Antibody targeting CD117

Abstract

The present disclosure relates to antibodies and antibody fragments specific to CD117. The antibody is an improved version of state-of-the-art antibodies. In addition to the strategy of sophisticated humanization, the antibody was also modified to remove some deleterious motifs within the CDR regions without losing beneficial properties. The antibody is useful for the treatment of diseases associated with CD117.

Description

Technical Field

[0001] The present disclosure relates to antibodies and antibody fragments specific for CD117. The antibodies are improved versions of state-of-the-art antibodies. In addition to the strategy of sophisticated humanization, the antibodies were also modified to remove some of the deleterious motifs within the CDR regions without losing beneficial properties. The antibodies are useful for the treatment of diseases associated with CD117.

[0002] Description of Funding The project leading to this application has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (Grant Agreement No. 818806).

Background Art

[0003] CD117 (also known as c-kit or stem cell factor receptor (SCFR)) is a single-pass transmembrane receptor tyrosine kinase that binds to the ligand stem cell factor (SCF). SCF activates the tyrosine kinase activity of CD117, resulting in the induction of CD117 homodimerization, which leads to signal transduction via both the PI3K-AKT and MAPK pathways (Kindblom et al., Am J. Path. 1998 152(5): 1259).

[0004] CD117 was first discovered as an oncogene and has been studied in the field of oncology (see, for example, Stankov et al. (2014) Curr Pharm Des. 20:2849-80). CD117 is highly expressed on hematopoietic stem cells (HSCs), and its ligand is stem cell factor, SCF. SCF is a growth factor that acts through CD117 and is required for the survival, proliferation, and differentiation of hematopoietic cells. The expression of CD117 on HSCs, together with its activity in the regulation of hematopoiesis, makes CD117 a potential target for a wide range of diseases across bone marrow conditioning (Russkamp et al. Exp. Hematol. (2021) 95:31-45; Czechowicz et al. Nat Commun (2019) 10:617). However, anti-CD117-based therapies that are effective for patient conditioning for transplantation, such as bone marrow transplantation, are still needed.

[0005] Several anti-CD117 moieties are known in the art, and some of them are currently under development. Antibody SR-1 was originally isolated from a hybridoma (WO 92 / 17505). Blood (2019) 133:2069-78 describes the use of SR-1 in blood and immune disorders driven by HSCs. A specific humanized version of SR-1 has been generated (WO 2007 / 127317; WO 2020 / 112687). WO 2020 / 033664 discloses a prototype SR-1 antibody for use in bispecific and multispecific constructs (the sequences of the variable heavy and variable light chains are identical to the sequences disclosed in US Patent Application Publication No. 2020 / 0165337A1). J. Clin. Oncol. (2021) Vol. 39 No. 15 Meeting Abstract 2021 ASCO Annual Meeting, abstract 7035 discloses the results of a Phase I clinical trial of antibody JSP191. Blood (2019) 134 (Suppl. 1):4428 discloses a study on the anti-CD117 antibody FSI-174. Anti-CD117 drug conjugates are described in WO 2016 / 020791. Other anti-CD117 antibodies are described in WO 2015 / 050959 and WO 2019 / 084064. A specific anti-CD117 antibody, such as antibody 104D2 Dianova (#117PE-100T), is also commercially available. These and other anti-CD117 moieties may be used in the context of the present disclosure. WO 2021 / 041945 discloses genomic alterations of antigens containing CD117 generated by base editing. However, the characteristics of these antigen changes are not known, and in particular, the biological functions of the mutants have not been tested.

Prior Art Documents

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Summary of the Invention

[0008] The present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, and the humanized antibody or antibody fragment has at least one mutation in which asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid or tyrosine. In certain embodiments, the humanized antibody or antibody fragment has one or more of the following additional mutations: a) The first asparagine in the HCDR2 region (SEQ ID NO: 5) is substituted with serine, b) The second asparagine in the LCDR3 region (SEQ ID NO: 9) is substituted with lysine, or c) Aspartic acid in the LCDR3 region (SEQ ID NO: 9) is substituted with glutamic acid.

[0009] The present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment a) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 95, b) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 81, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 113, c) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 113, d) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 71, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, e) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, f) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, or g) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9 comprising.

[0010] The present disclosure relates to a humanized antibody or antibody fragment, and the humanized antibody or antibody fragment a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98, b) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 99, c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100, d) a variable heavy chain of SEQ ID NO: 70 and a variable light chain of SEQ ID NO: 98, e) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 97, f) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 61, or g) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 62 comprising.

[0011] The present disclosure also relates to a humanized antibody or antibody fragment that inhibits the binding of SCF to CD117, the SCF-dependent proliferation of CD117-positive cells, and / or the SCF-dependent phosphorylation of CD117.

[0012] The present disclosure also relates to a humanized antibody or antibody fragment that binds to CD117 with generally the same affinity as an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0013] The present disclosure also relates to a humanized antibody or antibody fragment that binds to CD117 with a higher affinity than an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0014] The present disclosure also relates to a humanized antibody or antibody fragment that binds to the same epitope on CD117 as an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0015] The present disclosure also relates to a humanized antibody or antibody fragment that is cross-reactive with cynomolgus monkeys.

[0016] The present disclosure also relates to a humanized antibody or antibody fragment as disclosed herein for use in medicine, preferably for the treatment of cancer, such as blood cancers (e.g., AML) or solid cancers (e.g., mast cell carcinoma, testicular stromal carcinoma, gastrointestinal stromal carcinoma, melanoma, breast cancer, and lung cancer), or for bone marrow preconditioning.

[0017] The present disclosure also relates to a nucleic acid or nucleic acid composition comprising a nucleic acid sequence or sequences encoding a humanized antibody or antibody fragment of the present disclosure.

[0018] The present disclosure also relates to a vector comprising a nucleic acid or nucleic acid composition encoding a humanized antibody or antibody fragment of the present disclosure.

[0019] The present disclosure also relates to a host cell comprising a vector or nucleic acid or nucleic acid composition of a humanized antibody or antibody fragment of the present disclosure.

[0020] The present disclosure also relates to a pharmaceutical composition comprising a humanized antibody or antibody fragment of the present disclosure and a pharmaceutically acceptable carrier or excipient.

Brief Description of the Drawings

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[0022] Definitions The present disclosure relates to antibodies that specifically bind to CD117, and to the use of such antibodies, particularly for therapeutic use.

[0023] The term "CD117" refers to a protein also known as KIT, c-Kit, SCFR, PBT or MASTC. Human CD117 has the following amino acid sequence (UniProt P10721, updated on June 28, 2023): MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTD PGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYH RLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSS SVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSAN VTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWE DYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDR LVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDS SAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIV MILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAGAF GKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVNLLGAC TIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNE YMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGM AFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPES IFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMY DIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSV GSTASSSQPLLVHDDV (SEQ ID NO: 1)

[0024] As used herein, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds that interact with an antigen. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., Igd, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

[0025] As used herein, the term "antibody fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, stabilization of spatial distribution). Examples of binding fragments include: a monovalent fragment consisting of the Fab fragment, VL, VH, CL, and CH1 domains; a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region, the F(ab)2 fragment; the Fd fragment consisting of the VH domain and the CH1 domain; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and isolated complementarity determining regions (CDRs), among others. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that enables them to be made as a single protein chain such that the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also to be included within the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136). Antibody fragments can be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies).An antibody fragment can be incorporated into a single-chain molecule containing a pair of tandem Fv segments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Patent No. 5,641,870).

[0026] The structure and position of immunoglobulin variable domains, such as CDRs, can be defined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991), ed. Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948; Annals of the New York Academy of Sciences, 764, 47-49 (1995); Nucleic Acids Research, 25, 206-211 (1997).

[0027] As used herein, "human antibody" or "human antibody fragment" refers to an antibody and antibody fragment having a variable region in which both the framework region and the CDR region are derived from sequences of human origin. Human antibodies can also be isolated from synthetic libraries or transgenic mice (e.g., Xenomouse, OmniMouse, Harbour Mouse, ATX-Gx Mouse, Trianni Mouse), provided that each system results in an antibody having a variable region in which both the framework and CDR regions are derived from sequences of human origin. Further, when the antibody contains a constant region, the constant region is also derived from such a sequence. Human origin includes, for example, human germline sequences, or human germline sequences or mutant versions of antibodies containing consensus framework sequences derived from human framework sequence analysis, as described in Knappik et al., (2000) J Mol Biol 296:57-86).

[0028] "Humanized antibody" or "humanized antibody fragment" is defined herein as an antibody molecule having a constant antibody region derived from a sequence of human origin and a variable antibody region or a part thereof, or only the CDR, derived from another species. For example, CDRs can be transplanted into a humanized antibody, where the CDRs of the variable domain are of non-human origin, while one or more frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin.

[0029] The term "chimeric antibody" or "chimeric antibody fragment" is defined herein as an antibody molecule having a constant antibody region derived from or corresponding to a sequence found in one species and a variable antibody region derived from another species. Preferably, the constant antibody region is derived from or corresponds to a sequence found in humans, and the variable antibody region (e.g., VH, VL, CDR or FR region) is derived from a sequence found in a non-human animal, such as a mouse, rat, rabbit or hamster.

[0030] The term "isolated antibody" or "isolated antibody fragment" refers to an antibody or antibody fragment that is substantially free of other antibodies or antibody fragments having different antigen specificities. Additionally, an isolated antibody or antibody fragment may be substantially free of other cellular materials and / or chemical substances. Thus, in some embodiments, the provided antibodies are isolated antibodies that are separated from antibodies having different specificities. Isolated antibodies can be monoclonal antibodies. Isolated antibodies can be recombinant monoclonal antibodies. However, an isolated antibody that specifically binds to a target epitope, isoform or variant may have cross-reactivity with other related antigens, for example, derived from other species (e.g., species homologs).

[0031] As used herein, the term "recombinant antibody" or "recombinant antibody fragment" includes any antibody or antibody fragment that is prepared, expressed, made or isolated by means that do not occur in nature. For example, an antibody isolated from a host cell transformed to express the antibody, an antibody selected and isolated from a recombinant combinatorial human antibody library, and an antibody prepared, expressed, made or isolated by any other means that includes splicing of all or part of the human immunoglobulin genes, sequences for other DNA sequences, or an antibody isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for a human immunoglobulin gene or a hybridoma prepared therefrom. Preferably, such recombinant antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, in the case of using an animal that is transgenic for a human Ig sequence, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are related to, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo, and are derived from human germline VH and VL sequences. Recombinant antibodies can be monoclonal antibodies.

[0032] As used herein, the term "monoclonal" has the meaning typically ascribed in the art, i.e., an antibody or antibody fragment (or its corresponding functional fragment) arising from a single clone of antibody-producing cells that recognizes a single epitope on the antigen to which it binds.

[0033] As used herein, since binding specificity is a relative rather than an absolute property, an antibody "binds specifically to", "specifically binds to", "is specific to / for" or "specifically recognizes" an antigen such as human CD117 if such an antibody is capable of distinguishing such an antigen from one or more reference antigens. For example, a standard ELISA assay or a standard flow cytometry assay can be performed. Scoring can be done by standard chromogenicity (e.g., with hydrogen peroxide, a secondary antibody containing horseradish peroxidase and tetramethylbenzidine), or by binding of a secondary antibody labeled with PE or another dye or marker. The reaction in a particular well is scored, for example, by the optical density (OD) at 450 nm, or by the mean or median fluorescence intensity (MFI) in flow cytometry. A typical background (= negative reaction) can be 0.1 OD and a typical positive reaction can be 1 OD. The background and positive reaction MFI depend greatly on the instrument settings. The positive / negative difference can exceed 10-fold. Typically, determination of binding specificity is done by using a set of about 3 to 5 unrelated antigens such as milk powder, BSA, transferrin rather than a single reference antigen. For flow cytometry, various antigen-negative cells can be used. However, an antibody that specifically binds to an antigen can have cross-reactivity with each corresponding orthologous antigen from other species (e.g., species homologs). In certain embodiments, such cross-reactivity to orthologous antigens is more preferred.

[0034] As used herein, an antibody is "cross-reactive" or "cross-reacts" if it binds to an orthologous antigen from another species. For example, an antibody is cross-reactive if it binds to human CD117 and cynomolgus monkey CD117.

[0035] As used herein, the term "affinity" refers to the strength of the interaction between a polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts with its target through weak non-covalent forces at multiple sites; the more interactions, the stronger the affinity.

[0036] The term "epitope" includes any proteinaceous region that is specifically recognized by an antibody or an antibody fragment thereof, or otherwise interacts with a molecule. Generally, an epitope is a chemically active surface group of a molecule, such as an amino acid or a carbohydrate or a sugar side chain, and can generally have specific three-dimensional structural properties as well as specific charge properties. As will be understood by those skilled in the art, substantially anything to which an antibody can specifically bind can be an epitope.

[0037] The term "domain" or "protein domain" refers to a region of a polypeptide chain of a protein that forms a functional unit and / or independently forms a three-dimensional structure.

[0038] A "composition" or the compositions of the disclosure can be used for therapeutic or prophylactic applications. Accordingly, the disclosure includes a pharmaceutical composition containing an antibody or an antibody fragment as disclosed herein, and a pharmaceutically acceptable carrier or excipient therefor. In related aspects, the disclosure provides methods for treating inflammatory diseases, autoimmune diseases, hematological malignancies and potentially other diseases. Such methods include administering to a subject in need thereof an effective amount of a pharmaceutical composition containing an antibody or an antibody fragment as described herein.

[0039] The present disclosure provides a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antibody fragment as disclosed herein. As used herein, "therapeutically effective amount" or "effective amount" refers to the amount of the anti-CD117 antibody necessary to induce the desired biological response. According to the present disclosure, a therapeutically effective amount is the amount of the anti-CD117 antibody necessary to treat and / or prevent a disease.

[0040] As used herein, "administered" or "administration" includes, but is not limited to, delivery of a drug in injectable form, e.g., by intravenous, intramuscular, intradermal or subcutaneous routes or mucosal routes, e.g., as a nasal spray or aerosol for inhalation, or as an ingestible solution, capsule or tablet. Preferably, administration is by injectable form.

[0041] As used herein, "treatment", "treat" or "treating" etc. refer to a clinical intervention that attempts to alter the natural course of a disease in a subject being treated and can be performed for prophylaxis or during the course of a clinical pathology. Desired effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of any direct or indirect pathological consequence of a disease, prevention of metastasis, reduction of the rate of disease progression, improvement or alleviation of a disease state, and remission or improvement of prognosis. In some embodiments, the antibody or antibody fragment according to the present disclosure is used to delay the onset of a disease or to slow the progression of a disease.

[0042] "Preventing" or "prevention" refers to reducing the risk of acquiring or developing a disease (i.e., not causing at least one clinical symptom of the disease in a subject who may be exposed to an agent that causes the disease or is susceptible to the disease prior to its onset). "Prevention" also refers to methods aimed at preventing the onset of a disease or its symptoms, or delaying the onset of a disease or its symptoms.

[0043] "Subject" or "species", as used in this context, refers to any mammal, including rodents such as mice or rats, and primates such as cynomolgus monkeys (Macaca fascicularis), marmoset monkeys (Callithrix jacchus), rhesus monkeys (Macaca mulatta) or humans (Homo sapiens). Preferably, the subject is a primate, most preferably a human.

[0044] The term "effector function" refers to biological activities resulting from the Fc region of an antibody that vary depending on the antibody isotype. Non-limiting examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and direct cell activation or direct cell inhibition.

[0045] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cell injury in which cytotoxic effector cells, which are bound to an antibody that is bound to an Fc receptor (FcR) present on certain cytotoxic cells (e.g., NK cells, neutrophils and macrophages), can specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. The initial cells for mediating ADCC, NK cells, express only FcyRIII, while monocytes / macrophages express FcyRI, FcyRII and FcyRIII.

[0046] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated when the first component of the complement system (C1q) binds to an antibody (of the appropriate subclass) of the present disclosure and it binds to its cognate antigen.

[0047] "Antibody-dependent cell phagocytosis" or "ADCP" refers to the mechanism of elimination of antibody-coated target cells by internalization by phagocytic cells such as macrophages or dendritic cells.

[0048] Throughout this specification, unless the context requires otherwise, the words "comprise", "have" and "include", and their respective variations such as "comprises", "comprising", "has", "having", "includes" and "including", are to be understood to mean including the stated element or integer or group of elements or integers but not to mean excluding any other element or integer or group of elements or integers.

[0049] As used herein, the terms "engineered" or "modified" include the manipulation of nucleic acids or polypeptides by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides or some combination of these techniques). Preferably, the antibodies or antibody fragments according to the present disclosure are engineered or modified to improve one or more properties such as antigen binding, stability, half-life, effector function, immunogenicity, safety, etc.

[0050] As used herein, the term "variant" refers to a polypeptide that differs from a reference polypeptide by one or more modifications, such as amino acid substitutions, insertions, or deletions. Variant polypeptides typically retain most of the properties of the reference polypeptide, such as binding to a target antigen, but introduce novel additional features or properties. For example, a variant polypeptide may have a higher affinity for a target antigen compared to the reference polypeptide, or a variant polypeptide may be a humanized version of the reference polypeptide.

[0051] As used herein, the term "amino acid mutation" is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications may be made, so long as the final construct has the desired feature, such as reduced binding to an Fc receptor. Deletions and insertions of amino acid sequences include N-terminal and / or C-terminal deletions and insertions of amino acid residues. A particular amino acid mutation is an amino acid substitution. Amino acid substitutions include substitutions by non-natural amino acids or by natural amino acid derivatives of the twenty standard amino acids. Amino acid mutations can be made using genetic or chemical methods well known in the art. Genetic methods can include, for example, site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods other than genetic modification, such as chemical modification, for changing the side chain groups of amino acid residues may also be useful. In this specification, various names may be used to denote the same amino acid mutation. For example, the substitution of glycine at position 237 of the antibody Fc region with alanine may be denoted as 237A, G237, G237A, or Gly237Ala.

[0052] As used herein, the term "EC50" refers to the concentration of an antibody or antibody fragment that induces a response in an assay that is intermediate between the baseline and the maximum value. Thus, this corresponds to the antibody or ligand concentration at which 50% of the maximum effect is observed.

[0053] As used herein, the term "Ka" refers to the association rate of a particular antibody-antigen interaction.

[0054] As used herein, the term "Kd" refers to the dissociation rate of a particular antibody-antigen interaction. The Kd value for an antibody can be determined using methods well established in the art.

[0055] As used herein, the term "KD" refers to the dissociation constant of a particular antibody-antigen interaction, obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration. A preferred method for determining the Kd of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore system, or by using biolayer interferometry with an Octet BLI instrument.

[0056] The terms "inhibition" or "inhibit" or "reduction" or "reduce" or "neutralization" or "neutralize" refer to a decrease or cessation of any phenotypic characteristic (such as binding or biological activity or function), or a decrease or cessation in the incidence, degree or likelihood of that characteristic. "Inhibition", "reduction" or "neutralization" need not be complete, so long as it is detectable using an appropriate assay. In some embodiments, "reduce" or "inhibit" or "neutralize" means the ability to cause a decrease of 20% or more. In another embodiment, "reduce" or "inhibit" or "neutralize" means the ability to cause a decrease of 50% or more. In yet another embodiment, "reduce" or "inhibit" or "neutralize" means the ability to cause an overall decrease of 75%, 85%, 90%, 95% or more.

[0057] As used herein, the term "antagonist" antibody refers to an antibody or antibody fragment that interacts with an antigen and partially or completely inhibits or neutralizes the biological activity or function of the target antigen or any other phenotypic property.

[0058] A "wild-type" protein is a version or variant of a protein as found in nature. The amino acid sequence of a wild-type protein, such as the Fc region of a human IgG1 antibody, is the amino acid sequence of the protein as it exists in nature. Due to allotypic differences, there may be two or more amino acid sequences for a wild-type protein. For example, there are several allotypes of the native human IgG1 heavy chain constant region (see, e.g., Jeffries et al. (2009) mAbs 1:1).

[0059] The "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain. The Fc region of an immunoglobulin generally includes two constant domains, the CH2 domain and the CH3 domain. The boundaries of the Fc region of the IgG heavy chain can vary slightly, but the human IgG heavy chain Fc region is usually defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region follows the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Various Fc modifications are commonly used. For reviews, see, for example, Antibodies (2020) 9:64. The silencing functions include (numbering follows the EU index) the LALA (L234A / L235A), PA-LALA (L234A / L235A / P329A), and PG-LALA (L234A / L235A / P329G) mutations, as well as the AEASS mutation (L234A / L235E / G237A / A330S / P331S). The preferred FC modification is PA-LALA.

[0060] Embodiments of the present invention Polypeptide In certain embodiments, the disclosure relates to an antibody or antibody fragment specific for CD117. In certain embodiments, the disclosure relates to an antibody or antibody fragment specific for human CD117. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific for CD117. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific for human CD117.

[0061] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0062] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, said antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, wherein said humanized antibody or antibody fragment has at least one mutation in which asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid or tyrosine.

[0063] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, said antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, wherein said humanized antibody or antibody fragment has at least one mutation in which asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid or tyrosine, and said humanized antibody or antibody fragment has one or more of the following further mutations: a) the first asparagine in the HCDR2 region (SEQ ID NO: 5) is substituted with serine, b) the second asparagine in the LCDR3 region (SEQ ID NO: 9) is substituted with lysine, and / or c) aspartic acid in the LCDR3 region (SEQ ID NO: 9) is substituted with glutamic acid.

[0064] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment as described above, said humanized antibody or antibody fragment a) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, b) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, c) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, d) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 71, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, e) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, f) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, or g) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9 comprising.

[0065] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment as described above, wherein the humanized antibody or antibody fragment a) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 98, b) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 99, c) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 100, d) the variable heavy chain of SEQ ID NO: 70 and the variable light chain of SEQ ID NO: 98, e) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 97, f) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 61, or g) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 62 and comprises.

[0066] In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific for human CD117, said antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, wherein said humanized antibody or antibody fragment has at least two of the following mutations: a) asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid or tyrosine, and b) aspartic acid in the LCDR3 region (SEQ ID NO: 9) is substituted with glutamic acid.

[0067] In certain embodiments, said humanized antibody or antibody fragment specific for human CD117 has one or more of the following additional mutations: a) the second asparagine in the LCDR3 region (SEQ ID NO: 9) is substituted with lysine, and / or b) the first asparagine in the HCDR2 region (SEQ ID NO: 5) is substituted with serine.

[0068] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, said antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, wherein said humanized antibody or antibody fragment has two of the following mutations: a) asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid, and b) aspartic acid in the LCDR3 region (SEQ ID NO: 9) is substituted with glutamic acid.

[0069] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, said antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, wherein said humanized antibody or antibody fragment has three of the following mutations: a) asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with tyrosine, b) the second asparagine in the LCDR3 region (SEQ ID NO: 9) is substituted with lysine, and c) aspartic acid in the LCDR3 region (SEQ ID NO: 9) is substituted with glutamic acid.

[0070] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, said antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, wherein said humanized antibody or antibody fragment has two of the following mutations: a) asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid, b) The second asparagine in the LCDR3 region (SEQ ID NO: 9) is replaced with lysine, and c) The aspartic acid in the LCDR3 region (SEQ ID NO: 9) is replaced with glutamic acid.

[0071] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, said antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, wherein said humanized antibody or antibody fragment has two of the following mutations: a) The first asparagine in the HCDR2 region (SEQ ID NO: 5) is replaced with serine, b) The asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced with glutamic acid, and c) The aspartic acid in the LCDR3 region (SEQ ID NO: 9) is replaced with glutamic acid.

[0072] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, said antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, wherein this humanized antibody or antibody fragment comprises a mutation in which the asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced with glutamic acid, histidine, glutamine, or serine.

[0073] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, and the humanized antibody or antibody fragment comprises a mutation in which the second asparagine in the LCDR3 region (SEQ ID NO: 9) is substituted with lysine or arginine.

[0074] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, and the humanized antibody or antibody fragment comprises a mutation in which the aspartic acid in the LCDR3 region (SEQ ID NO: 9) is substituted with glutamic acid.

[0075] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, and the humanized antibody or antibody fragment comprises the following two mutations: the asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid, histidine, glutamine or serine, and the second asparagine in the LCDR3 region (SEQ ID NO: 9) is substituted with lysine or arginine.

[0076] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, and the humanized antibody or antibody fragment comprises two of the following mutations: The asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid, histidine, glutamine or serine, and The aspartic acid in the LCDR3 region (SEQ ID NO: 9) is substituted with glutamic acid.

[0077] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, and the humanized antibody or antibody fragment comprises two of the following mutations: The second asparagine in the LCDR3 region (SEQ ID NO: 9) is substituted with lysine or arginine, and The aspartic acid in the LCDR3 region (SEQ ID NO: 9) is substituted with glutamic acid.

[0078] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, and the humanized antibody or antibody fragment comprises three of the following mutations: The asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid, histidine, glutamine or serine, The second asparagine in the LCDR3 region (SEQ ID NO: 9) is replaced with lysine or arginine, and the aspartic acid in the LCDR3 region (SEQ ID NO: 9) is replaced with glutamic acid.

[0079] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, and the humanized antibody or antibody fragment comprises a mutation in which the first leucine in the LCDR2 region (SEQ ID NO: 8) is replaced with lysine, histidine, glutamic acid, serine, or glutamine.

[0080] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, and the humanized antibody or antibody fragment comprises the following four mutations: the asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced with glutamic acid, histidine, glutamine, or serine, the second asparagine in the LCDR3 region (SEQ ID NO: 9) is replaced with lysine or arginine, the aspartic acid in the LCDR3 region (SEQ ID NO: 9) is replaced with glutamic acid, and the first leucine in the LCDR2 region (SEQ ID NO: 8) is replaced with lysine, histidine, glutamic acid, serine, or glutamine.

[0081] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, said antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, wherein said humanized antibody or antibody fragment comprises a mutation in which the first asparagine in the HCDR2 region (SEQ ID NO: 5) is substituted with serine or glutamine.

[0082] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, said antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, wherein said humanized antibody or antibody fragment comprises the following four mutations: the asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid, histidine, glutamine, or serine, the second asparagine in the LCDR3 region (SEQ ID NO: 9) is substituted with lysine or arginine, the aspartic acid in the LCDR3 region (SEQ ID NO: 9) is substituted with glutamic acid, and the first asparagine in the HCDR2 region (SEQ ID NO: 5) is substituted with serine or glutamine.

[0083] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117, said antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, wherein said humanized antibody or antibody fragment comprises the following five mutations: the asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid, histidine, glutamine, or serine, The second asparagine in the LCDR3 region (SEQ ID NO: 9) is replaced with lysine or arginine, The aspartic acid in the LCDR3 region (SEQ ID NO: 9) is replaced with glutamic acid, The first leucine in the LCDR2 region (SEQ ID NO: 8) is replaced with lysine, histidine, glutamic acid, serine or glutamine, and The first asparagine in the HCDR2 region (SEQ ID NO: 5) is replaced with serine or glutamine.

[0084] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment a) a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, b) a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, c) a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, d) a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 71, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, e) a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, f) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, g) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 121, and the LCDR3 region of SEQ ID NO: 115, h) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 123, and the LCDR3 region of SEQ ID NO: 115, i) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 125, and the LCDR3 region of SEQ ID NO: 115, j) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 127, and the LCDR3 region of SEQ ID NO: 115, or k) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113 comprising.

[0085] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment a) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, b) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, c) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, or d) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 71, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95 comprising.

[0086] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the antibody or antibody fragment comprises a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95.

[0087] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113.

[0088] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113.

[0089] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 71, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 95.

[0090] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 45, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 113.

[0091] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 44, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 113.

[0092] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 109, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 113.

[0093] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 115.

[0094] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 107, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 115.

[0095] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 107, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 121, and an LCDR3 region of SEQ ID NO: 115.

[0096] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 107, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 123, and an LCDR3 region of SEQ ID NO: 115.

[0097] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 107, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 125, and an LCDR3 region of SEQ ID NO: 115.

[0098] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 107, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 127, and an LCDR3 region of SEQ ID NO: 115.

[0099] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113.

[0100] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98, b) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 99, c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100, d) a variable heavy chain of SEQ ID NO: 70 and a variable light chain of SEQ ID NO: 98, e) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 105, f) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 105, g) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 117, h) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 118, i) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 119, j) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 120, or k) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 100 comprises.

[0101] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98, b) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 99, c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100, or d) a variable heavy chain of SEQ ID NO: 70 and a variable light chain of SEQ ID NO: 98 comprises.

[0102] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98.

[0103] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 99.

[0104] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100.

[0105] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 70 and a variable light chain of SEQ ID NO: 98.

[0106] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 101.

[0107] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 102.

[0108] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 103.

[0109] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 105.

[0110] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 105.

[0111] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 117.

[0112] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 118.

[0113] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 119.

[0114] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 120.

[0115] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 100.

[0116] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises six CDRs of one of the antibodies disclosed in Table 18.

[0117] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises six CDRs as defined by Kabat of one of the antibodies disclosed in Table 18.

[0118] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises six six CDRs as defined by one of the antibodies disclosed in Table 18 according to IMGT.

[0119] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises one variable heavy chain and one variable light chain of an antibody disclosed in Table 18.

[0120] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is a monoclonal antibody or antibody fragment.

[0121] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is a recombinant antibody or antibody fragment.

[0122] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is of the IgG isotype.

[0123] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is of the IgG1 class.

[0124] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is of the human IgG1 class.

[0125] The isolated antibodies or antibody fragments according to the present disclosure may or may not be fused to one or more other amino acid residues, polypeptides or moieties. Such fusion proteins can be prepared by any suitable method including genetic or chemical approaches. The linked moieties may contain a secretion or leader sequence, a sequence that aids in detection, expression, isolation or purification, or a sequence that, for example, results in an improvement in protein stability during recombinant production. Non-limiting examples of possible moieties include β-galactosidase, glutathione-S-transferase, luciferase, T7 polymerase fragment, secretion signal peptide, antibody or antibody fragment, toxin, cytokine, chemokine, reporter enzyme, a moiety capable of binding to a metal ion such as a polyhistidine tag, a tag suitable for detection and / or purification, a homo- or hetero-association domain, a moiety that improves the solubility of a protein, or a moiety that includes an enzyme cleavage site.

[0126] Thus, the isolated antibodies or antibody fragments according to the present disclosure may optionally contain one or more moieties for binding to other targets or target proteins of interest. It should be apparent that such additional moieties may or may not provide additional functionality to the antibody and may or may not modify the properties of the isolated antibodies or antibody fragments according to the present disclosure.

[0127] Nucleic acid In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment a) a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, b) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, c) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, d) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 71, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, e) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, f) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, g) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 121, and the LCDR3 region of SEQ ID NO: 115, h) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 123, and the LCDR3 region of SEQ ID NO: 115, i) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 125, and the LCDR3 region of SEQ ID NO: 115, j) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 127, and the LCDR3 region of SEQ ID NO: 115, k) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, l) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, m) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, or n) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9 comprising.

[0128] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment is a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98, b) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 99, c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100, d) a variable heavy chain of SEQ ID NO: 70 and a variable light chain of SEQ ID NO: 98, e) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 105, f) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 105, g) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 117, h) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 118, i) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 119, j) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 120, k) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 100, l) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 97, m) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 61, or n) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 62 and comprises.

[0129] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises six six CDRs as defined by one Kabat of the antibodies disclosed in Table 18.

[0130] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises six six CDRs as defined by one IMGT of the antibodies disclosed in Table 18.

[0131] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises one variable heavy chain and one variable light chain of the antibodies disclosed in Table 18.

[0132] In one embodiment, the nucleic acid composition and / or the nucleic acid sequence and / or the plurality of nucleic acid sequences are isolated.

[0133] Vector In one embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a humanized or antibody fragment specific for human CD117 according to the present disclosure.

[0134] In one embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the humanized antibodies or antibody fragments specific for human CD117 disclosed in Table 18.

[0135] Host cell In one embodiment, the present disclosure provides a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a humanized antibody or antibody fragment specific for human CD117 according to the present disclosure.

[0136] In one embodiment, the present disclosure refers to a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the humanized antibodies or antibody fragments specific for human CD117 disclosed in Table 18.

[0137] In one embodiment, the host cell according to the present disclosure is capable of expressing a humanized antibody or antibody fragment specific for human CD117 encoded by the vector composition or nucleic acid composition.

[0138] In a further embodiment, the host cell is an isolated host cell. In a further embodiment, the host cell is a mammalian cell. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a CHO cell. In one embodiment, the cell is a HEK cell. In another embodiment, the cell is a PERC.6 cell. In one embodiment, the cell is an HKB11 cell.

[0139] Those skilled in the art will recognize that the nucleic acid sequence or sequences encoding the heavy and / or light chains of the antibodies or antibody fragments of the present disclosure can be cloned into different vectors or the same vector.

[0140] Vectors can be introduced into suitable host cells, such as prokaryotic (e.g., bacterial) cells or eukaryotic (e.g., yeast or mammalian) cells, by methods well known in the art (e.g., "Current Protocol in Molecular Biology", Ausubel et al. (eds.), Greene Publishing Assoc and John Wiley Interscience, New York, 1989 and 1992). A number of cloning vectors are known to those skilled in the art, and the selection of an appropriate cloning vector is a matter of choice. The gene can be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operator (collectively referred to herein as "control" elements) such that the nucleic acid sequence encoding the desired protein is transcribed into RNA in a host cell transformed with the vector containing this expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. When expressed in a host cell, the antibodies or antibody fragments of the present disclosure are obtained. These steps can be accomplished in a variety of ways, as known to those skilled in the art. Generally, such steps typically involve transforming or transfecting a suitable host cell with a nucleic acid composition or vector composition or infectious particle encoding the antibody or antibody fragment. Further, such steps typically involve culturing the host cell under conditions suitable for the growth (replication, growth) of the host cell and culturing under conditions suitable for the production (expression, synthesis) of the encoded antibody or antibody fragment. Culturing of the host cell under conditions suitable for growth or expression is typically achieved in the presence of a medium containing components suitable for inducing cell growth or expression. In certain embodiments, the method for the production of the antibodies or antibody fragments of the present disclosure further comprises isolating and purifying the produced antibodies or antibody fragments from the host cell or the medium. If the expression system secretes the protein into the growth medium, the protein can be purified directly from the medium. If the protein is not secreted, the protein is isolated from the cell lysate or recovered from the cell membrane fraction. The selection of appropriate growth conditions and recovery methods is within the skill of those in the art.Next, the antibodies or antibody fragments of the present disclosure can be purified by several techniques known to those skilled in the art.

[0141] In one embodiment, the present disclosure refers to a method of producing a humanized antibody or antibody fragment specific for human CD117 of any of the antibodies disclosed in Table 18. In one embodiment, a method of making an isolated antibody or antibody fragment according to the present disclosure is provided, the method comprising culturing a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the antibody or antibody fragment according to the present disclosure under conditions suitable for the expression of the antibody or antibody fragment, and isolating the antibody or antibody fragment from the host cell or the host cell culture medium. The antibodies or antibody fragments isolated as described herein can be purification techniques known in the art such as high performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The conditions used to purify a particular antibody or antibody fragment depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor or antigen to which the antibody or antibody fragment binds can be used. For example, a matrix having Protein A or Protein G can be used for affinity chromatography purification of the antibody or antibody fragment according to the present disclosure. The purity of the antibody or antibody fragment can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high pressure liquid chromatography, etc.

[0142] Specificity In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117. In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117 disclosed in Table 18. In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for a polypeptide encoded by the amino acid sequence of SEQ ID NO: 1. In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0143] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117 and cynomolgus CD117.

[0144] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment cross-competes with antibody SR-1 for binding to CD117. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment cross-competes for binding to CD117 with an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0145] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to the same epitope on CD117 as antibody SR-1. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to the same epitope on CD117 as an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0146] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment retains the binding specificity of antibody SR-1. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment retains the binding specificity of an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0147] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to CD117 with substantially the same affinity as antibody SR-1. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to CD117 with substantially the same affinity as an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0148] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to CD117 with the same affinity as antibody SR-1. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to CD117 with the same affinity as an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0149] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to CD117 with at least the same affinity as antibody SR-1. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to CD117 with at least the same affinity as an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0150] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to CD117 with a higher affinity than antibody SR-1. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to CD117 with a higher affinity than an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0151] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to CD117 with a higher affinity than antibody SR-1. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to CD117 with a higher affinity than an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0152] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment binds to CD117 with an affinity at least 2-fold higher than that of an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, and the humanized antibody or antibody fragment comprises the following two mutations: a) asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid, histidine, glutamine or serine, and b) aspartic acid in the LCDR3 region (SEQ ID NO: 9) is substituted with glutamic acid. In certain embodiments, the antibody or antibody fragment binds to CD117 with an affinity 3-fold, at least 5-fold or at least 10-fold higher for CD117.

[0153] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment has a lower number of highly critical deamidation sequence motifs than antibody SR-1. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment has a lower number of highly critical deamidation sequence motifs than an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0154] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment has a smaller number of highly important sequence motifs for chemical cleavage than antibody SR-1. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment has a smaller number of highly important sequence motifs for chemical cleavage than an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0155] Biological function In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment is equivalent to antibody SR-1 in terms of biological function. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment is equivalent to an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9 in terms of biological function.

[0156] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment inhibits the biological function of CD117 to at least the same extent as antibody SR-1. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment inhibits the biological function of CD117 to at least the same extent as an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0157] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment that inhibits the binding of SCF to CD117. The binding of SCF to CD117 is important for the transmission of functional signals by CD117. The binding of CD117 to SCF can be measured by generally known systems such as the Octet system or as described in the following experimental section of this specification. In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment inhibits the binding of SCF to CD117 to at least the same extent as an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.

[0158] In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment inhibits the binding of SCF to CD117 more strongly than an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.

[0159] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment that inhibits SCF-dependent proliferation of CD117-positive cells. SCF-dependent proliferation can be assayed by any commonly used assay such as cell number, colony-forming unit (CFU) assay, cell titer glo (CTG; Promega), and carboxyfluorescein succinimidyl ester (CFSE) cell proliferation assay, or as described in the experimental section below in this specification, and can be tested by those skilled in the art. In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, said antibody or antibody fragment inhibiting SCF-dependent proliferation of CD117-positive cells to at least the same extent as an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0160] In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, said antibody or antibody fragment inhibiting SCF-dependent proliferation of CD117-positive cells with an IC50 lower than that of an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9. In certain embodiments, the IC50 is at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold lower than that of an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0161] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment that inhibits SCF-dependent phosphorylation of CD117. SCF-dependent phosphorylation is another function of CD117. SCF-dependent phosphorylation can be measured, for example, in cells expressing CD117, such as TF-1 cells, as described in the experimental section below herein. In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, said antibody or antibody fragment inhibiting SCF-dependent phosphorylation of CD117 to at least the same extent as an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.

[0162] In other embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, said antibody or antibody fragment inhibiting SCF-dependent phosphorylation of CD117 more strongly than an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.

[0163] Effector function The Fc region of an immunoglobulin generally confers desirable pharmacokinetic properties of the antibody, such as an extended half-life in serum, and the ability to induce effector functions through binding to Fc receptors expressed on cells. On the other hand, binding to Fc receptors can also lead to unwanted activation of certain cell surface receptors, which can lead to unwanted cytokine release and severe side effects upon systemic administration.

[0164] Thus, for a particular therapeutic situation, it is desirable to reduce or abrogate the normal binding of the wild-type Fc region of an antibody, such as a wild-type IgG Fc region, to one or more or all of the Fc receptors and / or its binding to complement components such as C1q in order to reduce or abrogate the ability of the antibody to induce effector functions. For example, it may be desirable to reduce or abrogate the binding of the Fc region of an antibody to one or more or all of the Fcγ receptors such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa. Effector functions can include, but are not limited to, one or more of the following: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, binding to NK cells, binding to macrophages, binding to monocytes, binding to polymorphonuclear cells, direct signaling to induce apoptosis, cross-linking of target-binding antibodies, dendritic cell maturation or T cell priming.

[0165] Reduction or abrogation of the binding of the Fc region to Fc receptors and / or C1q is typically achieved by mutating the wild-type Fc region, such as an IgG1 Fc region, more specifically a human IgG1 Fc region, resulting in a variant or modified Fc region of the wild-type Fc region, such as a mutated human IgG1 Fc region. Substitutions that result in a decrease in binding may be useful. Non-conservative amino acid substitutions, i.e., substituting one amino acid with another having different structural and / or chemical properties, are preferred for reducing or abrogating the binding properties of the Fc region to Fc receptors.

[0166] Thus, in one embodiment, an isolated antibody or antibody fragment specific for human CD117 according to the present disclosure comprises a mutant Fc region in which binding to Fc receptors and / or C1q is reduced or abrogated when compared to the wild-type Fc region. In such an embodiment, the isolated antibody or antibody fragment according to the present disclosure comprises a mutant Fc region that reduces or abrogates the ability of the antibody to induce effector functions. In a further embodiment, the isolated antibody or antibody fragment according to the present disclosure does not substantially induce effector functions.

[0167] In certain embodiments, the effector function is one or more selected from the group consisting of CDC, ADCC, and ADCP. In one embodiment, the effector function is ADCC. In one embodiment, the effector function is CDC. In one embodiment, the effector function is ADCP. In one embodiment, the isolated antibody or antibody fragment according to the present disclosure does not substantially induce ADCC and / or CDC and / or ADCP. In one embodiment, the isolated antibody or antibody fragment according to the present disclosure does not induce ADCC or ADCP in vitro.

[0168] In one embodiment, the mutated Fc region of the isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions that reduce or abrogate the binding of the mutated Fc region to one or more Fc receptors and / or C1q when compared to the wild-type Fc region. In one embodiment, the mutated Fc region of the isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions that reduce or abrogate the ability of the antibody to induce effector functions when compared to the wild-type Fc region. In certain embodiments, the one or more amino acid substitutions can reduce the binding affinity of the mutated Fc region to one or more Fc receptors and / or C1q by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold or even at least 50-fold when compared to the wild-type Fc region. In alternative embodiments, the one or more amino acid substitutions can reduce the ability of the isolated antibody or antibody fragment according to the present disclosure to induce effector functions by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold or even at least 50-fold when compared to the wild-type Fc region.

[0169] In one embodiment, the mutated Fc region of the isolated antibody or antibody fragment according to the present disclosure does not substantially bind to one or more Fc receptors and / or C1q. In one embodiment, the mutated Fc region of the antibody according to the present disclosure substantially abrogates the ability of the antibody to induce effector functions. In one embodiment, the antibody or antibody fragment according to the present disclosure does not substantially induce effector functions. In one embodiment, the effector function is ADCC and / or ADCP and / or CDC. In one embodiment, the antibody or antibody fragment according to the present disclosure does not substantially induce effector functions, which means that the level of induced effector function does not significantly exceed the background measured in the absence of the antibody.

[0170] In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an Fcy receptor. In one embodiment, the Fc receptor is human FcyRIIIa, FcyRI, FcyRIIa and / or FcyRIIb.

[0171] In one embodiment, the isolated antibody or antibody fragment according to the present disclosure comprises a mutated human IgG1 Fc region, which comprises one or more amino acid substitutions compared to the wild-type human IgG1 Fc region. In one embodiment, the one or more amino acid substitutions reduce or abrogate the binding of the mutated Fc region to Fc receptors and / or C1q and / or reduce the ability of the antibody to induce effector functions when compared to the wild-type Fc region.

[0172] Various Fc modifications are commonly used. For reviews, see, e.g., Antibodies(2020)9:64. Silencing functions include the LALA (L234A / L235A), PA-LALA (L234A / L235A / P329A) and PG-LALA (L234A / L235A / P329G) mutations (numbering according to the EU index), as well as the AEASS mutation (L234A / L235E / G237A / A330S / P331S).

[0173] In certain embodiments, the present disclosure relates to a humanized antibody specific for CD117, wherein the humanized antibody has a silenced Fc region. In certain embodiments, the present disclosure relates to a humanized antibody specific for CD117, wherein the humanized antibody or antibody fragment is in the AEASS format.

[0174] In certain embodiments, the present disclosure relates to a humanized antibody specific for CD117, wherein the humanized antibody comprises an Fc region with at least the following modifications: L234A, L235E, G237A, A330S, P331S (numbering according to the EU index). In certain embodiments, the present disclosure relates to a humanized antibody specific for CD117, wherein the humanized antibody comprises an Fc region with the following modifications: L234A, L235E, G237A, A330S, P331S (numbering according to the EU index).

[0175] In certain embodiments, the present disclosure relates to a humanized antibody specific for CD117, wherein the humanized antibody or antibody fragment is in the PA-LALA format. In certain embodiments, the present disclosure relates to a humanized antibody specific for CD117, wherein the humanized antibody comprises an Fc region having at least the following modifications: L234A, L235A, P329A (numbering follows the EU index). In certain embodiments, the present disclosure relates to a humanized antibody specific for CD117, wherein the humanized antibody comprises an Fc region having the following modifications: L234A, L235A, P329A (numbering follows the EU index).

[0176] In certain embodiments, the present disclosure relates to a humanized antibody specific for CD117, wherein the humanized antibody or antibody fragment is in the PG-LALA format. In certain embodiments, the present disclosure relates to a humanized antibody specific for CD117, wherein the humanized antibody comprises an Fc region having at least the following modifications: L234A, L235A, P329G (numbering follows the EU index). In certain embodiments, the present disclosure relates to a humanized antibody specific for CD117, wherein the humanized antibody comprises an Fc region having the following modifications: L234A, L235A, P329G (numbering follows the EU index).

[0177] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody is a) a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, b) a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, c) a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, d) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 71, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, e) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, f) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, g) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 121, and the LCDR3 region of SEQ ID NO: 115, h) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 123, and the LCDR3 region of SEQ ID NO: 115, i) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 125, and the LCDR3 region of SEQ ID NO: 115, j) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 127, and the LCDR3 region of SEQ ID NO: 115, k) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, l) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, m) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, or n) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9 and comprising, wherein the Fc region of the humanized antibody is silenced.

[0178] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody a) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, b) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, c) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, d) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 71, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, e) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, f) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, g) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 121, and the LCDR3 region of SEQ ID NO: 115, h) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 123, and the LCDR3 region of SEQ ID NO: 115, i) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 125, and the LCDR3 region of SEQ ID NO: 115, j) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 127, and the LCDR3 region of SEQ ID NO: 115, k) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, l) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, m) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, or n) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9 comprising wherein the Fc region of the humanized antibody comprises modified L234A, L235E, G237A, A330S, and P331S (numbering follows the EU index).

[0179] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody a) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, b) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, c) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, d) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 71, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, e) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, f) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, g) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 121, and the LCDR3 region of SEQ ID NO: 115, h) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 123, and the LCDR3 region of SEQ ID NO: 115, i) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 125, and the LCDR3 region of SEQ ID NO: 115, j) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 127, and the LCDR3 region of SEQ ID NO: 115, k) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, l) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, m) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, or n) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9 comprising wherein the Fc region of the humanized antibody comprises modified L234A, L235A, and P329A (numbering follows the EU index).

[0180] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody a) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, b) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, c) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, d) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 71, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, e) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, f) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 115, g) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 121, and the LCDR3 region of SEQ ID NO: 115, h) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 123, and the LCDR3 region of SEQ ID NO: 115, i) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 125, and the LCDR3 region of SEQ ID NO: 115, j) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 127, and the LCDR3 region of SEQ ID NO: 115, k) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 107, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, l) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, m) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, or n) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9 comprising, Here, the Fc region of the humanized antibody contains modified L234A, L235A, and P329G (numbering follows the EU index).

[0181] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98, b) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 99, c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100, d) a variable heavy chain of SEQ ID NO: 70 and a variable light chain of SEQ ID NO: 98, e) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 105, f) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 105, g) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 117, h) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 118, i) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 119, j) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 120, k) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 100, l) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 97, m) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 61, or n) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 62 and wherein the Fc region of the humanized antibody is silenced.

[0182] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98, b) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 99, c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100, d) Variable heavy chain of SEQ ID NO: 70 and variable light chain of SEQ ID NO: 98, e) Variable heavy chain of SEQ ID NO: 22 and variable light chain of SEQ ID NO: 105, f) Variable heavy chain of SEQ ID NO: 106 and variable light chain of SEQ ID NO: 105, g) Variable heavy chain of SEQ ID NO: 106 and variable light chain of SEQ ID NO: 117, h) Variable heavy chain of SEQ ID NO: 106 and variable light chain of SEQ ID NO: 118, i) Variable heavy chain of SEQ ID NO: 106 and variable light chain of SEQ ID NO: 119, j) Variable heavy chain of SEQ ID NO: 106 and variable light chain of SEQ ID NO: 120, k) Variable heavy chain of SEQ ID NO: 106 and variable light chain of SEQ ID NO: 100, l) Variable heavy chain of SEQ ID NO: 22 and variable light chain of SEQ ID NO: 97, m) Variable heavy chain of SEQ ID NO: 22 and variable light chain of SEQ ID NO: 61, or n) Variable heavy chain of SEQ ID NO: 22 and variable light chain of SEQ ID NO: 62 and comprising, wherein the Fc region of the humanized antibody comprises modified L234A, L235E, G237A, A330S and P331S (numbering follows the EU index).

[0183] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody a) Variable heavy chain of SEQ ID NO: 22 and variable light chain of SEQ ID NO: 98, b) Variable heavy chain of SEQ ID NO: 22 and variable light chain of SEQ ID NO: 99, c) Variable heavy chain of SEQ ID NO: 22 and variable light chain of SEQ ID NO: 100, d) Variable heavy chain of SEQ ID NO: 70 and variable light chain of SEQ ID NO: 98, e) Variable heavy chain of SEQ ID NO: 22 and variable light chain of SEQ ID NO: 105, f) Variable heavy chain of SEQ ID NO: 106 and variable light chain of SEQ ID NO: 105, g) Variable heavy chain of SEQ ID NO: 106 and variable light chain of SEQ ID NO: 117, h) Variable heavy chain of SEQ ID NO: 106 and variable light chain of SEQ ID NO: 118, i) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 119, j) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 120, k) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 100, l) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 97, m) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 61, or n) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 62 comprising, wherein the Fc region of the humanized antibody comprises modified L234A, L235A and P329A (numbering is according to the EU index).

[0184] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody a) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 98, b) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 99, c) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 100, d) The variable heavy chain of SEQ ID NO: 70 and the variable light chain of SEQ ID NO: 98, e) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 105, f) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 105, g) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 117, h) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 118, i) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 119, j) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 120, k) The variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 100, l) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 97, m) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 61, or n) The variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 62 comprising wherein the Fc region of the humanized antibody comprises modified L234A, L235A and P329G (numbering according to the EU index).

[0185] Therapeutic methods The antibodies and antibody fragments of the present disclosure, or pharmaceutical compositions incorporating the same, can be used for the treatment of various conditions. For example, such antibodies can be used in the ablation of endogenous hematopoietic stem and progenitor cells (HSPC) in a subject in need of ablation of endogenous hematopoietic stem and progenitor cells (HSPC). Ablation of endogenous HSPC is the first step in stem cell replacement therapy. Stem cell replacement therapy generally involves reducing or eliminating endogenous HSPC that are defective in some way and replacing them with replenishing HSPC. The replenishing HSPC can be autologous, allogeneic or xenogeneic. Endogenous HSPC can be defective as a result of a genetic mutation that impairs function or expression (e.g., sickle cell anemia or thalassemia), as a result of a blood cancer, or as a result of damage from chemotherapy used in the treatment of cancer. Endogenous HSPC can also be replaced in conjunction with organ transplantation because endogenous HSPC can cause an immune attack on the graft.

[0186] Antibodies against CD117 can also be used in the treatment of cancers that express CD117. Such cancers include blood cancers, such as AML, and solid tumors, such as mast cell cancer, testicular stromal cancer, gastrointestinal stromal cancer, melanoma, breast cancer and lung cancer. The expression of CD117 is preferably at a higher level than that of histocompatible normal control cells as determined by immunohistochemical assay.

[0187] The isolated antibodies or antibody fragments according to the present disclosure can also be used in other therapeutic methods such as the treatment of inflammatory diseases, autoimmune diseases, urticaria, nodular prurigo, eosinophilic esophagitis, mastocytosis, hematological malignancies and potentially other diseases.

[0188] In certain embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for medical use. In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of cancer. In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of AML. In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of solid cancer. In certain embodiments, the solid cancer is mast cell cancer, testicular stromal cancer, gastrointestinal stromal cancer, melanoma, breast cancer or lung cancer.

[0189] In certain embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of diseases associated with the unwanted presence of CD117. In certain embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of diseases associated with the unwanted presence of CD117-positive cells.

[0190] In one embodiment, the disease to be treated is a proliferative disease. In certain embodiments, the disease is cancer. Non-limiting examples of cancer include hematological malignancies such as chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), myelodysplastic syndromes, mastocytosis, etc., and non-hematological malignancies such as bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, sarcoma, skin cancer, squamous cell carcinoma, bone cancer, melanoma, renal cell carcinoma and kidney cancer, etc.

[0191] In one embodiment, the present disclosure provides a method for the treatment of a disease.

[0192] In one embodiment, the present disclosure provides a method for the treatment of a disease, comprising administering to a patient the antibody or antibody fragment of the present disclosure.

[0193] In one embodiment, the present disclosure provides a method for the treatment of a disease, comprising administering to a subject in need of treatment of the disease the antibody or antibody fragment of the present disclosure.

[0194] In one embodiment, the present disclosure provides a method for preventing a disease.

[0195] In one embodiment, the present disclosure provides a method for preventing a disease, comprising administering to a subject an antibody or antibody fragment of the present disclosure.

[0196] In one embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for treating a disease. In one embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use in treating a disease. In one embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use in treating a disease in a subject in need of treatment for the disease.

[0197] In one embodiment, the present disclosure provides the use of an isolated antibody or antibody fragment according to the present disclosure for the manufacture of a medicament. In one embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use as a medicament. In one embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use in medicine. In one embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use as a medicament for treating a subject in need of treatment.

[0198] In one embodiment, the present disclosure provides an isolated antibody or antibody fragment specific for human CD117 according to the present disclosure for use in a method of treating a subject having a disease, comprising administering to the subject a therapeutically effective amount of an antibody or antibody fragment according to the present disclosure.

[0199] In one embodiment, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent. The subject in need of treatment is typically a mammal, more specifically a human. For use in a method of treatment, the isolated antibody or antibody fragment according to the present disclosure is formulated, administered, and dosed in a manner consistent with good medical practice.

[0200] Pharmaceutical composition In one embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or antibody fragment according to the present disclosure and a pharmaceutically acceptable carrier or excipient.

[0201] The antibody is administered in an effective regimen that achieves the intended purpose, such as a reduction in endogenous HSPC or cancer cells expressing CD117, in terms of dosage, route of administration, and frequency of administration. In some examples, efficacy can be observed in an individual patient compared to historical controls or past experience in the same patient. In other examples, efficacy can be demonstrated in preclinical studies or in clinical trials in a population of treated patients compared to a control population of untreated patients.

[0202] The pharmaceutical composition may further comprise at least one other pharmaceutically active compound. The pharmaceutical composition according to the present disclosure can be used for the diagnosis, prevention, and / or treatment of diseases associated with the undesirable presence of CD117, particularly human CD117. The pharmaceutical composition according to the present disclosure can be used for the diagnosis, prevention, and / or treatment of diseases associated with the undesirable presence of CD117-positive cells, particularly CD117-positive human cells. In particular, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure suitable for prophylactic, therapeutic, and / or diagnostic use in mammals, more specifically humans.

[0203] Generally, the antibody or antibody fragment according to the present disclosure can be formulated as a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure, at least one pharmaceutically acceptable carrier or excipient, and optionally one or more additional pharmaceutically active compounds. Such formulations can be suitable for oral, parenteral, topical administration, or administration by inhalation. Thus, a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure can be administered parenterally, for example, intravenously, intramuscularly, or subcutaneously. Alternatively, the antibodies of the present invention can be administered by a non-parenteral route, such as orally or topically. In a preferred embodiment, the pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure is administered intravenously or subcutaneously.

[0204] In particular, the antibodies or antibody fragments according to the present disclosure can be used in combination with one or more pharmaceutically active compounds that are used or can be used for the prevention and / or treatment of diseases involving a target antigen of interest, and as a result, a synergistic effect may or may not be obtained. Examples of such compounds, as well as the routes, methods, and pharmaceutical formulations or compositions for administering them, will be apparent to the clinician.

[0205] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and / or treatment of diseases associated with the unwanted presence of CD117, particularly human CD117. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and / or treatment of diseases associated with the unwanted presence of CD117-positive cells, particularly CD117-positive human cells. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use as a medicament. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and / or treatment of autoimmune and / or inflammatory diseases and / or cancer.

[0206] In one embodiment, the present disclosure provides a method for treating an autoimmune and / or inflammatory disease and / or cancer in a subject in need of treatment of an autoimmune and / or inflammatory disease and / or cancer using a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure.

[0207] There is further provided a method for manufacturing an antibody or antibody fragment according to the present disclosure in a form suitable for in vivo administration, the method comprising: (a) obtaining an antibody or antibody fragment by the method according to the present disclosure; and (b) formulating said antibody or antibody fragment together with at least one pharmaceutically acceptable carrier or excipient, whereby a preparation of the antibody or antibody fragment is formulated for in vivo administration. The pharmaceutical composition according to the present disclosure comprises a therapeutically effective amount of one or more antibodies or antibody fragments according to the present disclosure dissolved in a pharmaceutically acceptable carrier or excipient.

[0208] Diagnostic use In one embodiment, the present disclosure provides the use of an isolated antibody or antibody fragment specific for human CD117 according to the present disclosure for the diagnosis of a disease. In one embodiment, the present disclosure provides the use of an antibody or antibody fragment according to the present disclosure for the detection of CD117, particularly human CD117. In one embodiment, the present disclosure provides a method for detecting CD117 in a subject or sample, the method comprising contacting said subject or sample with an isolated antibody or antibody fragment specific for human CD117 of the present disclosure. In one embodiment, the present disclosure provides a method for diagnosing a disease in a subject, the method comprising contacting said subject or sample with an isolated antibody or antibody fragment according to the present disclosure. The antibody can also be used to determine the CD117 expression level in patient-derived cells. The CD117 expression level can, for example, serve as a therapeutic biomarker for patient stratification.

Examples

[0209] Example 1: Anti-CD117 antibody of the present invention Antibody SR-1 is a mouse antibody first described in Broudy et al, Blood (1992) 79: 338-46. The sequence of SR-1 is disclosed, for example, in US Patent Application Publication No. 20200165337A1 (see SEQ ID NOs: 1 and 5 therein).

[0210] The sequences of the variable chains and CDRs are shown in the following table.

Table 1

[0211] The humanization of SR-1 is described in International Publication No. WO2007 / 127317 and International Publication No. WO2020 / 112687. International Publication No. WO2007 / 127317 utilizes a classical humanization approach by CDR grafting to produce an antibody called AMG191. No further modification was made to AMG191. Also, International Publication No. WO2020 / 112687 utilizes a relatively classical approach, with specific modifications introduced into the framework region. Therefore, such antibodies still have drawbacks such as being prone to fragmentation of the humanized antibody and the occurrence of unnecessary post-translational modification site(s). Such sites are present within the CDRs of SR-1, and since modifications within the CDRs are expected to affect antibody properties such as affinity, neither of the aforementioned humanization approaches was aimed at removing these sites. Through a sophisticated approach, the inventors of the present application succeeded in humanizing SR-1 while simultaneously removing unnecessary post-translational modifications and fragmentation sites. Surprisingly, the generated antibody also did not compromise the affinity and activity of the antibody. This was achieved by a stepwise approach, as will be further detailed in the following examples.

[0212] Example 2: Humanization of Mouse Antibody SR-1 The humanization process relied on a combination of the latest research on antibody structure and CDR grafting technology combined with the latest database of mature human IgG sequences. Several human framework sequences were identified to be used as the "acceptor" framework for the target CDR sequences. All acceptor sequences are derived from mature human IgG. As a result, the humanized sequences are expected to be non-immunogenic and retain the canonical structure of the CDR loops.

[0213] Example 2.1: Design of Humanized Variants of the Variable Heavy Chain The most closely related human germline gene V region identified in Homo sapiens was IGHV1-3 * 01: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 16)

[0214] A database of human IgG sequences was searched using the BLAST search algorithm for comparison with the mouse VH domain, and candidate human variable domains were selected from the top 200 BLAST results. These were narrowed down to three candidates based on combinations of framework homology, maintaining important framework residues and canonical loop structures.

[0215] The three acceptor frameworks are as follows: >QEP24303: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYGISWVRQAPGQGLEWMGWISTYNGDTNYAQKLRGRVTMTTDTSTSTAYMELRSLTSDDTAVYYCARDRLSTGTTFYYWGQGTLVTVSS (SEQ ID NO: 17) >ADWO8092: QVQLVESGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQAPGQRLEWMGEINPDNGHTNYNEKFKSRVTITVDKSASTAYMELSSLRSEDTAVYYCAREADYSYGAFDIWGPGTTVTVSS (SEQ ID NO: 18) >AKU38660: QVQLVQSGAEVRKPGASVKLSCKASGYTFTRYSMHWVRQAPGQGLEWMGIlNPSGGSTSYAQNFQGRVTLTRDTSTSTVYMELSSLRSEDTAVYYCTRDRLRNWFDPWGQGTLVTVSS (SEQ ID NO: 19)

[0216] For the purpose of obtaining more stable products, two additional frameworks were designed based on homology and sequence to allow for larger arrays: >IGHV3-23 * O3: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 20) >IGHV5-51 * 01: EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCAR (SEQ ID NO: 21)

[0217] Using the CDRs of the mouse VH of SR-1 transplanted into the aforementioned acceptor framework, the following humanized variants were selected and synthesized: >VH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTMTTDTSTSTAYMELRSLTSDDTAVYYCARERDTRFGNWGQGTLVTVSA (SEQ ID NO: 22) >VH2 QVQLVESGAEVKKPGASVKLSCKASGYTFTSYNMHWVRQAPGQRLEWMGVIYSGNGDTSYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVTVSA (SEQ ID NO: 23) >VH3 QVQLVQSGAEVRKPGASVKLSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTLTRDTSTSTVYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVTVSA (SEQ ID NO: 24) >VH4 EVQLLESGGGLVKPGASLRLSCAASGYTFTSYNIHWVRQAPGQGLEWVSAIYSGNVDTSYSQKFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERDTRFGNWGQGTLVTVSA (SEQ ID NO: 25) >VH5 EVQLVQSGAEVKKPGESLKISCKGSGYTFTSYNMHWVRQMPGKGLEWMGVIYSGNADTSYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARERDTRFGNWGQGTLVTVSA (SEQ ID NO: 26)

[0218] The SEQ ID NOs of the variable heavy chains containing those CDRs are shown in the following table.

Table 2

[0219] The alignment of the humanized mutants with the original mouse SR-1 sequence is shown in Figure 1. The homology of the humanized mutants with the original mouse VH sequence is shown in the following table.

Table 3

[0220] Example 2.2: Design of humanized mutants of variable light chains The most closely related human germline gene V region identified in Homo sapiens was IGKV3D-11 * 02: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWH (SEQ ID NO: 32)

[0221] A database of human IgK sequences was searched using the BLAST search algorithm for comparison with the mouse VL domain, and candidate human variable domains were selected from the top 200 BLAST results. These were narrowed down to two candidates based on combinations of framework homologies, maintaining important framework residues and canonical loop structures.

[0222] The two acceptor frameworks are as follows: >ADU57829: EIVLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIKS (SEQ ID NO: 33) >CAB46458: DIVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPYTFGQGTKLEIK (SEQ ID NO: 34)

[0223] The latter framework (CAB46458) was used in two approaches - one with a framework as shown above (VL2) and one with additional mutations introduced (VL3). Also, three additional variants were selected based on homology: >IGKV3-15 * 01 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP (SEQ ID NO: 35) >IGKVl-39 * 01 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP (SEQ ID NO: 36) >IGKV3D-11 * 01 EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWH (SEQ ID NO: 37)

[0224] Using the CDR of the mouse VL of SR-1 transplanted into the aforementioned acceptor framework, the following humanized variants were selected and synthesized: >VL1 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIK (SEQ ID NO: 38) >VL2 DIVMTQSPSTLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQNNEDPYTFGQGTKLEIK (SEQ ID NO: 39) >VL3 DIVMTQSPSTLSASVGDRVTITCRASESVDIYGSSFMHWYQQKPGQPPKLLIYLASNLESGVPSRFSGSGSGTEFTLTISSLEADDVATYYCQQNNEDPYTFGGGTKLEIK (SEQ ID NO: 40) >VL4 EIVMTQSPATLSVSPGERATLSCRASESVDIYGQSFMHWYQQKPGQAPRLLIYLASNLESGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQNDEEPYTFGGGTKLEIK (SEQ ID NO: 41) >VL5 DIQMTQSPSSLSASVGDRVTITCRASESVDIYGNAFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANEEPYTFGGGTKLEIK (SEQ ID NO: 42) >VL6 EIVLTQSPATLSLSPGERATLSCRASESVDIYGQSFMHWYQQKPGQAPRLLIYLASNLESGlPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQNNENPYTFGGGTKLEIK (SEQ ID NO: 43)

[0225] The SEQ ID NOs of the variable light chains containing those CDRs are shown in the following table.

Table 4

[0226] The alignment of the humanized mutants with respect to the original mouse SR-1 sequence is shown in Figure 2. The homology of the humanized mutants with respect to the original mouse VL sequence is shown in the following table.

Table 5

[0227] Example 2.3: Expression and Purification of Humanized Mutants DNA encoding the amino acid sequences of the respective antibodies was synthesized and cloned into the mammalian transient expression plasmid pETE V2 (Fusion Antibodies, Northern Ireland). Antibodies were expressed using a CHO-based transient expression system, and the resulting antibody-containing cell culture supernatant was clarified by centrifugation and filtration. Antibodies were purified from the cell culture supernatant using state-of-the-art chromatography equipment via affinity chromatography. The purified antibodies were buffer-exchanged with phosphate-buffered saline. When determined by reducing and denaturing sodium dodecyl sulfate polyacrylamide gel, the purity of these antibodies was determined to be >95%. Antibody concentration was determined by measuring the absorbance at 280 nm.

[0228] Five humanized variable heavy chains were tested in combination with five humanized variable light chains, yielding a total of 25 combinations. Of these 25 combinations, 9 could not be produced. These 9 combinations included all combinations containing either the humanized heavy chain VH4 or the humanized light chain VL6. The remaining 16 combinations could be produced in sufficient amounts for further characterization.

[0229] Example 2.4: Affinity Determination of Humanized Variants An IgG antibody was immobilized on a biosensor using an appropriate capture surface, and the binding of soluble antigen to the immobilized antibody was monitored by BLI (Octet). The resulting sensorgrams were analyzed using the provided software (Fortebio). As the antigen, human recombinant CD117 (c-Kit) from Sino Biological was used (Catalog No. 11996-H08H, Lot No. LC14SE0812). As a preliminary experiment, a series of experiments were conducted to optimize the assay parameters. The following parameters were selected.

Table 6

[0230] In the main experiment, a kinetic assay was performed by first capturing IgG using an anti-human Fc biosensor. Subsequently, the mAb capture biosensor was immersed in wells containing different concentrations of antigen (association phase), followed by a dissociation phase in running buffer. To enable double-reference correction, the IgG capture sensor was immersed in wells containing only buffer, and the blank sensor was also immersed in wells containing antigen. By referring to this, a means was provided to compensate for both the natural dissociation of captured IgG and the non-specific binding of antigen to the sensor surface. The steps were carried out at 25 °C with a constant oscillation of 1000 rpm. A new sensor was used for each sample. The dissociation rate constant (Kd) was calculated using ForteBio Data Analysis software. All consumables used were those recommended by ForteBio.

[0231] All samples were diluted with freshly prepared running buffer. Using the described capture method, antibody variants were immobilized on the surface of a series of biosensors. Antigen was passed over the surface to generate a binding response. Binding data for the IgG:antigen interaction were collected on the biosensor at 25 °C. To globally fit the results and obtain the best values for ka, kd, and KD, a dilution series of antigen was used in the association phase. Response data for the binding of antigen to IgG immobilized on the surface were fitted to a 1:1 binding model. The kinetic parameters are summarized in the following table. HC0 LC0 refers to mouse SR-1.

Table 7

[0232] Five combinations of antibodies, namely VH1 / VL1, VH1 / VL2, VH1 / VL3, VH3 / VL1, and VH5 / VL1, showed dissociation constants KD within a 2-fold range compared to the original antibody SR-1 (HC0 LC0). Three combinations of antibodies, namely VH1 / VL1, VH1 / VL2, and VH1 / VL3, actually showed improved dissociation constants KD compared to the original antibody SR-1.

[0233] Example 2.5: Evaluation of the Biophysical Characteristics of Five Preselected Antibodies Protein aggregation along the non-native pathway has been found to be caused by the instability of the protein's higher-order structure or the partial unfolding of the protein. Due to the unfolding of the protein, hydrophobic core residues are exposed and then combine with other partially unfolded monomers to form dimers, which ultimately act as nucleation points for large-scale aggregation. Therefore, the higher-order structural stability indicates the tendency of the protein towards non-native aggregation. Thermal stability is accepted as a feasible method for monitoring the higher-order structural stability of proteins through the determination of its melting point (Tm).

[0234] Differential scanning fluorimetry (DSF) enables the monitoring of higher-order structural stability. From multiple studies, highly correlated data have been found between differential scanning calorimetry (DSC) and DSF, suggesting that DSF can successfully monitor the thermal stability of proteins and ultimately the tendency towards non-native aggregation. DSF uses a fluorescent dye that is quenched in an aqueous environment but fluoresces under hydrophobic conditions. Therefore, under heat exposure, the antibody loses its higher-order structural stability, begins to unfold, and hydrophobic core residues are exposed, which can be measured as an increase in the fluorescent signal. Subsequently, the melting temperature (dF / dT curve) of the protein of interest can be determined using a temperature-versus-fluorescence (melting profile) plot.

[0235] Many antibody molecules exhibit a biphasic melting profile. From published studies, it has been shown that mAbs exhibit characteristic unfolding profiles as a result of the melting transitions of their individual domains: CH2, CH3, and Fab. The thermal stability of IgG molecules is affected by all of these domains. The DSF curves reported for antibodies show two clearly distinguishable transitions: the lower temperature corresponds to the unfolding of the CH2 domain (Tm1), and the higher temperature corresponds to the melting of the CH3-Fab domains (Tm2). The Fab melting transition is generally clearly defined by a peak that is up to two to three times larger than the peaks of CH2 or CH3.

[0236] For DSF analysis, 5 μl of Sypro Orange (diluted 1 / 200 in water; Sigma) and 45 μl of a 0.3 mg / ml solution of the antibody to be tested were added to a tube (Bio-Rad; TLS0831). The tube was sealed with an optical flat cap (Bio-Rad; TCS0803) and heated in an i-Cycler iQ5 real-time PCR detection system (Bio-Rad) from 20 °C to 95 °C in 0.5 °C increments. Fluorescence changes in the wells of the plate were simultaneously monitored with a charge-coupled (CCD) camera. The excitation and emission wavelengths were 485 nm and 575 nm, respectively. The temperature midpoint Tm for the protein unfolding transition was calculated using Bio-Rad iQ5 software.

[0237] For size exclusion chromatography (SEC), samples were diluted to a final concentration of 0.1 mg / ml using phosphate-buffered saline (PBS). Highly purified antibody samples were independently loaded onto a Superdex 200 increase 10 / 300 GL gel filtration column. 50 μl of the sample was injected and the column flow rate was maintained at 0.75 ml / min. The separation and equilibration steps were performed at 19 °C in phosphate-buffered saline. Protein peaks were monitored using absorbance at 214 nm, and spectra were analyzed using the Unicorn evaluation software package (Cytiva).

[0238] Melting curves were successfully generated for all samples. All of the antibodies tested showed a single-phase transition melting profile. The Tm values are summarized in the table below.

[0239] In addition, SEC analysis was successfully performed for all samples. The peak quality for SEC analysis was good for all samples. All antibodies eluted as a single major peak. From column calibration, the major peak corresponds to monomeric IgG.

Table 8

[0240] Example 2.6: Summary and selection of final candidates for further modification All combinations except those containing VH4 and / or VL6 were successfully expressed and purified. From SDS-PAGE analysis, all antibodies showed sufficient levels of purity. Under reducing conditions, both the heavy and light chains of the antibody are visible and are observed at predicted molecular weights of ~50 and 25 kDa, respectively. Under non-reducing conditions, a single major band and several minor bands are observed. Additional bands (impurities) are probably the result of non-glycosylated IgG and IgG degradation products (e.g., single [partial] light chain, combination of two heavy chains and one light chain, two heavy chains, two heavy chains and one light chain).

[0241] In kinetic (Octet) analysis, the combinations of VH1 / LC1, VH1 / LC2, VH1 / LC3, VH3 / LC1 and VH5 / LC1 showed binding characteristics similar to those of the control antibody HCO LCO and dissociation constants within two-fold of the control antibody.

[0242] The biophysical properties of all antibodies were as good as those of the original SR-1 antibody.

[0243] For further modification, antibody VH1 / LC1, which showed an affinity of 0.79 nM, almost twice as high as that of the original SR-1 antibody (1.47 nM), was selected.

[0244] Example 3: Removal of individual sites prone to PTM or fragmentation Example 3.1: Design of modified humanized variants In the next modification step, antibodies were screened for sites that tend to be Fv glycosylation, deamidation, isomerization, fragmentation or other types of post-translational modification (PTM) sequence hotspots. Some such sites were identified in the CDRs of the antibody. See the following table. [Table 9]

[0245] To remove these harmful amino acid motifs, variants of antibody VH1 / VL1 were generated. The approach to individual changes was rationalized by the analysis as described below.

[0246] In silico calculations of descriptors based on sequence and structure were performed to identify suitable variants that enhance developability, binding affinity, and solubility while preserving the structural integrity and biological activity of the antibody. For this purpose, physicochemical descriptors were evaluated against a dataset of therapeutic antibodies retrieved from the TABS database (https: / / tabs.craic.com / ) and a second dataset consisting of a paired antibody repertoire from the Observed Antibody Space database (http: / / opig.stats.ox.ac.uk / webapps / oas / ). Sequence alignment, antibody numbering, and CDR canonical structures were annotated during the alignment of variable domain sequences to an isotype-specific hidden Markov model (HMM) constructed as described in Nat Protoc (2014) 9:2771-83. A template-based approach for the framework, canonical structure modeling for the CDRs, and a random forest machine learning modeling approach for the heavy chain loop H3 were used to construct a three-dimensional model based on the methods described in Nucleic Acid Res (2017) 45:W17-W23 and Bioinformatics (2014) 30:2733-40. For VH / VL packing, a template-based approach based on the overall similarity and identity at residue position L44 was used as described in FEBS J. (2011) 278:2858-66. Among the sequence descriptors, the antibody solubility profile was calculated as a linear combination of different physicochemical properties such as the hydrophobicity of each amino acid, the electrostatic charge at neutral pH, and the alpha helix and beta strand tendencies (Nat Struct Biol (1996) 3:842-8, J Mol Biol (1994) 238:693-708). To account for the influence of neighboring amino acids, the inventors used a sliding window approach, in which the solubility descriptor for each residue was averaged over a window of seven consecutive amino acids and corrected for the occurrence of polar / nonpolar aggregation tendency patterns (J Mol Biol (2000) 296:961-8; Methods Mol Biol (2022) 2313:57-113).During the modification stage, the inventors did not impose specific target values on local and global solubility, but the inventors excluded variants that led to the formation of covalent aggregates (cysteine) and solvent-exposed hydrophobic patches when evaluated by the relative solvent exposure per residue calculated in the 3D model of the paired VH / VL domains (F1000Res (2016) 5:189). Furthermore, variants that affected the higher-order structure of the CDR and thus the antibody binding mode were also excluded. This was done by evaluating the contribution of the modification sites to any of the established canonical structures (Nat Protocol (2014) 9:2771-83) and by using residue-specific probability scores (Bioinformatics (2013) 29:2285-91) to evaluate the predicted binding modes of native and modified molecules. Additionally, the inventors excluded / prioritized variants that led to an increase / decrease in immunogenic risk when evaluated by comparing the in-silico calculated immunogenicity profile with that of the natural human repertoire and a large dataset of known immunogenic therapeutic antibodies (ADA frequency). For this purpose, the inventors used a re-adaptation of the NetMHCIIpan neural network algorithm (Nucl Acids Res (2020) 48:W449-W454) to screen native and mutant antibody sequences for the presence of class II-restricted HLA ligands and putative T cell epitopes. The inventors calculated the overall and local immunogenicity profiles corrected by the presence of shared HLA ligands for the human germline that were thought to be tolerogenic or immunotolerogenic. Variants that did not carry neoepitopes (new HLA conjugates vs native) or cross-reactive epitopes (ligands recognized by multiple HLA alleles) were given a higher priority, and variants that showed a high epitope content in regions of the antibody that showed a low immunogenicity score in the reference antibody dataset were given a lower priority.

[0247] The following table summarizes the new variants.

Table 10

[0248] The sequences of the new VH chain and VL chain are shown below. >VL1.2 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGQSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV (SEQ ID NO: 56) >VL1.3 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGRSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV (SEQ ID NO: 57) >VL1.4 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGSSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV (SEQ ID NO: 58) >VL1.5 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGKSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV (SEQ ID NO: 59) >VL1.6 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGFSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV (SEQ ID NO: 60) >VL1.7 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV (SEQ ID NO: 61) >VL1.8 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGYSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV (SEQ ID NO: 62) >VL1.9 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNEDPYTFGQGTKLEIKRTV (SEQ ID NO: 63) >VL1.10 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRNEDPYTFGQGTKLEIKRTV (SEQ ID NO: 64) >VL1.11 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQENEDPYTFGQGTKLEIKRTV (SEQ ID NO: 65) >VL1.12 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEDPYTFGQGTKLEIKRTV (SEQ ID NO: 66) >VL1.13 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEDPYTFGQGTKLEIKRTV (SEQ ID NO: 67) >VL1.14 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEPPYTFGQGTKLEIKRTV (SEQ ID NO: 68) >VL1.15 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEEPYTFGQGTKLEIKRTV (SEQ ID NO: 69) >VH1.2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGSGDTSYNQKFKGRVTMTTDTSTSTAYMELRSLTSDDTAVYYCARERDTRFGNWGQGTLVTVSA (SEQ ID NO: 70)

[0249] The SEQ ID NOs of the variable light chains containing those CDRs are shown in the following table.

Table 11

[0250] Example 3.2: Testing of the Modified Humanized Mutants The modified mutants were first subjected to the same type of analysis and characterization as the humanized mutants. For the experimental setup, refer to Example 2.

[0251] All mutants could be expressed and purified, but the yields were not high enough for all mutants for all assays, especially differential scanning fluorimetry (DSF). Four antibodies showed an increase in the dissociation constant KD of more than twofold when compared to the original SR-1 antibody (VH1 / VL1.3, VH1 / VL1.5, VH1 / LC1.10, and VH1 / VL1.11). Five of the antibodies, namely VH1 / LC1.2, VH1 / LC1.4, VH1 / LC1.7, VH1 / LC1.8, and VH1 / LC1.15, showed a dissociation constant KD that was even lower than that of the original antibody SR-1. Three mutants had a monomer content of less than 85% (HC1 / LC1.2, HC1 / LC1.10, and HC1 / LC1.11). In DSF, some antibodies showed a slightly lower TM1. For some antibodies, TM2 could also be measured. The results are summarized in the following table.

Table 12

[0252] Example 3.3: Summary and Selection of Candidates for Further Modification In summary, it was possible to remove undesirable motifs from the sequences of the humanized variants of SR-1. This was particularly surprising since the sequence motifs were located within the CDR regions. Nevertheless, the new variants were nearly identical and retained the ability to bind to the target antigen, and some variants even had increased affinity, with antibodies VH1 / VL1.7 and VH1 / VL1.8 showing the highest affinity. At the same time, the biophysical properties of the binders remained within an acceptable range.

[0253] For further modification, five antibodies were selected (VH1 / VL1.7, VH1 / VL1.8, VH1 / VL1.13, VH1 / VL1.15, and VH1.2 / VL1), which as a whole showed the most promising properties for further derivatization.

[0254] Example 4: Removal of Multiple Sites Prone to PTM or Fragmentation Example 4.1: Design of Further Modified Humanized Variants Motivated by the successful removal of harmful sequence motifs from the CDRs of the humanized variants of SR-1, the inventors then investigated whether it was also possible to remove multiple such motifs without losing the affinity or functionality of the binder.

[0255] In addition to the sites modified in Example 3, the YL motif in the variable light chain was also modified. The following variants were generated.

[0256] Table 13 ("---" indicates no change in the site): [Table 13]

[0257] Variable light chains VL1.28 and VL1.29 each contain additional mutations from L to K and from N to R, respectively.

[0258] The sequences of the new VH chain and VL chain are shown below. >VL1.16 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGYSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEEPYTFGQGTKLEIKRTV (SEQ ID NO: 97) >VL1.17 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEEPYTFGQGTKLEIKRTV (SEQ ID NO: 98) >VL1.18 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGYSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV (SEQ ID NO: 99) >VL1.19 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV (SEQ ID NO: 100) >VL1.25 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGQSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV (SEQ ID NO: 101) >VL1.26 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGSSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV (SEQ ID NO: 102) >VL1.27 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGHSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV (SEQ ID NO: 103) >VL1.28 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGSSFMHWYQQKPGKAPKLLIYKASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV (SEQ ID NO: 104) >VL1.29 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNREEPYTFGQGTKLEIKRTV (SEQ ID NO: 105) >VH1.4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGQGDTSYNQKFKGRVTMTTDTSTSTAYMELRSLTSDDTAVYYCARERDTRFGNWGQGTLVTVSA (SEQ ID NO: 106)

[0259] The sequence numbers of the variable light chains containing those CDRs are shown in the following table.

Table 14

[0260] Example 4.2: Testing of Further Modified Humanized Variants The modified variants were first subjected to the same type of analysis and characterization as the humanized variants. For the experimental setup, refer to Example 2.

[0261] All variants could be expressed and purified in sufficient yields. The two antibodies showed an increase in the dissociation constant K D by more than two-fold (VH1 / VL1.27 and VH1 / VL1.28) when compared to the original SR-1 antibody. Five of the new antibodies, namely VH1 / VL1.16, VH1 / VL1.17, VH1 / VL1.19, VH1.2 / VL1.16 and VH1.2 / VL1.17, showed a dissociation constant K D that was at least two-fold lower than the dissociation constant KD of the original antibody SR-1. The monomer content of all antibodies exceeded 92%. By SEC, TM2 could be measured for all antibodies. The results are summarized in the following table.

Table 15

[0262] Example 4.3: Summary and Selection of Candidates for Further Modification In summary, it was further possible to remove some undesirable motifs from the sequences of the humanized variants of SR-1. At the same time, the affinity of many variants was even higher than that of the original SR-1 antibody, and the biophysical properties of the binders remained within the range of SR.1.

[0263] Several antibodies are considered particularly preferred (VH1 / VL1.17, VH1 / VL1.19, VH1 / VL1.27 and VH1.4 / VL1.29) and exhibit the most promising properties. In each of these antibodies, at least two harmful sequence motifs were removed: the deamidation site of LCDR1 (NS to ES in VH1 / VL1.17, VH1 / VL1.19 and VH1.4 / VL1.29 and NS to HS in VH1 / VL1.27), and the isomerization / fragmentation site of LCDR3 (DP to EP in all binders mentioned). Furthermore, the deamidation site of LCDR3 was changed from NN to NK in VH1 / VL1.19 and VH1 / VL1.27 and from NN to NR in VH1.4 / VL1.29. Also, in the binder VH1.4 / VL1.29, the deamidation site of HCDR2 was removed (NG to QG).

[0264] Also particularly preferred are antibodies having an affinity equal to or higher than that of antibody SR-1, namely VH1 / VL1.17, VH1 / VH1.18, VH1 / VL1.19, VH1.2 / VL1.17 and VH1 / VL1.16.

[0265] Example 5: Further Sequence Variations Example 5.1: Design of Further Modified Humanized Variants Based on the results and experience obtained so far, further sequence variations were designed. These mutations are aimed at further combining additional mutations and also enhancing hydrophilicity.

[0266] The following variants were prepared. Table 16 ("---" indicates that there was no change at that site): [Table 16]

[0267] Furthermore, the binders VH1.4 / VL1.30, VH1.4 / VL1.31, VH1.4 / VL1.32 and VH1.4 / VL1.33 each contain an L-to-H, L-to-H, L-to-H and L-to-H mutation, respectively, to improve hydrophilicity.

[0268] The sequences of the new VH and VL chains are shown below. >VL1.30 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYHASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNREEPYTFGQGTKLEIK (SEQ ID NO: 117) >VL1.31 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYEASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNREEPYTFGQGTKLEIK (SEQ ID NO: 118) >VL1.32 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYSASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNREEPYTFGQGTKLEIK (SEQ ID NO: 119) >VL1.33 EIVLTQSPSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYQASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNREEPYTFGQGTKLEIK (SEQ ID NO: 120)

[0269] The SEQ ID numbers of the variable light chains containing those CDRs are shown in the following table.

Table 17

[0270] Example 5.2: Testing of Further Modified Humanized Variants The modified variants were first subjected to the same type of analysis and characterization as the humanized variants. See Example 2 for the experimental setup.

[0271] All variants can be expressed and purified in sufficient yields. The antibodies exhibit an affinity as measured by the dissociation constant K within the range of the original SR-1 antibody. D Many antibodies even have an improved affinity. The biophysical properties of the antibodies are good.

[0272] Example 5.3: Summary and Selection of Candidates for Further Modification In summary, it was further possible to remove some undesirable motifs from the sequences of the humanized variants of SR-1. At the same time, the affinity of many variants was even higher than that of the original SR-1 antibody. At the same time, the biophysical properties of the binders remained within the range of the case of SR-1.

[0273] Certain specific antibodies are considered particularly preferred (VH1 / VL1.17, VH1 / VL1.19, VH1 / VL1.27 and VH1 / LC1.29) and exhibit the most promising properties. In each of these antibodies, harmful sequence motifs were removed: the deamidation site of LCDR1 (NS to ES in VH1 / VL1.17, VH1 / VL1.19 and VH1.4 / VL1.29, and NS to HS in VH1 / VL1.27), the deamidation site of LCDR3 (NN to NK in VH1 / VL1.19 and VH1 / VL1.27, and NN to NR in VH1.4 / VL1.29), and the isomerization / fragmentation site of LCDR3 (DP to EP in all of the above-mentioned binders). Further, in the binder VH1.4 / VL1.29, the deamidation site of HCDR2 was removed (NG to QG).

[0274] Example 6: Summary of the sequences of the antibodies disclosed in this patent The following table summarizes the important antibodies made and tested in this disclosure. The VH and VL sequences of the antibodies are shown. Each VH sequence, VL sequence and CDR sequence can be found in the previous examples. [Table 18] JPEG2025521496000019.jpg152148

[0275] Example 7: Antibody-mediated inhibition of SCF-dependent proliferation SCF-dependent proliferation is a function of CD117. The humanized antibodies of this disclosure were tested for their ability to inhibit SCF-dependent proliferation compared to the original SR-1 antibody and several other prior art antibodies. In addition to the above SR-1 antibody, the antibodies in Table 19 were tested and compared with the antibodies of this disclosure. [Table 19] JPEG2025521496000021.jpg242170

[0276] NegCtrl is a human IgG1 control antibody specific for the chicken lysosome team. AMG191 and FSI-174 are human IgG1 antibodies specific for human CD117. AMG191 is a humanized derivative of SR-1, but the 6 CDRs are identical to SR-1, that is, AMG191 still contains important sites of the CDRs removed in the humanized antibodies of the present disclosure. FSI-174 is also a humanized derivative of SR-1. Compared with SR-1, FSI-174 has two amino acid differences in HCDR2 and one amino acid difference in LCDR1. By introducing these changes to bring them closer to the human germline sequences, the human-likeness of the antibodies was enhanced. One of the changes introduced into FSI-174 simultaneously removes the deamidation site in LCDR1. However, the LCDR3 of FSI-174 is identical to the LCDR3 of SR-1, that is, the deamidation hotspots and isomerization / fragmentation hotspots in LCDR3 still exist in FSI-174. The CDRs (by Kabat) of SR-1, AMG191, and FSI-174 are shown in Table 20.

Table 20

[0277] SCF-dependent proliferation was measured as follows: TF-1 cells were seeded at a cell concentration of 150,000 cells / ml on sterile leukocyte culture-treated plates in RPMI medium + 10% FCS. The cells were treated with 100 ng / ml of SCF and dose settings of various CD117 antibodies or isotype control antibodies at different concentrations. After 3 days of incubation, the CellTiter-Glo 2.0 assay (Promega) was performed, the luminescence was read with a luminometer (Envision, Perkin Elmer), and the IC50 was determined. The results are shown in Table 21.

Table 21

[0278] Some of the generated antibodies showed an IC50 within or even less than the range of the original SR-1 antibody and its known humanized derivatives in this SCF-dependent proliferation assay.

[0279] Example 8: Antibody-mediated inhibition of SCF-dependent phosphorylation SCF-dependent phosphorylation is another function of CD117. TF-1 cells expressing CD117 are used to measure the antibody-mediated inhibition of SCF-dependent phosphorylation. TF-1 cells were seeded at a concentration of 1 million cells / mL in 2 mL of medium (RPMI 1640 supplemented with GlutaMAX + 10% heat-inactivated FBS) in a 6-well plate. After pre-treating the cells with antibodies at 0.005, 0.05, and 0.5 μg / mL for 30 minutes, 100 ng / mL of recombinant human SCF (1 mg / mL PeproGMP® recombinant human SCF) was added for 5 minutes. The cells were harvested, washed with ice-cold PBS, and then resuspended in 500 μL of lysis buffer (lysis buffer from Signaling containing protease inhibitors and phosphatase inhibitors). The cells were snap-frozen in liquid nitrogen, thawed twice at 37 °C, and then frozen at -80 °C until further use. The undiluted samples were thawed and processed according to the manufacturer's protocols of the PathScan® Phospho-c-Kit (Tyr719) Sandwich ELISA Kit (Cell Signaling) and the Human c-Kit (CD117) ELISA Kit (Abcam). Absorbance (OD_450nm) was evaluated using Envision (Perkin Elmer). The same antibodies as in Example 7 were tested. The results are shown in Figure 3.

[0280] As can be seen, most of the antibodies of the present disclosure tested inhibited SCF-dependent phosphorylation at least as strongly as the SR-1 antibody and the humanized derivatives AMG191 and FSI-174.

[0281] Example 9: Inhibition of SCF binding in TF-1 cells In this experiment, it was tested whether the antibodies of the present disclosure were able to inhibit the binding of SCF to CD117.

[0282] For this purpose, TF-1 wild-type cells were seeded at a cell concentration of 100,000 cells / well. Fc receptor blocking was performed using 25 μL of TruStain FcX (BioLegend). Next, the cells were treated with the antibody to be tested at a fixed concentration of 50 μg / ml. After 15 minutes, a mixture of various concentrations of biotinylated SCF (Acro-Biosystems) and streptavidin PE (Invitrogen) was added. After an additional 30 minutes, the cells were washed and live dead staining via 7AAD was performed for 10 minutes using 0.5 μg / mL of 7-aminoactinomycin D (7AAD) (BioLegend), and then the cells were analyzed by flow cytometry. The following antibodies: NegCtrl, SR-1, AMG191, FSI-174, Dufour, Matterhorn, Piz Bernina, Allalinhorn, Piz Zupo, Eiger, Piz Argient, Piz Roseg, Bietschhorn and Bellavista were tested. The results are shown in Figure 4.

[0283] Except for Piz Roseg and Piz Argient, all of the antibodies of the present disclosure effectively inhibited the binding of SCF to CD117. The NegCtrl antibody (anti-chicken lysozyme) did not inhibit the binding of SCF to CD117.

[0284] Example 10: Binding of antibodies to CD117 in TF-1 cells The binding of the antibodies of the present disclosure to CD117 on TF-1 cells was measured. TF-1 wild-type cells were seeded at a concentration of 100,000 cells / well. Fc receptor blocking was performed for 15 minutes using 25 μL of TruStain FcX (BioLegend). Next, the cells were treated with the antibody to be tested at various concentrations (0.0001 to 5 μg / ml). After a further 30 minutes, the cells were washed and then a secondary antibody was added to the cells (IgG(H+L) cross-adsorbed goat anti-human, Alexa Fluor® 488). After 30 minutes and two additional washing steps, live dead staining via 7AAD was performed for 10 minutes using 0.5 μg / mL of 7-aminoactinomycin D, and then the cells were analyzed by flow cytometry. The results are shown in Figure 5.

[0285] With the exception of Piz Roseg and Piz Argient, all of the antibodies of the present disclosure strongly bound to CD117 on TF-1 cells. Binding could also be detected for Piz Roseg and Piz Argient, but only at higher antibody concentrations. The NegCtrl antibody (anti-chicken lysozyme) showed no binding to TF-1 cells.

[0286] Example 11: Inhibition of SCF binding in hematopoietic stem cells In this experiment, it was tested whether the antibodies of the present disclosure are capable of inhibiting the binding of CD117 to SCF in HSPCs (CD34+CD38− cells) and HSCs (CD34+CD38−CD90+CD45RA− cells).

[0287] The experiments were conducted in low cytokine medium and high cytokine medium. The low cytokine medium consists of StemPro medium (Gibco), StemPro nutrients (Gibco), 50 ng / ml LDL (STEMcell Technologies), 1% penicillin / streptomycin (Thermo Fisher), 1% glutamine (Thermo Fisher), 20 ng / ml Flt3 (Miltenyi Biotec), 50 ng / ml TPO (Milteny Biotec) and various concentrations of SCF (Milteny Biotec). The high cytokine medium consists of the same components + 50 ng / ml IL6 (Milteny Biotec), 10 ng / ml IL3 (Milteny Biotec), 10 ng / ml IL2 (Milteny Biotec), 20 ng / ml IL7 (Milteny Biotec), 50 ng / ml IL11 (Milteny Biotec), 3 ng / ml EPO (STEMcell Technologies) and 20 ng / ml GM-CSF (Milteny Biotec). Antibodies NegCtrl, SR-1 and Allalinhorn were tested. The results are shown in Figure 6.

[0288] The same results were obtained for both test cell populations and both media. Both antibodies SR-1 and Allalinhorn completely and to the same extent inhibited the binding of SCF to CD117. The NegCtrl antibody (anti-chicken lysozyme) did not inhibit the binding of SCF to CD117.

[0289] Example 12: Inhibition of proliferation in HSPC and HSC SCF-dependent proliferation was also measured in mobilized CD34+ HSPC isolated from peripheral blood (CliniMACS Prodigy, Miltenyi Biotech). Cells were seeded at a cell concentration of 75,000 cells / ml in a sterile leukocyte culture-treated plate with a transparent bottom in complete Stemspan AOF medium containing cytokines (100 ng / ml SCF; FLT3, TPO, and IL-3). Cells were treated with various concentrations of the antibodies of the present disclosure. After 3 days of incubation, the CellTiter-Glo 2.0 assay (Promega) was performed, luminescence was read with a luminometer (Envision, Perkin Elmer), and the IC50 was determined. The results are shown in Table 22.

Table 22

[0290] The results obtained with CD34+ HSPC correlate well with those obtained with TF-1 cells. Some of the antibodies generated showed IC50 values within or even below the range of the original SR-1 antibody and its known humanized derivatives in this SCF-dependent proliferation assay.

[0291] For human CD34+ HSC (different donors), in a sterile leukocyte culture-treated plate, cells are seeded at a cell density of 75,000 cells / ml in complete Stemspan AOF medium containing cytokines: 100 ng / ml SCF, 100 ng / ml TPO, 60 ng / ml IL-3, and 100 ng / ml FLT-3 ligand. Next, HSC are treated with various CD117 antibodies or isotype control antibodies at different concentrations, and the plates are incubated for 3 days. After 3 days, the CellTiter-Glo 2.0 assay (Promega) is performed, and luminescence is read with a luminometer (e.g., Envision plate reader).

[0292] Example 13: HSC depletion experiment The source of human HSC depletion in mice using various mAbs was Pang et al. (Blood (2019) 133:2069-78).

[0293] One million HSPCs were injected into NBSGW mice (Jackson Laboratories). At 8, 10, 12, and 14 days after cell injection, the mice were administered 25 mg / kg of the antibody Allalinhorn i.v. per dose. After 16 weeks, the mice were euthanized, and blood, spleen, and bone marrow were analyzed by FACS. The results for bone marrow HSC depletion are shown in Figure 10. The figure shows the in vivo depletion of HSCs after injection of Allalinhorn compared to animals administered an isotype control antibody. HSCs were identified by FACS as live / hCD45+ / CD34+ / CD38- / CD45RA- / CD90+.

[0294] In another experiment, NSG mice (Jackson Laboratories) were sub-lethally irradiated 1 day before injection of one million gene-edited HSPCs carrying the E73K mutation of CD117. At 8, 10, 12, and 14 days after cell injection, the mice were administered 25 mg / kg of antibody Allalinhorn or 25 mg / kg of an isotope control antibody i.v. After 16 weeks, the mice were euthanized, and blood, spleen, and bone marrow were analyzed by FACS.

[0295] The results for bone marrow are shown in Figure 11. CD117+ bone marrow cells were identified by FACS as live / hCD45+ / CD33+ / CD117+. Antibody clone 104D2 was used to identify CD117+ cells, which does not interfere with the binding of SR-1 and Allalinhorn to CD117. Clone 104D2 was used as an expression control. Based on double staining with anti-CD117 clone SR-1 and anti-CD117 clone 104D2, cells were classified as unedited (104D2+ and SR-1+) or E73K edited (104D2+ but SR-1-).

[0296] Figure 11 shows the depletion of unedited cells in mice administered with antibody Allaninhorn compared to mice administered with an isotype control antibody. In contrast, as a result of the injection of Allaninhorn, enrichment of E73K mutant cells occurred compared to animals given the isotype control antibody.

[0297] Example 14: Removal of important sites of the CDR has a beneficial effect on antibody stability. The beneficial effect of the removal of important sites is tested by subjecting the antibody to different stress conditions such as heat stress and pH stress.

[0298] For heat stress, the antibody solution is incubated in the dark at 50 °C for 5 days. For pH stress conditions, the antibody solution is diluted to 1 mg / ml in PBS and the buffer is exchanged with either 20 mM citrate buffer pH 3 or 20 mM Tris buffer pH 9. The samples at low and high pH are then incubated in the dark at room temperature for 1, 3 and 7 days. At the end of the incubation period, the stress-loaded samples are neutralized by diluting 1:2 with 200 mM phosphate buffer pH 7.4 and stored at -80 °C. All samples are then analyzed by high performance size exclusion chromatography (HP-SEC), cation exchange chromatography (CEX), CE-SDS and / or RP-HPLC and peptide mapping to identify chemical and physical degradation. To assess the effect of forced degradation on the biological activity of the molecule, measurement of binding to CD117 is also performed on the stress-loaded antibody.

[0299] Example 15: Antibody-mediated inhibition of HSPC colony formation The effect of the antibody on HSPC colony formation was tested as follows. HSPCs were placed on methylcellulose containing hematopoietic cytokine (MethoCult GF H84435) (500 cells per 35 mm dish, in duplicate), and grown at 37 °C, 5% CO2 for 14 days in the presence of anti-CD117 antibodies SR-1, Dufour, and Allalinhorn. The antibodies were added once to the medium at the indicated final concentrations (0.1, 1, and 10 μg / mL) at the time of plating. Colony numbers were counted manually using an inverted microscope (and scoring dish with grid).

[0300] The results are shown in Figure 7. All three anti-CD117 antibodies tested led to a dose-dependent decrease in colonies. The decrease in colony formation was most prominent with the antibody Allalinhorn.

[0301] Example 16: SCF-Dependent Phosphorylation of CD117 TF-1 cells (wild type, knockout, and mutants E73K, D121K, and S123K of CD117) were seeded at a concentration of 1 million cells / mL in 2 mL of medium (RPMI 1640 supplemented with GlutaMAX + 10% heat-inactivated FBS) in 6-well plates. Prior to cell harvest, the cells were treated with 1 - 20 ng / mL of recombinant human SCF (PeproGMP recombinant human SCF at 1 mg / mL) for 5 minutes. After washing the cells with ice-cold PBS, the cells were resuspended in 500 μL of lysis buffer (lysis buffer from Signaling containing protease inhibitors and phosphatase inhibitors). The cells were snap-frozen in liquid nitrogen, thawed twice at 37 °C, and then frozen at -80 °C until further use. The undiluted samples were thawed and processed according to the manufacturer's protocol for the PathScan Phospho-c-Kit (Tyr719) Sandwich ELISA Kit (Cell Signaling). Absorbance (OD_450nm) was evaluated using Envision (Perkin Elmer).

[0302] Exemplary results of the variant E73K are shown in Figure 8. Wild-type TF-1 cells and the E73K, D121K, and S123K variants of CD117 showed SCF-dependent phosphorylation at position Tyr719 of CD117. The phosphorylation levels of knockout cells were at background levels.

[0303] Example 17: Preserved Signaling of CD117 Variants in the Presence of Blocking Antibodies TF-1 cells (wild-type and variants E73K, E73Y, D121K, S123F, and S123K of CD117) were seeded at a concentration of 1 million cells / mL in 2 mL of medium (RPMI 1640 supplemented with GlutaMAX + 10% heat-inactivated FBS) in a 6-well plate. The cells were pretreated with 0.5 μg / mL of antibody Piz Bernina or Allalinhorn for 30 minutes, and then 100 ng / mL of recombinant human SCF (1 mg / mL PeproGMP recombinant human SCF) was added for 5 minutes. The cells were harvested, washed once with ice-cold PBS, and resuspended in 500 μL of lysis buffer (lysis buffer from Signaling containing protease inhibitors and phosphatase inhibitors). Next, the cells were snap-frozen in liquid nitrogen, thawed twice at 37°C, and then frozen at -80°C until further use. The undiluted samples were thawed and processed according to the manufacturer's protocol of the PathScan Phospho-c-Kit (Tyr719) Sandwich ELISA Kit (Cell Signaling) or the Human c-Kit (CD117) ELISA Kit (Abcam). Absorbance (OD_450nm) was evaluated using Envision (Perkin Elmer).

[0304] Exemplary results of the variants D121K and S123K are shown in Figure 9. SCF-dependent CD117 phosphorylation in wild-type TF-1 cells was blocked by the antibodies Piz Bernina or Allalinhorn, while there was no effect in TF-1 cells with the D121K and S123K mutations of CD117 phosphorylation. The same was observed for the variants E73K, E73Y, and S123F (data not shown).

[0305] Example 18: CD117 variants are resistant to treatment with ADCs In this experiment, DF-1 cells were used. DF-1 cells lack CD117. The selected CD117 variants (E73Y, D121K, S123K) were transfected into the cells. The transfected cells were treated with ADCs (both antibody Piz Bernina and Allalinhorn conjugated to teserine) at concentrations of 0.1, 1 and 10 μg / ml. After 48 hours, the cells were analyzed by FACS.

[0306] The results are shown in Figure 12. D121K A5 and D121K A6 represent two different batches of plasmid for the same variant. Both ADCs tested, Piz Bernina-teserine and Allalinhorn-teserine, resulted in efficient depletion of wild-type DF-1 cells, but DF-1 cells transfected with the D121K or S123K variants of CD117 were not depleted.

[0307] Example 19: Further HSC depletion experiments The source of human HSC depletion in mice using various mAbs was Pang et al. (Blood (2019) 133:2069-78).

[0308] One million HSPCs from two different donors carrying the E73K, S123K or D121K variants of CD117 were injected into NBSGW mice (Jackson Laboratories). The mouse control group was given unedited electroporation control HSPCs. Seven, nine, eleven and twelve days after cell injection, the mice were administered the antibody Allalinhorn at a dose of 4 mg / kg i.v. per dose. After 16 weeks, the mice were euthanized and blood, spleen and bone marrow were analyzed by FACS. The results for depletion of unedited CD117 cells and enrichment of edited CD117 cells are shown in Figure 13. The antibody clone 104D2 was used to identify CD117+ cells, which does not interfere with the binding of SR-1 and Allalinhorn to CD117. Clone 104D2 was used as an expression control. Based on double staining with anti-CD117 clone SR-1 and anti-CD117 clone 104D2, cells were classified as unedited (104D2+ and SR-1+) or edited (104D2+ but SR-1-).

[0309] Figure 13 shows the depletion of unedited CD34+ progenitor cells (gated as live / hCD45+ / CD34+ / CD38-) in mice given the antibody Allalinhorn compared to mice given an isotype control antibody. In contrast, as a result of the injection of Allalinhorn, enrichment of the E73K, D121K and S123K edited mutant cells occurred compared to animals given an isotype control antibody.

Claims

1. A humanized antibody or antibody fragment specific for human CD117, comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, wherein the asparagine in the LCDR1 region (SEQ ID NO: 7) is substituted with glutamic acid or tyrosine, and having at least one mutation, the humanized antibody or antibody fragment.

2. The following further mutations: a) The first asparagine in the HCDR2 region (SEQ ID NO: 5) is substituted with serine, b) The second asparagine in the LCDR3 region (SEQ ID NO: 9) is substituted with lysine, or c) The aspartic acid in the LCDR3 region (SEQ ID NO: 9) is substituted with glutamic acid, The humanized antibody or antibody fragment according to claim 1, having one or more of the above.

3. a) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, b) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, c) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 113, d) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 71, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, e) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 95, f) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, or g) A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9 The humanized antibody or antibody fragment according to claim 1 or 2, comprising the same.

4. a) A variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98, b) A variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 99, c) A variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100, d) A variable heavy chain of SEQ ID NO: 70 and a variable light chain of SEQ ID NO: 98, e) A variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 97, f) A variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 61, or g) A variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 62 The humanized antibody or antibody fragment according to any one of claims 1 to 3, comprising the same.

5. The humanized antibody according to any one of claims 1 to 4, wherein the antibody is of the human IgG1 class and is optionally a silenced IgG1 antibody such as an antibody having the PA-LALA mutation.

6. The humanized antibody or antibody fragment according to any one of claims 1 to 5, which inhibits SCF binding to CD117, SCF-dependent proliferation of CD117-positive cells, and / or SCF-dependent phosphorylation of CD117.

7. The humanized antibody or antibody fragment according to any one of claims 1 to 6, which binds to CD117 with at least the same affinity as an antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO:

9.

8. An antibody or antibody fragment comprising a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, and the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, which binds to an epitope on CD117 and is the same as the humanized antibody or antibody fragment according to any one of claims 1 to 7.

9. The humanized antibody or antibody fragment according to any one of claims 1 to 8, which is a monoclonal antibody or antibody fragment.

10. The humanized antibody or antibody fragment according to any one of claims 1 to 9, for use in medicine.

11. The humanized antibody or antibody fragment according to any one of claims 1 to 10, wherein the use in medicine is the treatment of cancer such as blood cancer or solid cancer, or the treatment of inflammatory diseases, autoimmune diseases, urticaria, nodular prurigo, eosinophilic esophagitis or mastocytosis.

12. A nucleic acid composition comprising one or more nucleic acid sequences encoding the humanized antibody or antibody fragment according to any one of claims 1 to 9.

13. A vector comprising the nucleic acid composition according to claim 12.

14. A host cell comprising the vector according to claim 13 or the nucleic acid composition according to claim 12.

15. A pharmaceutical composition comprising the humanized antibody or antibody fragment according to claims 1 to 9 and a pharmaceutically acceptable carrier or excipient.

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