True human antibodies specific to interleukin-1-alpha

Abstract

The fully human monoclonal antibody comprises (i) an antigen-binding variable region that exhibits very high binding affinity for IL-1α, and (ii) a constant region that is effective both in binding C1q but activating the complement system, and in binding to multiple different Fc receptors.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to Canadian Patent Application No. 3,095,740, filed on 7 October 2020, which is incorporated herein by reference in its entirety.

[0002] Statement regarding federally sponsored research Not applicable.

[0003] Field of Invention This invention generally relates to the fields of immunology and antibodies (Ab). [Background technology]

[0004] Interleukin-1-alpha (IL-1α) is a pro-inflammatory cytokine that plays a role in several different activities, including inflammation, immune responses, tumor metastasis, and hematopoiesis. IgG autoantibodies against IL-1α occur spontaneously in the general human population and are thought to be beneficial in several different diseases involving sterile inflammation. [Overview of the project]

[0005] The amino acid sequences encoding the light chain and heavy chain variable regions of a monoclonal Ab (mAb) that binds to human IL-1α with high affinity have been discovered. Accordingly, what is described herein is a purified human mAb comprising (i) an antigen-binding variable region that exhibits very high binding affinity to human IL-1α, and (ii) a light chain variable region containing the amino acid sequence of SEQ ID NO: 1 (or its CDR) and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2 (or its CDR).

[0006] Also described herein is a set of isolated nucleic acids comprising a first nucleic acid encoding the heavy chain of a human mAb that specifically binds to IL-1α, and a second nucleic acid encoding the light chain of a human mAb that specifically binds to human IL-1α. The first nucleic acid may encode the amino acid sequence of SEQ ID NO: 1 (or its CDR), and the second nucleic acid may encode the amino acid sequence of SEQ ID NO: 2 (or its CDR).

[0007] In another embodiment, the following is described herein: an expression vector comprising both a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 (or its CDR) and a nucleic acid encoding the amino acid sequence of SEQ ID NO: 2 (or its CDR). Also described herein is a set of expression vectors comprising a first expression vector encoding the amino acid sequence of SEQ ID NO: 1 (or its CDR) and a second expression vector encoding the amino acid sequence of SEQ ID NO: 2 (or its CDR).

[0008] In addition, described herein are isolated host cells (e.g., mammalian cells such as CHO cells) comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 (or its CDR) and a nucleic acid encoding the amino acid sequence of SEQ ID NO: 2 (or its CDR).

[0009] Unless otherwise defined, all technical terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which this invention pertains. Commonly understood definitions of biological terms can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.

[0010] In this specification, the words “a” or “an” preceding a noun refer to one or more specific nouns. For example, the phrase “antibody” refers to “one or more antibodies.”

[0011] The term “antibody” or “Ab” means any immunoglobulin (e.g., human, rodent, cartilaginous fish, or camelid antibody) or its conjugate that specifically binds to an antigen (e.g., human IL-1α). Various types of Abs are known to those skilled in the art. Non-limiting examples of Abs include monoclonal Abs (e.g., full-length Abs), polyclonal Abs, multispecific Abs (e.g., bispecific Abs), single-stranded Abs (e.g., single-domain Abs, camelid Abs, and cartilaginous fish Abs), chimeric (e.g., humanized) Abs, and fully human Abs (i.e., true human Abs), such as those that can be found in or induced in humans. The term antibody also encompasses Ab conjugates (e.g., stabilizing proteins, labels, or therapeutic agents (e.g., any therapeutic agent described herein or known in the art)).

[0012] The term “antigen-binding fragment” means any portion of a full-length Ab containing at least one variable domain capable of specifically binding to an antigen [e.g., a variable domain of a mammalian (e.g., human, mouse, rat, rabbit, or goat) heavy or light chain immunoglobulin, a camelid variable antigen-binding domain (VHH), or a novel antigen receptor (Ig-NAR) domain of cartilaginous fish immunoglobulin]. For example, the antigen-binding fragments described herein may comprise at least a portion of an Ab Fc region that is sufficient to mediate antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC) in mammals (e.g., humans) and / or conjugate to a therapeutic agent (e.g., any therapeutic agent described herein or known in the art). As another example, the antigen-binding fragments described herein may comprise at least a portion of an Ab Fc region that does not mediate ADCC and / or CDC in mammals (e.g., humans). Non-limiting examples of Ab fragments include Fab, Fab', F(ab')2, Fv fragments, diabodies, linear antibodies, and multispecific Abs formed from Ab fragments. Additional Ab fragments containing at least one camelid VHH domain or at least one cartilaginous fish Ig-NAR domain include minibodies, microantibodies, sub-nanoantibodies, and nanoantibodies, as well as any other forms of Ab described in U.S. Patent Application Publication No. 2010 / 0092470.

[0013] The term "human antibody" refers to an antibody (Ab) encoded by nucleic acids present in the human genome (e.g., reconstituted human immunoglobulin heavy or light chain loci). In some embodiments, human Abs are produced in mammalian (e.g., human) cell cultures (e.g., Chinese hamster ovary cell lines). In some embodiments, human Abs are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human Abs are produced in bacterial or yeast cells.

[0014] The term “single-chain antibody” refers to a single polypeptide containing at least one variable binding domain capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein and known in the art (see, for example, the antibody described in U.S. Patent Application Publication No. 2010 / 0092470).

[0015] If Ab or its antigen-binding fragment binds to a specific antigen, such as human IL-1α, but to a lesser extent recognizes and binds to other molecules in the sample (e.g., neither recognizes nor binds), then Ab or its antigen-binding fragment "specifically binds" to that antigen or "specifically" "binds" to that antigen (through the epitope described herein, where the full-length antibody includes incorporating light and heavy chain variable regions). In some embodiments, Ab or its antigen-binding fragment is in phosphate-buffered saline at a concentration of 1 × 10⁻⁶ -10 M or less (for example, 1 × 10) -11 Less than M or 1 × 10 -12 The Ab or antigen-binding fragment selectively binds to the epitope with affinity (KD) less than M (e.g., determined by surface plasmon resonance). The ability of the Ab or antigen-binding fragment to specifically bind to the protein epitope may be determined by methods known in the art or by any of those methods described herein.

[0016] The term “complementarity-determining region” or “CDR” refers to a region within Ig (heavy or light Ig) that forms part of the antigen-binding site in Ab or its antigen-binding fragment. As is known in the art, heavy Ig contains three CDRs, namely CDR1, CDR2, and CDR3, and light Ig also contains three CDRs, namely CDR1, CDR2, and CDR3. In any Ab or its antigen-binding fragment, the three CDRs from heavy Ig and the three CDRs from light Ig together form the antigen-binding site in Ab or its antigen-binding fragment. The Kabat-Bat database is a system used in the art to number the CDR sequences present in light Ig or heavy Ig.

[0017] Methods and materials similar or identical to those described herein can be used in the practice or testing of the present invention, but appropriate methods and materials are described below. All applications and publications mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the specific embodiments discussed below are illustrative only and not limiting.

[0018] Embodiments of the present disclosure are described herein with reference to the accompanying figures, which are merely examples.

Brief Description of the Drawings

[0019] [Figure 1] FIG. 1 is a graph showing the results of an Octet Red96 assay indicating that the binding affinity (KD) of XIA14 for IL-1α was 9.29×10−11. [Figure 2] FIG. 2 is a graph showing the results of an Octet Red96 assay indicating that the binding affinity (KD) of XIA14 for the neonatal Fc receptor (FcRn) was 2.83×10−7.

Modes for Carrying Out the Invention

[0020] This specification describes compositions and methods related to fully human (true human) mAbs that contain antigen-binding variable regions that exhibit very high binding affinity for IL-1α. The preferred embodiments described below illustrate the application of these compositions and methods. Nevertheless, from the description of these embodiments and based on the description provided below, other aspects of the present invention can be created and / or practiced.

[0021] Methods involving conventional immunological and molecular biological techniques are described herein. Immunological methods (e.g., detection and localization of antigen-ab complexes, immunoprecipitation, immunoblotting, and assays for similar methods) are generally known in the art and are described in method papers such as *Current Protocols in Immunology*, Coligan et al., ed., John Wiley & Sons, New York. Molecular biological techniques are described in detail in papers such as *Molecular Cloning: A Laboratory Manual*, 2nd ed., vol. 1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; and *Current Protocols in Molecular Biology*, Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York. Ab methods are described in *Handbook of Therapeutic Abs*, Dubel, S., ed., Wiley-VCH, 2007. Cell culture techniques are generally well-known in this field and are described in detail in methodological papers such as Culture of Animal Cells: A Manual of Basic Technique, 4th edition, by R. Ian Freshney, Wiley-Liss, Hoboken, NJ, 2000; and General Techniques of Cell Culture by Maureen A. Harrison and Ian F. Rae, Cambridge University Press, Cambridge, UK, 1994. Methods for protein purification are discussed in Guide to Protein Purification: Methods in Enzymology, Vol. 182, Deutscher MP, ed., Academic Press, San Diego, Calif., 1990.

[0022] A fully human mAb comprising (i) an antigen-binding variable region exhibiting very high binding affinity to human IL-1α, and (ii) a light chain variable region containing the amino acid sequence of SEQ ID NO: 1 (or its CDR) and a heavy chain containing the amino acid sequence of SEQ ID NO: 2 (or its CDR). The light chain and heavy chain variable regions described herein (together forming a Fab) can be conjugated to an Fc or a portion thereof using conventional molecular biological techniques to fuse a desired Fc portion to the Fab or antigen-binding fragment. By this method, full-length immunoglobulins such as human IgG1 (e.g., IgG1a or IgG1b), IgG2 (e.g., IgG2a or IgG2b), IgG3 (e.g., IgG3a or IgG3b), IgG4 (e.g., IgG4a or IgG4b), IgD, IgA (e.g., IgA1 and IgA2), IgE, or IgM (e.g., dimers, pentamers and hexamers) (and different allotypes thereof) incorporating the light chain and heavy chain variable regions described herein can be produced.

[0023] The mAbs described herein may be affinity-matured to increase their binding specificity by known methods such as VH and VL domain shuffling (Marks et al. Bio / Technology 10:779-783, 1992), random mutagenesis of the hypervariable region (HVR) and / or framework residues (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.154(7):3310-9, 1995; and Hawkins et al., J.Mol.Biol.226:889-896, 1992), or modified by other methods. Amino acid sequence variants of Ab may be prepared by introducing appropriate changes into the nucleotide sequence encoding Ab. In addition, modifications to the nucleic acid sequence encoding mAb may be altered (e.g., without altering the amino acid sequence of the mAb) to enhance mAb production in a particular expression system (e.g., intron removal and / or codon optimization for a given expression system). The mAbs described herein may also be modified by conjugation to another protein (e.g., another mAb) or a non-protein molecule. For example, an mAb may be conjugated to a water-soluble polymer such as polyethylene glycol or carbon nanotubes (see, e.g., Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005). See U.S. Patent Application No. 11 / 754,899.

[0024] Amino acid mutations may be introduced within specific regions of these IgG subclasses. Possible amino acid mutations include, for example, those that enhance binding to the FcK receptor (as described in, e.g., Proc.Natl.Acad.Sci.USA103(11):4005-4010,2006;MAbs 1(6):572-579,2009;US Patent Application Publications 2010 / 0196362; 2013 / 0108623; 2014 / 0171623; 2014 / 0093496; and 2014 / 0093959), or those that enhance or reduce binding to FcRn (e.g., J.Biol.Chem.276(9):6591-6604,2001;Int (As described in Immunol. 18(12): 1759-1769, 2006; and J. Biol. Chem. 281(33): 23514-23524, 2006).

[0025] The two types of H chains are heterogeneously associated to produce a bispecific Ab. Techniques such as the knobs-into-holes technique (e.g., described in J.Immunol.Methods 248(1-2):7-15, 2001; and J.Biol.Chem. 285(27):20850-20859, 2010), electrostatic repulsion technique (e.g., described in brochure WO06 / 106905), and seedbody technique (e.g., described in Protein Eng.Des.Sel. 23(4):195-202, 2010) may be used for the heterogeneous association of the two types of H chains via the CH3 domain. Any of the Abs described herein may have modified or deficient glycans. Examples of antibodies with modified sugar chains include glycosylated antibodies (e.g., described in pamphlet WO99 / 54342), antibodies with defucosylated sugar chains (e.g., described in pamphlets WO00 / 61739, WO02 / 31140, WO06 / 067847, and WO06 / 067913), and antibodies with sugar chains containing bifid GlcNAc (e.g., described in pamphlet WO02 / 79255). Known methods for generating glycosylated IgG antibodies include introducing a mutation into asparagine at EU numbering position 297 in the heavy chain (J. Clin. Pharmacol. 50(5):494-506, 2010), and generating IgG using Escherichia coli (J. Immunol. Methods 263(1-2):133-147, 2002; and J. Biol. Chem. 285(27):20850-20859, 2010). Furthermore, heterogeneity in IgG due to C-terminal lysine deletion and heterogeneity due to disulfide bond mispairing in the hinge region of IgG2 can be reduced by introducing amino acid deletions / substitutions (e.g., described in brochure WO09 / 041613). Any Ab or antigen-binding fragment described herein contains at least one (e.g., one, two, three, four, five, or six) amino acids that are not present in the corresponding human Ab (e.g., amino acids that are not present in the CDR, e.g., added, inserted, or substituted).Any of the Abs or antigen-binding fragments described herein may also have at least one deleted amino acid (e.g., compared to the corresponding human Ab), such as a deletion from the N- or C-terminus of the light or heavy chain, or a deletion of amino acids from a constant domain (e.g., the Fc domain).

[0026] Preferably, to ensure that a high-titer human IL-1α-specific mAb can be administered to a subject with minimal adverse effects, the mAb composition of the present invention is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 99.9 wt% or higher purity (excluding any excipients). The mAb composition of the present invention may contain only one type of mAb (i.e., that produced from a single clone of B lymphocyte strain). In addition to the human IL-1α mAb, the Ab composition of the present invention may also contain other mAbs that specifically bind to antigens other than human IL-1α.

[0027] To modify or enhance function, the mAb may be another conjugated molecule such as a cytotoxin or a detectable label. The human IL-1α-specific mAb may be conjugated with one or more cytotoxins to more effectively kill cells expressing IL-1α. The cytotoxin for use in the present invention can be any cytotoxic agent (e.g., a molecule that can kill cells after contacting the cells) that can be conjugated to a human IL-1α-specific mAb. Examples of cytotoxins include radionuclides (e.g., 35 S, 14 C, 32 P, 125 I, 131 I, 90 Y, 89 Zr, 201 Tl, 186 Re, 188 Re, 57 Cu, 213 Bi, and 211Examples of cytotoxins include, but are not limited to, conjugated radionuclides and chemotherapeutic agents. Further examples of cytotoxins include antimetabolites (e.g., 5-fluorouracil (5-FU), methotrexate (MTX), fludarabine, etc.), microtubule inhibitors (e.g., vincristine, vinblastine, colchicine, taxanes (paclitaxel and docetaxel, etc.), etc.), alkylating agents (e.g., cyclophasphamide, melphalan, bischloroethylnitrosourea (BCNU), etc.), and platinum compounds (e.g., cisplatin (also known as cDDP)). Examples of other cytotoxic agents include, but are not limited to, carboplatin, oxaliplatin, JM-216, CI-973, etc., anthracyclines (e.g., doxorubicin, daunorubicin, etc.), antibiotic preparations (e.g., mitomycin C), topoisomerase inhibitors (e.g., etoposide, tenoposide, and camptothecin), or rutin, diphtheria toxin (DT), Pseudomonas aeruginosa exotoxin (PE) A, PE40, abrin, saporin, pokeweed virus protein, ethidium bromide, glucocorticoids, anthrax toxin, and others. See, for example, U.S. Patent No. 5,932,188.

[0028] Human IL-1α specific mAbs can also be conjugated to detectable labels. Useful detectable labels in this invention include biotin or streptavidin, magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, and similar), and radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C, 32 P, 111 In, 97 Ru, 67 Ga, 68 Ga, or 72Examples include radiopaque materials such as metals for radioimaging, paramagnetic contrast agents for nuclear magnetic resonance imaging, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Means for detecting such labels are well known to those skilled in the art. For example, radioactive labels may be detected using photographic film or a scintillation counter. Fluorescent markers may also be used and can be detected using a photodetector to detect the emitted light. Enzyme labels are typically detected by providing the enzyme with a substrate and detecting the reaction products produced by the action of the enzyme on the substrate, while colorimetric labels are detected simply by visualizing the colored labels.

[0029] The present invention also encompasses nucleic acid molecules encoding mAbs specific to human IL-1α. The same nucleic acid molecule may encode both the heavy and light chains of a human IL-1α-specific mAb, but a set of two different nucleic acid molecules, one encoding the heavy chain and the other the light chain, may also be used. Any other suitable nucleic acids encoding the amino acid sequences of the mAbs described herein may also be used.

[0030] For mAb generation, nucleic acid molecules encoding heavy and light chains may be incorporated into an expression vector in an orientation such that such nucleic acid molecules are operably ligated to expression regulatory sequences, such as transcription and translation regulatory sequences. Examples of expression vectors include plasmid-derived vectors, as well as virus-derived vectors such as adenoviruses, adeno-associated viruses, and retroviruses. Nucleic acid molecules encoding light and heavy chains may be incorporated into one vector or different vectors. The vectors of the present invention may also include regulatory sequences such as promoters and / or enhancers (see U.S. Patents No. 5,168,062, 4,510,245, and 4,968,615), selection markers, or sequences encoding affinity tags (to facilitate purification) or detectable labels.

[0031] For mAb generation, the vector of the present invention can be introduced into a suitable host cell, such as a prokaryotic cell like bacteria, or preferably a eukaryotic cell like a mammalian, plant, or yeast host cell. Examples of methods for introducing heterogeneous polynucleotides into host cells include the use of a viral vector, electroporation, encapsulation of polynucleotides into liposomes, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, Agrobacterium-mediated transformation, microparticle gun transformation, and direct microinjection of DNA into the nucleus. Mammalian cell lines are currently preferred for mAb expression from the vector. Examples of mammalian host cells include Chinese hamster ovary (CHO) cells (e.g., DG44CHO cell line or CHO-K1 cell line), HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), NS0 cells, SP2 cells, HEK-293T cells, 293FreeStyle cells, and NIH-3T3 cells. mAbs may also be expressed in transgenic animals or plants. See, for example, U.S. Patents No. 5,827,690; No. 5,756,687; No. 5,750,172; No. 5,741,957; No. 6,046,037; and No. 5,959,177.

[0032] The Ab and antigen-binding fragments described herein may be formulated as pharmaceutical compositions comprising the Ab and antigen-binding fragments and at least one pharmaceutically acceptable carrier (e.g., a non-natural pharmaceutically acceptable carrier). Non-limiting examples of pharmaceutically acceptable carriers include sterile water, physiological saline, stabilizers, excipients, antioxidants (e.g., ascorbic acid), buffers (e.g., phosphates, citrates, histidine, and other organic acids), preservatives, surfactants (e.g., PEG and Tween), chelating agents (e.g., EDTA or EGTA), and binders. Additional examples of pharmaceutically acceptable carriers include low molecular weight polypeptides, proteins (e.g., serum albumin and gelatin), amino acids (e.g., glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine, and lysine), sugars and carbohydrates (e.g., polysaccharides and monosaccharides), and sugar alcohols (e.g., mannitol and sorbitol). When preparing aqueous solutions for injection, isotonic solutions containing physiological saline, glucose, and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used, if necessary, in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol and PEG), and nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer 188, and HCO-50). By mixing hyaluronidase into the formulation, larger volumes of fluid can be administered subcutaneously (see, for example, Expert. Opin. Drug. Deliv. 4(4):427-440, 2007).

[0033] The Ab and antigen-binding fragments provided herein may be encapsulated, for example, in microcapsules (e.g., hydroxymethylcellulose, gelatin, and poly(methyl methacrylate)) or incorporated as components of colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, “Remington's Pharmaceutical Science 16th edition”, Oslo Ed. (1980)). Methods for preparing pharmaceutical compositions as controlled-release pharmaceutical agents are also well known, and such methods may be applied to the Ab and antigen-binding fragments of the present invention (see, for example, Langer et al., J. Biomed. Mater. Res. 15:267-277, 1981; Langer, Chemtech. 12:98-105, 1982; U.S. Patent No. 3,773,919; European Patent Application Publication No. 58,481; Sidman et al., Biopolymers 22:547-556, 1983; and European Patent No. 133,988).

[0034] The pharmaceutical compositions provided herein may be formulated for intravenous, intra-arterial, intradermal, subcutaneous, intramuscular, intraperitoneal, or oral administration. [Examples]

[0035] Example 1 - Discovery of heavy chain and light chain variable region sequences of anti-human IL-1α Ab

[0036] Plasma and peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors. The presence of anti-IL-1α antibodies in plasma was confirmed by bead-based flow cytometry analysis using streptavidin magnetic beads conjugated with biotinylated recombinant human IL-1α. PBMCs were isolated from donor blood using Histopaque® 1077 and Accuspin® test tubes, and cells were isolated from a subset of the PBMCs. RNA was extracted from PBMCs and B cells using conventional methods, and cDNA was prepared from RNA. PCR was performed on cDNA using the procedure and primers described in U.S. Patent No. 9,453,217. Since the reactivity in plasma was isotyped to be predominantly from the IgG4 subclass, with some signaling from the IgG1 subclass, the reverse primers used were selected to specifically amplify IgG4 and IgG1. Both kappa and lambda light chain libraries were prepared. The phage library was generated from IgG4-kappa overlap, and three sound pannings were performed without any phage amplification between rounds. An input library diversity of 0.65e12 was found. After round 2, half of the clones were counted on the plate, and the remainder were subjected to an additional round of panning. After three rounds of panning, 38 clones remained. ELISA-based screening was performed on the phage supernatant of recombinant human IL-1α coated ELISA plates using an anti-FLAG antibody for the detection of bound phages, and positive clones were identified. One of these, designated XIA14, was selected and sequenced. The amino acid sequences of the variable regions of the light and heavy chains of the human monoclonal antibody designated XIA14 are as follows, and the CDR (determined by IMGT / DomainGapAlign; Ehrenmann, F., Lefranc, M.-P. Cold Spring Harb Protoc., 2011(6):737-749(2011).DOI:10.1101 / pdb.prot5636.PMID:21632775.) is shown in bold and underlined: TIFF0007877306000001.tif39170

[0037] Thus, the XIA14 light chain comprises CDR1 having the amino acid sequence QSVLYSSNNKNY [SEQ ID NO: 3]; CDR2 having the amino acid sequence WAS [SEQ ID NO: 4]; and CDR3 having the amino acid sequence QQYYSSPYT [SEQ ID NO: 5]. Similarly, the XIA14 heavy chain comprises CDR1 having the amino acid sequence GGRFTNYA [SEQ ID NO: 6]; CDR2 having the amino acid sequence IIPIFDET [SEQ ID NO: 7]; and CDR3 having the amino acid sequence ATGSNSYYGLY [SEQ ID NO: 8].

[0038] Example 2 - Characterization of XIA14

[0039] The results of the Octet Red 96 assay showed that the binding affinity (KD) of XIA14 to IL-1α was 9.29 × 10⁶. -11 This was shown (see Figure 1). The results of the Octet Red 96 assay showed that the binding affinity (KD) of XIA14 to the neonatal Fc receptor (FcRn) was 2.83 × 10⁻¹⁴. -7 This was demonstrated (see Figure 2).

[0040] Capability assays based on HUVEC are used for the IC of XIA14. 50 This showed that it was 5.0 ng / ml. In short, 0.2 × 10 6HUVEC cells (Corning® 354151) / ml were seeded in a 96-well flat-bottom plate. XIA14 molecules were diluted to concentrations ranging from 610 pg / ml to 100 μg / ml. The diluted XIA14 was administered to the HUVEC cells in the 96-well plate at a final concentration range of 61 pg / ml to 10 μg / ml. 0.5 ng / ml hIL-1α was applied to each well in the 96-well plate, and the assay plate was incubated at 37°C / 5% CO2 for 18 hours. HUVEC cells were stained with anti-ICAM-1 antibody (eBioscience 12-0549, clone HA58), and ICAM-1 expression levels were determined using flow cytometry. Data analysis was performed using FlowJo, and IC50 was calculated using KaleidaGraph.

[0041] Other Embodiments Although the present invention has been described in detail, it should be understood that the foregoing description is intended to be illustrative and does not limit the scope of the invention, and that the scope of the invention is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A composition comprising a purified human monoclonal antibody that specifically binds to interleukin-1-alpha (IL-1α) and comprises a light chain variable region amino acid sequence including the CDRs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and a heavy chain variable region amino acid sequence including the CDRs of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

8.

2. The composition according to claim 1, wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 1, and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 2.

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