True human antibodies specific for interleukin 1 alpha (IL-1 alpha)

Abstract

The fully human monoclonal Ab comprises: (i) an antigen-binding variable region that exhibits very high binding affinity for IL-1 alpha; and (ii) a constant region that is effective in both activating the complement system by Clq binding and binding to several different Fc receptors.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Canadian Patent Application No. 3,095,740, filed on October 7, 2020, the entire contents of which are hereby incorporated by reference.

[0003] Statement on Federally Funded Research

[0004] not applicable. Invention Field

[0005] This invention relates generally to the fields of immunology and antibodies (Abs).

[0006] background

[0007] IL-1α is a pro-inflammatory cytokine that plays a role in many different activities, including inflammation, immune response, tumor metastasis, and hematopoiesis. IgG autoantibodies against IL-1α are naturally present in the general population and are believed to be beneficial for many different diseases involving aseptic inflammation.

[0008] Overview

[0009] Amino acid sequences encoding the light chain variable region and heavy chain variable region of a monoclonal Ab (mAb) that binds to human IL-1α with high affinity have been discovered. Therefore, this paper describes purified human mAbs containing an antigen-binding variable region that (i) exhibits a very high binding affinity for human IL-1α and (ii) comprises 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).

[0010] This article also describes a set of isolated nucleic acids comprising a first nucleic acid encoding a heavy chain of a human mAb that specifically binds to IL-1α and a second nucleic acid encoding a light chain of a human mAb that specifically binds to human IL-1α. The first nucleic acid is capable of encoding the amino acid sequence of SEQ ID NO:1 (or its CDR) and the second nucleic acid is capable of encoding the amino acid sequence of SEQ ID NO:2 (or its CDR).

[0011] On the other hand, this document describes expression vectors comprising both nucleic acids encoding the amino acid sequence of SEQ ID NO:1 (or its CDR) and nucleic acids encoding the amino acid sequence of SEQ ID NO:2 (or its CDR). This document also describes 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).

[0012] Additionally, this article describes isolated host cells (e.g., mammalian cells, such as CHO cells) containing nucleic acids encoding the amino acid sequence of SEQ ID NO:1 (or its CDR) and nucleic acids encoding the amino acid sequence of SEQ ID NO:2 (or its CDR).

[0013] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art 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.

[0014] As used in this article, the word "a" or "an" preceding a noun indicates one or more specific nouns. For example, the phrase "an antibody" means "one or more antibodies".

[0015] The term “antibody” or “Ab” means any immunoglobulin (e.g., human, rodent, cartilaginous fish, or camel antibody) or its conjugate that specifically binds to an antigen (e.g., human IL-1α). A wide variety of Abs are known to those skilled in the art. Non-limiting examples of Abs include: monoclonal Abs (e.g., including full-length Abs), polyclonal Abs, multispecific Abs (e.g., bispecific Abs), single-chain Abs (e.g., single-domain Abs, camel Abs, and cartilaginous fish Abs), chimeric (e.g., humanized) Abs, and fully human Abs (i.e., true human Abs, including Abs that can be found or induced in the human body). The term antibody also includes Ab conjugates (e.g., Abs conjugated to a stabilizing protein, a label, or a therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art).

[0016] 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., variable domains of mammalian (e.g., human, mouse, rat, rabbit, or goat) heavy or light chain immunoglobulins, camel-like variable antigen-binding domains (VHH), or cartilaginous fish immunoglobulin neoantigen receptor (Ig-NAR) domains]. For example, the antigen-binding fragment described herein may include at least a portion of the Ab's Fc region sufficient to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) and / or conjugate with a therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art). As another example, the antigen-binding fragment described herein may include at least a portion of the Ab's 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. Other Ab fragments containing at least one camel VHH domain or at least one cartilaginous fish Ig-NAR domain include mini-bodies, micro-antibodies, subnano-antibodies, and nanobodies, as well as any other form of Ab described in U.S. Patent Application Publication No. 2010 / 0092470.

[0017] The term "human antibody" refers to an Ab encoded by nucleic acids present in the human genome (e.g., rearranged 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.

[0018] The term "single-chain antibody" refers to a single polypeptide comprising at least one variable binding domain capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein and are known in the art (see, for example, the antibody described in U.S. Patent Publication No. 2010 / 0092470).

[0019] When an Ab or its antigen-binding fragment binds to a specific antigen (e.g., human IL-1α) but recognizes and binds to other molecules in the sample to a lesser extent (e.g., does not recognize and bind to) it, the Ab or its antigen-binding fragment "specifically binds" or "binds specifically" to that antigen (via an epitope of a full-length antibody containing both a light chain variable region and a heavy chain variable region as described herein). In some embodiments, the Ab or its antigen-binding fragment is in phosphate-buffered saline solution at a concentration equal to or less than 1 × 10⁻⁶. -10 M (for example, less than 1×10) -11 M or less than 1 x 10 -12 The affinity (KD) of the antigen-binding fragment (e.g., determined by surface plasmon resonance) binds selectively to the epitope. The ability of the antigen or antigen-binding fragment to specifically bind to the protein epitope can be determined using methods known in the art or any of those described herein.

[0020] The term "complementarity-determining region" or "CDR" refers to the region within an Ig (heavy chain Ig or light chain Ig) that forms the antigen-binding site in an Ab or its antigen-binding fragment. As is known in the art, heavy chain Ig contains three CDRs: CDR1, CDR2, and CDR3, and light chain Ig contains three CDRs: CDR1, CDR2, and CDR3. In any Ab or its antigen-binding fragment, the three CDRs from the heavy chain Ig and the three CDRs from the light chain Ig together form the antigen-binding site in the Ab or its antigen-binding fragment. The Kabat database is a system in the art for numbering CDR sequences present in light chain Ig or heavy chain Ig.

[0021] Although similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All applications and publications mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, the specific embodiments discussed below are illustrative only and not intended to be limiting. Brief description of the attached diagram

[0023] Schemes of this disclosure will now be described by way of example only with reference to the accompanying drawings.

[0024] Figure 1 This graph shows the results of the Octet Red 96 assay, which indicates that XIA14 has a binding affinity (KD) of 9.29 × 10⁻⁶ for IL-1α. -11 .

[0025] Figure 2This graph shows the results of the Octet Red 96 assay, which indicates that XIA14 has a binding affinity (KD) of 2.83 × 10⁻⁶ for the neonatal Fc receptor (FcRn). -7 .

[0026] Detailed description

[0027] This document describes compositions and methods relating to fully human (true human) mAbs comprising an antigen-binding variable region exhibiting very high binding affinity for IL-1α. Preferred embodiments described below illustrate the adaptation of these compositions and methods. Nevertheless, based on the description of these embodiments, other aspects of the invention can be manufactured and / or practiced on the basis of the description provided below.

[0028] This article describes methods involving conventional immunological and molecular biological techniques. Immunological methods (e.g., assays for the detection and localization of antigen-Ab complexes, immunoprecipitation, immunoblotting, etc.) are generally known in the art and described in methodological literature such as Current Protocols in Immunology, edited by Coligan et al., John Wiley & Sons, New York. Molecular biological techniques are described in detail in literature such as Molecular Cloning: A Laboratory Manual, 2nd Edition, vols. 1–3, edited by Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 and Current Protocols in Molecular Biology, edited by Ausubel et al., Greene Publishing and Wiley-Interscience, New York. Ab methods are described in Handbook of Therapeutic Abs, edited by Dubel, S., Wiley-VCH, 2007. Cell culture techniques are generally known in the art and are described in detail in methodological literature such as R. Ian Freshney's *Culture of Animal Cells: A Manual of Basic Technique*, 4th edition, Wiley-Liss, Hoboken, NJ, 2000, and Maureen A. Harrison and Ian F. Rae's *General Techniques of Cell Culture*, Cambridge University Press, Cambridge, UK, 1994. Methods for protein purification are discussed in *Guide to Protein Purification: Methods in Enzymology*, Vol. 182, edited by Deutscher MP, *Academic Press*, San Diego, Calif., 1990.

[0029] The complete human mAb contains an antigen-binding variable region that (i) exhibits a very high binding affinity for human IL-1α and (ii) comprises 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). The light chain and heavy chain variable regions described herein (which together form a Fab) can be linked to an Fc or a portion thereof using conventional molecular biology techniques to fuse the desired Fc moiety to the Fab or antigen-binding fragment. In this way, full-length immunoglobulins containing the light chain variable region and heavy chain variable region described herein can be prepared, 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 the various allotypes described above).

[0030] The mAbs described herein can be maturated to enhance or otherwise alter their binding specificity using known methods such as VH and VL domain shuffling (Marks et al., Bio / Technology 10:779-783, 1992), random mutagenesis of hypervariable regions (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). Amino acid sequence variants of Abs can be prepared by introducing appropriate changes into the nucleotide sequence encoding the Ab. Additionally, modifications to the nucleic acid sequence encoding a mAb can be made (e.g., without altering the amino acid sequence of the mAb) to enhance mAb production in certain expression systems (e.g., intron elimination and / or codon optimization for a given expression system). The mAbs described herein can also be modified by conjugation to another protein (e.g., another mAb) or a non-protein molecule. For example, mAbs can be conjugated to water-soluble polymers such as polyethylene glycol or carbon nanotubes (see, for example, Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005). See U.S. Patent Application No. 11 / 754,899.

[0031] Amino acid mutations can be introduced into the constant regions of these IgG subclasses. Such mutations can include, for example, mutations that enhance binding to the FcK receptor (as described in, for example, Proc. Natl. Acad. Sci. USA 103(11):4005-4010, 2006; Mabs 1(6):572-579, 2009; US 2010 / 0196362; US 2013 / 0108623; US 2014 / 0171623; US 2014 / 0093496; and US 2014 / 0093959), or amino acid mutations that enhance or degrade binding to FcRn (as described in, for example, J. Biol. Chem. 276(9):6591-6604, 2001; Int Immunol. 18(12):1759-1769, 2006; and (As described in J.Biol.Chem.281(33):23514-23524, 2006).

[0032] Two types of H chains undergo heterologous association to produce bispecific Abs. Knob-in-hole techniques (such as those described, for example, in J. Immunol. Methods 248(1-2):7-15,2001 and J. Biol. Chem. 285(27):20850-20859,2010), electrostatic repulsion techniques (such as those described, for example, WO 06 / 106905), and SEEDbody techniques (such as those described, for example, in Protein Eng. Des. Sel. 23(4):195-202,2010) can be used to heterologously associate two types of H chains via the CH3 domain. Any of the Abs described herein can be an Ab with modified sugar chains or defective sugar chains. Examples of antibodies with modified glycan chains include glycosylated engineered antibodies (as described in, for example, WO 99 / 54342), antibodies with defucosylated glycan chains (as described in, for example, WO00 / 61739, WO 02 / 31140, WO 06 / 067847 and WO 06 / 067913), and antibodies with glycan chains containing bisecting GlcNAc (as described in, for example, WO02 / 79255). Known examples of methods for generating IgG antibodies with glycan defects include methods that introduce a mutation into asparagine at EU number position 297 in the heavy chain (J. Clin. Pharmacol. 50(5):494-506, 2010), and methods that generate IgG using E. coli (J. Immunol. Methods 263(1-2):133-147, 2002; and J. Biol. Chem. 285(27):20850-20859, 2010). Furthermore, the heterogeneity associated with the deletion of C-terminal lysine in IgG and the heterogeneity associated with disulfide bond mismatches in the hinge region of IgG2 can be reduced by introducing amino acid deletions / substitutions (as described, for example, in WO 09 / 041613). Any of the Abs or antigen-binding fragments 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., added, inserted, or substituted amino acids, e.g., not within the CDR). Any of the Abs or antigen-binding fragments described herein may also have at least one missing amino acid (e.g., compared to the corresponding human Ab), for example, a deletion from the N-terminus or C-terminus of the light or heavy chain, or a deletion from a constant structural domain (e.g., the Fc domain).

[0033] Preferably, to ensure that high-titer human IL-1α-specific mAbs can be administered to subjects with minimal adverse effects, the purity (excluding any excipients) of the mAb compositions 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%, or higher percentages by weight. The mAb compositions of the present invention may comprise only a single type of mAb (i.e., a mAb produced by a single clonal B lymphocyte line). In addition to human IL-1α mAbs, the Ab compositions of the present invention may also comprise other mAbs that specifically bind to antigens other than human IL-1α.

[0034] To alter or enhance the function of mAbs, they can be conjugated to another molecule, such as a cytotoxin or a detectable marker. Human IL-1α-specific mAbs can be conjugated to one or more cytotoxins to more effectively kill cells expressing IL-1α. The cytotoxins used in this invention can be any cytotoxic agent capable of conjugating to human IL-1α-specific mAbs (e.g., molecules capable of killing cells upon contact with them). Examples of cytotoxins include, but are not limited to, radionuclides (e.g., radioisotopes). 35 S, 14 C 32 P, 125 I, 131 I, 90 Y、 89 Zr、 201 Tl、 186 Re、 188 Re、 57 Cu、 213 Bihe 211At), conjugated radionuclides, and chemotherapeutic agents. Other examples of cytotoxins include, but are not limited to, antimetabolites (e.g., 5-fluorouracil (5-FU), methotrexate (MTX), fludarabine, etc.), antimicrotubule agents (e.g., vincristine, vinblastine, colchicine, taxanes (such as paclitaxel and docetaxel, etc.), alkylating agents (e.g., cyclophosphamide, melphalan, dichloroethylnitrosourea (BCNU), etc.), platinum-based agents (e.g., cisplatin (also known as cDDP), carboplatin, oxaliplatin, JM-216, CI-973, etc.), anthracyclines (e.g., doxorubicin, daunorubicin, etc.), antibiotics (e.g., mitomycin-C), topoisomerase inhibitors (e.g., etoposide, teniposide, and camptothecin), or other cytotoxic agents such as ricin, diphtheria toxin (DT), pseudomonad exotoxin (PE)A, PE40, abrin, saporin, and pokeweed virus protein. Viral proteins, ethidium bromide, glucocorticoids, anthrax toxin, etc. See, for example, U.S. Patent No. 5,932,188.

[0035] Human IL-1α-specific mAbs can also be conjugated to detectable markers. Detectable markers that can be used in this invention include biotin or streptoacidin, magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, etc.), and radioactive markers (e.g., 3 H, 125 I, 35 S, 14 C 32 P, 111 In、 97 Ru、 67 Ga、 68 Ga or 72 Radioactive contrast agents, such as metals used for radiographic imaging, paramagnetic agents used for magnetic resonance imaging, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, are known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or a scintillation counter. Fluorescent markers can also be used, and these can be detected by detecting the emitted illuminance using a photodetector. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction products produced by the enzyme's action on the substrate, and colorimetric labels are detected by simple visual observation of the colored label.

[0036] This invention also includes nucleic acid molecules encoding mAbs specific to human IL-1α. Although the same nucleic acid molecule can simultaneously encode both the heavy and light chains of a human IL-1α-specific mAb, a set of two different nucleic acid molecules, one encoding the heavy chain and the other encoding the light chain, can also be used. Any other suitable nucleic acid encoding the amino acid sequence of the mAb described herein can also be used.

[0037] For mAb generation, nucleic acid molecules encoding heavy and light chains can be integrated into expression vectors in a manner in which such nucleic acid molecules are operatively linked to expression control sequences, such as transcription and translation control sequences. Examples of expression vectors include plasmid-derived vectors and viral-derived vectors (such as adenovirus, adeno-associated virus, and retrovirus). Nucleic acid molecules encoding light and heavy chains can be integrated into a single vector or different vectors. The vectors of the present invention may also include regulatory sequences, such as promoters and / or enhancers (see U.S. Patent Nos. 5,168,062, 4,510,245, and 4,968,615), selectable markers, or sequences encoding affinity tags (for facilitating purification) or detectable tags.

[0038] To generate mAbs, the vector of the present invention can be introduced into suitable host cells, such as prokaryotic cells (e.g., bacteria) or preferably eukaryotic cells (e.g., mammalian, plant, or yeast host cells). Examples of methods for introducing heterologous polynucleotides into host cells include the use of viral vectors, electroporation, encapsulation of polynucleotides in liposomes, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, Agrobacterium-mediated transformation, biolistic transformation, and direct microinjection of DNA into the nucleus. For expressing mAbs from vectors, mammalian cell lines are currently preferred. Examples of mammalian host cells include Chinese hamster ovary (CHO) cells (e.g., DG44CHO or CHO-K1 cell lines), HeLa cells, young hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), NSO cells, SP2 cells, HEK-293T cells, 293Freestyle cells, and NIH-3T3 cells. mAb can also be expressed in transgenic animals or plants. See, for example, U.S. Patent Nos. 5,827,690; 5,756,687; 5,750,172; 5,741,957; 6,046,037; and 5,959,177.

[0039] The Ab and antigen-binding fragments described herein can be formulated into 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. Further examples of pharmaceutically acceptable carriers include low molecular weight peptides, 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, physiological saline and isotonic solutions containing glucose and other excipients (such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride) can be used, and if desired, can be combined with appropriate solubilizers (such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol and PEG)) and nonionic surfactants (such as polysorbate 80, polysorbate 20, poloxamer 188, and HCO-50). By incorporating hyaluronidase into the formulation, larger fluid volumes can be administered subcutaneously (see, for example, Expert. Opin. Drug. Deliv. 4(4):427-440, 2007).

[0040] The Ab and antigen-binding fragments provided herein can be encapsulated, for example, in microcapsules (e.g., microcapsules made of hydroxymethyl cellulose, 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", edited by Oslo (1980)). Methods for preparing pharmaceutical compositions into controlled-release agents are also well known, and such methods can 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. EP 58,481; Sidman et al., Biopolymers 22:547-556, 1983; and EP 133,988).

[0041] The pharmaceutical compositions provided herein can be formulated for intravenous, intra-arterial, intradermal, subcutaneous, intramuscular, intraperitoneal, or oral administration. Example

[0042] Example 1 - Discovery of heavy chain and light chain variable region sequences of anti-human IL-1αAb

[0043] Plasma and peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors. The presence of anti-IL-1α antibodies in the plasma was confirmed by bead-based flow cytometry analysis using streptomycin antibiotin-conjugated magnetic beads with biotinylated recombinant human IL-1α. Histopaque was used. TM 1077 and Accuspin TM PBMCs were isolated from donor blood, and cells were isolated from a portion of the PBMCs. RNA was extracted from the PBMCs and B cells using standard methods, and cDNA was prepared from the RNA. PCR was performed on the cDNA using the procedure and primers described in U.S. Patent No. 9,453,217. The reverse primers used were selected to specifically amplify IgG4 and IgG1 because the reactivity in plasma was isotyped to be predominantly from the IgG4 subclass, with some signal from the IgG1 subclass. Both κ and λ light chain libraries were prepared. Phage libraries were generated from IgG4-κ overlap and subjected to three rounds of panning without any phage amplification between rounds. The diversity of the input library was found to be 0.65e12. After the second round, half of the clones were counted on the plate, and the remainder were panned again. After three rounds of panning, 38 clones were retained. ELISA-based screening was performed on phage supernatant on ELISA plates coated with recombinant human IL-1α, and the bound phages were detected with anti-FLAG antibody to identify positive clones. One of them (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 of the designated XIA14 are shown below, where 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:

[0044] XIA14_Light Chain

[0045]

[0046] XIA14_Relink

[0047]

[0048] The XIA14 light chain therefore has the following: CDR1 with the amino acid sequence QSVLYSSNNKNY [SEQ ID NO:3], CDR2 with the amino acid sequence WAS [SEQ ID NO:4], and CDR3 with the amino acid sequence QQYYSSPYT [SEQ ID NO:5]. Similarly, the XIA14 heavy chain therefore has the following: CDR1 with the amino acid sequence GGRFTNYA [SEQ ID NO:6], CDR2 with the amino acid sequence IIPIFDET [SEQ ID NO:7], and CDR3 with the amino acid sequence ATGNSYYGLY [SEQ ID NO:8].

[0049] Characterization of Example 2-XIA14

[0050] The results measured by Octet Red 96 showed that the binding affinity (KD) of XIA14 for IL-1α was 9.29 × 10⁻⁶. -11 (see Figure 1 The results of Octet Red 96 assays showed that XIA14 had a binding affinity (KD) of 2.83 × 10⁻⁶ for the neonatal Fc receptor (FcRn). -7 (see Figure 2 ).

[0051] HUVEC-based efficacy assays indicate that XIA14 IC 50 It is 5.0 ng / ml. In short, 0.2 × 10⁻⁶ 6 HUVEC cells (Corning) TM 354151) / ml was seeded into 96-well flat-bottom plates. XIA14 molecules were diluted to concentrations ranging from 610 pg / ml to 100 μg / ml. The diluted XIA14 was administered to HUVEC cells in the 96-well plates at final concentrations ranging from 61 pg / ml to 10 μg / ml. 0.5 ng / ml hIL-1α was applied to each well of 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 by flow cytometry. Data analysis was performed using FlowJo, and IC50 was calculated using KaleidaGraph.

[0052] Other implementation plans

[0053] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims. sequence list <110> Ex Biotech <120> True human antibodies specific to interleukin-1α (IL-1α) <130> PAT 109601W-90 <150> CA 3,095,740 <151> 2020-10-07 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 114 <212> PRT <213> Homo sapiens <400> 1 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro His Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Ser Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg <210> 2 <211> 118 <212> PRT <213> Homo sapiens <400> 2 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Arg Phe Thr Asn Tyr 20 25 30 Ala Ile Leu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Leu 35 40 45 Gly Gly Ile Ile Pro Ile Phe Asp Glu Thr Asp His Ala Gln Asp Phe 50 55 60 Gln Asp Arg Leu Thr Ile Thr Val Asp Glu Ser Met Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Thr Gly Ser Asn Ser Tyr Tyr Gly Leu Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 3 <211> 12 <212> PRT <213> Homo sapiens <400> 3 Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 4 <211> 3 <212> PRT <213> Homo sapiens <400> 4 Trp Ala Ser 1 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <400> 5 Gln Gln Tyr Tyr Ser Ser Pro Tyr Thr 1 5 <210> 6 <211> 8 <212> PRT <213> Homo sapiens <400> 6 Gly Gly Arg Phe Thr Asn Tyr Ala 1 5 <210> 7 <211> 8 <212> PRT <213> Homo sapiens <400> 7 Ile Ile Pro Ile Phe Asp Glu Thr 1 5 <210> 8 <211> 11 <212> PRT <213> Homo sapiens <400> 8 Ala Thr Gly Ser Asn Ser Tyr Tyr Gly Leu Tyr 1 5 10

Claims

1. A pharmaceutical composition comprising a purified human monoclonal antibody that specifically binds to interleukin-1α (IL-1α) and comprises a light chain variable region amino acid sequence and a heavy chain variable region amino acid sequence, wherein the light chain variable regions CDR1, CDR2, and CDR3 are composed of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively, and the heavy chain variable regions CDR1, CDR2, and CDR3 are composed of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

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

2.

3. A group of isolated nucleic acids, comprising (i) a first nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of a purified human monoclonal antibody that specifically binds to interleukin-1α (IL-1α), wherein CDR1, CDR2, and CDR3 of the light chain variable region are composed of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and (ii) a second nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of the heavy chain variable region of the purified human monoclonal antibody that specifically binds to interleukin-1α (IL-1α), wherein CDR1, CDR2, and CDR3 of the heavy chain variable region are composed of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

4. The isolated nucleic acid according to claim 3, wherein the first nucleic acid encodes the amino acid sequence of SEQ ID NO:1, and the second nucleic acid encodes the amino acid sequence of SEQ ID NO:

2.

5. An isolated host cell comprising (i) a first nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of a purified human monoclonal antibody that specifically binds to interleukin-1α (IL-1α), wherein CDR1, CDR2, and CDR3 of the light chain variable region are composed of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively, and (ii) a second nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of the heavy chain variable region of the purified human monoclonal antibody that specifically binds to interleukin-1α (IL-1α), wherein CDR1, CDR2, and CDR3 of the heavy chain variable region are composed of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

6. The isolated host cell according to claim 5, wherein the first nucleic acid encodes the amino acid sequence of SEQ ID NO:1, and the second nucleic acid encodes the amino acid sequence of SEQ ID NO:

2.

7. The isolated host cell according to claim 5 or claim 6, wherein the host cell is a mammalian cell.

8. The isolated host cell according to claim 7, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

9. An expression vector comprising (i) a first nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of a light chain variable region of a purified human monoclonal antibody that specifically binds to interleukin-1α (IL-1α), wherein CDR1, CDR2, and CDR3 of the light chain variable region are composed of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and (ii) a second nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of a heavy chain variable region of the purified human monoclonal antibody that specifically binds to interleukin-1α (IL-1α), wherein CDR1, CDR2, and CDR3 of the heavy chain variable region are composed of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

10. The expression vectors according to claim 9, wherein the first nucleic acid encodes the amino acid sequence of SEQ ID NO:1, and the second nucleic acid encodes the amino acid sequence of SEQ ID NO:2.

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