSA: fraction V of bovine serum albumin from bovine serum; Roche Applied Sciences; catalog number 10735086001.

Twin 20: Twin 20; Carl Roth; catalog number 9127.2.

TMB: TMB solution; Invitrogen; catalog number SB02.

HCl: 1M hydrochloric acid Titripur; Merck; catalog number 1090571000.

ELISA Buffer: PBS, 0.5% BSA, 0.05% Tween.

Wash Buffer: PBS, 0.1% Tween.

Blocking buffer: PBS, 2% BSA, 0.05% Tween.

Samples: 1:4 dilution in ELISA buffer.

Procedure.

1. Add 12.5 µl of 0.25 µl/ml or 1 µl/ml of protein diluted in PBS to a NUNC Maxisorp 384-well plate and incubate for 1 hour at room temperature.

2. Wash 3 times with 90 µl wash buffer.

3. Add 90 µl blocking buffer to each well and incubate for 1 hour at room temperature.

4. Wash 3 times with 90 µl wash buffer. Plates can be stored dry for up to 6 weeks at 20°C, sealed in aluminum foil.

5. Add 12.5 µl of standard antibody in 1:2 dilution steps or sample (diluted with ELISA buffer) and incubate for 1 hour at room temperature.

6. Wash 3 times with 90 µl wash buffer.

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7. Add 12.5 µl 1:5000 POD antibody in ELISA buffer and incubate for 1 hour at room temperature.

8. Wash 6 times with 90 µl wash buffer.

9. Add 15 µl of TMB.

10. Add 15 µl HCl after 15 min staining.

11. Measure absorbance at 450 nm/620 nm.

Example 4 Suppression of NFkB expression by stably transfected A549-NFkBRE-Luc cells after IL-1 (α/β) stimulation.

The principle of analysis.

Stably transfected A549-NFkB-RE-Luc cells (Signesis) are pipetted into a 384-well plate and incubated overnight. On day 2, anti-IL1R3 antibodies are allowed to bind to stably transfected A549-NFkB-RE-Luc cells, which are then stimulated with the addition of IL-1 (α or β). This leads to transcription of the luciferase gene due to the activation of the NFkB signaling pathway, which can be measured by cell lysis and by the addition of luciferin.

Check whether antibodies can suppress the activation of the NFkB pathway and, therefore, reduce the luminescent signal.

Materials.

Plates: sterile 384-well white flat bottom polystyrene TC-treated microplates, low rim; Corning; catalog number 3570.

Proteins: IL-1a (P026 09); recombinant human IL-1a/IL-1F1; 10 µg/ml; R&D Systems; part number 200-LA-002 IL-1e (P026 10); recombinant human IL-1e/IL-1F2; 25 µg/ml; R&D Systems; part number 200-LB-005.

Standard Ab: MAB-15-0115; MAB Discovery GmbH; 2.51 mg/ml; working concentration 10 mcg/ml.

Cells: stably transfected A549-NFkB-RE-Luc cells; signosis; catalog number SL-0014.

Medium: DMEM; PAN; catalog number P04-04510.

FCS: low IgG fetal bovine serum from South Africa; PAN; catalog number 1552-P120909.

Penicillin/Streptomycin: 10,000 units penicillin/ml; 10 mg streptomycin/ml; PAN Biotech; catalog number P06-07100.

Releasing agent: Trypsin-EDTA 1x; PAN; catalog number P10-023100 (4 ml for T175/2 ml for T75, ~8 min 37°C).

Cell media: DMEM, 10% FCS, 1% penicillin/streptomycin.

Detection Kit: Steady-Glo™ Luciferase Assay System; Promega; catalog number E2510.

Procedure.

1. Cultivate stably transfected A549-NFkB-RE-Luc cells (1.7E+04 cells/cm ² for 3 days, 2.28E+04 cells/cm ² for 2 days) in cell medium. No more than 10 passages!

2. 40,000 stably transfected A549-NFkB-RE-Luc cells in 25 µl of media per well (conc=1.6 x 106 cells/ml) are transferred to a 384-well white treated flat bottom cell culture plate.

Incubate overnight at 37°C/5% CO ₂ .

3. Aspirate the medium from the plate and add 10 µl of the sample or standard in the medium to the plate using the CyBio robot pipette (software: Remove medium and transfer samples in folder P026/NFKB). Incubate for 1 hour at 37°C/5% CO ₂ .

4. Add 10 µl of IL-1 (α or β) in media to the plate using the CyBio robot pipette (software: Transfer from reservoir in folder P026/NFkB) (working concentration: 0.2 ng/ml, assay concentration: 0.1 ng/ml) and incubated for 5 hours at 37°C/5% CO ₂ .

Prior to step 4, dissolve Steady-Glo support in Steady-Glo buffer according to Steady-Glo protocol and equilibrate this solution and assay plate at room temperature.

5. Add 20 µl Steady-Glo mix, mix thoroughly to ensure proper cell lysis. Incubate at room temperature, 10 min.

6. Determine the relative units of luminescence in each well using a microplate reader set to an integration time of 500 ms (software: Lumineszenz-384).

Example 5 IL-6 secretion by A549 cells after IL-1 (α/β) stimulation.

The principle of analysis.

A549 cells are pipetted into a 384-well plate and incubated with anti-IL1R3 antibodies. The cells are then stimulated with IL-1 (α or β) and IL-6 is secreted into the assay medium. The amount of IL-6 is measured by ELISA.

Materials.

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Assay kit: DuoSet ELISA for human IL-6; catalog number DY206-05 (R&D Systems); The DuoSet consists of an immobilized human anti-III-6 antibody (part 840113), an antibody identifying human IL-6 (part 840114), human IL-6 (part 840115) and streptavidin-HRP (part 8939755).

Plates: 384-well transparent cell culture plates; Corning; catalog number 3701; Maxisorp 384-well plates; NUNC; catalog number 464718.

PP plate: 120 µl 384-well deep transparent Diamond plate; Axigen (Corning); part number P-384-12OSQ-C.

Proteins: IL-1a; recombinant human IL-1-a/IL-1F1; R&D Systems; part number 200-LA-002 ΓΕ-1β; recombinant human IL-1e/IL-1F2; R&D Systems; part number 200LB-005 rhIL1-ra/IL-1F3; R&D Systems; catalog number No. 280-RA-010.

Standard Ab: human anti-IL-1RAcP/IL-1R3 antibody; R&D Systems; part number AF676 or AF676-SP.

Medium: DMEM; PAN; catalog number P04-04510.

FCS: low IgG fetal bovine serum from South Africa; PAN; catalog number 1552-P120909.

Penicillin/Streptomycin: 10,000 units penicillin/ml; 10 mg streptomycin/ml; PAN Biotech; catalog number P06-07100.

PBS: packaged buffers, pre-mixed PBS buffer, 10x; Roche Applied Sciences; catalog number 11666789001.

BSA: fraction V of bovine serum albumin from bovine serum; Roche Applied Sciences; catalog number 10735086001.

Twin 20: Twin 20; Carl Roth; catalog number 9127.2.

TMB: TMB solution; Invitrogen; catalog number SB02.

HCl: 1M hydrochloric acid Titripur; Merck; catalog number 1090571000.

ELISA Buffer: PBS, 0.5% BSA, 0.05% Tween.

Wash Buffer: PBS, 0.1% Tween.

Blocking buffer: PBS, 2% BSA, 0.05% Tween.

Cell media: DMEM, 10% FCS, 1% penicillin/streptomycin.

Procedure.

cell stimulation.

1. Inoculate 6000 A549 cells in 25 µl of medium per well (concentration=2.4 x 10 ⁵ cells/ml) in a cell culture plate. Incubate overnight at 37°C/5% CO ₂ .

2. Aspirate the medium from the plate and add 12.5 µl of sample or standard in medium to the plate. Incubate for 3 hours at 37°C/5% CO2.

3. Add 12.5 µl of IL-1 (α or β) to the plate (working solution: 0.2 ng/ml, assay concentration 0.1 ng/ml) and incubate for 48 hours at 37°C/5% CO2 .

4. Aspirate the medium and transfer to a coated and blocked ELISA plate (step 9). Alternatively, supernatants can be stored at -80° C. in a PP plate for one week.

Preparation of the ELISA plate.

5. Dilute the capture antibody in PBS to a concentration of 2 µg/mL. Immediately add capture antibodies to a 384-well Maxisorp plate at 12.5 µl per well. The plate is sealed and incubated for 1 hour at room temperature.

6. Aspirate media from each well and wash with wash buffer, repeat this process two more times for a total of three washes. Wash by filling each well with wash buffer (90 µl) using an automatic washer. Complete removal of fluid at each stage is essential for good performance. After the last wash, remove the remaining wash buffer by aspiration or by inverting the plate and blotting it with clean paper towels.

7. Block the plates by adding 90 µl of blocking buffer to each well. Incubate at room temperature for at least 1 hour.

8. Repeat the aspiration/washing process as in step 6. The plates are now ready for sample addition. Coated and blocked plates can be stored at -20°C dry for one month.

analysis procedure.

9. Add 12.5 µl of pure sample or IL-6 standard diluted in ELISA buffer (EB) to each well. Cover with a lid and incubate for 1 h at room temperature.

10. Repeat the aspiration/washing process as in step 6 of preparing the ELISA plate.

11. Add 12.5 µl of detection antibody diluted in EB to each well. Cover with a lid and incubate for 1 h at room temperature.

12. Repeat the aspiration/washing process as in step 6 of preparing the ELISA plate.

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13. Add 12.5 µl 1:40 diluted streptavidin-HRP in ELISA buffer to each well.

Cover the plate with a lid and incubate for 20 minutes at room temperature. Avoid direct light on the plate.

14. Repeat the aspiration/washing process as in step 6 of preparing the ELISA plate.

15. Add 15 µl of substrate solution (TMB) to each well. Incubate for 20 min at room temperature.

16. Add 15 µl stop solution (HCl, 1M) to each well. Gently tap the plate to ensure thorough mixing.

17. Immediately determine the absorbance in each well using a microplate reader set at 450 nm. If there is a wavelength correction, set the wavelength to 540 nm or 570 nm (program: TMB stop 384 Cytokine). Readings taken directly at 450 nm without correction may be higher and less accurate.

Example 6 Determination of binding characteristics for huIL1RaP-specific antibodies by competition assay.

The principle of analysis.

NUNC Maxisorp 384-well microtiter plates are coated with Can04 reference antibody. During this time, the His-tagged target protein is pre-incubated with the second antibody to be tested and the anti-HIS-POD antibody. This pre-incubated mixture is then added to the assay plate and after sufficient time for color to develop, the absorbance at 450 nm/620 nm is measured.

Materials.

Plates: NUNC Maxisorp 384-well plates; catalog number 464718.

Coating Ab: Can04 (MAB Discovery GmbH, CEP Ab No. 184, concentration 1 mg/mL, assay concentration 100 ng/mL).

Protein: huIL1RaP-His protein (P026_01; Fusion_1_Chain_A labeled huIL1RaP-His homodimer; GeneArt, concentration 3 mg/ml, assay concentration 62.5 ng/ml).

Standard Ab: Can04 (see Ab for coating, initial working concentration 3 µg/ml).

Negative control: Her2 antibody (LifeSpan, catalog number LS-C95808/26358, concentration 225 μg/ml, initial working concentration 3 μg/ml).

Identification Ab: anti-HIS POD antibody (peroxidase-conjugated monoclonal anti-polyhistidine antibody, Sigma, No. A7058, concentration 7.5 mg/ml, assay concentration: 3.33 μg/ml).

PBS: packaged buffers, pre-mixed PBS buffer, 10x; Roche Applied Sciences; catalog number 11666789001.

BSA: fraction V of bovine serum albumin from bovine serum; Roche Applied Sciences; catalog number 10735086001.

Twin 20: Twin 20; Sigma Aldrich; catalog number P1379.

TMB: TMB solution; Merck; catalog number CL07.

HCl: 1M hydrochloric acid Titripur; Merck; catalog number 1090571000.

ELISA Buffer: PBS, 0.5% BSA, 0.05% Tween.

Wash Buffer: PBS, 0.05% Tween.

Blocking buffer: PBS, 2% BSA, 0.05% Tween.

Procedure.

12. Prepare the pre-incubation mixture and incubate for 2 hours at room temperature.

A. Pre-incubation (in 384-well plate).

i. Mix 10 µl dilution series of secondary antibody (1:2 dilution, initial working concentration 3 µg/ml) in ELISA or BLANK buffer with ii. 10 µl His-tagged protein (assay concentration 62.5 ng/ml) and iii. 10 µl of anti-HIS POD antibody (assay concentration: 3.33 µg/ml) and incubated for 1 hour at room temperature.

13. Meanwhile, coat the NUNC Maxisorp plate with 20 µl of coating antibody (Can04, assay concentration 100 ng/ml) in PBS and incubate for 1 hour at room temperature.

14. Wash 3 times with 90 µl wash buffer.

15. Block with 90 µl of blocking buffer for 1 hour at room temperature.

16. Wash 3 times with wash buffer.

17. Add 20 µl of pre-incubated mixture to ELISA buffer for 1 hour at room temperature.

18. Wash 6 times with wash buffer.

19. Add 25 µl of TMB.

20. Add 25 µl HCl after sufficient color development.

21. Measure absorbance at 450 nm/620 nm.

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Example 7 Reverse screening for IL12.

The principle of analysis.

IL12 binding is used as a reverse screen. The HER proteins are labeled with a linker, huFc and His (HER1 has no His tag), similar to the IL12 protein. Antibodies that bind to the label are positive in both assays, while antigen-specific antibodies only bind to HER proteins and not to IL12.

Materials.

Plates: NUNC Maxisorp 384-well plates; catalog number 464718.

Proteins: recombinant human IL-12 ReI Fc chimera; R&D Systems; catalog number No. 839-B1; concentration for analysis 0.5 µg/ml.

Standard Ab: ΤΕ-12Κβ1 antibody; Genetex; part number GTX103917; 1 mg/ml; initial concentration for analysis 500 ng/ml (then 1:2 dilutions).

Identifying Ab: peroxidase-linked species-specific anti-rabbit IgG (donkey) Fab2 fragment (ECL); G.E.; part number NA9340; dilution for analysis: 1:5000.

Samples: dilution in ELISA buffer depends on the project (a 1:2 dilution is recommended for highly concentrated IgG).

Procedure.

1. Add 12.5 µl of 0.5 µl/ml HER protein in PBS to a 384-well NUNC Maxisorp plate and incubate for 1 hour at room temperature.

2. Wash 3 times with wash buffer.

3. Add 90 µl blocking buffer to each well and incubate for 1 hour at room temperature.

4. Wash 3 times with wash buffer. Plates sealed with aluminum foil can be frozen for several weeks at -20°C.

5. Add 12.5 µl of standard antibody at a 1:2 dilution or sample diluted in ELISA buffer and incubate for 1 hour at room temperature (frozen plates should be thawed shortly before sample application).

6. Wash 3 times with wash buffer.

7. Add 12.5 µl 1:5000 POD antibody in ELISA buffer and incubate for 1 hour at room temperature.

8. Wash 6 times with wash buffer.

9. Add 15 µl of TMB.

10. Add 15 µl HCl after sufficient staining has developed (depends on project, IL12 assay is no shorter than other assays).

11. Measure absorbance at 450 nm/620 nm.

Example 8 Cell Binding Assay.

A549 and NIH-3T3 cells were cultured in DMEM+10% FCS. HEK-293 cells were cultured in DMEM+15% FCS, and cells and SK-MEL-30 in RPMI+10% FBS. Cells were harvested with Accumax (Sigma), washed with PBS and resuspended in staining buffer (BD Pharmingen). Ahtu-IL-1R3 antibodies were incubated with cells in staining buffer for 30 min at 4°C at a concentration of 10 μg/ml. For the binding assay (EC ₅₀ ) of SK-MEL-30 cells, cells were incubated with a 1:2 dilution series starting at 20 μg/ml. Cells were washed with staining buffer and incubated with a goat anti-human secondary antibody labeled with Alexa-488 (Dianova) for 30 min at 4°C. Cells were washed with staining buffer and resuspended in buffer containing DRAQ7 stained dead cells (Abcam) diluted 1:100. Cells were analyzed using a Sampler BD Accuri C6 flow cytometer. A fit curve was generated and EC ₅₀ calculated using Excel (Microsoft) and XLfit (IDBS).

Example 9 Biochemical ELISA assay of human IL-1R3.

Nunc Maxisorp 384-well plates were coated with recombinant Fc-labeled hIL-1R3 (Ser21Glu359) at 0.25 μg/ml in PBS for 60 minutes at room temperature. The plates were washed three times with wash buffer (PBS 0.1% Tween) and blocked with PBS, 0.2% BSA, 0.05% Tween for 60 minutes at room temperature. After three washes with wash buffer, antibodies were added to the ELISA buffer (PBS, 0.5% BSA, 0.05% Tween) at concentrations from 6 to 0.03 μg/ml (1:3 dilution series) and incubated for 60 min at room temperature. The plates were washed three times with wash buffer followed by incubation with peroxidase-linked species-specific F(ab) ₂ anti-human IgG (goat, AbD Serotec) at a 1:5000 dilution in ELISA buffer for 60 minutes at room temperature. Before adding the TMB substrate solution (Invitrogen, 15 μl/well), the plates were washed six times with wash buffer. After a 5 minute incubation stop solution (1M HCl, 15 μl/well) was added and the absorbance was measured (450 nm/620 nm) using a Tecan M1000 plate reader. Using Excel (Microsoft) and XLfit (IDBS), fit curves were built and EC ₅₀ calculated.

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Example 10 IL-1a and IL-Ιβ functional neutralization assay.

A-549-NFkB-RE-Luc (Signosis) were cultured in DMEM, 10% FCS, 1% penicillin/streptomycin for 5 days before being plated in 384-well white flat-bottom polystyrene cell culture plates (Corning) at cells 40,000 cells/well in 25 µl of medium. Cells were incubated overnight at 37°C/5% CO ₂ . The medium was removed by aspiration and monoclonal or polyclonal (goat anti-human IL-1R3, AF676, R&D Systems) antibodies were added at various concentrations in 10 μl of medium and incubated for 60 min at 37°C/5% CO ₂ . Recombinant human IL-1a or IL-1e proteins (R&D Systems) were added to 10 μl of medium to a final concentration of 0.1 ng/ml and the plates were incubated for 5 hours at 37°C/5% CO ₂ . 20 µl Steady-Glo™ solution (Promega) was added to each well, mixed thoroughly and the plates were incubated for 10 min at room temperature until luminescence was measured using a Tecan M1000 plate reader. Using Excel (Microsoft) and XLfit (IDBS), fit curves were built and EC ₅₀ calculated.

Example 11 IL-1a and IL-1β functional neutralization assay - IL6 A-54 release assay 9.

A549 cells were plated at 6000 cells/well in 25 μl of medium in 384-well processed cell culture plates (Corning) in DMEM, 10% FCS, 1% penicillin/streptomycin. Cells were incubated overnight at 37°C/5% CO ₂ . The medium was removed by aspiration, and monoclonal or polyclonal (goat anti-human IL-1R3, AF676, R&D Systems) antibodies were added at various concentrations in a volume of 12.5 μl medium and incubated for 3 h at 37°C/5% CO ₂ . Recombinant human IL-1a or IL-1e proteins (R&D Systems) were added to 12.5 µl of medium to a final concentration of 0.1 ng/ml and the plates were incubated for 48 hours at 37°C/5% CO ₂ . Human IL-6 secreted levels were measured in the cell supernatant using the Human IL-6 DuoSet ELISA kit (R&D Systems, Cat. No. DY206-05) according to the manufacturer's instructions. Using Excel (Microsoft) and XLfit (IDBS), fit curves were built and EC ₅₀ calculated.

Example 12 IL-33 functional neutralization assay.

HEK-Blue™ IL-33 cells (InvivoGen) were cultured in DMEM, 10% FCS for 5 days before being plated in 384-well clear, flat-bottomed, processed cell culture microplates (Corning) at a density of 25,000 cells/well in 15 µl of medium. Various concentrations of monoclonal or polyclonal (goat anti-human IL-1R3, AF676, R&D Systems) antibodies were added to 5 μl of medium and the plates were incubated for 60 min at 37°C/5% CO ₂ . Recombinant human IL-33 protein (R&D Systems) was added to 5 μl of medium to a final concentration of 5 ng/ml and the plates were incubated overnight at 37° C./5% CO ₂ . 5 μl of cell supernatants were transferred to clear, flat-bottomed NBS polystyrene microplates (Corning) containing 20 μl of 2xQUANTI-Blue reagent (InvivoGen). The plates were incubated at 37° C. for 45 minutes and the absorbance was measured at 655 nm using a Tecan M1000 plate reader. Using Excel (Microsoft) and XLfit (IDBS), fit curves were built and EC ₅₀ calculated.

Example 13 IL-36 functional neutralization assay.

HEK293/17-IF cells (MAB Discovery GmbH) were cultured in DMEM, 10% FCS, 20 μg/ml hygromycin for 5 days before being plated in 384-well white flat bottom polystyrene cell culture plates (Corning) at a density of 30,000 cells/well in 20 µl of medium. Cells were incubated overnight at 37°C/5% CO ₂ . The medium was removed by aspiration and various concentrations of monoclonal or polyclonal (goat anti-human IL-1R3, AF676, R&D Systems) antibodies were added in a volume of 10 μl of medium. The plates were incubated for 60 min at 37°C/5% CO ₂ . Recombinant human IL-36g protein (R&D Systems) was added to 10 μl of medium to a final concentration of 15 ng/ml and the plates were incubated overnight at 37°C/5% CO ₂ . 20 µl SteadyGlo™ solution (Promega) was added to each well, mixed thoroughly and the plates were incubated for 10 min at room temperature before measuring luminescence using a Tecan M1000 plate reader. Using Excel (Microsoft) and XLfit (IDBS), fit curves were built and EC ₅₀ calculated.

Example 14 Neutralization of IL-1a, IL-33 and IL-36a.

Anti-IL-1R3 antibody functions were evaluated for signaling in three different cell lines using either IL-1a, IL-33 or IL-36a to determine the effect of IL-1R3 on signaling pathways involving the three IL-1R3 receptors. 1 (IL-1R1, -R4 or -R6).

The A549 human lung epithelial cell line was stimulated with IL-1a as a model for IL-1 dependent diseases such as autoinflammatory diseases. The cell line was cultured in T75 flasks (37°C, 5% CO2) in complete F-12K media (10% FCS, 1% penicillin/streptomycin) and separated on average 2 times a week; the number of passages before analysis was no more than 15. A549 cells were seeded (50,000/well) in a 96-well flat-bottom plate, kept for 3 hours before pre-incubation for 1 hour with MAB-16-0030 (20 - 1 μg/ml) or IL-1Ra (10 µg/ml). Cells were then stimulated with human recombinant IL-1a (50 μg/ml, Peprotech) for 24 h prior to collection of supernatants and analysis for IL-6 production (Duoset ELISA, RnD Systems).

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The human mast cell line (HMC-1) was studied for IL-33 dependent induction of IL-8 production. The cell line was cultured in T75 flasks (37°C, 5% CO2) in Iscove's complete Dulbecco's medium (IMDM, 10% FCS, 1% penicillin/streptomycin) and separated on average 3 times a week with a cell density of no more than 2x10 ⁶ /ml; at the same time, the number of passages before analysis was no more than 15. HMC-1 cells were seeded (30,000/well) in a 96-well flat-bottomed plate, kept for 3 hours before pre-incubation for 1 hour with MAB-16-0030 (20-1 µg/ml) or IL-1Ra (10 µg/ml). Cells were then stimulated with recombinant human IL-33 (20 ng/ml, RnD systems) for 24 h prior to collection of supernatants and analysis for IL-8 production (Duoset ELISA, RnD Systems).

The effect on IL-36 signaling was studied using human keratinocyte (HaCaT) lines. The cell line was cultured in T75 flasks (37°C, 5% CO2) in complete DMEM (10% FCS, 1% penicillin/streptomycin) and separated on average 3 times/week; the number of passages before analysis was no more than 15. HaCaT cells were seeded (50,000/well) in a 96-well flat-bottomed plate, kept for 3 hours before pre-incubation for 1 hour with MAB-16-0030 (20-1 µg/well). ml) or IL-1Ra (10 µg/ml). Cells were then stimulated with recombinant human IL-36a (50 ng/ml, RnD systems) for 24 h before supernatant collection and analysis for IL-8 production (Duoset ELISA, RnD Systems).

Example 15 Viability and release of IL-6 by PBMCs.

The effect of anti-hIL-1R3 antibody MAB-16-0030 on the viability of unstimulated PBMCs (500,000/well) obtained from three healthy donors was studied using a conventional MTT reduction assay. Briefly, PBMC (200 μl) were incubated either with medium alone or with MAB-16-0030 (20 μg/ml). After 24 h, 3 and 5 days, the PBMCs were incubated for 2 h with MTT (20 μl) before measuring the absorbance at 570 nm on an ELISA reader. Using the known linearity between uptake and the number of viable cells converting MTT, the number of viable cells was calculated using only one medium as a control, taking its value as 100%. On the day of the MTT assay, PBMC supernatants from the same donors and incubated under the same conditions were collected and then analyzed for IL-6 production (Duoset ELISA, RnD systems) to assess any possible stimulatory effect of MAB-16-0030 antibody alone. .

Example 16 Functional blocking of PBMCs.

To assess the effect of MAB-16-0030 on human cells stimulated with various antigens, freshly isolated PBMCs from healthy donors were used. For all stimuli, experiments were performed using 500,000 PBMC/well, performing stimulation in a total volume of 200 μl. Cells were seeded and incubated with either medium alone, either MAB-16-0030 (20-1.1 μg/ml) or IL-1Ra (10 μg/ml) for 1 h prior to stimulation. Used the following incentives; LPS (10 ng/mL, 24 h, RPMI without FCS), anti-human CD3/CD28 (1.25 µg/mL, 0.5 µg/mL (eBioscience) 3 days, RPMI 10% FCS), IL12/-33 ( 2, 20 ng/ml (Peprotech, RnD Systems)) 3 days, RPM 10% FCS) or heat-inactivated Candida albicans (0.5x10 ⁶ /ml, 5 days, RPMI 10% FCS). After stimulation, supernatants were collected and analyzed by Duoset ELISA (RnD Systems) for cytokine production according to the manufacturer's protocol.

Example 17 Functional blocking of immune cells in whole blood.

Heat-inactivated Candida albicans was used to stimulate whole blood. Freshly collected blood from healthy donors (tubes with EDTA) was placed in microcentrifuge tubes (250 µl/tube) and pre-incubated with either medium alone (RPMI, no FCS) or MAB-16-0030 (20-0.1 µg/ml ), or IL-1Ra (10 µg/ml) for 1 hour before stimulation with Candida albicans (0.5x10 ⁶ /ml) with a final volume of 1 ml. After 24 h incubation (37° C., 5% CO ₂ ), supernatants were collected and analyzed for cytokine production by ELISA (Duoset, RnD Systems).

Example 18 Mixed Lymphocyte Culture Reactions (MLRs) PBMCs from healthy different donors were mixed 1:1 (250,000/donor) and incubated for 5 days (RPMI, 10% FCS) with either medium alone or MAB-16 -0030 (20-1 µg/ml) or IL-1Ra (10 µg/ml). Cytokine production was analyzed using a Quansys multiplex platform according to the manufacturer's protocol.

Description of drawings

Fig. 1 - sequences (amino acids represented by a one-letter code).

Complete sequences of variable regions (VR).

Heavy chain: VH complete: SEQ ID NO: 1-34 and SEQ ID NO: 173.

Light chain: VL complete: SEQ ID NO: 35-68 and SEQ ID NO: 174.

Complementarity determining regions (CDRs).

Heavy chain:

CDRH1: SEQ ID NO: 69-85;

CDRH2: SEQ ID NO: 86-102;

CDRH3: SEQ ID NO: 103-119.

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Light chain:

CDRL1: SEQ ID NO: 120-136;

CDRL2: SEQ ID NO: 137-153;

CDRL3: SEQ ID NOs: 154-170 and 175.

Constant Regions (CR).

Light chain: CR-L: SEQ ID NO: 171;

Heavy chain: CR-H: SEQ ID NO: 172.

In the figures below, AF676 is a commercial polyclonal antibody preparation purchased from the following link: https://www.mdsystems.com/products/human-il-1-racpiil-1-r3-antibody af676;

fig. 2 - ELISA of human IL-1R3.

384-well microtiter plates were coated with human IL-1R3 protein presenting the human extracellular domain of IL-1R3 (0.5 mg/ml, at least 1 hour). After a vigorous wash step followed by a blocking step, antibodies (12.5 μl per well) were added and incubated for 1 hour at room temperature. Unbound antibody was extensively washed. The amount of bound antibody was identified by incubating the microtiter plate with an anti-human peroxidase-labeled detection antibody (1 hour at room temperature). The peroxidase reaction was initiated by adding TMB and the absorbance was measured at 450 nm/620 nm.

To ensure specific binding to human IL-1R3, back-screening was performed in parallel with an IL12 protein with identical features. The analysis parameters were identical to those described above. B cell supernatants binding to human IL-1R3 but not to IL12 were considered active;

fig. 3 - analysis of HEK293 reporters.

HEK293T/17-FR cells were stably transfected with the pGL4.32[luc2P/NF-KB-RE/Hygro] vector (Promega) and plated in 384-well PDL Costar cell culture plates followed by a 30 min incubation with antibodies. Cells were then stimulated with IL-1e for 5 h before determining NF-kB activity using the Steady-Glo (Promega) luciferase assay kit according to the manufacturer's protocol;

fig. 4 - analysis of the reporter gene NFkB luciferase in a stable cell line A549.

Stably transfected A549-NFkB-RE-Luc cells (purchased from Signosis) were cultured for 3 days (1.7E+04 cells/cm ³ ). 384-well white flat-bottomed polystyrene TC treated microtiter plates (Corning) were filled with 4 x 10 ⁴ cells per well. After culturing for 10 h, the cells were incubated with antibodies for 1 h before stimulating the cells with 10 μl of IL-1e for an additional 5 h. each well in relation to unstimulated cells;

fig. 5 - cell binding assay: binding to IL-1R3 expressing cells.

Humanized anti-IL-1R3 IgG1-LALA antibodies were evaluated for their binding to cell lines with different IL-1R3 receptor densities by flow cytometry. Humanized anti-IL-1R3 IgG1-LALA antibodies bind to cell lines with low and high levels of IL-1R3 expression. The antibodies do not bind to mouse NIH-3T3 cells. The experiments were carried out in accordance with the method described in example 8;

fig. 6 - cell binding assay: cell binding in the SK-MEL-30 cell line highly expressed human IL-1R3.

Cell binding EC ₅₀ values for humanized anti-IL-1R3 IgG1-LALA antibodies were determined by binding to a highly expressed IL-1R3 cell line SK-MEL-30 by flow cytometry. Humanized anti-IL-1R3 IgG1-LALA antibodies MAB-16-0030 and MAB-160149 have cell binding levels of 307 and 306 ng/ml, respectively. The experiments were carried out in accordance with the method described in example 8;

fig. 7 - biochemical ELISA analysis of human IL-1R3.

Binding of humanized anti-IL-1R3 IgG1-LALA antibodies to recombinant human IL-1R3 protein was assessed by biochemical ELISA analysis. Exemplary antibodies show EC ₅₀ binding values of 16.3 and 29.1 ng/ml, respectively. The experiments were carried out in accordance with the method described in example 9;

fig. 8 - Suppression of IL-1a and IL-1b mediated NfKB signaling in A549-NFkB-RELuc cells.

Functional neutralization of IL-1a and IL-1b was assessed by reporter gene analysis using A549-NFkB-RE-Luc cells stimulated with 0.1 ng/ml IL-1a and IL-1b, respectively. Humanized anti-IL-1R3 IgG1-LALA antibodies have EC ₅₀ values superior to goat anti-human IL1-R3 polyclonal antibodies AF776 (R&D Systems). The experiments were carried out in accordance with the method described in example 10;

- 22 040391 fig. 9 - Analysis of the functional neutralization of IL-1a and IL-1P: Suppression of mediated IL-a and

IL-1b release of IL-6 by A-549 cells.

Neutralization of IL-a and IL-1b mediated IL-6 release by humanized anti-IL1R3 IgG1-LALA antibodies was assessed using A-549 cells. _EC50 values indicate that humanized anti-IL-lR3 IgG1-LALA antibodies outperform goat anti-human IL1R3 polyclonal antibodies AF676 (FIG. R&D Systems). The experiments were carried out in accordance with the method described in example 11;

fig. 10 - IL-33 Functional Neutralization Assay: Suppression of IL-33 mediated NfkB signaling in HEK-Blue-IL33TM cells.

Neutralization of IL-33 mediated cell signaling by humanized anti-[L-1R3 IgG1-LALA antibodies was assessed using IL-33 stimulated HEK-Blue-IL33™ cells containing the reporter gene (InvivoGen). EC ₅₀ demonstrate that humanized anti-[L-1R3 IgG1-LALA antibodies outperform goat anti-human IL-1R3 polyclonal antibodies AF676 (FIG. R&D Systems). The experiments were carried out in accordance with the method described in example 12;

fig. 11 - analysis of the functional neutralization of IL-36. Suppression of IL-36 mediated NfkB signaling in HEK-293/17-IF cells.

Neutralization of IL-36 mediated cell signaling by humanized anti-IL-1R3 IgG293-LALA antibodies was assessed using IL-33 stimulated HEK-Blue-IL33™ cells containing the reporter gene (InvivoGen). Humanized anti-IL-1R3 IgG1-LALA antibodies have EC ₅₀ values superior to goat anti-human IL1-R3 polyclonal antibodies AF776 (R&D Systems). The experiments were carried out in accordance with the method described in example 13;

fig. 12 - neutralization of the release of cellular cytokines mediated by IL-1a, IL-33 and IL-36a.

Neutralization of cellular release of cytokines mediated by IL-1a, IL-33 and IL-36a was assessed using specific IL-1a, IL-33 and EE-36a-dependent cell systems. Inhibition of cytokine release by a typical humanized anti-IL-1R3 IgG1-LALA antibody of the present invention (MAB-16-0030) was evaluated and compared with IL-1Ra. While the antibody of the invention is able to suppress cytokine release mediated by all three stimuli, IL-1Ra only affects the release of IL-1a-mediated cytokine. The experiments were carried out according to the method described in example 14;

fig. 13 - Viability and IL-6 release from unstimulated PBMCs treated with humanized anti-IL-1R3 IgG1-LALA antibody.

Binding of antibodies to immune cells can lead to debilitating and detrimental effects, for example, by direct induction of apoptotic signaling pathways, stimulation of excessive cytokine release, or antibody effector function, antibody-dependent cell-mediated cytotoxicity (ADCC). To rule out the direct effect of humanized anti-IL-1R3 IgG1-LALA antibody on the viability of PBMCs, the viability of PBMCs from three donors and IL-6 release were examined after incubation with various concentrations of a typical humanized anti-IL-1R3 IgG1-LALA antibody by invention (MAV-160030) within 1, 3 and 5 days. MAB-16-0030 did not affect either viability or IL-6 release. These results confirm that humanized anti-IL-1R3 IgG1-LALA antibodies block IL-1R3 function on immune cells without causing significant cell depletion or deleterious effects at the cellular level. The experiments were carried out in accordance with the method described in example 15;

fig. 14 - functional blocking of PBMCs activated by various stimuli.

To determine whether humanized anti-IL-1R3 IgG1-LALA antibodies suppressed activation of PBMCs stimulated with specific or complex stimuli, PBMCs from 10 donors were stimulated with LPS heat-inactivated Candida albicans, IL-12/IL-33, or antiCD3/CD28 . An exemplary humanized anti-IL-1R3 IgG1-LALA antibody of the invention is capable of suppressing cytokine release mediated by all tested stimuli. The experiments were carried out in accordance with the method described in example 16;

fig. 15 - functional blocking of immune cells in whole blood activated by Candida albicans.

To test whether the humanized anti-IL-1R3 IgG1-LALA antibody suppresses immune cell activation in whole blood, whole blood from 8 donors was stimulated with heat-inactivated Candida albicans. An exemplary humanized anti-IL-1R3 IgG1-LALA antibody of the invention shown in Figure 1 is capable of inhibiting Candida-induced IL-6 cytokine release. The experiments were carried out in accordance with the method described in example 17;

fig. 16 - blocking the release of cytokines in mixed lymphocyte reactions (MLR).

The ability of humanized anti-IL-1R3 IgG1-LALA antibodies to block the release of various cytokines was evaluated in mixed lymphocyte reactions (MLR) using PBMCs obtained from healthy, different donors. An exemplary humanized anti-IL-1R3 IgG1-LALA antibody of the invention

Claims (10)

The line shown in the drawing is able to suppress the release of IFNy, IL-6, TNF-α, IL-13, IL-17 and IL-10. The experiments were carried out in accordance with the method described in Example 18. (1) VL regions of SEQ ID NO: 60; (1) VH regions of SEQ ID NO: 26; (1) VL regions of SEQ ID NO: 60; (1) VH regions of SEQ ID NO: 26; (1) CDR1H with SEQ ID NO: 77, CDR2H with SEQ ID NO: 94, CDR3H with SEQ ID NO: 111, CDR1L with SEQ ID NO: 128, CDR2L with SEQ ID NO: 145 and CDR3L with SEQ ID NO: 162 ; 1. A humanized antibody that specifically binds to IL-1R3, or a fragment or derivative thereof, containing at least a portion of said antibody that is sufficient to provide Gk-N3-binding specificity, wherein the antibody, or fragment or derivative thereof, contains three heavy chain CDRs and three light chain CDRs selected from the group consisting of: (2) VL regions of SEQ ID NO: 174; and (3) VL regions of SEQ ID NO: 64. (2) VH regions of SEQ ID NO: 28; and (3) VH regions of SEQ ID NO: 30. (2) VL regions of SEQ ID NO: 174; and (3) VL regions of SEQ ID NO: 64. (2) VH regions of SEQ ID NO: 28; and (3) VH regions of SEQ ID NO: 30. 2. The humanized antibody, antibody fragment or derivative of claim 1, wherein the antibody comprises a heavy chain variable region (VH) that is at least 90% identical to a VH region selected from the group consisting of: (2) CDR1H with SEQ ID NO: 79, CDR2H with SEQ ID NO: 96, CDR3H with SEQ ID NO: 113, CDR1L with SEQ ID NO: 130, CDR2L with SEQ ID NO: 147 and CDR3L with SEQ ID NO: 175 ; and (3) CDR1H of SEQ ID NO: 81, CDR2H of SEQ ID NO: 98, CDR3H of SEQ ID NO: 115, CDR1L of SEQ ID NO: 132, CDR2L of SEQ ID NO: 149, and CDR3L of SEQ ID NO: 166. 3. A humanized antibody, antibody fragment or derivative according to claim 1 or 2, wherein the antibody contains a light chain variable region (VL) that is at least 90% identical to a VL region selected from the group consisting of: 4. The humanized antibody, antibody fragment or derivative of claim 2, wherein the antibody comprises a heavy chain variable region (VH) containing an amino acid sequence selected from the group consisting of: 5. The humanized antibody, antibody fragment or derivative of claim 3, wherein the antibody comprises a light chain variable region (VL) containing an amino acid sequence selected from the group consisting of: 6. Humanized antibody, fragment or derivative of an antibody according to any one of claims 1 to 5, which inhibits: (i) IL-1R3 induced NFkB activity; (ii) NFkB activity stimulated by IL-1a, IL-1e, IL-33 and/or IL-36; and/or (iii) NFkB activity stimulated by a complex selected from the group consisting of IL1p/IL-1R1/IL-1RAcP, IL-1a/IL-1R1/IL-1RAcP, IL-33/ST2/IL-1RACP and /or IL-36/IL-36R/IL-1RAcP. 7. A humanized antibody, antibody fragment or antibody derivative according to any one of the preceding claims, wherein said antibody inhibits, at a concentration of 10 μg/ml, NFkB expression in lysates of A549-NFkB-RE-Luc cells stimulated with 0.1 ng/ml human IL- 1a, human IL-1e, IL33 and/or IL-36, 50% or more, preferably 60% or more, preferably 80% or more, or preferably 90% or more, and more preferably 95 % or more relative to the results of the same assay without the specified antibody. 8. A humanized antibody, antibody fragment or antibody derivative according to any one of the preceding claims, wherein said antibody inhibits IL-1a, IL-1e, IL-33 and/or IL-36 stimulated, respectively, luciferase activity in HEK293T/17 cells transfected with luciferase under the control of the NF-kB reporter gene. 9. A humanized antibody, antibody fragment or antibody derivative according to any one of the preceding claims, wherein said antibody contains at least amino acid substitutions in L234A and L235A of a human IgG1 Fc fragment or S228P and L235E of a human IgG4 Fc fragment. 10. Use of a humanized antibody according to any one of the preceding claims for the treatment of an IL-1R3 mediated disease, in particular, wherein the IL-1R3 mediated disease is selected from the group consisting of lung cancer, non-small cell lung cancer (NSCL), bronchioloalveolar lung cancer, cancer bone, pancreatic cancer, skin cancer, head or neck cancer, skin or -