Anti-human CD117 monoclonal antibody and application thereof

Abstract

The invention provides an anti-human CD117 monoclonal antibody and an application thereof. The anti-human CD117 monoclonal antibody comprises 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, the amino acid sequence of the CDR-H1 is as shown in SEQ ID NO: 1, the amino acid sequence of the CDR-H2 is as shown in SEQ ID NO: 2, the amino acid sequence of the CDR-H3 is as shown in SEQ ID NO: 3, the amino acid sequence of the CDR-L1 is as shown in SEQ ID NO: 4, the amino acid sequence of the CDR-L2 is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementary determining regions is as shown in SEQ ID NO: 6. The amino acid sequence of the CDR-L3 is as shown in SEQ ID NO: 6. Compared with the existing known anti-human CD117 monoclonal antibody, the antibody disclosed by the invention shows better cellular level biology, and is expected to show a good clinical effect in the aspect of preventing and treating related diseases.

Description

Technical Field

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

[0002] Chronic urticaria (CU) is a common disease that is primarily driven by mast cells and has a significant negative impact on the patient's quality of life. The signs and symptoms of CU are caused by the activation and degranulation of skin mast cells (MCs) and the subsequent release of mediators that cause sensory nerve activation, vasodilation, extravasation, and recruitment of circulating inflammatory cells (including eosinophils and basophils). Mast cells (MCs) are bone marrow-derived cells that are present in many organs and tissues of the human body. They contain a large number of pre-existing and newly formed secretory granules and have unique pleiotropic effects. 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 mainly distributed 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 its structure consists of an extracellular region, a transmembrane region, and a cytoplasmic region. The extracellular region includes five immunoglobulin-like (Immunoglobulin, Ig) domains. The first three domains are important sites for CD117 to bind to its ligand stem cell factor (Stem cell factor, SCF), and the last two domains are structures required for receptor dimerization. SCF is mainly secreted by endothelial cells, fibroblasts, and bone marrow stromal cells. Most SCF appears in the form of transmembrane proteins, but the extracellular part can be cut and released to exist in the solution as a non-covalently linked dimer. When CD117 binds to SCF, it homodimerizes and activates its intrinsic tyrosine kinase activity, which then triggers the initiation of multiple signal transduction pathways, namely 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 gives the SCF / c-Kit system activity complexity. When the c-kit gene is abnormally expressed in mast cells, the c-Kit protein can be spontaneously phosphorylated even without binding to SCF, causing abnormal activation of the SCF / c-Kit signal, regulating the maturation, migration and proliferation, degranulation, and release of inflammatory mediators of mast cells, mediating pathological inflammatory responses, and participating in the pathological process of chronic inflammatory and autoimmune diseases such as urticaria, nodular prurigo, and eosinophilic esophagitis.

[0003] Targeted inhibition of the SCF / c-Kit signaling pathway has good application prospects in the prevention and treatment of spontaneous and induced urticaria, and has huge market potential. According to relevant studies, Celldex Therapeutics' Barzolvolimab (CDX-0159) has now entered the clinical phase 2 research stage. It is a humanized monoclonal antibody that can bind to the receptor tyrosine kinase CD117, which is necessary for maintaining mast cell function and survival, with high specificity, and can potentially inhibit its activity; in the Phase II clinical trial of moderate to severe chronic spontaneous urticaria (CSU), it can produce rapid, deep and lasting responses, and has good safety, providing 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, CDR-H3 and three light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, wherein:

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

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

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

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

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

[0012] The amino acid sequence of CDR-L3 (in this specification, CDR-L3 means 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 as shown in SEQ ID No: 7

[0015] (EVQLVESGGGLVQPGGSLRLSCAASGFSFSNKDVMGWVRQAPGKGLEWIAGIYTGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLFYSNYYNLWGQGTLVTVSS), 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:7;

[0016] The amino acid sequence of the light chain variable region is as shown in SEQ ID No: 8

[0017] (DIQMTQSPSSVSASVGDRVTITCQASESISNYLSWYQQKPGKAPERLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNTYVSSGSITFGGGTKVEIK), 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.

[0018] 3. The monoclonal antibody according to item 1 or 2, comprising a heavy chain and a light chain, wherein:

[0019] The amino acid sequence of the heavy chain is as shown in SEQ ID No: 10

[0020] (EVQLVESGGGLVQPGGSLRLSCAASGFSFSNKDVMGWVRQAPGKGLEWIAGIYTGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLFYSNYYNLWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), 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;

[0021] The amino acid sequence of the light chain is as shown in SEQ ID No: 11

[0022] 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:11.

[0023] 4. An isolated nucleic acid encoding the monoclonal antibody according to any one of items 1 to 3.

[0024] 5. A host cell comprising the nucleic acid described in item 4.

[0025] 6. A method for producing a monoclonal antibody, wherein the method comprises culturing the host cell according to item 5 to thereby produce the monoclonal antibody according to any one of items 1 to 3.

[0026] 7. A pharmaceutical composition, comprising the monoclonal antibody according to any one of items 1 to 3 and a pharmaceutically acceptable carrier.

[0027] 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.

[0028] 9. The pharmaceutical composition according to item 8, wherein the diseases related to signal transduction mediated by human CD117 protein for treatment include immune system diseases, digestive system diseases, blood and lymphatic system diseases.

[0029] 10. The pharmaceutical composition according to item 9, wherein the immune system diseases include eosinophilic esophagitis, chronic urticaria and prurigo.

[0030] 11. Use of the monoclonal antibody described in any one of items 1 to 3 in the preparation of a medicament for treating diseases related to signal transduction mediated by human CD117 protein.

[0031] 12. The use according to item 11, wherein the diseases related to signal transduction mediated by human CD117 protein for treatment include immune system diseases, digestive system diseases, blood and lymphatic system diseases, preferably, the immune system diseases include eosinophilic esophagitis, chronic urticaria and prurigo.

[0032] Effects of the Invention

[0033] The present application provides a new anti-human CD117 monoclonal antibody, whose humanized antibody biological activity and affinity are comparable to or better than the control antibody CD117 monoclonal antibody CDX-0159 currently in the phase III clinical study (CD117 monoclonal antibody CDX-0159 is a monoclonal antibody drug targeting CD117 developed by Celldex Therapeutics, which can produce rapid, deep and lasting responses in the phase 1b clinical trial of moderate to severe chronic spontaneous urticaria, and has good safety). The monoclonal antibody provided in the present application shows better biological activity than CDX-0159 at the cellular level, and is expected to show good clinical effects in the prevention and treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1This is a schematic diagram of the nucleic acid electrophoresis results of the QX013N transient expression plasmid. Among them, 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.

[0035] Figure 2 This is a flowchart of transient expression.

[0036] Figure 3 Schematic diagram of the electrophoresis detection results of QX013N (HZD524-61).

[0037] Figure 4 Schematic diagram of the results of QX013N neutralization activity analysis (cell proliferation method - M07e cells).

[0038] Figure 5 Schematic diagram of the results of QX013N neutralization activity analysis (reporter gene method - NF-kB signal transduction).

[0039] Figure 6 Schematic diagram of the results of QX013N neutralization activity analysis (FACS Blocking method-LUVA cells).

[0040] Figure 7 Schematic diagram of the binding activity of QX013N and CDX-0159 with porcine CD117.

[0041] Figure 8 Schematic diagram of the signal value effect of QX013N and CDX-0159 in neutralizing the binding of human SCF to human CD117.

[0042] The accompanying drawings are used to better understand the present application and do not constitute an improper limitation on the present application. DETAILED DESCRIPTION

[0043] The present application is described in further detail below in conjunction with specific embodiments. The embodiments provided in the present application are to enable a more thorough understanding of the present application and to enable the scope of the present application to be fully communicated to those skilled in the art. The various details of the embodiments are to aid understanding and should be considered as merely exemplary. Therefore, those of ordinary skill in the art should 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, the description of known functions and structures is omitted in the following description.

[0044] 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.

[0045] Generally speaking, the terms used in this specification have the following meanings.

[0046] In this specification, an "isolated" antibody is an antibody that has been separated from the 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).

[0047] In this specification, "monoclonal antibody" means an antibody derived from a colony of substantially homologous antibodies, that is, each antibody constituting the colony is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced in the production process of monoclonal antibody products), such variants are usually present in trace amounts. Different from polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody colony, and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using all or part of a transgenic animal comprising a human immunoglobulin locus, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0048] 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.

[0049] In the present specification, human CD-117 protein (also referred to as C-kit in some cases) refers to a membrane receptor derived from humans, and the amino acid sequence of its extracellular region is shown in SEQ ID NO: 9, wherein the underlined portion represents a signal peptide.

[0050] SEQ ID NO:9:

[0051]

[0052]

[0053] In the present specification, "anti-human CD-117 monoclonal antibody" means 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.

[0054] The anti-human CD-117 monoclonal antibody of the present application may not bind to proteins unrelated to the target. Here, "unrelated proteins" refers to proteins other than human CD-117 as a target; here, "unrelated" means: when the binding ability of the anti-human CD-117 monoclonal antibody 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 antibody of the present application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

[0055] 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 ).

[0056] The experimental results show that the anti-human CD-117 monoclonal antibody of the present application can specifically bind to human CD-117.

[0057] The anti-human CD-117 monoclonal antibody of the present application can bind to porcine CD-117, and may or may not bind to CD-117 of, for example, marmosets, cynomolgus monkeys, pigs, dogs, rabbits, rats, mice, guinea pigs, etc.

[0058] 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 and CD-117.

[0059] 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), CDR-H3 (heavy chain CDR3) and three light chain complementary determining regions CDR-L1 (light chain CDR1), CDR-L2 (light chain CDR2), 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.

[0060] In a specific embodiment, the anti-human CD-117 monoclonal antibody described in the present application comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein:

[0061] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 7

[0062] (EVQLVESGGGLVQPGGSLRLSCAASGFSFSNKDVMGWVRQAPGKGLEWIAGIYTGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLFYSNYYNLWGQGTLVTVSS), or having 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:7;

[0063] The amino acid sequence of the light chain variable region is as shown in SEQ ID No: 8

[0064] or having 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.

[0065] In a specific embodiment, the anti-human CD-117 monoclonal antibody described in the present application comprises a heavy chain and a light chain, wherein:

[0066] 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 with the amino acid sequence shown in SEQ ID No: 10;

[0067] 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.

[0068] SEQ ID NO: 10

[0069]

[0070] SEQ ID NO: 11

[0071]

[0072] Among them, SEQ ID NO: 10 and 11 are all humanized sequences. Through CDR transplantation and amino acid reversion mutations that potentially affect the structure of the CDR region in the framework region, and the activity after Fc transformation is consistent with the parent rabbit antibody, the degree of humanization reaches 97.0%. In this specification, "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that usually contain nucleic acid molecules, but the nucleic acid molecules are present outside the chromosome or at a chromosomal location different from its natural chromosomal location.

[0073] As used herein, "isolated nucleic acid encoding an anti-KIT 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.

[0074] In this specification, "vector" means 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 as well as vectors that integrate into the genome of a host cell into which it has 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."

[0075] 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 offspring of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and the offspring derived therefrom (regardless of the number of generations). Offspring may not be identical to the parental cell in terms of nucleic acid content, but may contain mutations. Offspring of mutants screened or selected for the initially transformed cells with the same function or biological activity are included in this specification.

[0076] In the present specification, "pharmaceutical composition" means a preparation which is in a form that enables the biological activity of the active ingredient contained therein to exert its effect and which does not contain additional components that are unacceptably toxic to the subject to which the preparation is to be administered.

[0077] In this specification, "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than an active ingredient, which is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0078] 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).

[0079] 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, which 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. (Nature Biotechnology) 23: 1117-1125 (2005). See also, for example, the XENOMOUSETM technology described in U.S. Pat. Nos. 6,075,181 and 6,150,584; the XENOMOUSETM technology described in U.S. Pat. No. 5,770,429; Technology; US Patent No. 7,041,870 describes Technology, and U.S. Patent Application Publication No. US2007 / 0061900 described The human variable regions from intact antibodies generated by such animals can be further modified, for example, by combining with a different human constant region.

[0080] 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, e.g., 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).

[0081] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries, and such variable domain sequences can then be combined with the desired human constant domains.

[0082] 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, various 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).

[0083] In certain phage display methods, the 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). Phages 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 without 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.

[0084] 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 techniques for generating multispecific antibodies include, but are not limited to, the recombinant co-expression of two pairs of immunoglobulin heavy chains-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, e.g., U.S. Pat. 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. 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, 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.

[0085] The monoclonal antibodies described herein also include engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576).

[0086] 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.

[0087] 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. The amino acid sequence variants of the antibody may be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues in the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution 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 may 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.

[0088] Example

[0089] The content of this application will be described below in conjunction with specific embodiments, but the scope of this application is not limited thereto.

[0090] 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 conventional methods unless otherwise specified, and those skilled in the art can undoubtedly implement the scheme and obtain corresponding results according to the contents of the examples.

[0091] Example 1 Preparation of anti-human CD117 monoclonal antibody QX013N

[0092] The human CD117 antigen produced by Quanxin Biotechnology was used as an immunogen to immunize New Zealand rabbits. The monoclonal antibodies with antigen binding specificity were obtained using B cell cloning technology, and then the monoclonal antibodies that bind to human CD117 and have the ability to inhibit human CD117 activity were screened. The target clones were selected through analysis and screening by Binding ELISA and Blocking ELISA. The above immunization and screening processes were entrusted to commercial companies.

[0093] Five clones were selected for recombinant expression and sequencing. It was determined that clone 524 had the best cell neutralization activity. Therefore, clone 524 was humanized. NCBI IgBlast was used to perform homology alignment of human IgG germline sequences, and IGHV3-66*01 was selected as a heavy chain CDR transplantation template, and 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 a 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 mutations were performed in 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.

[0094] The gene of the heavy chain (SEQ ID NO: 10) was synthesized by GenScript, and the heavy chain gene was obtained by double digestion with HindIII and EcoRI, and the gene of the light chain variable region (SEQ ID NO: 8) was obtained by PCR amplification. 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.

[0095] The results of nucleic acid electrophoresis detection of the double-enzyme-digested heavy chain gene fragment, PCR-amplified light chain variable region gene fragment and double-enzyme-digested plasmid are as follows: Figure 1 As shown. Figure 1From the results, we can see that the double enzyme digestion results of the antibody heavy chain and the PCR amplification results of the light chain variable region and the results of double enzyme digestion of the heavy chain and light chain expression plasmids show that the plasmid size of the heavy chain and light chain is about 5000bp, the full length of the heavy chain is about 1410bp, and the light chain variable region is about 447bp.

[0096] 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, dilute the cells to a density of 6×10 6 cells / mL, 25mL of cells were placed in a 125mL shake flask and waited for transfection. The transfection and expression process was as follows Figure 2 shown.

[0097] On the 6th day after transfection, the culture supernatant was harvested and purified by Protein A. The purified antibody was detected by SDS-PAGE electrophoresis and named QX013N (HZD524-61). The results of protein electrophoresis for detecting the antibody were as follows: Figure 3 As shown. Protein electrophoresis was detected using denaturing reducing gel. Figure 3 The results showed that there were two bands with sizes of approximately 50 kDa and 25 kDa, respectively, which were consistent with the theoretical molecular weights of the heavy chain (49.2 kDa) and the light chain (23.4 kDa).

[0098] Example 2 Equilibrium dissociation constant (K D )

[0099] Biacore T200 was used to detect the affinity of QX013N (HZD524-61) to human KIT, and all processes were carried out at 25°C. A commercial Sensor chip CM5 chip was used to fix an appropriate amount of antibodies 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. It flowed 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 antibody binding rate constant k a , the dissociation rate constant k d and the dissociation equilibrium constant K D value.

[0100] In addition, the affinity of QX013N (HZD524-61) was compared with that of CDX-0159, a monoclonal antibody against human CD117 that is currently in Phase 2 clinical trials. The detection method for known antibodies was the same as that for QX013N, and the results are shown in Table 1. CDX-0159 was constructed based on the sequence provided by patent NZ630363B, and was transiently transfected into ExpiCHO-S cells to obtain the product.

[0101] Table 1 Affinity of anti-human CD117 monoclonal antibodies binding to human CD117

[0102]

[0103] The data in the table are: each sample was tested three times and the average value was calculated.

[0104] Example 3 QX013N and CDX-0159 neutralize the proliferation activity of recombinant human SCF-induced M07E cells

[0105] M07e cells were used to determine the activity of QX013N and CDX-0159 in neutralizing the cell proliferation induced by recombinant human SCF. 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. The mixture was 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. The cell proliferation was detected using a multifunctional microplate reader and a dose-effect curve was drawn (eg Figure 4 The neutralizing activity of the antibodies was then analyzed.

[0106] Figure 4 The results shown in the figure showed that QX013N had the activity of neutralizing the proliferation of M07e cells induced by recombinant human SCF, with an IC 50 The value is 50.55 ng / mL, and the IC of the control antibody CDX-0159 50 The value was 55.11 ng / mL, and QX013N was better than the control antibody CDX-0159 in neutralizing human SCF-induced M07e cell proliferation activity.

[0107] Example 4 QX013N and CDX-0159 neutralize NF-kB signaling activity induced by recombinant human SCF in HEK293 cells overexpressing human CD117 reporter gene

[0108] HEK293 reporter cells overexpressing human CD117 were used to determine the effect of QX013N and CDX-0159 on the intracellular NF-kB signaling activity mediated by recombinant human SCF through CD117. HEK293 cells overexpressing human CD117 were plated at 4 × 10 5 Cells were plated into 96-well plates and then cultured overnight at 37°C and 5% CO2. Antibodies were diluted to a concentration range of 0 to 40 μg / mL and added to the cells for 1 hour. 400 ng / mL of recombinant human SCF was added and then cultured for 24 hours at 37°C and 5% CO2. One-Lite Luciferase Assay System detection reagent (Vazyme, DD1203-02) was added to detect cells, and SoftMax was used to fit the four-parameter curve to calculate the sample IC 50 The neutralizing activity of the antibody was analyzed. Figure 5 shown.

[0109] Figure 5 The results shown in the figure 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 value is 9.70ng / mL, and the IC of the control antibody CDX-0159 50 The value was 13.63 ng / mL, and QX013N neutralized human SCF-induced reporter gene activity better than the control antibody CDX-0159.

[0110] Example 5 QX013N and CDX-0159 neutralize the binding activity of recombinant human SCF to CD117 on the surface of LUVA cell membrane

[0111] 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 Biotin was added. TMAlexa Fluor 488 detection reagent, cells were washed with PBS and resuspended, flow cytometry was used for detection, and GraphPad was used to fit the four-parameter curve and calculate the sample IC 50 The results were as follows: Figure 6 shown.

[0112] Figure 6 The results shown in the figure show that QX013N can neutralize the binding of recombinant human SCF to CD117 on the surface of LUVA cell membrane, 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.

[0113] Example 6 Detection of Binding Activity of QX013N and CDX-0159 to Porcine CD117

[0114] Binding ELISA was used to detect the binding activity of QX013N and CDX-0159 with 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, and 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 an ELISA reader to read the absorbance value at 450nm (the absorbance value at 650nm 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 the figure below. Figure 7 shown.

[0115] Figure 7 The results shown show that QX013N can bind to porcine CD117, but CDX-0159 does not. This result suggests that QX013N and CDX-0159 recognize different epitopes.

[0116] Example 7 Difference in signal values ​​between QX013N and CDX-0159 in neutralizing the binding of human SCF to human CD117

[0117] ELISA was used to detect the difference in binding signal values ​​between human SCF and human CD117 neutralized by QX013N and CDX-0159. Human SCF was diluted to 1 μg / mL and added to the ELISA plate and coated overnight. A series of antibody dilutions ranging from 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 ELISA 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 ELISA plate was placed in an ELISA reader to read the absorption value at 450 nm (the absorption value at 650 nm was used as a reference), and a signal value effect curve was drawn to analyze the difference in binding signal values ​​between human SCF and human CD117 neutralized by the antibody. The signal value effect curve is shown as follows: Figure 8 shown.

[0118] Figure 8 The results shown show that when the antibody concentration is 0, the binding signal value of human CD117 and human SCF is 100%; when the antigen-antibody binding is at saturation, the signal value of QX013N neutralizing human SCF and human CD117 is 55.6%; CDX-0159 neutralizing human SCF and human CD117 is 30.5%. This result suggests that QX013N and CDX-0159 recognize different epitopes.

[0119] The above is only a preferred embodiment of the present application and does not limit the present application in any form. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the content of the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. An anti-human CD117 monoclonal antibody, comprising three heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and three light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, wherein: The amino acid sequence of CDR-H1 is shown in SEQ ID No: 1; The amino acid sequence of CDR-H2 is shown in SEQ ID No: 2; The amino acid sequence of CDR-H3 is shown in SEQ ID No: 3; The amino acid sequence of CDR-L1 is shown in SEQ ID No: 4; The amino acid sequence of CDR-L2 is shown in SEQ ID No: 5; The amino acid sequence of CDR-L3 is shown in SEQ ID No:

6.

2. The monoclonal antibody according to claim 1, 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, 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. A host cell comprising the nucleic acid of claim 4 .

6. A method for producing a monoclonal antibody, 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. The pharmaceutical composition according to claim 7, wherein The pharmaceutical composition is used for treating diseases related to signal transduction mediated by human CD117 protein.

9. The pharmaceutical composition according to claim 8, wherein The diseases related to signal transduction mediated by human CD117 protein for treatment include immune system diseases, digestive system diseases, and blood and lymphatic system diseases.

10. The pharmaceutical composition according to claim 9, wherein The immune system diseases include eosinophilic esophagitis, chronic urticaria and prurigo.

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

12. The use according to claim 11, wherein The diseases related to signal transduction mediated by human CD117 protein for treatment include immune system diseases, digestive system diseases, blood and lymphatic system diseases. Preferably, the immune system diseases include eosinophilic esophagitis, chronic urticaria and prurigo.

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