Anti-human CD117 monoclonal antibody and use thereof

Abstract

The present invention provides an anti-human CD117 monoclonal antibody and a use thereof. The anti-human CD117 monoclonal antibody of the present invention comprises three heavy chain complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3 having amino acid sequences as shown in SEQ ID NOs. 1-3, respectively, and three light chain complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3 having amino acid sequences as shown in SEQ ID NOs. 4-6, respectively. The antibody shows good activity at cytology level.

Description

Anti-human CD117 monoclonal antibody and its application Technical Field

[0001] The present application relates to the field of antibody drugs. Specifically, the present application relates to monoclonal antibodies targeting human CD117 and their applications. Background Art

[0002] Chronic urticaria (CU) is a common disease primarily driven by mast cells, with a significant negative impact on patients' quality of life. The signs and symptoms of CU are caused by the activation and degranulation of cutaneous mast cells (MCs), and the subsequent release of mediators that trigger sensory nerve activation, vasodilation, extravasation, and recruitment of circulating inflammatory cells (including eosinophils and basophils). Mast cells (MCs) are bone marrow-derived cells present in many organs and tissues of the human body. They contain a large number of pre-existing and newly formed secretory granules and possess unique pleiotropic properties. CD117 (also known as c-Kit) is a cell membrane protein encoded by the c-kit proto-oncogene and belongs to the type III receptor tyrosine kinase family. It is primarily expressed on the surface of hematopoietic stem cells, mast cells, and most gastrointestinal stromal tumor cells, melanoma cells, and other cells. The CD117 protein contains 976 amino acids and is composed of an extracellular region, a transmembrane region, and a cytoplasmic region. The extracellular region includes five immunoglobulin-like (Ig) domains. The first three domains are important sites for CD117 to bind to its ligand, stem cell factor (SCF), while the last two domains are required for receptor dimerization. SCF is primarily secreted by endothelial cells, fibroblasts, and bone marrow stromal cells. Most SCF appears as a transmembrane protein, but the extracellular portion can be cleaved and released as a non-covalently linked dimer in solution. When CD117 binds to SCF, it homodimerizes and activates its intrinsic tyrosine kinase activity, subsequently triggering the initiation of multiple signal transduction pathways, including the phosphatidylinositol 3-kinase (PI3-K) pathway, the Janus kinase pathway, the signal transducer and activator of transcription (STAT) pathway, and the mitogen-activated protein kinase (MAPK) pathway. The physiological effects of c-Kit in controlling cell survival, proliferation, differentiation, and migration depend on the activation of specific or overlapping pathways, which confers complexity to the activity of the SCF / c-Kit system. When the c-kit gene is abnormally expressed in mast cells, the c-Kit protein can be spontaneously phosphorylated even in the absence of SCF binding, resulting in abnormal activation of the SCF / c-Kit signal, regulating the maturation, migration and proliferation of mast cells, degranulation, and release of inflammatory mediators, mediating pathological inflammatory responses, and participating in the pathological process of chronic inflammatory and autoimmune diseases such as urticaria, prurigo nodularis, and eosinophilic esophagitis.

[0003] Targeted inhibition of the SCF / c-Kit signaling pathway has promising applications in the prevention and treatment of spontaneous and induced urticaria, and holds enormous market potential. Research indicates that Celldex Therapeutics' Barzolvolimab (CDX-0159), currently in Phase 2 clinical trials, is a humanized monoclonal antibody that binds with high specificity to the receptor tyrosine kinase CD117, essential for mast cell function and survival, and can potentially inhibit its activity. In a Phase II clinical trial for moderate to severe chronic spontaneous urticaria (CSU), it produced rapid, profound, and durable responses with a favorable safety profile, offering a new treatment option for patients with chronic urticaria. Summary of the Invention

[0004] The purpose of the present application is to provide a novel anti-human CD117 monoclonal antibody, a pharmaceutical composition comprising the monoclonal antibody, and the pharmaceutical use of the monoclonal antibody.

[0005] Specifically, this application involves the following:

[0006] 1. An anti-human CD117 monoclonal antibody, comprising three heavy chain complementary determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein:

[0007] The amino acid sequence of CDR-H1 (CDR-H1 in this specification represents heavy chain CDR1) is shown in SEQ ID No: 1 (NKDVMG);

[0008] The amino acid sequence of CDR-H2 (CDR-H2 in this specification represents heavy chain CDR2) is shown in SEQ ID No: 2 (GIYTGSGSTYYASWAKG);

[0009] The amino acid sequence of CDR-H3 (CDR-H3 in this specification represents heavy chain CDR3) is shown in SEQ ID No: 3 (DLFYSNYYNL);

[0010] The amino acid sequence of CDR-L1 (CDR-L1 in this specification represents light chain CDR1) is shown in SEQ ID No: 4 (QASESISNYLS);

[0011] The amino acid sequence of CDR-L2 (CDR-L2 in this specification represents light chain CDR2) is shown in SEQ ID No: 5 (KASTLAS);

[0012] The amino acid sequence of CDR-L3 (CDR-L3 in this specification represents light chain CDR3) is shown in SEQ ID No: 6 (QNTYVSSGSIT).

[0013] 2. The monoclonal antibody according to item 1, comprising a heavy chain variable region and a light chain variable region, wherein:

[0014] The amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 7 (EVQLVESGGGLVQPGGSLRLSCAASGFSFSNKDVMGWVRQAPGKGLEWIAGIYTGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLFYSNYYNLWGQGTLVTVSS), or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID No: 7;

[0015] The amino acid sequence of the light chain variable region is shown in SEQ ID No:8 (DIQMTQSPSSVSASVGDRVTITCQASESISNYLSWYQQKPGKAPERLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNTYVSSGSITFGGGTKVEIK), or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID No:8.

[0016] 3. The monoclonal antibody according to item 1 or 2, comprising a heavy chain and a light chain, wherein:

[0017] The amino acid sequence of the heavy chain is as SEQ ID No:10(EVQLVESGGGLVQPGGSLRLSCAASGFSFSNKDVMGWVRQAPGKGLEWIAGIYTGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLFYSNYYNLW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No: 10;

[0018] The amino acid sequence of the light chain is shown in SEQ ID No: 11 (DIQMTQSPSSVSASVGDRVTITCQASESISNYLSWYQQKPGKAPERLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNTYVSSGSITFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC), or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID No: 11.

[0019] 4. An isolated nucleic acid encoding the monoclonal antibody according to any one of items 1 to 3.

[0020] 5. A host cell comprising the nucleic acid according to item 4.

[0021] 6. A method for producing a monoclonal antibody, comprising culturing the host cell according to item 5 to thereby produce the monoclonal antibody according to any one of items 1 to 3.

[0022] 7. A pharmaceutical composition, comprising the monoclonal antibody according to any one of items 1 to 3 and a pharmaceutically acceptable carrier.

[0023] 8. The pharmaceutical composition according to item 7, wherein the pharmaceutical composition is used to treat diseases related to signal transduction mediated by human CD117 protein.

[0024] 9. The pharmaceutical composition according to item 8, wherein the diseases treated for signal transduction-related diseases mediated by human CD117 protein include immune system diseases, digestive system diseases, and blood and lymphatic system diseases.

[0025] 10. The pharmaceutical composition according to item 9, wherein the immune system disease includes eosinophilic esophagitis, chronic urticaria and prurigo.

[0026] 11. Use of the monoclonal antibody according to any one of items 1 to 3 in the preparation of a medicament for treating diseases associated with signal transduction mediated by human CD117 protein.

[0027] 12. The use according to item 11, wherein the diseases treated for signal transduction-related diseases mediated by human CD117 protein include immune system diseases, digestive system diseases, blood and lymphatic system diseases, and preferably, the immune system diseases include eosinophilic esophagitis, chronic urticaria and prurigo.

[0028] 13. A method for treating a disease associated with signal transduction mediated by human CD117 protein, comprising the step of administering a therapeutically effective amount of the monoclonal antibody according to item 1 to a patient suffering from the disease.

[0029] Effects of the Invention

[0030] The present application provides a new anti-human CD117 monoclonal antibody, which has better affinity and biological activity for human CD117 protein than the CD117 monoclonal antibodies in the prior art, such as CDX-0159 (CDX-0159 is a monoclonal antibody drug targeting CD117 developed by Celldex Therapeutics, which can produce rapid, deep and lasting responses in a Phase 1b clinical trial of moderate to severe chronic spontaneous urticaria and has good safety). The monoclonal antibody provided in this application shows biological activity at the cellular level that is superior to the CD117 monoclonal antibodies in the prior art, and is expected to show good clinical effects in the prevention and treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a schematic diagram of the nucleic acid electrophoresis results of the QX013N transient expression plasmid. In particular, M: Marker; Band 1: 524VH-Hu35, HindIII / EcoRI; Band 2: pQXHC, HindIII / EcoRI; Band 3: PCR product 524VK-Hu17; Band 4: pQX2.3, HindIII / BsiWI.

[0032] Figure 2 is a flowchart of transient expression.

[0033] FIG3 is a schematic diagram of the electrophoresis detection results of QX013N (HZD524-61).

[0034] FIG4 is a schematic diagram of the results of the QX013N neutralization activity analysis (cell proliferation assay - M07e cells).

[0035] FIG5 is a schematic diagram showing the results of the neutralization activity analysis of QX013N (reporter gene method - NF-kB signal transduction).

[0036] FIG6 is a schematic diagram showing the results of QX013N neutralization activity analysis (FACS Blocking method-LUVA cells).

[0037] FIG7 is a schematic diagram of the binding activity of QX013N and CDX-0159 to porcine CD117.

[0038] FIG8 is a schematic diagram showing the signal value effect of QX013N and CDX-0159 in neutralizing the binding of human SCF to human CD117.

[0039] The accompanying drawings are used to better understand the present application and do not constitute an improper limitation to the present application. DETAILED DESCRIPTION

[0040] The present application is described in further detail below in conjunction with specific embodiments. The embodiments provided in this application are intended to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art. The various details of the embodiments are provided to aid understanding and should be considered as merely exemplary. Therefore, those of ordinary skill in the art will recognize that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0041] 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.

[0042] Generally speaking, the terms used in this specification have the following meanings.

[0043] 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). Methods for evaluating antibody purity are well known in the art, for example, see Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0044] In this specification, " monoclonal antibody " represents the antibody that derives 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 preparation), such variant is usually present in trace.Different from the polyclonal antibody preparation that generally includes the different antibodies for different determinants (epi-positions), every kind of monoclonal antibody of monoclonal antibody preparation 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 present invention 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 that comprises human immunoglobulin locus, and such method and other exemplary method for preparing monoclonal antibody are described herein.

[0045] 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.

[0046] In this specification, human CD-117 protein (sometimes also referred to as C-kit) refers to a membrane receptor derived from humans, the amino acid sequence of its extracellular region is shown in SEQ ID NO: 9, wherein the underlined portion represents a signal peptide.

[0047] In this specification, "anti-human CD-117 monoclonal antibody" refers to a monoclonal antibody that can bind to human CD-117 with sufficient affinity so that the monoclonal antibody can be used as a diagnostic agent and / or therapeutic agent targeting human CD-117.

[0048] The anti-human CD-117 monoclonal antibodies of the present application may not bind to proteins unrelated to the target. Here, "unrelated proteins" refers to proteins other than the target human CD-117; here, "no binding" means that, when the binding ability of the anti-human CD-117 monoclonal antibodies of the present application to human CD-117 as its target is taken as 100%, the binding ability of the anti-human CD-117 monoclonal antibodies of the present application to the unrelated proteins is less than 10%, for example, less than 5%, 1%, 0.1%, 0.01%, 0.001%, or 0.

[0049] The human CD-117 monoclonal antibody of the present application has an equilibrium dissociation constant (K) of ≤1 μM, ≤100 nM, ≤50 nM, or ≤40 nM. D ).

[0050] The experimental results show that the anti-human CD-117 monoclonal antibody of the present application can specifically bind to human CD-117.

[0051] The anti-human CD-117 monoclonal antibody of the present application can bind to CD-117 of, for example, marmosets, cynomolgus monkeys, rhesus monkeys, cats, dogs, and pigs, but may not bind to CD-117 of, for example, rats and mice.

[0052] The anti-human CD-117 monoclonal antibody of the present application is comparable to or superior to similar monoclonal antibody products reported on the market in many aspects of biological activity, including, for example, neutralizing the binding activity of recombinant human SCF to CD-117.

[0053] Specifically, the present application relates to an anti-human CD117 monoclonal antibody, which comprises three heavy chain complementary determining regions CDR-H1 (heavy chain CDR1), CDR-H2 (heavy chain CDR2), and CDR-H3 (heavy chain CDR3) and three light chain complementary determining regions CDR-L1 (light chain CDR1), CDR-L2 (light chain CDR2), and CDR-L3 (light chain CDR3), wherein: the amino acid sequence of CDR-H1 is shown in SEQ ID No: 1 (NKDVMG); the amino acid sequence of CDR-H2 is shown in SEQ ID No: 2 (GIYTGSGSTYYASWAKG); the amino acid sequence of CDR-H3 is shown in SEQ ID No: 3 (DLFYSNYYNL); the amino acid sequence of CDR-L1 is shown in SEQ ID No: 4 (QASESISNYLS); the amino acid sequence of CDR-L2 is shown in SEQ ID No: 5 (KASTLAS); the amino acid sequence of CDR-L3 is shown in SEQ ID No:6(QNTYVSSGSIT) shown.

[0054] In a specific embodiment, the anti-human CD-117 monoclonal antibody described herein comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein:

[0055] the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:7 (EVQLVESGGGLVQPGGSLRLSCAASGFSFSNKDVMGWVRQAPGKGLEWIAGIYTGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLFYSNYYNLWGQGTLVTVSS), or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID No:7;

[0056] The amino acid sequence of the light chain variable region is shown in SEQ ID No: 8 (DIQMTQSPSSVSASVGDRVTITCQASESISNYLSWYQQKPGKAPERLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNTYVSSGSITFGGGTKVEIK), or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No: 8.

[0057] In a specific embodiment, the anti-human CD-117 monoclonal antibody described herein comprises a heavy chain and a light chain, wherein:

[0058] The amino acid sequence of the heavy chain is as shown in SEQ ID No: 10, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID No: 10;

[0059] The amino acid sequence of the light chain is as shown in SEQ ID No: 11, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No: 11.

[0060] SEQ ID NOs: 10 and 11 are both humanized sequences. Through CDR grafting and framework region backmutations of amino acids potentially affecting CDR region structure, and after Fc modification, activity consistent with the parent rabbit antibody, the degree of humanization reached 97.0%. As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0061] In this specification, "isolated nucleic acid encoding human CD-117 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.

[0062] 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."

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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).

[0067] 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 challenge. These animals typically contain a portion or all of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or are present extrachromosomally 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™ technology described in U.S. Patent Nos. 6,075,181 and 6,150,584; the XENOMOUSE™ technology described in U.S. Patent No. 5,770,429. Technology; US Patent No. 7,041,870 describes Technology, and U.S. Patent Application Publication No. US2007 / 0061900 Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0068] 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).

[0069] 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.

[0070] 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).

[0071] In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which is 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.

[0072] 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.

[0073] The monoclonal antibodies described herein also include engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies" (see, eg, US 2006 / 0025576).

[0074] The antibodies herein may also include the multispecific antibodies described in WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, WO2010 / 145793, WO2011 / 117330, WO2012 / 025525, WO2012 / 025530, WO2013 / 026835, WO2013 / 026831, WO2013 / 164325 or WO2013 / 174873.

[0075] 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 the desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0076] Example

[0077] The content of this application will be described below with reference to specific embodiments, but the scope of this application is not limited thereto.

[0078] Unless otherwise specified, the reagents and instruments used in the following examples are conventional reagents and instruments in the art and can be obtained commercially. The experimental methods used are all conventional methods unless otherwise specified. Those skilled in the art can undoubtedly implement the described protocols and obtain the corresponding results based on the content of the examples.

[0079] Example 1 Preparation of anti-human CD117 monoclonal antibody QX013N

[0080] Quanxin Bio uses its own expressed human CD117 antigen as an immunogen to immunize New Zealand rabbits. Using B cell cloning technology, they generate monoclonal antibodies with antigen-specific binding. They then screen for monoclonal antibodies that bind to human CD117 and have inhibitory activity against it. Binding ELISA and blocking ELISA assays are used to analyze and screen the target clones. The immunization and screening process is outsourced to a commercial company.

[0081] Five clones were selected for recombinant expression and sequencing. Clone #524 demonstrated the highest neutralizing activity in cells. Therefore, clone #524 was 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 #524 (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 #524 (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; back mutations were performed on specific sites in the framework region and site-directed mutagenesis of the CDR region to obtain the variable region of the monoclonal antibody QX013N of the present application. Finally, the 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.

[0082] The heavy chain gene (SEQ ID NO: 10) was synthesized by GenScript and obtained by double digestion with HindIII and EcoRI. The light chain variable region gene (SEQ ID NO: 8) was amplified by PCR. The heavy chain expression plasmid pQXHC was double digested with HindIII and EcoRI; the light chain expression plasmid pQX2.3 was double digested with HindIII and BsiWI. The heavy chain gene was inserted into the heavy chain expression plasmid using T4 ligase to construct the heavy chain expression plasmid pQXHC-524VH-Hu35. The light chain PCR-amplified gene was inserted into the light chain expression plasmid using Infusion recombinase to construct the light chain expression plasmid pQX2.3-524VK-Hu17.

[0083] The results of nucleic acid electrophoresis for double-enzyme digestion of the heavy chain gene fragment, PCR-amplified light chain variable region gene fragment, and double-enzyme digestion of the plasmid are shown in Figure 1. As can be seen from the results in Figure 1, the double-enzyme digestion results of the antibody heavy chain, the PCR amplification results of the light chain variable region, and the double-enzyme digestion results of the heavy and light chain expression plasmids show that the plasmid size of the heavy and light chains is approximately 5000 bp, the full length of the heavy chain is approximately 1410 bp, and the light chain variable region is approximately 447 bp.

[0084] The heavy chain expression plasmid pQXHC-524VH-Hu35 (the amino acid sequence of the expressed heavy chain is shown in SEQ ID NO: 10) and the light chain expression plasmid pQX2.3-524VK-Hu17 (the amino acid sequence of the expressed light chain is shown in SEQ ID NO: 11) with the correct sequence were co-transfected into ExpiCHO-S cells. One day before transfection, ExpiCHO-S cells were diluted to 3×10 6 On the day of transfection, the cell density was diluted to 6×10 6 cells / mL, 25 mL of cells were placed in a 125 mL shake flask and ready for transfection. The transfection and expression process is shown in Figure 2.

[0085] Six days after transfection, the culture supernatant was harvested and purified using Protein A. The purified antibody, designated QX013N (HZD524-61), 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 (49.2 kDa) and light chain (23.4 kDa).

[0086] Example 2 Equilibrium dissociation constant (K D )

[0087] Biacore T200 was used to detect the affinity of QX013N (HZD524-61) to human CD117, and all processes were carried out at 25°C. A commercial Sensor chip CM5 chip was used to fix an appropriate amount of antibody by 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. Use the instrument's own analysis software to select the 1:1 binding model in the Kinetics option for fitting and calculate the binding rate constant k of the antibody. a , the dissociation rate constant k d and the dissociation equilibrium constant KD value.

[0088] In addition, the affinity of QX013N (HZD524-61) was compared with that of CDX-0159, a monoclonal antibody targeting human CD117 currently in Phase 2 clinical trials. The detection method for the known antibody was the same as that used for QX013N, and the results are shown in Table 1. CDX-0159 was produced in-house by transiently transfecting ExpiCHO-S cells with an expression plasmid constructed based on the sequence provided in patent NZ630363B.

[0089] Table 1 Affinity of anti-human CD117 monoclonal antibodies binding to human CD117

[0090] The data in the table are: each sample was tested three times and the average value was calculated.

[0091] Example 3 QX013N and CDX-0159 neutralize the proliferation activity of M07E cells induced by recombinant human SCF

[0092] The neutralization of cell proliferation induced by recombinant human SCF by QX013N and CDX-0159 was determined using M07e cells. M07E cells were plated at 4×10 4 Cells were plated into 96-well plates, and the antibody was diluted to a concentration range of 0 to 10 μg / mL. After adding the antibody to the cells and incubating for 1 hour, 50 ng / mL of recombinant human SCF was added and mixed, and the cells were cultured at 37°C and 5% CO2 for 24 hours. 100 μL of the cell suspension was collected and added to a black 96-well plate. 100 μL of Cell Counting-Lite 2.0 Luminescent Cell Viability Assay reagent (Vazyme, DD1101-02) was taken and mixed with the cell suspension. Cell proliferation was detected using a multifunctional microplate reader, and a dose-effect curve was drawn (as shown in Figure 4) to analyze the neutralizing activity of the antibody.

[0093] The results shown in Figure 4 show that QX013N has the activity of neutralizing the proliferation of M07e cells induced by recombinant human SCF, IC 50 The IC value of the control antibody CDX-0159 was 50.55 ng / mL. 50 The value was 55.11 ng / mL, and QX013N neutralized the proliferation activity of M07e cells induced by human SCF better than the control antibody CDX-0159.

[0094] Example 4 QX013N and CDX-0159 neutralize NF-κB signaling activity induced by recombinant human SCF in HEK293 cells overexpressing human CD117 reporter gene

[0095] HEK293 cells overexpressing human CD117 reporter gene were used to determine the effect of QX013N and CDX-0159 on the intracellular NF-κB signaling activity mediated by recombinant human SCF through CD117. HEK293 cells overexpressing human CD117 were plated at 4×10 cells per well. 5 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 40 μg / mL and added to the cells for 1 hour. Recombinant human SCF (400 ng / mL) was then added and cultured for 24 hours at 37°C and 5% CO2. One-Lite Luciferase Assay System reagent (Vazyme, DD1203-02) was added to detect the cells, and a four-parameter curve was fitted using SoftMax to calculate the sample IC. 50 The neutralizing activity of the antibody was analyzed using the values ​​of 5.

[0096] The results shown in Figure 5 show that QX013N can neutralize the NF-kB signal transduction induced by recombinant human SCF in HEK293 cells overexpressing human CD117 reporter gene. 50 The IC value of the control antibody CDX-0159 was 9.70 ng / mL. 50 The value was 13.63 ng / mL, and QX013N neutralized the human SCF-induced reporter gene activity better than the control antibody CDX-0159.

[0097] Example 5 QX013N and CDX-0159 neutralize the binding activity of recombinant human SCF to CD117 on the LUVA cell membrane

[0098] LUVA cells were used to determine the neutralization activity of QX013N and CDX-0159 in binding to recombinant human SCF and CD117 on the cell membrane. LUVA cells were plated at 5×10 5 The cells were plated in a U-shaped 96-well plate, and the antibody was diluted to a concentration range of 0 to 8 μg / mL. After adding the antibody to the cells and incubating at 4°C for 1 hour, 20 μL Bio SCF (10 μg / mL) was added to each well and incubated for 1 hour. The cells were washed twice with PBS and Anti-Biotina (eBioscience) was added. TM Alexa Fluor 488 detection reagent was used. Cells were washed with PBS and resuspended. Flow cytometry was used for detection. GraphPad was used to fit a four-parameter curve and calculate the sample IC. 50 The results are shown in Figure 6.

[0099] The results shown in Figure 6 show that QX013N can neutralize the binding of recombinant human SCF to CD117 on the surface of LUVA cell membranes, and its IC 50 The IC of the control antibody CDX-0159 was 175.9 ng / mL. 50 The neutralization activity of QX013N in neutralizing the binding of recombinant human SCF to CD117 on the surface of LUVA cell membrane is better than that of the control antibody CDX-0159.

[0100] Example 6 Detection of the Binding Activity of QX013N and CDX-0159 to CD117 of Other Species

[0101] Binding ELISA was used to detect the binding activity of QX013N and CDX-0159 to porcine CD117. Porcine CD117 was diluted to 1 μg / mL and added to the ELISA plate and coated overnight at 2-8°C. After blocking, a series of antibody dilutions diluted to a concentration range of 0 to 2 μg / mL and Goat-anti-human IgG HRP were added for incubation. Finally, the substrate was added for color development, and the stop solution was added to terminate the color development reaction. After termination, the ELISA plate was placed in a microplate reader to read the absorbance value at 450 nm (the absorbance value at 650 nm was used as a reference), and a dose-effect curve was drawn to analyze the binding specificity of the antibody. The dose-effect curve is shown in Figure 7.

[0102] The results shown in Figure 7 show that QX013N can bind to porcine CD117, but CDX-0159 does not. This result suggests that QX013N and CDX-0159 recognize different epitopes.

[0103] We also used the same method to detect the binding activity of QX013N to CD117 of other species. The experimental results showed that QX013N bound to CD117 of marmosets, crab-eating macaques, rhesus macaques, cats, and dogs, but not to CD117 of rats and mice.

[0104] Example 7 Differences in signal values ​​between QX013N and CDX-0159 in neutralizing the binding of human SCF to human CD117

[0105] ELISA was used to detect the difference in binding signal values ​​between QX013N and CDX-0159 in neutralizing human SCF and human CD117. Human SCF was diluted to 1 μg / mL and added to the enzyme-labeled plate and coated overnight. A series of antibody dilutions with a dilution concentration range of 0 to 20 μg / mL were mixed with Bio Human CD117 diluted to 200 ng / mL and incubated; after the incubation, the mixed antigen-antibody mixture was added to the blocked enzyme-labeled plate for incubation, and finally the substrate was added for color development, and the stop solution was added to terminate the color development reaction. After termination, the enzyme-labeled plate was placed in an enzyme reader to read the absorbance value at 450 nm (the absorbance value at 650 nm was used as a reference), and a signal value effect curve was drawn to analyze the difference in the binding signal value between the antibody and human SCF and human CD117. The signal value effect curve is shown in Figure 8.

[0106] The results shown in Figure 8 show that when the antibody concentration was 0, the binding signal value of human CD117 and human SCF was 100%. When the antigen-antibody binding reached saturation, the neutralization signal value of QX013N for human SCF and human CD117 binding was 55.6%, while the neutralization signal value of CDX-0159 for human SCF and human CD117 binding was 30.5%. These results suggest that QX013N and CDX-0159 recognize different epitopes.

[0107] The above description is merely a preferred embodiment of the present application and does not constitute any limitation on the present application. Any person skilled in the art may utilize the technical content disclosed above to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments based on the technical essence of the present application that do not depart from the technical solution of the present application shall still fall within the scope of protection of the technical solution of the present application.

Claims

1. A monoclonal antibody against human CD117, said monoclonal antibody comprising three heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and three light chain complementarity determining regions CDR-L1, CDR-L2, 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, said monoclonal antibody comprising 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 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No:7; The amino acid sequence of the light chain variable region is as shown in SEQ ID No:8 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No:

8.

3. The monoclonal antibody according to claim 1 or 2, said monoclonal antibody comprising a heavy chain and a light chain, wherein: The amino acid sequence of the heavy chain is as shown in SEQ ID No:10 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No:10; The amino acid sequence of the light chain is as shown in SEQ ID No:11 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No:

11.

4. An isolated nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 3.

5. A host cell comprising the nucleic acid according to claim 4.

6. A method for producing monoclonal antibodies, wherein, The method comprises culturing the host cell according to claim 5 to produce the monoclonal antibody according to any one of claims 1 to 3.

7. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the monoclonal antibody according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

8. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a medicament for treating diseases related to signal transduction mediated by human CD117 protein.

9. A method for treating diseases related to signal transduction mediated by human CD117 protein, which comprises the step of administering a therapeutically effective amount of the monoclonal antibody according to claim 1 to a patient suffering from said disease.

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