Anti-human interferon alpha receptor 1 monoclonal antibody and uses thereof

Abstract

Provided is an anti-human interferon α receptor 1 (IFNAR1) monoclonal antibody and use thereof. The anti-human interferon α receptor 1 (IFNAR1) monoclonal antibody of the present invention has the same binding affinity to human IFNAR1 as the anti-human IFNAR1 monoclonal antibody anifrolumab, and has the same neutralizing activity at the cellular level as anifrolumab, and is expected to be used for the prevention and treatment of related diseases.

Description

[Technical Field]

[0001] The present invention relates to the field of antibody drugs. Specifically, the present invention relates to a monoclonal antibody against human interferon alpha receptor 1 (IFNAR1) and uses thereof. [Background technology]

[0002] Type I interferons (IFNs) (IFNα, IFNβ, IFNω, and IFNτ) are a family of structurally related cytokines with antiviral, antitumor, and immunomodulatory properties (Hardy et al., Blood. 97:473, 2001; Cutrone and Langer, J. Biol. Chem. 276:17140, 2001). The human IFNα locus contains two subfamilies. The first subfamily consists of 14 nonallelic genes and four pseudogenes with at least 80% homology. The second subfamily (αII or ω) contains five pseudogenes and one functional gene, which shows 70% homology to the IFNα gene (Weissmann & Weber, Prog. Nucl. Acid Res. Mol. Biol., 33:251-300, 1986). Although IFNα subtypes have different specific activities, they share the same biosphere (Streuli et al., PNAS-USA 78:2848, 1981) and the same cellular receptor (Agnet M. et al., "Interferon 5", 1st Edition, Gresser, pp. 1-22, Academic Press, London, 1983).

[0003] Beta interferon (IFNβ) is encoded by a single gene that shares approximately 50% homology with IFNα.

[0004] All type I interferons bind to a cell surface receptor (IFNα receptor, IFNAR) consisting of two transmembrane proteins, IFNAR1 and IFNAR2. IFNAR1 has been reported to be required for high-affinity binding as well as for the specificity of the IFNAR complex (Cutrone & Langer, J. Biol. Chem. 276:17140, 2001). The functional differences between the various type I interferon subtypes remain unclear, but it is thought that each interacts with a different portion of the IFNAR receptor, potentially leading to diverse signaling outcomes (Cook et al. (1996) J. Biol. Chem. 271:13448). In particular, studies using mutant forms of IFNAR1 and IFNAR2 suggest that α and β interferons signal differently through the receptor by interacting differently with their corresponding chains (Lewerenz et al. (1998) J. Mol. Biol. 282:585).

[0005] Early functional studies of type I interferons focused on innate defense against viral infections (Haller et al., (1981) J. Exp. Med. 154:199; Lindenmann et al., (1981) Methods Enzymol. 78:181). However, recent studies suggest that type I interferons are potent immunoregulatory cytokines in the adaptive immune response. In particular, type I interferons have been shown to promote the differentiation of naive T cells along the Th1 pathway (Brinkmann et al., (1993) J. Exp. Med. 178:1655), enhance antibody production (Finkelman et al., (1991) J. Exp. Med. 174:1179), and support the functional activity and survival of memory T cells (Santini et al., (2000) J. Exp. Med. 191:1777; Tough et al., (1996) Science 272:1947).

[0006] Many studies suggest that IFNα can promote the maturation or activation of dendritic cells (DCs) (Santini, et al., (2000) J. Exp. Med. 191:1777; Luft et al., (1988) J. Immunol., 161:1947; Luft et al., (2002) Int. Immunol., 14:367; Radvanyi et al., (1999) Scand. J. Immunol., 50:499). Furthermore, increased type I interferon expression has been reported in many autoimmune diseases (Foulis et al., (1987) Lancet, 2:1423; Hooks et al., (1982) Arthritis Rheum 25:396; Hertzog et al., (1988) Clin. Immunol. Immunopathol. 48:192; Hopkins and Meager (1988) Clin. Exp. Immunol. 73:88; Arvin and Miller (1984) Arthritis Rheum. 27:582). The best-studied examples of this include insulin-dependent diabetes mellitus (IDDM) (Foulis (1987), supra) and systemic lupus erythematosus (SLE) (Hooks (1982), supra), both of which are associated with elevated IFN-α levels, as well as rheumatoid arthritis (RA) (Hertzog (1988), Hopkins and Meager (1988), Arvin and Miller (1984), supra), in which IFN-β may play a more important role.

[0007] Administration of interferon-α has been reported to exacerbate disease in patients with psoriasis and multiple sclerosis and to induce an SLE-like syndrome in patients without a history of autoimmune disease. Interferon-α has also been shown to induce glomerulonephritis in normal mice and promote the development of spontaneous autoimmune disease in NZB / W mice. Furthermore, IFN-α treatment has been shown to cause undesirable side effects, such as fever and neuropathy, in some cases. Therefore, there are pathological conditions in which inhibiting type I interferon activity could be beneficial for patients, and there is a need for agents that effectively inhibit type I interferon activity.

[0008] Anifrolumab, a monoclonal antibody drug targeting IFNAR1 developed by AstraZeneca, is intended for use in treating diseases such as systemic lupus erythematosus (Phase III clinical trials) and lupus nephritis (Phase II clinical trials). Summary of the Invention

[0009] The present invention aims to provide a novel anti-human interferon α receptor 1 monoclonal antibody, a pharmaceutical composition containing the monoclonal antibody, and pharmaceutical uses of the monoclonal antibody.

[0010] That is, the present invention includes the following.

[0011] 1. An isolated anti-human interferon alpha receptor 1 monoclonal antibody comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3), (a) the amino acid sequence of said CDR-H1 (herein, CDR-H1 refers to heavy chain CDR1) is set forth in SEQ ID NO: 1 (SYYMT); (b) the amino acid sequence of the CDR-H2 (herein, CDR-H2 refers to heavy chain CDR2) is set forth in SEQ ID NO: 2 (VINVYGGTYYASWAKG); (c) the amino acid sequence of the CDR-H3 (herein, CDR-H3 refers to heavy chain CDR3) is set forth in SEQ ID NO: 3 (EDVAVYMAIDL); (d) the amino acid sequence of the CDR-L1 (herein, CDR-L1 refers to light chain CDR1) is set forth in SEQ ID NO: 4 (QASQSISNQLS); (e) the amino acid sequence of said CDR-L2 (herein, CDR-L2 refers to light chain CDR2) is set forth in SEQ ID NO: 5 (DASSLAS); and (f) The monoclonal antibody, wherein the amino acid sequence of the CDR-L3 (herein, CDR-L3 represents light chain CDR3) is set forth in SEQ ID NO: 6 (LGIYGDGADDGIA).

[0012] 2. Contains a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 7, and the amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMTWVRQAPGKGLEWVSVINVYGGTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDVAVYMAIDLWGQGTLVTVSS; and Item 2. The monoclonal antibody according to Item 1, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8, and the amino acid sequence is AIQMTQSPSSLSASVGDRVTITCQASQSISNQLSWYQQKPGKAPKLLIYDASSLASGVPSRFSGSRSGTKFTLTISSLQPEDFATYYCLGIYGDGADDGIAFGGGTKVEIK.

[0013] 3. An isolated nucleic acid encoding the monoclonal antibody described in any one of the preceding claims.

[0014] 4. A host cell comprising the nucleic acid according to item 3.

[0015] The nucleic acid can be present on a vector. The vector can be of any type, for example, a recombinant vector such as an expression vector. Any of several host cells can be used. In one embodiment, the host cell is a prokaryotic cell, for example, E. coli. In another embodiment, the host cell is a eukaryotic cell, for example, a mammalian cell such as a Chinese hamster ovary (CHO) cell.

[0016] 5. A method for producing a monoclonal antibody, comprising culturing the host cell according to Item 4 to produce the monoclonal antibody according to any one of the preceding items.

[0017] The method includes expressing a recombinant vector encoding the anti-human interferon alpha receptor 1 monoclonal antibody in a suitable host cell, thereby producing the monoclonal antibody. In certain embodiments, the method includes culturing a host cell containing nucleic acid encoding the anti-human interferon alpha receptor 1 monoclonal antibody, thereby expressing the nucleic acid. The method may further include recovering the anti-human interferon alpha receptor 1 monoclonal antibody from the host cell culture or host cell culture medium.

[0018] 6. A pharmaceutical composition comprising the monoclonal antibody described in any one of the preceding claims and a pharmaceutically acceptable carrier.

[0019] The pharmaceutical composition may further comprise an additional therapeutic agent (eg, a different anti-human interferon alpha receptor 1 (IFNAR1) antibody).

[0020] 7. The pharmaceutical composition according to item 6, which is used to treat a disease associated with interferon-mediated signal transduction.

[0021] 8. The pharmaceutical composition according to Item 7, wherein the disease associated with interferon-mediated signaling is systemic lupus erythematosus, insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple sclerosis, psoriasis, autoimmune thyroiditis, rheumatoid arthritis, glomerulonephritis, HIV infection, AIDS, transplant rejection and / or graft-versus-host disease.

[0022] 9. Use of a monoclonal antibody according to any preceding claim in the preparation of a medicament for treating a disease associated with interferon-mediated signaling.

[0023] 10. The use according to item 9, wherein the disease associated with interferon-mediated signaling is systemic lupus erythematosus, insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple sclerosis, psoriasis, autoimmune thyroiditis, rheumatoid arthritis, glomerulonephritis, HIV infection, AIDS, transplant rejection and / or graft-versus-host disease.

[0024] 11. A method comprising administering the monoclonal antibody described in any one of the preceding claims or the pharmaceutical composition described in any one of the preceding claims to a subject in need thereof. Methods for treating diseases associated with interferon-mediated signaling.

[0025] 12. The method according to item 11, wherein the disease associated with interferon-mediated signaling is systemic lupus erythematosus, insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple sclerosis, psoriasis, autoimmune thyroiditis, rheumatoid arthritis, glomerulonephritis, HIV infection, AIDS, transplant rejection and / or graft-versus-host disease. [Effects of the Invention]

[0026] The present invention provides a novel anti-human interferon α receptor 1 (IFNAR1) monoclonal antibody, which has comparable binding affinity to IFNAR1 and comparable cellular neutralizing activity to an anti-human interferon α receptor 1 monoclonal antibody (anifrolumab) that has entered Phase III clinical trials.

[0027] The monoclonal antibody of the present invention exhibits neutralizing activity at the cellular level equivalent to that of anifrolumab (prepared according to the sequence expression disclosed in the patent), and is expected to show good clinical efficacy in the prevention and treatment of related diseases. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 shows the results of nucleic acid electrophoresis for constructing the HZD1203-45 transient expression plasmid, where M is a marker; band 1 is the PCR product 362VH-Hu6; band 2 is pHZDCH, HindIII / NheI; band 3 is the PCR product 362VH-Hu20; and band 4 is pHZDCK, HindIII / BsiWI. [Figure 2] FIG. 2 is a flow chart of transient transfection expression. [Figure 3] Figure 3 is an electrophoresis detection diagram of QX006N (HZD1203-45-IgG4.1). [Figure 4] FIG. 4 shows the activity of QX006N (HZD1203-45-IgG4.1) and anifrolumab to neutralize human interferon-induced STAT1 / 2 phosphorylation in HEK Blue IFNα / β cells. [Figure 5] FIG. 5 shows the activity of QX006N and anifrolumab in neutralizing human interferon inhibiting Daudi cell proliferation. [Figure 6] FIG. 6 shows the activity of QX006N and anifrolumab in neutralizing human interferon-induced release of CXCL10 / IP10 from whole blood. DETAILED DESCRIPTION OF THE INVENTION

[0029] Scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, except that in case of conflict, the definitions used herein shall control.

[0030] Generally speaking, the terms used herein have the following meanings:

[0031] As used herein, an "isolated" antibody refers to an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, and the purity is determined, for example, by electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For methods of assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0032] As used herein, a "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody in the population is identical and / or binds to the same epitope. Except for possible variant antibodies (e.g., including naturally occurring mutations or those that arise during the preparation of a monoclonal antibody), such variants are generally present in minor amounts. Unlike a typical polyclonal antibody preparation, which contains different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring the antibody to be prepared by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by several techniques, including, but not limited to, hybridoma, recombinant DNA, phage display, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. This application describes such methods, as well as other exemplary methods for preparing monoclonal antibodies.

[0033] As used herein, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between binding partner members (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed as an equilibrium dissociation constant (KD). Affinity can be measured by routine methods known in the art.

[0034] As used herein, human interferon alpha / beta receptor 1 (IFNAR1) refers to a membrane protein derived from humans, and the amino acid sequence of its extracellular domain is shown in SEQ ID NO: 9, where the underlined portion represents the signal peptide. SEQ ID NO:9: MMVVLLGATTLVLVAVAPWVLSAAAGG KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEVDSFTPFRKAQIG PPEVHLEAEDKAIVIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPP PENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLL PPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSK

[0035] As used herein, "anti-human interferon α receptor 1 monoclonal antibody" means a monoclonal antibody that can bind to human interferon α receptor 1 with sufficient affinity so that it can be used as a diagnostic and / or therapeutic agent targeting human interferon α receptor 1.

[0036] The anti-human interferon α receptor 1 (IFNAR1) monoclonal antibody of the present invention does not bind to a target, unrelated protein. Here, "unrelated protein" refers to a protein other than the target human interferon α receptor 1. Here, "does not bind" means that, when the binding ability of the anti-human interferon α receptor 1 (IFNAR1) monoclonal antibody of the present invention to its target human interferon α receptor 1 is taken as 100%, the binding ability of the anti-human interferon α receptor 1 (IFNAR1) monoclonal antibody of the present invention to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.

[0037] The anti-human interferon α receptor 1 (IFNAR1) monoclonal antibody of the present invention may not bind to interferon α receptor 1 of other animal species. Here, "other animal species" refers to animal species other than humans, such as marmosets, cynomolgus monkeys, pigs, dogs, rabbits, rats, mice, and guinea pigs. Here, "does not bind" means that, when the binding ability of the anti-human interferon α receptor 1 (IFNAR1) monoclonal antibody of the present invention to its target, human interferon α receptor 1, is taken as 100%, the binding ability of the anti-human interferon α receptor 1 (IFNAR1) monoclonal antibody of the present invention to interferon α receptor 1 of other animal species is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.

[0038] The anti-human interferon α receptor 1 monoclonal antibody of the present invention has an equilibrium dissociation constant (K D )

[0039] The experimental results demonstrate that the anti-human interferon α receptor 1 (IFNAR1) monoclonal antibody of the present invention can specifically bind to human interferon α receptor 1 (IFNAR1).

[0040] The anti-human interferon α receptor 1 (IFNAR1) monoclonal antibody of the present invention has many biological activities equivalent to those of anifrolumab, a monoclonal antibody against human IFNAR1 currently in Phase III clinical trials, including the activity of neutralizing human interferon-induced phosphorylation of STAT1 / 2 in cells, the activity of neutralizing human interferon-induced inhibition of Daudi cell growth, and the activity of neutralizing human interferon-induced release of CXCL10 / IP10 from human whole blood.

[0041] In one embodiment, the amino acid sequence of the heavy chain of the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody of the present invention is set forth in SEQ ID NO:10, and the amino acid sequence of the light chain is set forth in SEQ ID NO:11. SEQ ID NO: 10 EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMTWVRQAPGKGLEWVSVINVYGGTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDVAVYMAIDLWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 11 AIQMTQSPSSLSASVGDRVTITCQASQSISNQLSWYQQKPGKAPKLLIYDASSLASGVPSRFSGSRSGTKFTLTISSLQPEDFATYYCLGIYGDGADDGIAFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Among them, SEQ ID NOs: 10 and 11 are both humanized sequences.

[0042] As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in cells that ordinarily contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0043] As used herein, "an isolated nucleic acid encoding an anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of the antibody, including such nucleic acid molecules in a single vector or separate vectors, and including such nucleic acid molecules present in one or more locations in a host cell.

[0044] As used herein, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of nucleic acids that are operably linked to them. Such vectors are referred to herein as "expression vectors."

[0045] As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny (regardless of the number of passages). Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0046] As used herein, "pharmaceutical composition" refers to a composition-like product that is in a form that enables the biological activity of the active ingredient contained therein and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is administered.

[0047] As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical composition that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0048] In the present invention, a "monoclonal antibody" is generally a human antibody and can be prepared using techniques well known to those skilled in the art. For example, human antibodies are generally described in van Dijk, MA and van de Winkel, JG, Curr. Opin. Pharmacol. 5:368-374 (2001) and Lonberg, N., Curr. Opin. Immunol. 20:450-459 (2008).

[0049] Antibodies can be prepared by administering immunogens to transgenic animals that have been modified to stimulate the production of fully human antibodies or intact antibodies with human variable regions in response to antigenic challenge. These animals typically contain some or all of the human immunoglobulin loci, either replacing the endogenous immunoglobulin loci or present extrachromosomally or randomly integrated within the animal. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated; see Lonberg, N., Nat. Biotech. 23:1117-1125 (2005) for a review of methods for obtaining human antibodies from transgenic animals. See also, e.g., the XENOMOUSE™ technology described in U.S. Patent Nos. 6,075,181 and 6,150,584, the HUMAB® technology described in U.S. Patent No. 5,770,429, the K-MMOUSE® technology described in U.S. Patent No. 7,041,870, and the VELOCIMOUSE® technology described in U.S. Patent Application Publication No. US2007 / 0061900. The human variable regions of intact antibodies generated from such animals can be further modified, for example, by combination with different human constant regions.

[0050] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cells and mouse-human hybrid myeloma cells used to produce human monoclonal antibodies have been described (see, e.g., Kozbor, D., J. Immunol. 133:3001-3005 (1984); Brodeur, B. R. et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; and Boerner, P. et al., Immunol. 147:86-95 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li, J. et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Other methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4);265-268 (which describes human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers, H.P. and Brandlein, S., Histology and Histopathology 20:927-937 (2005) and Vollmers, H.P. and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27:185-191 (2005).

[0051] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries, and then combining such variable domain sequences with desired human constant domains.

[0052] Human antibodies can also be selected based on an autoantibody library. That is, human antibodies can be isolated by screening a combinatorial library for antibodies with one or more desired activities. For example, various methods are known in the art for creating phage display libraries and screening such libraries for antibodies with desired binding properties. This method is reviewed, for example, in Hoogenboom, H.R. et al., Methods in Molecular Biology 178:1-37 (2001), and further described, for example, in McCafferty, J. et al., Nature 348:552-554 (1990); Clackson, T. et al., Nature 352:624-628 (1991); Marks, J.D. et al., J.Mol.Biol. 222:581-597 (1992); Marks, J.D. and Bradbury, A., Methods in Molecular Biology 248:161-175 (2003); Sidhu, S.S. et al., J.Mol.Biol. 338:299-310 (2004); Lee, C.V. et al. al., J. Mol. Biol. 340:1073-1093 (2004); Fellouse, FA, Proc. Natl. Acad. Sci. USA 101:12467-12472 (2004); and Lee, CV et al., J. Immunol. Methods 284:119-132 (2004).

[0053] In some phage display methods, complete sets of VH and VL genes are cloned by polymerase chain reaction (PCR) and randomly recombined into a phage library, which is then screened for antigen-binding phages, as described in Winter, G. et al., Ann. Rev. Immunol. 12:433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, unimmunized repertoires (e.g., from humans) can be cloned to provide a single source of antibodies against multiple non-self and self antigens in the absence of immunization, as described in Griffiths, AD et al., EMBO J, 12:725-734 (1993). Finally, non-immunized libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, encoding highly variable CDR3 regions using PCR primers containing random sequences, and rearranging them in vitro, as described by Hoogenboom, H.R. and Winter, G., J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0054] The antibody may also be a multispecific antibody, such as a bispecific antibody. Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. Techniques for producing multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305:537-540 (1983); WO93 / 08829; and Traunecker, A. et al., EMBO J. 10:3655-3659 (1991)) and "protuberance-into-cavity" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced using various techniques, such as electrostatic steering effects to generate antibody Fc heterodimeric molecules (WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan, M. et al., Science 229:81-83 (1985)), using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny, S.A. et al., J. Immunol. 148:1547-1553 (1992)), use of "double antibody" technology to generate bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), the use of single-chain Fv (scFv) dimers (see, e.g., Gruber, M. et al. al., J. Immunol. 152:5368-5374 (1994)), and preparation of trispecific antibodies (see, for example, Tutt, A. et al., J. Immunol. 147:60-69 (1991)).

[0055] The monoclonal antibodies described herein also include engineered modified antibodies with three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576).

[0056] The antibodies herein also include the multispecific antibodies described in WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, and WO2010 / 145793, WO2011 / 117330, WO2012 / 025525, WO2012 / 025530, WO2013 / 026835, WO2013 / 026831, WO2013 / 164325, or WO2013 / 174873.

[0057] The monoclonal antibodies described herein may be antibody variants, for example, if it is desired to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, as long as the final construct possesses the desired properties, such as antigen binding. Thus, in certain embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutations include HVRs and FRs. For example, amino acid substitutions can be introduced into a target antibody and products with the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC, can be screened. [Example]

[0058] The present invention will be described in more detail below through examples, but it should be understood that the present invention is not limited to these examples.

[0059] Example 1 Preparation of anti-human interferon α receptor 1 monoclonal antibody QX006N Human interferon α receptor 1 (IFNAR1) for immunization of New Zealand rabbits was purchased from Shanghai Novoprotein Scientific Co., Ltd., and antigen-binding specific antibody clones were obtained using B cell cloning technology. Monoclonal antibodies binding to human IFNAR1 were then screened for human IFNAR1 inhibitory activity. Cell supernatants were first detected by binding ELISA to select clones that bound to human IFNAR1. Then, clones with human IFNAR1 inhibitory activity were selected using the HEK Blue IFN α / β reporter gene cell assay. The above immunization and screening processes were completed by a commercial company.

[0060] Thirty-seven clones were selected for recombinant expression and sequenced. 362# and 1203# were found to have the best cell-neutralizing activity, and the sequences of the two clones were highly similar. Therefore, 362# was first humanized, and 1203# was screened. 1203# was found to have superior activity, so the 1203# clone was humanized based on the humanization of 362#. Based on identity comparison of human IgG germline sequences (Germline) using NCBI IgBlast, IgGHV3-66*01 was selected as the heavy chain CDR-grafting template, and the CDR regions of the 1203# clone heavy chain (i.e., CDR-H1 (SEQ ID NO: 1), CDR-H2 (SEQ ID NO: 2), and CDR-H3 (SEQ ID NO: 3)) were grafted into the framework regions of IgGHV3-66*01. IGKV1-6*01 was selected as the light chain CDR-grafting template, and the CDR regions of the 1203# clone light chain (i.e., CDR-L1 (SEQ ID NO: 4), CDR-L2 (SEQ ID NO: 5), and CDR-L3 (SEQ ID NO: 6)) were grafted into the framework regions of IGKV1-6*01. Backmutations were performed at specific sites in the framework regions to obtain the variable regions of the monoclonal antibody QX006N of the present invention. Finally, the sequence of the humanized heavy chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence of the humanized light chain variable region is shown in SEQ ID NO: 8.

[0061] The heavy chain variable region gene (SEQ ID NO: 7) and light chain variable region gene (SEQ ID NO: 8) were amplified by PCR using the gene sequence of the 362# humanized antibody as a template. The heavy chain expression plasmid pHZDCH was double-digested with HindIII and NheI, and the light chain expression plasmid pHZDCK was double-digested with HindIII and BsiWI. The PCR-amplified genes were then inserted into the corresponding expression plasmids using infusion recombinase to construct the heavy chain expression plasmid pHZDCH-362VH-Hu6 and the light chain expression plasmid pHZDCK-362VK-Hu20. During the humanization process, the gene for the 1203# humanized antibody was numbered 362, and the protein was numbered 1203.

[0062] The results of detecting the double digestion of the plasmids by nucleic acid electrophoresis are shown in Figure 1. As can be seen from the results in Figure 1, the PCR amplification of the antibody heavy chain variable region and light chain variable region, and the double digestion of the heavy chain and light chain expression plasmids, show that the sizes of the heavy chain and light chain plasmids are approximately 10,000 bp, the light chain variable region is approximately 447 bp, and the heavy chain variable region is approximately 471 bp.

[0063] Humanized antibody HZD1203-45 was obtained by humanizing 1203#. To reduce the ADCC effect of the antibody, the human IgG1 constant region of the HZD1203-45 heavy chain expression plasmid pHZDCH-362VH-Hu6 was replaced with human IgG4 to obtain the heavy chain expression plasmid pHZDCH-362VH-Hu6-IgG4.1.

[0064] ExpiCHO-S cells were co-transfected with the sequence-correct heavy chain expression plasmid pHZDCH-362VH-Hu6-IgG4.1 and the sequence-correct light chain expression plasmid pHZDCK-362VK-Hu20. The day before transfection, 3 × 10 ExpiCHO-S cells were cultured. 6 The cells were diluted to 6 × 10 cells / ml and passaged before transfection. 6The cells were diluted to 125 ml / ml and 25 ml of cells were placed in a 125 ml shake flask for transfection. The transfection and expression process is shown in Figure 2.

[0065] Culture supernatants were collected 4 to 8 days after transfection and purified in one step using Protein A. The purified antibody was detected by SDS-PAGE electrophoresis and designated QX006N (HZD1203-45-IgG4.1). The amino acid sequence of the heavy chain is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain is shown in SEQ ID NO: 11. The results of protein electrophoresis of this antibody are shown in Figure 3. Protein electrophoresis was performed on a denaturing, reducing gel. As can be seen from the results shown in Figure 3, two bands were observed, with sizes of approximately 50 kDa and 25 kDa, respectively, which are consistent with the theoretical molecular weights of the heavy chain (48.9 kDa) and light chain (23.4 kDa).

[0066] Example 2 Equilibrium dissociation constant (K D ) measurement The affinity of QX006N (HZD1203-45-IgG4.1) to human IFNAR1 was detected using a Biacore T200 instrument. All processes were performed at 25°C. A commercially available Protein A chip was used, and an appropriate amount of antibody was immobilized by capture to achieve an Rmax of approximately 50 RU and a capture flow rate of 10 μl / min. The antigen was serially diluted, and the instrument flow rate was switched to 30 μl / min. The reference and antibody-immobilized channels were run in order of lowest to highest concentration, with buffer running as a negative control. After each binding and dissociation, the chip was regenerated with pH 1.5 glycine. Using the instrument's software, a 1:1 binding model was selected in the Kinetics option and fitted to determine the antibody binding rate constant, k a , dissociation rate constant k d , and the equilibrium dissociation constant K D The value of was calculated.

[0067] We also compared the affinity of QX006N (HZD1203-45-IgG4.1) with that of anifrolumab, a monoclonal antibody against human IFNAR1 that has already entered Phase III clinical trials. The detection method for the known antibody was the same as that for QX006N. The results are shown in Table 1. Anifrolumab was self-produced by transiently transfecting ExpiCHO-S cells with an expression plasmid constructed based on the 9D4 sequence provided by patent WO2009100309A2.

[0068] [Table 1]

[0069] Example 3: Activity of QX006N and Anifrolumab in Neutralizing Human Interferon-Induced STAT1 / 2 Phosphorylation in HEK Blue IFNα / β Cells The HEK Blue IFNα / β reporter cell line was used to measure the activity of QX006N in antagonizing interferon-mediated IFNAR1-mediated phosphorylation of the intracellular signaling molecule STAT1 / 2. Cells were cultured in culture medium at 4 × 10 per well. 4 Cells were added to a 96-well plate and cultured overnight at 37°C and 5% CO2. Serial dilutions of antibody concentrations ranging from 0 to 5 μg / ml were added to the cells, followed by 0.2 ng / ml of IFNα.2b. After 24 hours of culture at 37°C and 5% CO2, the cell culture supernatant was collected, 10% QUANTI-Blue™ detection reagent was added, and the cells were incubated for 1 hour at 37°C and 5% CO2. The OD630nm values were then measured, and a dose-response curve was generated to analyze the antagonistic activity of the antibody. The dose-response curve is shown in Figure 4.

[0070] As can be seen from the results shown in Figure 4, QX006N can inhibit human interferon-induced STAT1 / 2 phosphorylation in HEK Blue IFNα / β cells, and the IC50 The IC50 of anifrolumab's activity in inhibiting interferon-induced IFNα / β phosphorylation was 5.23 ng / ml in HEK Blue IFNα / β cells. 50 was 4.43ng / ml.

[0071] Example 4 Activity of QX006N and Anifrolumab in Neutralizing Human Interferon-Induced Inhibition of Daudi Cell Growth The activity of QX006N in antagonizing interferon-induced IFNAR1-induced cell proliferation was measured using the Daudi human lymphoma cell line. 4 × 10 cells per well in culture medium were cultured. 4 Cells were added to a 96-well plate and cultured overnight at 37°C in 5% CO2. Serial dilutions of antibody ranging from 0 to 20 μg / ml were added to the cells, followed by 0.8 ng / ml of IFNα.2b. After 72 hours of culture at 37°C in 5% CO2, the cell cultures were harvested, and cell proliferation was detected using CellTiter-Glo. A dose-response curve was generated to analyze the antagonistic activity of the antibodies. The dose-response curve is shown in Figure 5.

[0072] As can be seen from the results shown in Figure 5, QX006N can inhibit interferon-induced Daudi cell proliferation, and the IC of QX006N's activity in inhibiting interferon-induced Daudi cell proliferation was 50 The IC50 of anifrolumab was 29.9 ng / ml, indicating its activity in inhibiting interferon-induced Daudi cell proliferation. 50 was 31.7ng / ml.

[0073] Example 5 Activity of QX006N and Anifrolumab in Neutralizing Human Interferon-Induced Release of CXCL10 / IP10 from Whole Blood The activity of QX006N in antagonizing interferon-induced IFNAR1-induced CXCL10 / IP10 release was measured using human whole blood. Whole blood was added at 100 μl per well to a 96-well plate and temporarily stored at 37°C and 5% CO2. Serial dilutions of antibody ranging from 0 to 40 μg / ml were added to the whole blood, followed by 8 ng / ml IFNα.2b and 40 ng / ml TNF-α. After 48 hours of incubation at 37°C and 5% CO2, cell culture supernatants were collected and CXCL10 / IP10 expression in the supernatants was detected using a sandwich ELISA. A dose-response curve was generated to analyze the antagonistic activity of the antibody. The dose-response curve is shown in Figure 6.

[0074] As can be seen from the results shown in Figure 6, QX006N can inhibit interferon-induced release of CXCL10 / IP10 from whole blood, and the IC of QX006N's activity in inhibiting interferon-induced release of CXCL10 / IP10 from whole blood was 50 The IC50 of anifrolumab was 698 ng / ml, indicating its activity in inhibiting interferon-induced release of CXCL10 / IP10 from whole blood. 50 was 562ng / ml.

Claims

1. 1. An isolated anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3), (a) the amino acid sequence of said CDR-H1 is set forth in SEQ ID NO:1; (b) the amino acid sequence of said CDR-H2 is set forth in SEQ ID NO:2; (c) the amino acid sequence of said CDR-H3 is set forth in SEQ ID NO:3; (d) the amino acid sequence of said CDR-L1 is set forth in SEQ ID NO:4; (e) the amino acid sequence of the CDR-L2 is set forth in SEQ ID NO:5; and (f) A monoclonal antibody, wherein the amino acid sequence of the CDR-L3 is set forth in SEQ ID NO:

6.

2. comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:7; and The monoclonal antibody of claim 1 , wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:

8.

3. An isolated nucleic acid encoding the monoclonal antibody of claim 1 or 2.

4. A host cell comprising the nucleic acid of claim 3.

5. A method for producing a monoclonal antibody, comprising culturing the host cell of claim 4 to produce the monoclonal antibody of claim 1 or 2.

6. A pharmaceutical composition comprising the monoclonal antibody of claim 1 or 2 and a pharma- ceutically acceptable carrier.

7. 1. A pharmaceutical composition for use in the treatment of a disease associated with interferon-mediated signaling, comprising: The pharmaceutical composition of claim 6, wherein the disease associated with interferon-mediated signaling is systemic lupus erythematosus, insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple sclerosis, psoriasis, autoimmune thyroiditis, rheumatoid arthritis, glomerulonephritis, HIV infection, AIDS, transplant rejection and / or graft-versus-host disease.

8. 23. Use in the preparation of a medicament for treating a disease associated with interferon-mediated signaling, comprising: The disease associated with interferon-mediated signaling is selected from systemic lupus erythematosus, insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple sclerosis, psoriasis, autoimmune thyroiditis, rheumatoid arthritis, glomerulonephritis, HIV infection, AIDS, transplant rejection and / or graft-versus-host disease; Use of the monoclonal antibody according to claim 1 or 2.

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