Anti-human interleukin-33 monoclonal antibody and use thereof

Abstract

Provided in the present application are an anti-human interleukin-33 monoclonal antibody and the use thereof. The monoclonal antibody has a high affinity for human interleukin-33, has neutralizing activity, and can be used for preventing or treating related diseases mediated by human interleukin-33.

Description

Anti-human interleukin-33 monoclonal antibody and its application Technical Field

[0001] The present application relates to the field of antibody drug technology. Specifically, the present application relates to monoclonal antibodies against human interleukin-33 (hIL-33) and their applications. Background Art

[0002] Interleukin-33 (IL-33) is a key member of the IL-1 family and is expressed in a variety of cells, such as epithelial cells, fibroblasts, endothelial cells, smooth muscle cells, macrophages, and dendritic cells. Because the IL-33 gene does not contain a signal peptide, it cannot be secreted. However, when cells or tissues are damaged, it is released as an alarmin, alerting immune cells expressing IL-33 receptors, playing an important role in host defense, immune regulation, and inflammation (Cayrol C. et al., (2014) Curr. Opin. Immunol. 31C:31-37; Liew FY et al., (2016) Nat. Rev. Immunol. 16:676-689).

[0003] The IL-33 receptor is a heterodimeric molecule composed of ST2 (also known as IL1RL1) and IL-1R accessory protein (IL-1RAcP). ST2 is the receptor bound by IL-33, and IL-1RAcP is a shared component of the receptors of IL-1α, IL-1β, IL-1F6, IL1F8, and IL1F9. It is not required for binding but is essential for signal transduction (Schmitz J. et al., (2005) Immunity. 23: 479-490). After IL-33 binds to ST2, IL-1RAcP is recruited to form the IL-33 / ST2 / IL1RAcP ternary complex, which then induces signal transduction through the MyD88 adaptor, IRAK1 and IRAK4 kinases, and TRAF6, ultimately activating MAPK and NFκB transcription factors (Cayrol C. et al., (2018) Immunol Rev., 281(1): 154-168).

[0004] IL-33 has been shown to be associated with inflammatory diseases. It acts on immune cells expressing ST2, such as Th2 cells, eosinophils, mast cells, etc., driving the production of type 2 immune cytokines, especially IL-5 and IL-13, thereby triggering severe pathological changes in mucosal organs (Molofsky A. et al., (2015) Immunity. 42:1005–1019; Mjosberg JM et al., (2011) Nat. Immunol. 12:1055-1062). Inflammatory diseases associated with IL-33 include atopic dermatitis, asthma, chronic obstructive pulmonary disease, etc. (Savinko T. et al., (2012) J. Invest. Dermatol. 132:1392-1400; Prefontaine D. et al., (2010) J. Allergy. Clin. Immunol. 125:752-754; Byers D. et al., (2013) J. Clin. Invest. 123:3967–3982).

[0005] Itepekimab / REGN3500, a monoclonal antibody targeting interleukin-33, jointly developed by Regeneron and Sanofi, is intended for the treatment of inflammatory diseases such as chronic obstructive pulmonary disease (Phase III clinical trial) and asthma (Phase II clinical trial). Etokimab / ANB020, developed by AnaptysBio, is intended for the treatment of asthma, chronic sinusitis, and other diseases (Phase II clinical trial).

[0006] Summary of the Invention

[0007] The purpose of the present application is to provide a novel anti-human interleukin 33 (hIL-33) monoclonal antibody, a pharmaceutical composition comprising the monoclonal antibody, and the pharmaceutical use of the monoclonal antibody.

[0008] The specific technical solutions of this application are as follows:

[0009] 1. An isolated anti-human interleukin-33 monoclonal antibody, characterized in that it comprises three heavy chain complementary determining regions and three light chain complementary determining regions, wherein the three heavy chain complementary determining regions are CDR-H1, CDR-H2 and CDR-H3, and the three light chain complementary determining regions are CDR-L1, CDR-L2 and CDR-L3, wherein:

[0010] The amino acid sequence of CDR-H1 (CDR-H1 in this specification represents heavy chain CDR1) is shown in SEQ ID NO: 1 (SYHMI);

[0011] The amino acid sequence of CDR-H2 (CDR-H2 in this specification represents heavy chain CDR2) is shown in SEQ ID NO: 2 (VIYPNSNIYYATWAKG);

[0012] The amino acid sequence of CDR-H3 (CDR-H3 in this specification represents heavy chain CDR3) is shown in SEQ ID NO: 3 (TIYVHVYSALSI);

[0013] The amino acid sequence of CDR-L1 (CDR-L1 in this specification represents light chain CDR1) is shown in SEQ ID NO: 4 (QASESVLNEVS);

[0014] The amino acid sequence of CDR-L2 (CDR-L2 in this specification represents light chain CDR2) is shown in SEQ ID NO: 5 (FASKLAS);

[0015] The amino acid sequence of CDR-L3 (CDR-L3 in this specification represents light chain CDR3) is shown in SEQ ID NO: 6 (QQDWSMDNIDNA).

[0016] 2. The monoclonal antibody according to item 1, characterized in that it comprises a heavy chain variable region and a light chain variable region, wherein:

[0017] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7 (EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYHMIWVRQAPGKGLEWVGVIYPNSNIYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYVHVYSALSIWGQGTLVTVSS);

[0018] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8 (AFQMTQSPSSVSASVGDRVTITCQASESVLNEVSWYQQKPGKAPKLLIYFASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDWSMDNIDNAFGGGTKVEIK).

[0019] 3. An isolated nucleic acid, characterized in that it encodes the monoclonal antibody according to item 1 or 2.

[0020] 4. A host cell, characterized in that it contains the nucleic acid according to item 3;

[0021] 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 a variety of host cells can be used; in one embodiment, the host cell is a prokaryotic cell, for example, Escherichia coli (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.

[0022] 5. A method for producing a monoclonal antibody, characterized in that the method comprises culturing the host cell according to item 4 to produce the monoclonal antibody according to item 1 or 2;

[0023] The method comprises expressing a recombinant vector encoding the anti-human interleukin-33 (IL-33) monoclonal antibody in a suitable host cell, thereby producing the monoclonal antibody; in certain embodiments, the method comprises culturing a host cell containing a nucleic acid encoding the anti-human interleukin-33 (IL-33) monoclonal antibody, thereby expressing the nucleic acid; the method may further comprise recovering the anti-human interleukin-33 (IL-33) monoclonal antibody from the host cell culture or host cell culture medium.

[0024] 6. A pharmaceutical composition, characterized in that it comprises the monoclonal antibody according to item 1 or 2 and a pharmaceutically acceptable carrier;

[0025] The pharmaceutical composition may further comprise an additional therapeutic agent (eg, a different anti-human interleukin-33 (IL-33) antibody).

[0026] 7. The pharmaceutical composition according to item 6, characterized in that it is used to treat diseases related to human interleukin-33-mediated signal transduction.

[0027] 8. The pharmaceutical composition according to item 7, characterized in that the diseases related to human interleukin-33-mediated signal transduction are selected from any one or more of the following: asthma, chronic obstructive pulmonary disease (COPD), age-related macular degeneration (AMD), chronic sinusitis, atopic dermatitis, multiple sclerosis, arthritis and inflammatory bowel disease.

[0028] 9. Use of the monoclonal antibody according to item 1 or 2 in the preparation of a medicament for treating diseases associated with interleukin-33-mediated signal transduction.

[0029] 10. The use according to item 9, characterized in that the diseases related to human interleukin-33-mediated signal transduction are selected from any one or more of the following: asthma, chronic obstructive pulmonary disease (COPD), age-related macular degeneration (AMD), chronic sinusitis, atopic dermatitis, multiple sclerosis, arthritis and inflammatory bowel disease.

[0030] 11. A method for treating diseases related to human interleukin-33-mediated signal transduction, comprising: administering the monoclonal antibody or the pharmaceutical composition described above to a subject in need thereof.

[0031] 12. The method according to item 11, characterized in that the disease related to human interleukin-33-mediated signal transduction is selected from any one or more of the following: asthma, chronic obstructive pulmonary disease (COPD), age-related macular degeneration (AMD), chronic sinusitis, atopic dermatitis, multiple sclerosis, arthritis and inflammatory bowel disease.

[0032] Effects of the Invention

[0033] The present application provides a new anti-human interleukin-33 (IL-33) monoclonal antibody, which has a comparable affinity for binding to human interleukin-33 and a comparable neutralizing activity at the cellular level to that of Etokimab / ANB020, compared to the existing anti-human interleukin-33 (IL-33) monoclonal antibody (Etokimab / ANB020).

[0034] The monoclonal antibody drug (Itepekimab / REGN3500) targeting interleukin-33 developed by Sanofi is intended for the treatment of inflammatory diseases such as chronic obstructive pulmonary disease (clinical phase III) and asthma (clinical phase II). Etokimab / ANB020 developed by AnaptysBio is used for chronic sinusitis (clinical phase II).

[0035] The monoclonal antibody of the present application shows neutralizing activity comparable to that of Etokimab / ANB020 (prepared by expression according to the sequence disclosed in the patent) at the cellular level, and is expected to exhibit good clinical effects in preventing and treating related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 shows the results of nucleic acid electrophoresis for the construction of the QX007N (HZD78-70) transient expression plasmid. M: Marker; Band 1: PCR product 78VH-Hu25; Band 2: pQX2.1, HindIII / NheI; Band 3: PCR product 78VK-Hu3-CK; Band 4: pQX1, HindIII / BamHI.

[0037] Figure 2 is a flowchart of transient expression.

[0038] FIG3 is an electrophoresis detection diagram of QX007N (HZD78-70).

[0039] Figure 4 shows that QX007N (HZD78-70) and Etokimab / ANB020 neutralize recombinant human interleukin-33-induced HEK Blue TM Figure 3. Activation of NF-κB / AP-1 signaling in IL-33 cells.

[0040] Figure 5 shows that QX007N (HZD78-70) and Etokimab / ANB020 neutralize natural human interleukin-33-induced HEK Blue TM Figure 3. Activation of NF-κB / AP-1 signaling in IL-33 cells.

[0041] FIG6 is a graph showing the activity of QX007N (HZD78-70) and Etokimab / ANB020 in neutralizing the release of IL-5 from KU812 cells induced by recombinant human interleukin-33.

[0042] FIG7 is a graph showing the activity of QX007N (HZD78-70) and Etokimab / ANB020 in neutralizing the release of IFN-γ induced by recombinant human interleukin-33 from human whole blood. DETAILED DESCRIPTION

[0043] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0044] Generally speaking, the terms used in this specification have the following meanings.

[0045] As used herein, an "isolated" antibody is one that has been separated from components of its natural environment. In certain embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0046] In this specification, " monoclonal antibody " represents the antibody derived from the colony of substantially homologous antibody, that is, each antibody constituting the colony is identical and / or in conjunction with identical epi-position, except possible variant antibody (for example, containing naturally occurring mutation or producing in the production process of monoclonal antibody product), such variant is usually present in trace.Different from the polyclonal antibody product that generally includes the different antibodies for different determinants (epi-positions), every kind of monoclonal antibody of monoclonal antibody product is for the single determinant on antigen.Thus, the modifier " monoclonal " indicates that the antibody derives from the feature of substantially homologous antibody colony, and should not be construed as needing to produce the antibody by any ad hoc method.For example, the monoclonal antibody to be used according to the application can be prepared by various techniques, and the technology includes, but is not limited to hybridoma method, recombinant DNA method, phage display method and the method for using all or part of the transgenic animal comprising human immunoglobulin locus, and such method and other exemplary methods for preparing monoclonal antibody are described herein.

[0047] In this specification, "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 indicated, "binding affinity" as used in this specification refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D Affinity can be measured by common methods known in the art.

[0048] In this specification, human interleukin-33 (hIL-33) refers to hIL-33 located in the cell nucleus, which is hydrolyzed by proteases to form mature hIL-33, secreted outside the cell, and exerts the biological activity of hIL-33, which has the amino acid sequence shown in SEQ ID NO: 9.

[0049] SEQ ID NO: 9:

[0050]

[0051] In this specification, "anti-human interleukin-33 (hIL-33) monoclonal antibody" refers to a monoclonal antibody that can bind to human interleukin-33 with sufficient affinity so that the monoclonal antibody can be used as a diagnostic agent and / or therapeutic agent targeting human interleukin-33.

[0052] The anti-human interleukin-33 monoclonal antibodies of the present application do not bind to proteins unrelated to their target. Here, "unrelated proteins" refers to proteins other than the target human interleukin-33; "no binding" means that, with the binding capacity of the anti-human interleukin-33 (hIL-33) monoclonal antibodies of the present application to the target human interleukin-33 as 100%, the binding capacity of the anti-human interleukin-33 (hIL-33) monoclonal antibodies of the present application to the unrelated proteins is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.

[0053] The anti-human interleukin-33 (hIL-33) monoclonal antibodies of the present application can bind to interleukin-33 from humans and cynomolgus macaques, but may not bind to interleukin-33 from other animal species. Here, "other animal species" refers to animal species other than humans and cynomolgus macaques, such as pigs, dogs, rabbits, rats, mice, and guinea pigs. When determining the species specificity of the anti-human interleukin-33 (hIL-33) monoclonal antibodies of the present application, "no binding" means that, with the binding capacity of the anti-human interleukin-33 (hIL-33) monoclonal antibodies of the present application to human interleukin-33 as its target being taken as 100%, the binding capacity of the anti-human interleukin-33 (hIL-33) monoclonal antibodies of the present application to interleukin-33 from other animal species is less than 5%, for example, 4%, 3%, 2%, 1%, or 0%.

[0054] The human interleukin-33 monoclonal antibody of the present application has an equilibrium dissociation constant (K D ).

[0055] The experimental results show that the anti-human interleukin-33 (hIL-33) monoclonal antibody of the present application can specifically bind to human interleukin-33 (hIL-33).

[0056] The anti-human interleukin-33 (hIL-33) monoclonal antibody disclosed herein is comparable to or superior to similar marketed monoclonal antibodies in numerous biological activities, including neutralization of recombinant / natural human interleukin-33-induced NF-κB / AP-1 signaling in cells, neutralization of interleukin-33-induced IL-5 release in KU812 cells, and neutralization of interleukin-33-induced IFN-γ release in human whole blood.

[0057] In one embodiment, the amino acid sequence of the heavy chain of the anti-human interleukin-33 (hIL-33) monoclonal antibody of the present application is shown in SEQ ID NO: 10; the amino acid sequence of the light chain is shown in SEQ ID NO: 11.

[0058] SEQ ID NO: 10

[0059]

[0060] SEQ ID NO: 11

[0061]

[0062] Among them, SEQ ID NOs: 10 and 11 are both humanized sequences.

[0063] In this specification, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0064] In this specification, "isolated nucleic acid encoding anti-human interleukin-33 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 such nucleic acid molecules present in one or more locations in a host cell.

[0065] As used herein, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term encompasses vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0066] In this specification, "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and the progeny derived therefrom (regardless of the number of generations). Progeny may not be identical to the parental cell in terms of nucleic acid content, but may contain mutations. Mutant progeny with the same function or biological activity that have been screened or selected for the initially transformed cells are included in this specification.

[0067] In this specification, "pharmaceutical composition" means a preparation that is in a form that allows the biological activity of the active ingredient contained therein to take effect, and that contains no additional components that are unacceptably toxic to the subject to which the formulation is to be administered.

[0068] In this specification, "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0069] In this application, "monoclonal antibody" is generally a human antibody, which can be prepared using techniques 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).

[0070] Antibodies can be prepared by administering immunogens to transgenic animals that have been modified to stimulate the production of complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. These animals typically contain a portion or all of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or are present outside the chromosomes or randomly integrated into the animal. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23: 1117-1125 (2005). See also, for example, the XENOMOUSE described in U.S. Patent Nos. 6,075,181 and 6,150,584. TM Technology; US Patent No. 5,770,429 describes Technology; US Patent No. 7,041,870 describes Technology, and U.S. Patent Application Publication No. US 2007 / 0061900 Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0071] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cells for producing human monoclonal antibodies have been described (see, for example, Kozbor, D., J. Immunol. 133: 3001-3005 (1984); Brodeur, BR et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; Boerner, P. et al., J. Immunol. 147: 86-95 (1991)). Human antibodies produced via 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 in, for example, U.S. Pat. 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, HP and Brandlein, S., Histology and Histopathology 20: 927-937 (2005); Vollmers, HP and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27: 185-191 (2005).

[0072] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries; these variable domain sequences can then be combined with the desired human constant domains.

[0073] Human antibodies can also be selected based on self-antibody libraries, that is, human antibodies can be isolated by screening combinatorial libraries for antibodies with desired one or more activities. For example, a variety of methods for producing phage display libraries and screening such libraries for antibodies with desired binding characteristics are known in the art. This method is reviewed in, for example, Hoogenboom, HR et al., Methods in Molecular Biology 178: 1-37 (2001), and is further described in, for example, McCafferty, J. et al., Nature 348: 552-554 (1990); Clackson, T. et al., Nature 352: 624-628 (1991); Marks, JD et al., J. Mol. Biol. 222: 581-597 (1992); Marks, JD and Bradbury, A., Methods in Molecular Biology 248:161-175(2003); Sidhu, SS et al., J. Mol. Biol. 338: 299-310 (2004); Lee, CV et 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).

[0074] In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which are then screened for antigen-binding phage, as described in Winter, G. et al., Ann. Rev. Immunol. 12:433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, non-immunized repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a large number of non-self and also self-antigens in the absence of any immunization, as described by Griffiths, AD et al., EMBO J, 12:725-734 (1993). Finally, unimmunized libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom, HR 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.

[0075] The antibody can also be a multispecific antibody, such as a bispecific antibody. Bispecific antibodies are monoclonal antibodies with binding specificity to at least two different sites. The technology for generating multispecific antibodies includes but is not limited to the recombinant co-expression of two pairs of immunoglobulin heavy chain-light chains with different specificities (see Milstein, C. and Cuello, AC, Nature 305: 537-540 (1983); WO 93 / 08829; and Traunecker, A. et al., EMBO J. 10: 3655-3659 (1991)) and "node-into-hole" engineering (see, for example, U.S. Patent No. 5,731,168). It can also be achieved by engineering electrostatic manipulation effects for generating antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent 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, SA et al., J. Immunol. 148:1547-1553 (1992)); using "diabody" technology for generating bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M. et al., J. Immunol. 152:5368-5374 (1994)); and preparing trispecific antibodies (as described, e.g., in Tutt, A. et al., J. Immunol. 147:60-69 (1991)) to generate multispecific antibodies.

[0076] The monoclonal antibodies described herein also include engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies" (see, eg, US 2006 / 0025576).

[0077] The antibodies herein may also include the multispecific antibodies described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792, and WO 2010 / 145793, WO 2011 / 117330, WO 2012 / 025525, WO 2012 / 025530, WO 2013 / 026835, WO 2013 / 026831, WO 2013 / 164325, or WO 2013 / 174873.

[0078] The monoclonal antibodies described herein may also be antibody variants, for example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Suitable modifications may be introduced into the nucleotide sequence encoding the antibody, or amino acid sequence variants of the antibody may be prepared by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, as long as the final construct has the desired characteristics, such as antigen binding. Therefore, in certain embodiments, antibody variants with one or more amino acid substitutions are provided, and the sites of interest for substitution mutations include HVR and FR, for example, amino acid substitutions can be introduced into the antibody of interest and screened for products with desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0079] Example

[0080] Hereinafter, the present application will be described in more detail through examples. It should be understood that the present application is not limited to these examples.

[0081] Example 1 Preparation of anti-human interleukin-33 monoclonal antibody QX007N

[0082] Human interleukin-33 (hIL-33) was purchased from Shanghai Jinan Technology Co., Ltd. and used to immunize New Zealand rabbits. B cell cloning technology was used to obtain antigen-binding specific antibody clones, and then monoclonal antibodies that bind to human interleukin-33 and have human interleukin-33 inhibitory activity were screened. First, the cell supernatant was tested using Binding ELISA to select clones that bind to human interleukin-33; then HEK Blue was used to detect the clones that bind to human interleukin-33. TM The IL-33 reporter gene cell assay was used to select clones with human interleukin-33 inhibitory activity. The above immunization and screening processes were outsourced to a commercial company.

[0083] Twelve clones were selected for recombinant expression and sequencing. Cell-neutralizing activity was determined to be highest in 78#, which was then humanized. Human IgG germline sequence (Germline) homology alignment was performed using NCBI IgBlast, and IGHV3-66*01 was selected as the heavy chain CDR transplantation template. The CDR region of the heavy chain of clone #78 (i.e., CDR-H1 (SEQ ID No: 1), CDR-H2 (SEQ ID No: 2), and CDR-H3 (SEQ ID No: 3)) was transplanted into the framework region of IGHV3-66*01; IGKV1-12*01 was selected as the light chain CDR transplantation template, and the CDR region of the light chain of clone #78 (i.e., CDR-L1 (SEQ ID No: 4), CDR-L2 (SEQ ID No: 5), and CDR-L3 (SEQ ID No: 6)) was transplanted into the framework region of IGKV1-12*01; specific sites in the framework region were back-mutated to obtain the variable region of the monoclonal antibody QX007N of the present application. Finally, the amino acid sequence of the humanized heavy chain variable region is shown in SEQ ID NO: 7; the amino acid sequence of the humanized light chain variable region is shown in SEQ ID NO: 8.

[0084] The gene encoding the heavy chain variable region (SEQ ID NO: 7) and the full-length light chain (SEQ ID NO: 11) was amplified by PCR. The heavy chain expression plasmid pQX2.1 was digested with HindIII and NheI; the transient expression plasmid pQX1 was digested with HindIII and BamHI. The PCR-amplified genes were inserted into the corresponding expression plasmids using infusion recombinase, constructing the heavy chain expression plasmid pQX2.1-78VH-Hu25 and the light chain expression plasmid pQX2.2-78VK-Hu3. pQX2.2 refers to the pQX1 plasmid expressing the light chain.

[0085] The results of double enzyme digestion of the plasmid detected by nucleic acid electrophoresis are shown in Figure 1. The results in Figure 1 show the PCR amplification results of the antibody heavy chain variable region and the full-length light chain, as well as the double enzyme digestion results of the heavy and light chain expression plasmids. The plasmid size of the heavy and light chains is approximately 5000 bp, the heavy chain variable region is approximately 480 bp, and the full-length light chain is approximately 781 bp.

[0086] The correct heavy chain expression plasmid pQX2.1-78VH-Hu25 (the amino acid sequence of the full-length heavy chain expressed by it is shown in SEQ ID NO: 10) and the light chain expression plasmid pQX2.2-78VK-Hu3 (the amino acid sequence of the full-length light chain expressed by it is shown in SEQ ID NO: 11) were co-transfected into ExpiCHO-S cells. The day before transfection, ExpiCHO-S cells were diluted to 3×106 On the day of transfection, the cell density was diluted to 6×10 6 cells / ml, 25ml of cells were placed in a 125ml shake flask and ready for transfection. The transfection and expression process is shown in Figure 2.

[0087] On day 5 after transfection, the culture supernatant was harvested and purified using Protein A. The purified antibody, designated QX007N (HZD78-70), was analyzed by SDS-PAGE electrophoresis. The results of protein electrophoresis for this antibody are shown in Figure 3. Protein electrophoresis was performed on a denaturing reducing gel. Figure 3 shows two bands, approximately 50 kDa and 25 kDa, respectively, which are consistent with the theoretical molecular weights of the heavy chain (49.3 kDa) and light chain (23.4 kDa).

[0088] Example 2 Equilibrium dissociation constant (K D )

[0089] Biacore T200 was used to detect the affinity of QX007N (HZD78-70) to human interleukin-33, and all processes were carried out at 25°C. A commercial Protein A chip was used, and an appropriate amount of antibody was fixed by the capture method so that Rmax was around 50RU, and the capture flow rate was 10μl / min. The antigen was gradient diluted, and the instrument flow rate was switched to 30μl / min. The flow was passed through the reference channel and the channel of the fixed antibody in order from low to high concentration, and the buffer solution was used as a negative control. After each binding and dissociation was completed, the chip was regenerated with pH1.5 glycine. The 1:1 binding model in the Kinetics option was selected in the instrument's own analysis software for fitting, and the binding rate constant k of the antibody was calculated. a , the dissociation rate constant k d and the equilibrium dissociation constant K D value.

[0090] In addition, the affinity of QX007N (HZD78-70) was compared with that of Etokimab / ANB020, a monoclonal antibody against human interleukin-33 developed by AnaptysBio. The detection method for known antibodies was the same as that for QX007N, and the results are shown in Table 1. Etokimab / ANB020 was produced in-house by transiently transfecting ExpiCHO-S cells with an expression plasmid constructed based on the APE4909 sequence provided in patent WO2015106080A2.

[0091] Table 1 Affinity of anti-human interleukin-33 antibodies binding to human interleukin-33

[0092] Sample name k a (105 M -1 S -1 )k d (10 -4 S -1 )K D (10 -10 M)QX007N3.924.8112.28ANB 0204.243.698.69

[0093] In addition, based on the same detection method as mentioned above, we also found that Etokimab / ANB020 can bind to interleukin-33 of cynomolgus monkeys and rhesus monkeys, while QX007N (HZD78-70) can bind to interleukin-33 of cynomolgus monkeys but not to interleukin-33 of rhesus monkeys.

[0094] Example 3 Neutralization of HEK Blue Induced by Human Interleukin-33 TM Detection of NF-κB / AP-1 signal transduction activity in IL-33 cells

[0095] HEK Blue TM IL-33 cells were generated by stably transfecting human embryonic kidney cells HEK 293 with the human IL1RL1 gene, and the responses of TNF-α and IL-1β were blocked, so HEK-Blue TM IL-33 cells specifically respond to IL-33. Interleukin-33 binds to cell surface IL-1RL1 / IL-1RAcP, triggering a signaling cascade that leads to NF-κB / AP-1 signaling and the production of secreted alkaline phosphatase (SEAP). This allows for the detection of interleukin-33 biological activity or antibody screening.

[0096] Leveraging HEK Blue TM IL-33 cells were used to determine the neutralization activity of QX007N (HZD78-70) on human interleukin-33. TM IL-33 cells were cultured at 4 × 10 4 Cells were plated into 96-well plates and cultured overnight at 37°C and 5% CO2. Antibodies were diluted to concentrations ranging from 0 to 500 ng / ml, mixed with 2 ng / ml recombinant human interleukin-33 and incubated for 1 hour. After incubation, the cells were added and cultured at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was collected and added to QUANTI-Blue at a ratio of 1:10. TMThe cells were added to the detection reagent (InvivoGen, rep-qbs2) and reacted at 37°C for 1 hour. The OD was measured using a Varioskan LUX multifunctional microplate reader. 630nm The data were analyzed using a four-parameter curve fitting method using softMaxPro software (Figure 4), and the antagonistic activity of the antibody was further analyzed.

[0097] The results in Figure 4 show that QX007N (HZD78-70) can inhibit the induction of HEK Blue by recombinant human interleukin-33 TM NF-κB / AP-1 signal transduction in IL-33 cells, its IC 50 In contrast, for Etokimab / ANB020, the IC 50 It is 6.05ng / ml.

[0098] Example 4 Neutralization of HEK Blue Induced by Natural Human Interleukin-33 TM Detection of NF-κB / AP-1 signal transduction activity in IL-33 cells

[0099] Prepare natural human interleukin-33 and verify the neutralizing activity of QX007N (HZD78-70) on natural human interleukin-33. Culture HFL-1 cells and induce with 200ng / ml TNF-α for 24 hours. Collect the cells and lyse them by repeated freeze-thaw method. Collect the supernatant of cell lysate, which contains human interleukin-33 and detect the neutralizing activity of QX007N (HZD78-70). TM IL-33 cell-based validation activity.

[0100] HEK Blue TM IL-33 cells were cultured at 4 × 10 4 Cells were plated into 96-well plates and cultured overnight at 37°C and 5% CO2. After the antibody was diluted to a concentration range of 0 to 1000 ng / ml, the diluent and natural human interleukin-33 were added, mixed, and added to the cells. The cells were cultured at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was collected and added to QUANTI-Blue at a ratio of 1:10. TM The assay reagent was added and reacted at 37°C for 1 hour. The OD was measured using a Varioskan LUX multifunctional microplate reader. 630nm The data were analyzed using a 4-parameter curve fitting method using SoftMax Pro software (Figure 5), and the neutralizing activity of the antibody was further analyzed.

[0101] The results in Figure 5 show that QX007N (HZD78-70) can inhibit the natural human interleukin-33-induced HEK Blue TMNF-κB / AP-1 signal transduction in IL-33 cells, its IC 50 was 3.91 ng / ml; in contrast, for Etokimab / ANB020, the IC 50 2.5ng / ml.

[0102] Example 5 Detection of the activity of neutralizing IL-5 released by KU812 (human peripheral blood basophilic leukemia cells) induced by human interleukin-33

[0103] The activity of QX007N (HZD78-70) in neutralizing human interleukin-33 was evaluated by using human interleukin-33-induced IL-5 release from KU812 (human peripheral blood basophilic leukemia cells). KU812 cells (2×10 5 Cells / well) were added with antibodies and recombinant human interleukin-33 (final concentration 4 ng / ml) and cultured at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was collected and the expression level of IL-5 in the supernatant was detected by Human IL-5 DuoSet ELISA (R&D, DY205). The OD was measured using a Varioskan LUX multifunctional microplate reader. 450nm The data were analyzed using a 4-parameter curve fitting method using SoftMax Pro software (Figure 6), and the neutralizing activity of the antibody was further analyzed.

[0104] The results in Figure 6 show that QX007N (HZD78-70) can neutralize the activity of IL-5 release induced by human interleukin-33 in KU812 (human peripheral blood basophilic leukemia cells), and its IC 50 was 5.87 ng / ml. In contrast, for Etokimab / ANB020, the IC 50 It is 44ng / ml.

[0105] Example 6 Detection of the activity of neutralizing human interleukin-33 to induce IFN-γ release from human whole blood

[0106] The neutralizing activity of QX007N (HZD78-70) was further characterized using human whole blood mononuclear cells as the assay basis and IFN-γ as the assay indicator. Whole blood from healthy volunteers was plated (100 μL / well), followed by the addition of antibodies and recombinant human interleukin-33 (final concentration 4 ng / ml) and incubated at 37°C and 5% CO2 for 24 hours. The OD values ​​were measured using a Varioskan LUX multifunctional microplate reader. 450nm The data were analyzed using a 4-parameter curve fitting method using SoftMax Pro software (Figure 7), and the neutralizing activity of the antibody was further analyzed.

[0107] The results in Figure 7 show that QX007N (HZD78-70) can neutralize the activity of human interleukin-33 inducing IFN-γ release in human whole blood with an IC50 of 16 ng / ml. In contrast, for Etokimab / ANB020, the IC50 was determined using the same method. 50 It is 31.9ng / ml.

Claims

1. An isolated anti-human interleukin-33 monoclonal antibody, characterized in that, it comprises three heavy-chain complementarity-determining regions and three light-chain complementarity-determining regions. The three heavy-chain complementarity-determining regions are CDR-H1, CDR-H2 and CDR-H3, and the three light-chain complementarity-determining regions are CDR-L1, CDR-L2 and CDR-L3, wherein: The amino acid sequence of CDR-H1 is as shown in SEQ ID NO: 1; The amino acid sequence of CDR-H2 is as shown in SEQ ID NO: 2; The amino acid sequence of CDR-H3 is as shown in SEQ ID NO: 3; The amino acid sequence of CDR-L1 is as shown in SEQ ID NO: 4; The amino acid sequence of CDR-L2 is as shown in SEQ ID NO: 5; The amino acid sequence of CDR-L3 is as shown in SEQ ID NO:

6.

2. The monoclonal antibody according to claim 1, characterized in that, it comprises a heavy-chain variable region and a light-chain variable region, wherein, The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO: 7; The amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:

8.

3. An isolated nucleic acid, characterized in that, it encodes the monoclonal antibody according to claim 1 or 2.

4. A host cell, characterized in that, it comprises the nucleic acid according to claim 3.

5. A method for producing a monoclonal antibody, characterized in that, the method comprises culturing the host cell according to claim 4 to produce the monoclonal antibody according to claim 1 or 2.

6. A pharmaceutical composition, characterized in that, it comprises the monoclonal antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, it is used for treating diseases related to interleukin-33-mediated signal transduction.

8. The pharmaceutical composition according to claim 7, characterized in that, the diseases related to interleukin-33-mediated signal transduction are selected from any one or more of the following: asthma, chronic obstructive pulmonary disease, age-related macular degeneration, chronic rhinosinusitis, atopic dermatitis, multiple sclerosis, arthritis and inflammatory bowel disease.

9. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a drug for treating diseases related to interleukin-33-mediated signal transduction.

10. The use according to claim 9, characterized in that, the diseases related to interleukin-33-mediated signal transduction are selected from any one or more of the following: asthma, chronic obstructive pulmonary disease, age-related macular degeneration, chronic rhinosinusitis, atopic dermatitis, multiple sclerosis, arthritis and inflammatory bowel disease.