RU2846007C1 - Completely human antibody specific to interleukin 1-alpha - Google Patents

Abstract

FIELD: pharmaceuticals; chemistry.

SUBSTANCE: invention relates to pharmaceutics and chemistry, specifically to a pharmaceutical composition capable of specifically binding to interleukin 1-alpha (IL-1α), containing a purified human monoclonal antibody that specifically binds to interleukin 1-alpha (IL-1α), and a pharmaceutically acceptable carrier.

EFFECT: high IL-1α binding affinity.

2 cl, 2 dwg, 2 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Canadian Patent Application No. 3,095,679, filed October 7, 2020, the entire contents of which are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

No data.

FIELD OF INVENTION

This invention generally relates to the fields of immunology and antibodies (Ab).

PRIOR ART

Interleukin 1 alpha (IL-1α) is a proinflammatory cytokine that plays a role in a number of different activities, including inflammation, immune responses, tumor metastasis, and hematopoiesis. Immunoglobulin G (IgG) autoantibodies to IL-1α occur naturally in the general human population and are thought to have beneficial effects in a number of different diseases that involve sterile inflammation.

SUMMARY OF THE INVENTION

An amino acid sequence of a light and heavy chain variable region of a monoclonal Ab (mAb) that binds to human IL-1α with high affinity is disclosed. Accordingly, this application describes purified human mAbs comprising (1) an antigen-binding variable region that exhibits very high binding affinity to human IL-1α, and (2) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 (or its complementarity determining regions (CDRs)), and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (or its CDRs).

In addition, a set of isolated nucleic acids is described herein, 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 may encode the amino acid sequence of SEQ ID NO: 1 (or its CDRs), and the second nucleic acid may encode the amino acid sequence of SEQ ID NO: 2 (or its CDRs).

According to another aspect, expression vectors are described herein that include both a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 (or its CDRs) and a nucleic acid encoding the amino acid sequence of SEQ ID NO: 2 (or its CDRs). Furthermore, a set of expression vectors is described herein, comprising a first expression vector encoding the amino acid sequence of SEQ ID NO: 1 (or its CDRs) and a second expression vector encoding the amino acid sequence of SEQ ID NO: 2 (or its CDRs).

Also described herein is an isolated host cell (e.g., a mammalian cell such as a Chinese hamster ovary (CHO) cell) comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 (or a CDR thereof) and a nucleic acid encoding the amino acid sequence of SEQ ID NO: 2 (or a CDR thereof).

Unless otherwise specified, all technical terms used in this description have the same meaning as commonly understood by one skilled in the art to which this invention pertains. Generally accepted 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.

The use of a singular noun in this description means one or more specific nouns. For example, the term "antibody" means "one or more antibodies."

The term "antibody" or "Ab" refers to any immunoglobulin (e.g., human, rodent, cartilaginous fish, or camelid antibodies) or conjugate thereof 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 (including, e.g., full-length Abs), polyclonal Abs, multispecific Abs (e.g., bispecific Abs), single-chain Abs (e.g., single-domain Abs, camelid Abs, and cartilaginous fish Abs), chimeric (e.g., humanized) Abs, and fully human Abs, including those that can be found or raised in humans (i.e., fully human Abs). The term "antibody" also includes Ab-based conjugates (e.g., an Ab conjugated to a stabilizing protein, label, or therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art)).

By "antigen-binding fragment" is meant any portion of a full-length Ab that comprises at least one variable domain [e.g., a variable domain of a mammalian (e.g., human, mouse, rat, rabbit, or goat) immunoglobulin heavy or light chain, a variable antigen-binding domain of a camelid antibody heavy chain (VHH), or a cartilaginous fish immunoglobulin novel antigen receptor (Ig-NAR) domain] capable of specifically binding to an antigen. For example, an antigen-binding fragment described herein may include at least a portion of an Fc region of an Ab that is sufficient to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) in a mammal (e.g., human), and/or may be conjugated to a therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art). As another example, an antigen-binding fragment described herein can include at least a portion of an Fc region of an Ab that does not mediate ADCC and/or CDC in a mammal (e.g., a human). 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 comprising at least one camelid VHH domain or at least one cartilaginous fish Ig-NAR domain include minibodies, microbodies, subnanobodies, and nanobodies, and any of the other forms of Ab described in U.S. Patent Application Publication No. 2010/0092470.

By "human antibody" is meant an Ab that is encoded by a nucleic acid (e.g., a reshaped locus encoding a human immunoglobulin heavy or light chain) present in the human genome. In some embodiments, the human Ab is produced in a mammalian (e.g., human) cell culture (e.g., a Chinese hamster ovary cell line). In some embodiments, the human Ab is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, the human Ab is produced in a bacterial or yeast cell.

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

When binding to a specific antigen, the Ab or antigen-binding fragment thereof "specifically binds" or "binds specifically" to that antigen, e.g., human IL-1α (via an epitope that binds to a full-length antibody comprising the light and heavy chain variable regions described herein), but recognizes and binds to a lesser extent (e.g., does not recognize and does not bind) other molecules in the sample. In some embodiments, the Ab or antigen-binding fragment thereof selectively binds to the epitope with an affinity (dissociation constant (KD)) equal to or below 1×10 ^-10 M (e.g., below 1×10 ^-11 M or below 1×10 ^-12 M) in phosphate-buffered saline (e.g., as determined using surface plasmon resonance). The ability of an Ab or antigen-binding fragment to specifically bind to an epitope of a protein can be determined using any of the methods known in the art or the methods described herein.

By "complementarity determining region" or "CDR" is meant a region in an Ig (either an Ig heavy chain or an Ig light chain) that forms part of the antigen-binding site in an Ab or an antigen-binding fragment thereof. As is known in the art, an Ig heavy chain comprises three CDRs: CDR1, CDR2, and CDR3, respectively, and an Ig light chain comprises three CDRs: CDR1, CDR2, and CDR3. In any Ab or antigen-binding fragment thereof, the three CDRs of the Ig heavy chain and the three CDRs of the Ig light chain collectively form the antigen-binding site in the Ab or in the antigen-binding fragment thereof. The Kabat database is one system used in the art for numbering the amino acid residue sequences of the CDRs present in an Ig light chain or an Ig heavy chain.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All applications and publications mentioned in this specification are incorporated by reference in their entireties. In case of conflict, the description of the present invention, including definitions, will control. In addition, the specific embodiments discussed below are illustrative only and are not intended to be limiting.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings.

Fig. 1 is a graph showing the results of the assay using the Octet Red 96 system, which showed that the binding affinity (KD) of XIA13 to IL-1α was 6.25×10 ^-11 M.

Figure 2 is a graph showing the results of the Octet Red 96 assay, which showed that the binding affinity (KD) of XIA13 to neonatal Fc receptor (FcRn) was 2.08×10 ^-7 M.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are compositions and methods relating to fully (genuine) human mAbs comprising an antigen-binding variable region that exhibits very high binding affinity for IL-1α. The preferred embodiments described below illustrate adaptations of these compositions and methods. However, other aspects of the invention may be made and/or practiced from the description of these embodiments and based on the description below.

Methods are described herein that include conventional immunology and molecular biology techniques. In general, immunological techniques (e.g., assays for detecting and localizing antigen-Ab complexes, immunoprecipitation, immunoblotting, and the like) are known in the art and are described in methodological scientific works such as Current Protocols in Immunology, Coligan et al., ed., John Wiley & Sons, New York. Molecular biology techniques are described in detail in such scientific works as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001: and Current Protocols in Molecular Biology, Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York. Methods related to Abs are described in Handbook of Therapeutic Abs, Dubei, S., ed., Wiley-VCH, 2007. Cell culture methods are generally known in the art and are described in detail in methodological scientific texts such as Culture of Animal Cells: A Manual of Basic Technique, 4th edition, by R Ian Freshney, Wiley-Liss, Hoboken, N.J., 2000; and General Techniques of Cell Culture, by Maureen A Harrison and Ian F Rae, Cambridge University Press, Cambridge, UK, 1994. Protein purification methods are discussed in Guide to Protein Purification. Methods in Enzymology, Vol.182, Deutscher M P, ed., Academic Press, San Diego, Calif., 1990.

Disclosed is a fully human mAb comprising (1) an antigen-binding variable region that exhibits very high binding affinity for human IL-1α, and (2) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 (or its CDRs), and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (or its CDRs). The light and heavy chain variable regions (which together form a Fab) described herein can be fused to an Fc region or a portion thereof using conventional molecular biology techniques to fuse a desired Fc portion to a Fab or antigen-binding fragment. In this manner, 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., dimeric, pentameric, and hexameric) (and various allotypes of the above forms) can be generated that contain the light and heavy chain variable regions described herein.

The mAbs described herein can be affinity matured to enhance or otherwise alter their binding specificity using known techniques such as shuffling of the heavy chain variable (VH) and light chain variable (VL) domains (Marks et al., Bio/Technology, 10: 779–783, 1992), random mutagenesis of hypervariable regions (HVRs) 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. Immuno,. 154(7): 3310-9, 1995; and Hawkins et al., J. Mol. Biol., 226: 889-896, 1992). Amino acid sequence variants of an Ab can be produced by making appropriate changes in the nucleotide sequence encoding the Ab. In addition, modifications can be made to the nucleic acid sequences encoding the mAb (e.g., without changing the amino acid sequence of the mAb) to enhance production of the mAb in certain expression systems (e.g., by removing introns and/or optimizing codons 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-proteinaceous molecule. For example, it is possible to conjugate a mAb to a water-soluble polymer such as polyethylene glycol or a carbon nanotube (see, e.g., Kam et al., Proc. Natl. Acad. Sci. USA, 102: 11600-11605, 2005). See U.S. Patent Application Ser. No. 11/754,899.

Mutations leading to changes in amino acids can be introduced into the constant region of IgG of these subclasses. Mutations resulting in amino acid changes that may be introduced may be, for example, those that enhance binding to FcK receptors (Fc fragment containing a mutation resulting in the formation of a knob) (as described, for example, in Proc. Natl. Acad. Sci. U.S.A., 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 enhance or weaken binding to FcRn (as described, for example, in J. Biol., Chem., 276(9): 6591-6604, 2001; Int. Immunol., 18(12): 1759-1769, 2006; and J. Biol,. Chem., 281(33): 23514-23524, 2006).

To form a bispecific Ab, it is necessary to perform the binding of two types of heterologous H chains. There are: knob-into-hole technology (described, for example, in J. Immunol. Methods. 248(1-2): 7-15, 2001; and J. Biol. Chem. 285(27): 20850-20859, 2010), electrostatic repulsion technology (described, for example, in WO 06/106905), technology for producing antibodies with domains engineered by chain exchange (SEEDbody) (described, for example, in Protein Eng. Des. Sel. 23(4): 195-202, 2010), and such technologies can be applied to bind two types of heterologous H chains using the constant domain 3 of the heavy chain (CH3). Any of the Abs described herein may be an antibody with modified carbohydrate chains or with a partial amount of carbohydrate chains. Examples of Abs having modified carbohydrate chains include antibodies engineered by altering glycosylation (described, for example, in WO 99/54342), Abs with defucosylated carbohydrate chains (described, for example, in WO 00/61739, WO 02/31140, WO 06/067847, and WO 06/067913), and Abs having a carbohydrate chain branched at N-acetylglucosamine (GlcNAc) residues (described, for example, in WO 02/79255). Known examples of methods for producing IgG antibodies with a partial number of carbohydrate chains include a method of introducing a mutation resulting in the substitution of asparagine at position 297 of the heavy chain according to the numbering system corresponding to the European (EU) index (J. Clin. Pharmacol., 50(5): 494-506, 2010), and a method of producing IgG using E. coli (J. Immunol. Methods., 263(1-2): 133-147, 2002; and J. Biol. Chem., 285(27): 20850-20859, 2010). In addition, heterogeneity due to deletion of the C-terminal lysine residue in IgG and heterogeneity due to misclosure of disulfide bonds 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 include at least one amino acid (e.g., one, two, three, four, five or six amino acids) (e.g., an added, inserted or substituted amino acid, e.g., not within a CDR) that is not present in the corresponding human Ab. Any of the Abs or antigen-binding fragments described herein can also comprise a deletion of at least one amino acid (e.g., compared to the corresponding human Ab), for example, comprise a deletion from the N- or C-terminus of the light or heavy chain, or a deletion of an amino acid from a constant domain (e.g., an Fc domain).

Preferably, in order to allow the human IL-1α-specific mAb to be administered to a subject at high titers and with minimal adverse effects, the mAb compositions of the invention comprise 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 more percent by weight of the mAb alone (excluding any excipients). The mAb compositions of the invention may comprise only one type of mAb (i.e., a mAb derived from a single clone of a B lymphocyte line). In addition to mAbs to human IL-1α, the Ab-based compositions of the invention may also include other mAbs that specifically bind to antigens other than human IL-1α.

In order to modify or enhance its function, the mAb can be conjugated to another molecule, such as a cytotoxin or a detectable label. A mAb specific for human IL-1α can be conjugated to one or more cytotoxins to more effectively kill cells expressing IL-1α. Cytotoxins for use in the present invention can be any cytotoxic agent (e.g., a molecule that can kill a cell upon contact with the cell) that can be conjugated to a mAb specific for human IL-1α. Examples of cytotoxins include, but are not limited ^to , radionuclides (e.g., ^35S , ^14C , ^32P , ^125I , 131I, ^90Y , ^89Zr , ^201Ti , ^186Re , ^188Re , ^57Cu , ^213Bi , and ^211At ), conjugated radionuclides, and chemotherapeutic agents. Additional examples of cytotoxins include, but are not limited to, antimetabolites (e.g., 5-fluorouracil (5-FU), methotrexate (MTX), fludarabine, etc.), microtubule-active agents (e.g., vincristine, vinblastine, colchicine, taxanes (such as paclitaxel and docetaxel), etc.), alkylating agents (e.g., cyclophosphamide, melphalan, bischloroethylnitrosourea (BCNU), etc.), platinum-based agents (e.g., cisplatin (also called cis-dichlorodiamineplatinum(II) (cDDP)), carboplatin, oxaliplatin, JM-216, CI-973, etc.), anthracyclines (e.g., doxorubicin, daunorubicin, etc.), antibiotic agents (e.g. mitomycin C), topoisomerase inhibitors (e.g., etoposide, tenoside, and camptothecins), or other cytotoxic agents such as ricin, diphtheria toxin (DT), Pseudomonas exotoxin A (PE), PE40, abrin, saporin, pokeweed antiviral protein, ethidium bromide, glucocorticoid, anthrax toxin, and others. See, e.g., U.S. Patent No. 5,932,188.

A mAb specific for human IL-1α can also be conjugated to a detectable label. Detectable labels useful in the present invention include biotin or streptavidin, magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radioactive labels (e.g., ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, ³² P, ¹¹¹ In, ⁹⁷ Ru, ⁶⁷ Ga, ⁶⁸ Ga, or ⁷² As), radiocontrast agents such as metals for radioisotope imaging, paramagnetic agents for magnetic resonance imaging, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in enzyme-linked immunosorbent assay (ELISA)) and colorimetric labels such as colloidal gold beads or colored glass or plastic (e.g., polystyrene, polypropylene, latex, and etc.). The means for detecting such labels are well known to those skilled in the art. For example, radioactive labels can be detected using photographic film or scintillation counters. Fluorescent markers can also be used, and their detection can be accomplished using a photodetector to detect the emitted radiation. Enzyme labels are typically detected by adding a substrate to an enzyme and detecting the reaction product formed as a result of the enzyme acting on the substrate, while colorimetric labels are detected by simply visualizing the colored label.

The present invention also encompasses nucleic acid molecules encoding a mAb specific for human IL-1α. Although a single nucleic acid molecule may encode both the heavy and light chains of a mAb specific for human IL-1α, a set of two different nucleic acid molecules, one encoding the heavy chain and the other encoding the light chain, may also be used. Any other suitable nucleic acid that encodes the amino acid sequences of the mAb described herein may also be used.

To produce mAbs, nucleic acid molecules encoding the heavy and light chains may be inserted into an expression vector in an orientation in which such nucleic acid molecules are operably linked to expression control sequences, such as transcription and translation control sequences. Examples of expression vectors include plasmid-derived vectors and vectors derived from viruses such as adenoviruses, adeno-associated viruses, and retroviruses. Nucleic acid molecules encoding the light chain and the heavy chain may be inserted into the same vector or into different vectors. The vectors of the invention may also include regulatory sequences such as promoters and/or enhancers (see U.S. Patent No. 5,168,062, U.S. Patent No. 4,510,245, and U.S. Patent No. 4,968,615), selectable markers, or sequences encoding affinity tags (to facilitate purification) or a detectable label.

To produce mAbs, the vectors of the invention can be introduced into a suitable host cell, for example a prokaryotic cell such as a bacterium, or preferably a eukaryotic cell such as a mammalian, plant or yeast host cell. Examples of methods for introducing heterologous polynucleotides into host cells include the use of viral vectors, electroporation, encapsulation of the polynucleotide(s) 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. Currently, mammalian cell lines are preferred for expression of mAbs using vectors. Examples of mammalian host cells include Chinese hamster ovary (CHO) cells (e.g., DG44 CHO cell line (CHO deficient in dihydrofolate reductase (dhfr)), or CHO-K1 cell line (CHO, subclone K1)), HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2 cells), NS0 cells, SP2 cells, human embryonic kidney, 293T cell line (HEK-293T), FreeStyle™ 293 cells, and NIH-3T3 cells. The mAb can also be expressed in transgenic animals or plants. See, for example, U.S. Patents 5,827,690, 5,756,687, 5,750,172, 5,741,957, 6,046,037, and 5,959,177.

The Ab and antigen-binding fragments described herein can be formulated into pharmaceutical compositions that comprise the Ab and antigen-binding fragment and at least one pharmaceutically acceptable carrier (e.g., a non-naturally occurring pharmaceutically acceptable carrier). Non-limiting examples of pharmaceutically acceptable carriers include sterilized water, saline, stabilizers, excipients, antioxidants (e.g., ascorbic acid), buffers (e.g., phosphate, citrate, histidine, and other organic acids), antiseptics, surfactants (e.g., polyethylene glycol (PEG) and Tween), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA) or ethyleneglycoltetraacetic acid (EGTA)) and binders. Further examples of pharmaceutically acceptable carriers also 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). In preparing an aqueous injection solution, physiological saline and isotonic solutions containing glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride can be used, and if necessary in combination with appropriate solubilizers such as alcohol (e.g. ethanol), polyalcohols (e.g. propylene glycol and PEG) and nonionic surfactants (e.g. polysorbate 80, polysorbate 20, poloxamer 188 and HCO50). By adding hyaluronidase to the composition, a larger volume of fluid can be administered subcutaneously (see, for example, Expert. Opin. Drug Deliv., 4(4): 427-440, 2007).

For example, the Ab and antigen-binding fragments disclosed herein can be encapsulated in microcapsules (e.g., made from hydroxymethylcellulose, gelatin, and poly(methyl methacrylate)) or incorporated as components into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, e.g., "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 can be applied to the Ab and antigen-binding fragments of the present invention (see, e.g., Langer et al., J. Biomed. Mate., Res., 15: 267-277, 1981; Langer, Chemtech., 12: 98-105, 1982; U.S. Patent No. 3,773,919; European Patent Application Publication No. EP 58481; Sidman et al., Biopolymers, 22: 547-556, 1983; and EP 133988).

The pharmaceutical compositions provided herein may be prepared for intravenous, intraarterial, intradermal, subcutaneous, intramuscular, intraperitoneal, or oral administration.

EXAMPLES

Example 1. Detection of variable region sequences of heavy and light chains of Ab to human IL-1α

Plasma and peripheral blood mononuclear cells (PBMCs) were isolated from a healthy human donor. The presence of anti-IL-1α antibodies in plasma was confirmed by bead-based flow cytometric analysis using streptavidin magnetic beads conjugated with biotinylated recombinant human IL-1α. PBMCs were isolated from donor blood using Histopaque™ 1077 medium and Accuspin™ tubes, and a proportion of PBMC cells were collected. RNA was extracted from PBMCs as well as B cells using conventional methods, and cDNA was generated from the RNA. PCR was performed on this cDNA using the methodology and primers described in US Patent 9,453,217. The reverse primer used was chosen to selectively amplify IgG4 and IgG1 since isotyping of plasma reactivity revealed that it was mostly of the IgG4 subclass, with some signal from the IgG1 subclass. Libraries of both kappa and lambda light chains were prepared. Phage libraries were constructed from regions overlapping IgG4-kappa and three rounds of panning were performed without any phage amplification between rounds. The diversity of the input library was found to be 0.65⋅10 ¹² . 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 with phage suspension supernatants on ELISA plates coated with recombinant human IL-1α using an antibody to the FLAG peptide tag to detect bound phages and positive clones were identified. One of these, designated XIA13, was selected and its DNA was sequenced. The amino acid sequences of the light and heavy chain variable regions of the human monoclonal antibody designated XIA13 are shown below, together with the CDRs (determined using the Immunogenetics Global Tag Database (IMGT)/DomainGapAlign; Ehrenmann, F., Lefranc, M.-P. Cold Spring Harb. Protoc., 2011(6): 737–749 (2011). DOI:10.1101/pdb.prot5636. PMID:21632775.), which are shown in bold and underlined:

>XIA13_Light chain

>XIA13_Heavy Chain

Thus, the light chain of XIA13 comprises a CDR1 having the amino acid sequence QSVLYSSNNKNY (SEQ ID NO: 3), a CDR2 having the amino acid sequence WAS (SEQ ID NO: 4), and a CDR3 having the amino acid sequence QQYYSTPST (SEQ ID NO: 5). Similarly, the heavy chain of XIA13 comprises a CDR1 having the amino acid sequence GGRFTNYA (SEQ ID NO: 6), a CDR2 having the amino acid sequence IIPIFDET (SEQ ID NO: 7), and a CDR3 having the amino acid sequence ATGSNSYYGLY (SEQ ID NO: 8).

Example 2. Defining XIA13 characteristics

The results of the Octet Red 96 assay showed that the binding affinity (KD) of XIA13 to IL-1α was 6.25× ^10-11 M (see Fig. 1). The results of the Octet Red 96 assay showed that the binding affinity (KD) of XIA13 to neonatal Fc receptor (FcRn) was 2.08× ^10-7 M (see Fig. 2).

A potency assay using human umbilical vein endothelial cells (HUVEC) showed that the 50% inhibitory concentration ( _IC50 ) of XIA13 was 3.7 ng/mL. Briefly, 0.2 x ¹⁰⁶ HUVEC cells (Corning™ 354151)/mL were seeded in a 96-well flat-bottomed plate. XIA13 molecules were diluted to concentrations ranging from 610 pg/mL to 100 μg/mL. The diluted XIA13 solution was added to HUVEC cells in the 96-well plate at final concentration ranges of 61 pg/mL to 10 μg/mL. Human IL-1α (hIL-1α) was added to each well of a 96-well plate at a concentration of 0.5 ng/ml and the assay plate was incubated at 37°C/5% _CO2 for 18 h. HUVEC cells were stained with an antibody to intercellular adhesion molecule 1 (ICAM-1) (eBioscience 12-0549, clone HA58) and the expression level of ICAM-1 was determined using flow cytometry. Data analysis was performed using FlowJo software and _IC50 was calculated using KaleidaGraph.

Other incarnations

It should be understood that although the invention has been described in detail, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the claims below.

--->

SEQUENCE LIST

<110> XBIOTECH INC.

<120> FULLY HUMAN ANTIBODY SPECIFIC TO INTERLEUKIN 1-

ALPHA

<130> PAT 109600W-90

<150> CA 3,095,679

<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 Pro 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 Thr Pro Ser Thr Phe Gly Gln Gly Thr Lys Val 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 Thr Pro Ser 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 (2)

1. A pharmaceutical composition capable of specifically binding to interleukin 1-alpha (IL-1α), comprising a purified human monoclonal antibody that specifically binds to interleukin 1-alpha (IL-1α) and comprises an amino acid sequence of a light chain variable region comprising a CDR1 (complementarity determining region) having the amino acid sequence of SEQ ID NO: 3, a CDR2 having the amino acid sequence of SEQ ID NO: 4, and a CDR3 having the amino acid sequence of SEQ ID NO: 5, and an amino acid sequence of a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 6, a CDR2 having the amino acid sequence of SEQ ID NO: 7, and a CDR3 having the amino acid sequence of SEQ ID NO: 8, and a pharmaceutically acceptable carrier. 2. The pharmaceutical composition according to claim 1, wherein the variable region of the light chain has an amino acid sequence with SEQ ID NO: 1, and the variable region of the heavy chain has an amino acid sequence with SEQ ID NO: 2.