Antibodies targeting CD117

Abstract

The present disclosure relates to antibodies and antibody fragments specific for CD117. These antibodies are improved versions of prior art antibodies. In addition to complex humanized motion, the antibodies are engineered to remove several detrimental motifs within the CDR region without losing any beneficial characteristics. These antibodies are useful in the treatment of diseases associated with CD117.

Description

Technical Field

[0001] The present disclosure relates to antibodies and antibody fragments specific for CD117. These antibodies are improved versions of prior art antibodies. In addition to complex humanization exercises, the antibodies are engineered to remove several harmful motifs within the CDR regions without losing any beneficial properties. These antibodies can be used to treat diseases associated with CD117.

[0002] Statement on Funding

[0003] The project that gave rise to this application has received funding from the European Research Council (ERC) under the EU's Horizon 2020 research and innovation programme (grant agreement No 818806). Background Art

[0004] CD117 (also known as c-kit or stem cell factor receptor (SCRF)) is a single transmembrane receptor tyrosine kinase that binds to the ligand stem cell factor (SCF). SCF induces homodimerization of CD117, which activates its tyrosine kinase activity and signals through both the PI3K-AKT and MAPK pathways (Kindblom et al, Am J. Path. 1998 152(5): 1 259).

[0005] CD117 was originally discovered to be an oncogene and was studied in the field of oncology (e.g., see Stankovet al, (2014) Curr Pharm Des. 20: 2849-80). CD117 is highly expressed on hematopoietic stem cells (HSC), and its ligand is the 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 HSC and its activity in regulating hematopoiesis make CD117 a potential target for bone marrow regulation in a variety of diseases (Russkamp et al, Exp. Hematol. (2021) 95: 31-45; Czechowicz et al, Nat Commun (2019) 10: 617). However, there is still a need for anti-CD117-based therapies that can effectively regulate patients for transplantation, such as bone marrow transplantation.

[0006] Several anti-CD117 parts are known in the art, some of which are currently under development. Antibody SR-1 was originally isolated from a hybridoma (WO1992017505). Blood (2019) 133: 2069-78 describes the use of SR-1 in HSC-driven blood and immune disorders. Certain humanized versions of SR-1 have been generated (WO2007127317; WO2020112687). WO2020033664 discloses the original SR-1 antibody for bispecific and multispecific constructs (the sequences of the variable heavy chain and variable light chain are the same as those disclosed in US20200165337A1). 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 (Supplement 1): 4428 discloses a study on the anti-CD117 antibody FSI-174. Anti-CD117 drug conjugates are described in WO2016020791. Other anti-CD117 antibodies are described in WO2015050959 and WO2019084064. Certain anti-CD117 antibodies are also commercially available, such as antibody 104D2Dianova (#117PE-100T). These and other anti-CD117 portions can be used in the context of the present disclosure. WO2021041945 discloses genomic alterations of antigens (including CD117) produced by base editing. However, these antigenic changes have not been characterized, and in particular, the biological functions of the variants have not been tested. Summary of the invention

[0007] The present disclosure relates to a humanized antibody or antibody fragment specific to human CD117, wherein the antibody or antibody fragment comprises a variable heavy chain and a variable light chain, the variable heavy chain comprises 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 the variable light chain comprises 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 humanized antibody or antibody fragment has at least one mutation, wherein the asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid or tyrosine. In certain embodiments, the humanized antibody or antibody fragment has one or more of the following additional mutations:

[0008] a) the first asparagine in the HCDR2 region (SEQ ID NO: 5) is replaced by serine,

[0009] b) the second asparagine in the LCDR3 region (SEQ ID NO:9) is replaced by lysine, or

[0010] c) Aspartic acid in the LCDR3 region (SEQ ID NO: 9) was replaced by Glutamic acid.

[0011] The present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises:

[0012] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0013] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0014] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0015] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 71, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0016] e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0017] f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9,

[0018] g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO:4, a HCDR2 region of SEQ ID NO:5, a HCDR3 region of SEQ ID NO:6, and a variable light chain comprising a LCDR1 region of SEQ ID NO:81, a LCDR2 region of SEQ ID NO:8, and a LCDR3 region of SEQ ID NO:9.

[0019] The present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises:

[0020] a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98,

[0021] b) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 99,

[0022] c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100,

[0023] d) the variable heavy chain of SEQ ID NO: 70 and the variable light chain of SEQ ID NO: 98,

[0024] e) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 97,

[0025] f) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 61, or

[0026] g) the variable heavy chain of SEQ ID NO:22 and the variable light chain of SEQ ID NO:62.

[0027] The present disclosure also relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment inhibits the binding of SCF to CD117, the SCF-dependent proliferation of CD117-positive cells, and / or the SCF-dependent phosphorylation of CD117.

[0028] The present disclosure also relates to humanized antibodies or antibody fragments, wherein the humanized antibodies or antibody fragments bind to CD117 with at least the same affinity as an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0029] The present disclosure also relates to humanized antibodies or antibody fragments, wherein the humanized antibodies or antibody fragments bind to CD117 with a higher affinity than an antibody or antibody fragment comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0030] The present disclosure also relates to humanized antibodies or antibody fragments, wherein the humanized antibodies or antibody fragments bind to the same epitope on CD117 as an antibody or antibody fragment comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0031] The present disclosure also relates to humanized antibodies or antibody fragments, wherein the humanized antibodies or antibodies are cross-reactive with cynomolgus monkeys.

[0032] 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 a hematological cancer (e.g. AML) or a solid cancer (e.g. mast cell cancer, testicular stromal cancer, gastrointestinal stromal cancer, melanoma, breast cancer and lung cancer) or for use in regulating the bone marrow.

[0033] The present disclosure also relates to nucleic acids or nucleic acid compositions comprising a nucleic acid sequence or multiple nucleic acid sequences encoding the humanized antibodies or antibody fragments of the present disclosure.

[0034] The present disclosure also relates to vectors comprising a nucleic acid or nucleic acid composition encoding the humanized antibodies or antibody fragments of the present disclosure.

[0035] The present disclosure also relates to a host cell comprising a vector, or a nucleic acid, or a nucleic acid composition of the humanized antibody or antibody fragment of the present disclosure.

[0036] The present disclosure also relates to a pharmaceutical composition comprising the humanized antibody or antibody fragment of the present disclosure and a pharmaceutically acceptable carrier or excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Alignment of the five humanized variants with the original mouse VH sequence of SR-1 is shown. VHO is the mouse sequence and VH1-5 are humanized variants. CDRs are underlined. Key residues important for the VH / VL interface and canonical loop structure have been kept as unchanged as possible.

[0038] Figure 2 Alignment of the five humanized variants with the original mouse VL sequence of SR-1 is shown. VLO is the mouse sequence and VL1-6 are humanized variants. The CDRs are underlined. Key residues important for the VH / VL interface and canonical loop structure have been kept as unchanged as possible.

[0039] Figure 3 The results of the assay for antibody-mediated inhibition of SCF-dependent phosphorylation of CD117 are shown. From left to right, the following antibodies were tested: A-NegCtrl, B-SR-1, C-AMG191, D-FSI-174, E-Dufour, F-Matterhorn, G-PizBernina, H-Allalinhorn, I-Piz Zupo, J-Eiger, K-Piz Argient, L-Piz Roseg, M-Bietschhorn, N-Bellavista.

[0040] Figure 4 The results of experiments investigating whether the antibodies of the present disclosure can inhibit the binding of CD117 to SCF are shown. The following antibodies were tested: NegCtrl, SR-1, AMG191, FSI-174, Dufour, Matterhorn, Piz Bernina, Allalinhorn, Piz Zupo, Eiger, Piz Argient, Piz Roseg, Bietschhorn, and Bellavista. Except for Piz Roseg and Piz Argient, all antibodies of the present disclosure effectively inhibited the binding of SCF to CD117. NegCtrl antibody (anti-chicken lysozyme) did not inhibit the binding of SCF to CD117.

[0041] Figure 5 Results of experiments investigating the binding of antibodies of the present disclosure to CD117 on TF-1 cells are shown. All antibodies of the present disclosure, except PizRoseg and Piz Argient, strongly bind to CD117 on TF-1 cells. Binding of Piz Roseg and Piz Argient could also be detected, although only at higher antibody concentrations. NegCtrl antibody (anti-chicken lysozyme) did not show any binding to TF-1 cells.

[0042] Figure 6 The results of experiments investigating whether the antibodies of the present disclosure can inhibit the binding of CD117 to SCF in HSPC (CD34+CD38- cells) and HSC (CD34+CD38-CD90+CD45RA- cells) in low cytokine medium and high cytokine medium are shown. The same results were obtained for both tested cell populations and both culture media. Both antibodies SR-1 and Allalinhorn completely inhibited the binding of SCF to CD117 to the same extent. NegCtrl antibody (anti-chicken lysozyme) did not inhibit the binding of SCF to CD117.

[0043] Figure 7 Results of antibody-mediated inhibition of HSPC colony formation are shown. All three anti-CD117 antibodies tested resulted in a dose-dependent reduction in colonies. Antibody Allalinhorn had the most pronounced reduction in colony formation.

[0044] Figure 8 SCF-dependent phosphorylation of CD117 and E73K (CD117 variant) at Tyr719 of wild-type TF-1 cells is shown. The D121K and S123K variants of CD117 behaved similarly (data not shown).

[0045] Fig. 9 It was shown that SCF-dependent CD117 phosphorylation in wild-type TF-1 cells was blocked by antibodies Piz Bernina and Allalinhorn, whereas it was not affected in TF-1 cells with the D121K and S123K variants of CD117 phosphorylation. The same was observed for variants E73K, E73Y and S123F (data not shown).

[0046] Fig.10 The antibody Allalinhorn was shown to effectively deplete HSPCs in vivo.

[0047] Fig.11 It is shown that the binding of the isotope control antibody is not affected by E73K compared to the antibody Allalinhorn.

[0048] Fig.12 It is shown that the ADCs tested (Piz Bernina-teserine and Allalinhorn-teserine) deplete wild-type DF-1 cells, but not DF-1 cells transfected with the D121K or S123K variants of CD117. D121K A5 and D121K A6 represent two different batches of plasmids of the same variant.

[0049] Fig.13 Compared with mice receiving isotype control antibody, mice receiving antibody Allalinhorn Depletion of unedited CD34+ progenitor cells (gated as live / hCD45+ / CD34+ / CD38-) in mice injected with 100 μM dT cells compared to animals receiving an isotype control antibody. Allalinhorn Resulting in enrichment of cells with E73K, D121K, and S123K edited variants. DETAILED DESCRIPTION

[0050] definition

[0051] The present disclosure relates to antibodies that specifically bind to CD117, and uses of such antibodies, particularly therapeutic uses.

[0052] 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):

[0053] (SEQ ID No.1)

[0054] As used herein, the term "antibody" refers to a protein comprising at least two heavy chains (H) and two light chains (L), which are interconnected by disulfide bonds and interact with antigens. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (i.e., CH1, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementary determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. 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 chain and the heavy chain are divided into regions of structural and functional homology.

[0055] 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, stabilizing spatial distribution). Examples of binding fragments include, but are not limited to, Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bond in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of VH domains (Ward et al, (1989) Nature 341: 544-546); and isolated complementary determining regions (CDRs). In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by a synthetic linker using recombinant methods, which enables them to be made into a single protein chain, wherein the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, 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 intended to be included in the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those skilled 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 biscFvs (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23: 1126-1136). Antibody fragments can be grafted onto scaffolds based on polypeptides such as type III fibronectin (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monoclonal antibodies). Antibody fragments can be incorporated into single chain molecules comprising a pair of tandem Fv fragments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen binding sites (Zapata et al., (1995) Protein Eng. 8: 1057-1062; and U.S. Pat. No. 5,641,870).

[0056] The structure and location of immunoglobulin variable domains (e.g., 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 Department of Health and Human Services (1991), eds. 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).

[0057] As used herein, "human antibodies" or "human antibody fragments" are antibodies and antibody fragments with variable regions, wherein both the framework region and the CDR region are from sequences of human origin. Human antibodies can also be isolated from synthetic libraries or from transgenic mice (e.g., Xenomouse, Omni mice, Harbour mice, ATX-Gx mice, Trianni mice), provided that the corresponding system output of antibodies with variable regions, wherein both the framework region and the CDR region are derived from sequences of human origin. In addition, if the antibody contains a constant region, the constant region is also derived from this sequence. Human origin includes, for example, human germline sequences, or mutant versions of human germline sequences, or antibodies containing a consensus framework sequence derived from human framework sequence analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296:57-86.

[0058] "Humanized antibodies" or "humanized antibody fragments" are defined herein as antibody molecules having constant antibody regions derived from sequences of human origin, and variable antibody regions or portions thereof or only CDRs derived from another species. For example, a humanized antibody can be CDR-grafted, wherein the CDRs of the variable domains are from non-human sources, while one or more frameworks in the variable domains are of human origin, and the constant domains (if any) are of human origin.

[0059] The term "chimeric antibody" or "chimeric antibody fragment" is defined herein as an antibody molecule having constant antibody regions derived from or corresponding to sequences present in one species and variable antibody regions derived from another species. Preferably, the constant antibody regions are derived from or correspond to sequences present in humans, and the variable antibody regions (e.g., VH, VL, CDR or FR regions) are derived from sequences present in non-human animals (e.g., mice, rats, rabbits or hamsters).

[0060] 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 antigenic specificities. In addition, an isolated antibody or antibody fragment may be substantially free of other cellular materials and / or chemicals. Therefore, in some aspects, the antibody provided is an isolated antibody that has been separated from antibodies having different specificities. The isolated antibody may be a monoclonal antibody. The isolated antibody may be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds to an epitope, isotype or variant of a target may have cross-reactivity to other related antigens, such as antigens from other species (e.g., species homologs).

[0061] As used herein, the term "recombinant antibody" or "recombinant antibody fragment" includes all antibodies or antibody fragments prepared, expressed, produced or isolated by means that do not exist in nature. For example, antibodies isolated from host cells transformed to express antibodies, antibodies selected and isolated from recombinant, combined human antibody libraries, and antibodies prepared, expressed, produced or isolated by any other means involving splicing of all or part of human immunoglobulin genes, sequences or other DNA sequences, or antibodies isolated from animals (such as mice) transgenic or transchromosomal human immunoglobulin genes or hybridomas prepared therefrom. Preferably, such recombinant antibodies have variable regions in which the framework regions and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic cell mutagenesis is performed), so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences derived from and related to human germline VH and VL sequences, and may not naturally exist in the human germline antibody library in vivo. The recombinant antibody can be a monoclonal antibody.

[0062] As used herein, the term "monoclonal" has the meaning generally given to it in the art, i.e., an antibody or antibody fragment (or its corresponding functional fragment) produced by a single clone of antibody-producing cells and recognizing a single epitope on the bound antigen.

[0063] As used herein, an antibody "binds specifically to", "specifically binds to" is "specific" or "specifically recognizes" an antigen (such as human CD117) if the antibody is able to distinguish between the antigen and one or more reference antigens, because binding specificity is not an absolute, but a relative property. For example, a standard ELISA assay or a standard flow cytometry assay can be performed. Scoring can be performed by standard chromogenic methods (e.g., a second antibody with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide) or by combining a second antibody labeled with PE or another dye or marker. The reaction in certain wells is scored by, for example, optical density (OD) at 450nm or by average or median fluorescence intensity (MFI) in flow cytometry. A typical background (= negative reaction) can be 0.1OD; a typical positive reaction can be 1OD. Background and positive reaction MFI are highly dependent on instrument settings. Positive / negative differences may be greater than 10 times. Typically, the determination of binding specificity is not by using a single reference antigen, but by using a group of about three to five unrelated antigens (e.g., milk powder, BSA, transferrin, etc.). For flow cytometry, various antigen-negative cells can be used. However, antibodies that specifically bind to antigens may have cross-reactivity to corresponding orthologous antigens from other species (e.g., species homologs). In some embodiments, this cross-reactivity to orthologous antigens is even preferred.

[0064] As used herein, an antibody is "cross-reactive" or "cross-reactive" if it binds to an orthologous antigen from another species. For example, if an antibody binds to human CD117 and cynomolgus monkey CD117, the antibody is cross-reactive.

[0065] As used herein, the term "affinity" refers to the strength of interaction between a polypeptide and its target at a single site. Within each site, the binding region of a polypeptide interacts with its target at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0066] The term "epitope" includes any protein region that is specifically recognized by an antibody or its antibody fragment or otherwise interacts with a molecule. Typically, an epitope is a chemically active surface grouping of a molecule, such as amino acids or carbohydrates or sugar side chains, and typically may have specific three-dimensional structural characteristics as well as specific charge characteristics. As will be appreciated by those skilled in the art, virtually anything that an antibody can specifically bind to may be an epitope.

[0067] The term "domain" or "protein domain" refers to a region in a protein polypeptide chain that forms a functional unit and / or independently forms a three-dimensional structure.

[0068] The "compositions" of the present disclosure can be used for therapeutic or preventive applications. Therefore, the present disclosure includes pharmaceutical compositions containing antibodies or antibody fragments as disclosed herein and thus containing pharmaceutically acceptable carriers or excipients. In a related aspect, the present disclosure provides a method for treating inflammatory diseases, autoimmune diseases, hematological malignancies, and potentially other diseases. This method contains the step of administering to a subject in need thereof an effective amount of a pharmaceutical composition containing an antibody or antibody fragment described herein.

[0069] The present disclosure provides methods of treatment, including administering a therapeutically effective amount of an antibody or antibody fragment as disclosed herein to a subject in need of such treatment. As used herein, "therapeutically effective amount" or "effective amount" refers to the amount of the anti-CD117 antibody required for eliciting a desired biological response. According to the present disclosure, a therapeutically effective amount is the amount of the anti-CD117 antibody necessary for treating and / or preventing a disease.

[0070] "Administered" or "administration" includes, but is not limited to, delivering the drug by an injectable form, such as, for example, an intravenous, intramuscular, intradermal or subcutaneous route, or a mucosal route, for example, as a nasal spray or aerosol for inhalation, or as an ingestible solution, capsule or tablet. Preferably, administration is by an injectable form.

[0071] As used herein, "treatment", "treat" or "treating" and the like refer to clinical interventions that attempt to alter the natural course of a disease in a treated subject, and may be used for prevention or performed during clinical pathology. The desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, antibodies or antibody fragments according to the present disclosure are used to delay the development of a disease or slow the progression of a disease.

[0072] "Preventing" or "prevention" refers to reducing the risk of developing a disease or a disease (i.e., preventing a subject who may be exposed to a disease-causing agent or who is susceptible to such a disease prior to the onset of the disease from developing at least one clinical symptom of the disease). "Prevention" also refers to methods intended to prevent the onset of a disease or its symptoms or to delay the onset of a disease or its symptoms.

[0073] "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), marmosets (Callithrix jacchus), rhesus monkeys (Macaca mulatta), or humans (Homo sapiens). Preferably, the subject is a primate, most preferably a human.

[0074] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with 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 cellular phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and direct cell activation or direct cell inhibition.

[0075] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bound to Fc receptors (FcRs) present on certain cytotoxic cells (such as NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. The primary cells that mediate ADCC (i.e., NK cells) express only FcyRIII, while monocytes / macrophages express FcyRI, FcyRII, and FcyRIII.

[0076] "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 by binding of the first component of the complement system (CIq) to an antibody of the present disclosure (of the appropriate subclass) bound to its cognate antigen.

[0077] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells are eliminated by internalization by phagocytes such as macrophages or dendritic cells.

[0078] Throughout the specification, unless the context requires otherwise, the words “comprise,” “have,” and “include,” and corresponding variations such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” will be understood to imply the inclusion of stated elements, or integers, or groups of elements or integers but not the exclusion of any other elements, or integers, or groups of elements or integers.

[0079] As used herein, the term "engineered" or "modified" includes manipulation of nucleic acids or polypeptides by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by 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.

[0080] As used herein, "variant" refers to a polypeptide that differs from a reference polypeptide by one or more modifications (e.g., amino acid substitutions, insertions, or deletions). Variant polypeptides typically retain most of the properties of a reference polypeptide, such as binding to a target antigen, but introduce new additional features or properties, such as a variant polypeptide having a higher affinity for a target antigen than a reference polypeptide, or a variant polypeptide being a humanized version of a reference polypeptide.

[0081] As used herein, the term "amino acid mutation" refers to amino acid replacement, deletion, insertion and modification. As long as the final construct has the desired characteristics, such as reducing the binding to the Fc receptor, any combination of replacement, deletion, insertion and modification can be performed. Amino acid sequence deletion and insertion include N-terminal and / or C-terminal deletion and insertion of amino acid residues. Specific amino acid mutations are amino acid replacements. Amino acid replacements include replacement by non-natural amino acids or by naturally occurring amino derivatives of twenty standard amino acids. Amino acid mutations can be produced using genes or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It can be imagined that methods (such as chemical modification) for changing the side chain groups of amino acid residues by methods other than genetic engineering can also be used. Various names can be used herein to indicate the same amino acid mutation. For example, the glycine at position 237 of the antibody Fc region is replaced with alanine and can be expressed as 237A, G237, G237A or Gly237Ala.

[0082] As used herein, the term "EC50" refers to the concentration of an antibody or antibody fragment that induces a response in an assay halfway between baseline and maximum. Thus, it represents the concentration of antibody or ligand at which 50% of the maximum effect is observed.

[0083] As used herein, the term "Ka" refers to the binding rate of a particular antibody-antigen interaction.

[0084] As used herein, the term "Kd" refers to the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using methods well known in the art.

[0085] As used herein, the term "KD" refers to the dissociation constant of a specific antibody-antigen interaction, which is obtained by 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 bio-layer interferometry using an Octet BLI instrument.

[0086] The term "inhibition" or "inhibit", or "reduction" or "reduce", or "neutralization" or "neutralize" refers to a reduction or cessation of any phenotypic characteristic, such as binding or biological activity or function, or to a reduction or cessation of the incidence, degree or likelihood of that characteristic. "Inhibition", "reduction" or "neutralization" need not be complete, as long as it can be detected using an appropriate assay. In some embodiments, "reduction" or "inhibition" or "neutralization" refers to the ability to cause a 20% or greater reduction. In another embodiment, "reduction" or "inhibition" or "neutralization" means the ability to cause a 50% or greater reduction. In yet another embodiment, "reduction" or "inhibition" or "neutralization" means the ability to cause an overall reduction of 75%, 85%, 90%, 95% or greater.

[0087] 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 or any other phenotypic characteristic of the target antigen.

[0088] A "wild-type" protein is a version or variant of a protein found in nature. The amino acid sequence of a wild-type protein (e.g., the Fc region of a human IgG1 antibody) is the amino acid sequence of the protein as it occurs in nature. Due to allotypic differences, a wild-type protein may have more than one amino acid sequence. For example, there are several naturally occurring allotypes of the constant region of the heavy chain of human IGg1 (see, e.g., Jeffries et al. (2009) mAbs 1:1).

[0089] "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, namely the CH2 domain and the CH3 domain. Although the boundaries of the Fc region of the IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may be present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region is according to the EU numbering system, also referred to as 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 generally used. For a review, see, for example, Antibodies (2020) 9: 64. Silent functions include (according to EU index numbering) LALA (L234A / L235A), PA-LALA (L234A / L235A / P329A) and PG-LALA (L234A / L235A / P329G) mutations, and AEASS mutation (L234A / L235E / G237A / A330S / P331S). The preferred FC modification is PA-LALA.

[0090] Embodiments of the present invention

[0091] Peptides

[0092] In certain embodiments, the present disclosure relates to antibodies or antibody fragments specific to CD117. In certain embodiments, the present disclosure relates to antibodies or antibody fragments specific to human CD117. In certain embodiments, the present disclosure relates to humanized antibodies or antibody fragments specific to CD117. In certain embodiments, the present disclosure relates to humanized antibodies or antibody fragments specific to human CD117.

[0093] 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.

[0094] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment has at least one mutation in which asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid or tyrosine.

[0095] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment has at least one mutation, wherein asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid or tyrosine, and wherein the humanized antibody or antibody fragment has one or more of the following additional mutations:

[0096] a) the first asparagine in the HCDR2 region (SEQ ID NO: 5) is replaced by serine,

[0097] b) the second asparagine in the LCDR3 region (SEQ ID NO:9) is replaced by lysine, and / or

[0098] c) Aspartic acid in the LCDR3 region (SEQ ID NO: 9) was replaced by Glutamic acid.

[0099] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment as described above, wherein the humanized antibody or antibody fragment comprises:

[0100] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0101] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0102] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0103] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 71, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0104] e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0105] f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9,

[0106] g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO:4, a HCDR2 region of SEQ ID NO:5, a HCDR3 region of SEQ ID NO:6, and a variable light chain comprising a LCDR1 region of SEQ ID NO:81, a LCDR2 region of SEQ ID NO:8, and a LCDR3 region of SEQ ID NO:9.

[0107] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment as described above, wherein the humanized antibody or antibody fragment comprises:

[0108] a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98,

[0109] b) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 99,

[0110] c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100,

[0111] d) the variable heavy chain of SEQ ID NO: 70 and the variable light chain of SEQ ID NO: 98,

[0112] e) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 97,

[0113] f) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 61, or

[0114] g) the variable heavy chain of SEQ ID NO:22 and the variable light chain of SEQ ID NO:62.

[0115] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment has at least two of the following mutations:

[0116] a) the asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid or tyrosine, and

[0117] b) Aspartic acid in the LCDR3 region (SEQ ID NO: 9) was replaced by glutamic acid.

[0118] In certain embodiments, the humanized antibody or antibody fragment specific for human CD117 further has one or more of the following mutations:

[0119] a) the second asparagine in the LCDR3 region (SEQ ID NO:9) is replaced by lysine, and / or

[0120] b) The first asparagine in the HCDR2 region (SEQ ID NO: 5) was replaced by serine.

[0121] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment has two of the following mutations:

[0122] a) the asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid, and

[0123] b) Aspartic acid in the LCDR3 region (SEQ ID NO: 9) was replaced by glutamic acid.

[0124] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment has three of the following mutations:

[0125] a) the asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by tyrosine,

[0126] b) the second asparagine in the LCDR3 region (SEQ ID NO:9) is replaced by lysine, and

[0127] c) Aspartic acid in the LCDR3 region (SEQ ID NO: 9) was replaced by Glutamic acid.

[0128] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment has two of the following mutations:

[0129] a) the asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid,

[0130] b) the second asparagine in the LCDR3 region (SEQ ID NO:9) is replaced by lysine, and

[0131] c) Aspartic acid in the LCDR3 region (SEQ ID NO: 9) was replaced by Glutamic acid.

[0132] 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 a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment has two of the following mutations:

[0133] a) the first asparagine in the HCDR2 region (SEQ ID NO: 5) is replaced by serine,

[0134] b) the asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid, and

[0135] c) Aspartic acid in the LCDR3 region (SEQ ID NO: 9) was replaced by Glutamic acid.

[0136] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises a mutation whereby asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid, histidine, glutamine, or serine.

[0137] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises a mutation whereby the second asparagine in the LCDR3 region (SEQ ID NO: 9) is replaced by lysine or arginine.

[0138] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises a mutation whereby aspartic acid in the LCDR3 region (SEQ ID NO: 9) is replaced by glutamic acid.

[0139] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises two of the following mutations:

[0140] The asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid, histidine, glutamine or serine, and

[0141] The second asparagine in the LCDR3 region (SEQ ID NO: 9) was replaced with lysine or arginine.

[0142] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises two of the following mutations:

[0143] The asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid, histidine, glutamine or serine, and

[0144] The aspartic acid in the LCDR3 region (SEQ ID NO: 9) was replaced by glutamic acid.

[0145] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises two of the following mutations:

[0146] The second asparagine in the LCDR3 region (SEQ ID NO:9) is replaced by lysine or arginine, and

[0147] The aspartic acid in the LCDR3 region (SEQ ID NO: 9) was replaced by glutamic acid.

[0148] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises three of the following mutations:

[0149] The asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid, histidine, glutamine or serine,

[0150] The second asparagine in the LCDR3 region (SEQ ID NO:9) is replaced by lysine or arginine, and

[0151] The aspartic acid in the LCDR3 region (SEQ ID NO: 9) was replaced by glutamic acid.

[0152] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises a mutation whereby the first leucine in the LCDR2 region (SEQ ID NO: 8) is replaced by lysine, histidine, glutamic acid, serine, or glutamine.

[0153] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises four of the following mutations:

[0154] The asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid, histidine, glutamine or serine,

[0155] The second asparagine in the LCDR3 region (SEQ ID NO: 9) is replaced by lysine or arginine,

[0156] The aspartic acid in the LCDR3 region (SEQ ID NO:9) is replaced by glutamic acid, and

[0157] The first leucine in the LCDR2 region (SEQ ID NO: 8) is replaced by lysine, histidine, glutamic acid, serine or glutamine.

[0158] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises a mutation whereby the first asparagine in the HCDR2 region (SEQ ID NO: 5) is replaced by serine or glutamine.

[0159] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises four of the following mutations:

[0160] The asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid, histidine, glutamine or serine,

[0161] The second asparagine in the LCDR3 region (SEQ ID NO: 9) is replaced by lysine or arginine,

[0162] The aspartic acid in the LCDR3 region (SEQ ID NO:9) is replaced by glutamic acid, and

[0163] The first asparagine in the HCDR2 region (SEQ ID NO: 5) was replaced by serine or glutamine.

[0164] 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 and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises five of the following mutations:

[0165] The asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid, histidine, glutamine or serine,

[0166] The second asparagine in the LCDR3 region (SEQ ID NO: 9) is replaced by lysine or arginine,

[0167] The aspartic acid in the LCDR3 region (SEQ ID NO:9) is replaced by glutamic acid,

[0168] The first leucine in the LCDR2 region (SEQ ID NO: 8) is replaced by lysine, histidine, glutamic acid, serine or glutamine, and

[0169] The first asparagine in the HCDR2 region (SEQ ID NO: 5) was replaced by serine or glutamine.

[0170] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises:

[0171] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0172] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0173] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0174] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 71, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0175] e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0176] f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0177] g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 121, and a LCDR3 region of SEQ ID NO: 115,

[0178] h) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 123, and a LCDR3 region of SEQ ID NO: 115,

[0179] i) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 125, and a LCDR3 region of SEQ ID NO: 115,

[0180] j) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 127, and a LCDR3 region of SEQ ID NO: 115, or

[0181] k) a variable heavy chain comprising a HCDR1 region of SEQ ID NO:4, a HCDR2 region of SEQ ID NO:107, a HCDR3 region of SEQ ID NO:6, and a variable light chain comprising a LCDR1 region of SEQ ID NO:79, a LCDR2 region of SEQ ID NO:8, and a LCDR3 region of SEQ ID NO:113.

[0182] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises:

[0183] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0184] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0185] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0186] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO:4, a HCDR2 region of SEQ ID NO:71, a HCDR3 region of SEQ ID NO:6, and a variable light chain comprising a LCDR1 region of SEQ ID NO:79, a LCDR2 region of SEQ ID NO:8, and a LCDR3 region of SEQ ID NO:95.

[0187] 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 and a variable light chain, the 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 the 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.

[0188] 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 and a variable light chain, the 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 the 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.

[0189] 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 and a variable light chain, the 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 the 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.

[0190] 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 and a variable light chain, the variable heavy chain comprising the HCDR1 region of SEQ ID NO:4, the HCDR2 region of SEQ ID NO:71, the HCDR3 region of SEQ ID NO:6, and the 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.

[0191] 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 and a variable light chain, the 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 the variable light chain comprising the LCDR1 region of SEQ ID NO:45, the LCDR2 region of SEQ ID NO:8, and the LCDR3 region of SEQ ID NO:113.

[0192] 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 and a variable light chain, the 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 the variable light chain comprising the LCDR1 region of SEQ ID NO:44, the LCDR2 region of SEQ ID NO:8, and the LCDR3 region of SEQ ID NO:113.

[0193] 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 and a variable light chain, the 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 the variable light chain comprising the LCDR1 region of SEQ ID NO:109, the LCDR2 region of SEQ ID NO:8, and the LCDR3 region of SEQ ID NO:113.

[0194] 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 and a variable light chain, the 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 the 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.

[0195] 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 and a variable light chain, the variable heavy chain comprising the HCDR1 region of SEQ ID NO:4, the HCDR2 region of SEQ ID NO:107, the HCDR3 region of SEQ ID NO:6, and the 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.

[0196] 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 and a variable light chain, the variable heavy chain comprising the HCDR1 region of SEQ ID NO:4, the HCDR2 region of SEQ ID NO:107, the HCDR3 region of SEQ ID NO:6, and the 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.

[0197] 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 and a variable light chain, the variable heavy chain comprising the HCDR1 region of SEQ ID NO:4, the HCDR2 region of SEQ ID NO:107, the HCDR3 region of SEQ ID NO:6, and the 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.

[0198] 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 and a variable light chain, the variable heavy chain comprising the HCDR1 region of SEQ ID NO:4, the HCDR2 region of SEQ ID NO:107, the HCDR3 region of SEQ ID NO:6, and the 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.

[0199] 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 and a variable light chain, the variable heavy chain comprising the HCDR1 region of SEQ ID NO:4, the HCDR2 region of SEQ ID NO:107, the HCDR3 region of SEQ ID NO:6, and the 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.

[0200] 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 and a variable light chain, the variable heavy chain comprising the HCDR1 region of SEQ ID NO:4, the HCDR2 region of SEQ ID NO:107, the HCDR3 region of SEQ ID NO:6, and the 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.

[0201] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises:

[0202] a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98,

[0203] b) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 99,

[0204] c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100,

[0205] d) the variable heavy chain of SEQ ID NO: 70 and the variable light chain of SEQ ID NO: 98,

[0206] e) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 105,

[0207] f) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 105,

[0208] g) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 117,

[0209] h) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 118,

[0210] i) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 119,

[0211] j) the variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 120, or

[0212] k) the variable heavy chain of SEQ ID NO: 106 and the variable light chain of SEQ ID NO: 100.

[0213] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises:

[0214] a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98,

[0215] b) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 99,

[0216] c) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 100, or

[0217] d) the variable heavy chain of SEQ ID NO: 70 and the variable light chain of SEQ ID NO: 98.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises 6 CDRs of one of the antibodies disclosed in Table 18.

[0233] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises the six CDRs defined by Kabat of one of the antibodies disclosed in Table 18.

[0234] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises the six CDRs defined by IMGT of one of the antibodies disclosed in Table 18.

[0235] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises the variable heavy chain and variable light chain of one of the antibodies disclosed in Table 18.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment belongs to the IgG1 class.

[0240] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment belongs to the human IgG1 class.

[0241] According to the antibody or antibody fragment of separation of the present disclosure, it may or may not be fused with one or more other amino acid residues, polypeptides or parts. This fusion protein can be prepared in any suitable manner, including genetic methods or chemical methods. The connecting portion may contain a secretory sequence or a leader sequence, a sequence that helps to detect, express, separate or purify, or a sequence that imparts increased protein stability (e.g., in a recombinant production process). Non-limiting examples of potential parts include beta-galactosidase, glutathione-S-transferase, luciferase, T7 polymerase fragment, secretion signal peptide, antibody or antibody fragment, toxin, cytokine, chemokine, reporter enzyme, a part that can bind metal ions such as a polyhistidine tag, a tag suitable for detection and / or purification, a homologous or heterologous binding domain, a part that increases protein solubility, or a part that comprises an enzymatic cleavage site.

[0242] Therefore, 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 clear that such additional moieties may or may not provide additional functions to the antibody, and may or may not modify the properties of the isolated antibodies or antibody fragments according to the present disclosure.

[0243] Nucleic Acids

[0244] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises:

[0245] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0246] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0247] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0248] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 71, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0249] e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0250] f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0251] g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 121, and a LCDR3 region of SEQ ID NO: 115,

[0252] h) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 123, and a LCDR3 region of SEQ ID NO: 115,

[0253] i) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 125, and a LCDR3 region of SEQ ID NO: 115,

[0254] j) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 127, and a LCDR3 region of SEQ ID NO: 115,

[0255] k) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0256] 1) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0257] m) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9,

[0258] n) a variable heavy chain comprising a HCDR1 region of SEQ ID NO:4, a HCDR2 region of SEQ ID NO:5, a HCDR3 region of SEQ ID NO:6, and a variable light chain comprising a LCDR1 region of SEQ ID NO:81, a LCDR2 region of SEQ ID NO:8, and a LCDR3 region of SEQ ID NO:9.

[0259] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises:

[0260] a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98,

[0261] b) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 99,

[0262] c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100,

[0263] d) the variable heavy chain of SEQ ID NO: 70 and the variable light chain of SEQ ID NO: 98,

[0264] e) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 105,

[0265] f) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 105,

[0266] g) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 117,

[0267] h) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 118,

[0268] i) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 119,

[0269] j) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 120,

[0270] k) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 100,

[0271] 1) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 97,

[0272] m) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 61, or

[0273] n) the variable heavy chain of SEQ ID NO:22 and the variable light chain of SEQ ID NO:62.

[0274] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises the six CDRs defined by Kabat of one of the antibodies disclosed in Table 18.

[0275] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises the 6 CDRs defined by IMGT of one of the antibodies disclosed in Table 18.

[0276] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD117, wherein the antibody or antibody fragment comprises a variable heavy chain and a variable light chain of one of the antibodies disclosed in Table 18.

[0277] In one embodiment, the nucleic acid composition and / or the nucleic acid sequence and / or the plurality of nucleic acid sequences are isolated.

[0278] Carrier

[0279] In one embodiment, the present disclosure provides a vector composition comprising a vector or multiple vectors, the vector comprising a nucleic acid composition, the nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding a humanized antibody or antibody fragment specific to human CD117 according to the present disclosure.

[0280] In one embodiment, the present disclosure provides a vector composition comprising a vector or multiple vectors, the vector comprising a nucleic acid composition, the nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding any one of the humanized antibodies or antibody fragments specific to human CD117 disclosed in Table 18.

[0281] Host cells

[0282] In one embodiment, the present disclosure provides a host cell comprising a vector composition, the vector composition comprising a vector or multiple vectors, the vector comprising a nucleic acid composition, the nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding a humanized antibody or antibody fragment specific to human CD117 according to the present disclosure.

[0283] In one embodiment, the present disclosure provides a host cell comprising a vector composition, the vector composition comprising a vector or multiple vectors, the vector comprising a nucleic acid composition, the nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding a humanized antibody or antibody fragment specific to human CD117 disclosed in Table 18.

[0284] 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 a vector composition or a nucleic acid composition.

[0285] In another embodiment, the host cell is an isolated host cell. In another 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 a HKB11 cell.

[0286] Those skilled in the art will recognize that the nucleic acid sequence or multiple nucleic acid 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 into the same vector.

[0287] The vector can be introduced into a suitable host cell, such as a prokaryotic cell (e.g., a bacterium) or a eukaryotic cell (e.g., a yeast or a mammal) by methods well known in the art (see, for example, "Current Protocol in Molecular Biology", Ausubel et al. (eds.), Greene Publishing Assoc and John Wiley Interscience, New York, 1989 and 1992). Many cloning vectors are known to those skilled in the art, and selecting 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 an optional operator (collectively referred to herein as "control" elements) so that the nucleic acid sequence encoding the desired protein is transcribed into RNA in a host cell transformed by a vector containing the expression construct. The coding sequence may or may not contain a signal peptide or a leader sequence. After expression in the host cell, the antibody or antibody fragment of the present disclosure is obtained. As known to those skilled in the art, these steps can be implemented in different ways. In general, such steps typically include transforming or transfecting a suitable host cell with a nucleic acid composition or a vector composition or an infectious particle encoding an antibody or antibody fragment. In addition, such steps are generally included in the culture of the host cell under conditions suitable for the host cell proliferation (amplification, growth), and the culture step under conditions suitable for producing (expressing, synthesizing) the encoded antibody or antibody fragment. Cultivating host cells under conditions suitable for proliferation or expression is generally completed in the presence of a culture medium containing components suitable for cell growth or induced expression. In a particular embodiment, the production method of the antibody or antibody fragment of the present disclosure also includes the step of separating and purifying the antibody or antibody fragment produced from the host cell or culture medium. If the expression system secretes the protein into the growth medium, the protein can be directly purified from the culture medium. If the protein is not secreted, separate from the cell lysate or recover the protein from the cell membrane fraction. The selection of appropriate growth conditions and recovery methods is within the technical scope of the art. The antibody or antibody fragment of the present disclosure can then be purified by a variety of techniques known to those skilled in the art.

[0288] In one embodiment, the present disclosure relates to a method for producing a humanized antibody or antibody fragment specific to human CD117 of any one of the antibodies disclosed in Table 18. In one embodiment, a method for producing an antibody or antibody fragment separated according to the present disclosure is provided, wherein the method includes culturing a host cell comprising a carrier composition under conditions suitable for expression of the antibody or antibody fragment, and separating the antibody or antibody fragment from the host cell or host cell culture medium, wherein the carrier composition includes one or more carriers, and the carrier includes a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding the antibody or antibody fragment according to the present disclosure. As described herein, the separated antibody or antibody fragment can be purified using 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 for purifying a specific antibody or antibody fragment will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and are obvious to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens bound to antibodies or antibody fragments can be used. For example, for affinity chromatography purification of antibodies or antibody fragments according to the present disclosure, a matrix with protein A or protein G can be used. The purity of antibodies or antibody fragments can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like.

[0289] Specificity

[0290] 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 an 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 an amino acid sequence of SEQ ID NO: 1.

[0291] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD117 and cynomolgus CD117.

[0292] 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 with an antibody or antibody fragment comprising a variable heavy chain and a variable light chain for binding to CD117, the 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 the 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.

[0293] 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 and a variable light chain, the 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 the 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.

[0294] 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 and a variable light chain, the 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 the 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.

[0295] 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 about 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 about the same affinity as an antibody or antibody fragment comprising a variable heavy chain and a variable light chain, the 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 the 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.

[0296] 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 and a variable light chain, the 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 the 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.

[0297] 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 and a variable light chain, the 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 the 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.

[0298] 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 and a variable light chain, the 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 the 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.

[0299] 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 and a variable light chain, the 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 the 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.

[0300] 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 CD117 with at least two times greater affinity than an antibody or antibody fragment comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, and wherein the humanized antibody or antibody fragment comprises two of the following mutations:

[0301] a) the asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by glutamic acid, histidine, glutamine or serine, and

[0302] b) Aspartic acid in the LCDR3 region (SEQ ID NO: 9) is replaced by glutamic acid. In certain embodiments, the antibody or antibody fragment binds to CD117 with a three-fold higher affinity, at least five-fold higher affinity, or at least ten-fold higher affinity.

[0303] 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 key deamidation sequence motif less than the number 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 has a key deamidation sequence motif less than the number of antibodies or antibody fragments comprising a variable heavy chain and a variable light chain, the 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 the 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.

[0304] 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 key sequence motif for chemical cleavage that is less than the number 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 has a key sequence motif for chemical cleavage that is less than the number of antibodies or antibody fragments comprising a variable heavy chain and a variable light chain, the 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 the 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.

[0305] Biological Function

[0306] 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 functionally equivalent to 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 is functionally equivalent to an antibody or antibody fragment comprising a variable heavy chain and a variable light chain, the 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 the 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.

[0307] 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 and a variable light chain, the 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 the 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.

[0308] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment inhibits the binding of SCF to CD117. The binding of SCF to CD117 is important for CD117 to transmit functional signals. The binding of CD117 to SCF can be measured by a known system, such as the Octet system, or as described in the experimental section below. 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 and a variable light chain, the 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 the 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.

[0309] 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 to a greater extent than an antibody or antibody fragment comprising a variable heavy chain and a variable light chain, the 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 the 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.

[0310] In certain embodiments, the disclosure relates to humanized antibodies or antibody fragments, wherein the humanized antibodies or antibody fragments inhibit the SCF-dependent proliferation of CD117-positive cells. SCF-dependent proliferation can be tested by any commonly used assay by those skilled in the art, such as cell counting, colony forming unit (CFU) assay, cell titer luminescence (CTG; Promega) and carboxyfluorescein succinimidyl ester (CFSE) cell proliferation assay, or as described in the experimental section below. In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment inhibits the SCF-dependent proliferation of CD117-positive cells to the same extent as an antibody or antibody fragment comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising SEQ ID NO: 4 HCDR1 region, SEQ ID NO: 5 HCDR2 region, SEQ ID NO: 6 HCDR3 region, and the variable light chain comprising SEQ ID NO: 7 LCDR1 region, SEQ ID NO: 8 LCDR2 region and SEQ ID NO: 9 LCDR3 region.

[0311] 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 SCF-dependent proliferation of CD117-positive cells with an IC50 lower than that of an antibody or antibody fragment comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain comprises 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 the variable light chain comprises 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 an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO:4, a HCDR2 region of SEQ ID NO:5, and a HCDR3 region of SEQ ID NO:6, and a variable light chain comprising a LCDR1 region of SEQ ID NO:7, a LCDR2 region of SEQ ID NO:8, and a LCDR3 region of SEQ ID NO:9.

[0312] In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment inhibits the 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), such as described in the experimental section below. In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment inhibits the SCF-dependent phosphorylation of CD117 to the same extent as an antibody or antibody fragment comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising SEQ ID NO: 4 HCDR1 region, SEQ ID NO: 5 HCDR2 region, SEQ ID NO: 6 HCDR3 region, and the variable light chain comprising SEQ ID NO: 7 LCDR1 region, SEQ ID NO: 8 LCDR2 region and SEQ ID NO: 9 LCDR3 region.

[0313] In other embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for CD117 as disclosed herein, wherein the antibody or antibody fragment inhibits SCF-dependent phosphorylation of CD117 to a greater extent than an antibody or antibody fragment comprising a variable heavy chain and a variable light chain, the 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 the 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.

[0314] Effector function

[0315] The Fc region of an immunoglobulin generally confers favorable pharmacokinetic properties to the antibody, such as a prolonged half-life in serum and the ability to induce effector functions by binding to Fc receptors expressed on cells. On the other hand, binding to Fc receptors may also lead to the undesired activation of certain cell surface receptors, resulting in undesired cytokine release and severe side effects upon systemic administration.

[0316] Therefore, for certain treatment situations, it is desirable to reduce or eliminate the normal binding of the wild-type Fc region of an antibody (e.g., a wild-type IgG Fc region) to one or more or all Fc receptors and / or to complement components (e.g., C1q) to reduce or eliminate the ability of the antibody to induce effector functions. For example, it may be desirable to reduce or eliminate the binding of the Fc region of an antibody to one or more or all Fcy receptors (e.g., FcyRI, FcyRIla, FcyRIIb, FcyRIIIa). Effector functions may 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 presenting cell uptake of antigens, binding to NK cells, binding to macrophages, binding to monocytes, binding to polymorphonuclear cells, direct signaling that induces apoptosis, cross-linking of targeted binding antibodies, dendritic cell maturation, or T cell activation.

[0317] Reducing or eliminating the binding of the Fc region to Fc receptors and / or to C1q is usually achieved by mutating a wild-type Fc region, such as an IgG1 Fc region, more particularly a human IgG1 Fc region, generating a variant of the wild-type Fc region or an engineered Fc region (e.g., a variant human IgG1 Fc region). Substitutions that result in reduced binding may be useful. In order to reduce or eliminate the binding properties of the Fc region to Fc receptors, non-conservative amino acid substitutions (i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties) are preferred.

[0318] Therefore, in one embodiment, according to the present disclosure, the isolated antibody or antibody fragment specific for human CD117 comprises a variant Fc region, and when compared with the wild-type Fc region, the binding of the variant Fc region to Fc receptors and / or C1q is reduced or eliminated. In such an embodiment, according to the present disclosure, the isolated antibody or antibody fragment comprises a variant Fc region, which reduces or eliminates the ability of the antibody to induce effector function. In another embodiment, according to the present disclosure, the isolated antibody or antibody fragment does not substantially induce effector function.

[0319] 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 antibody or antibody fragment separated according to the present disclosure does not substantially induce ADCC and / or CDC and / or ADCP. In one embodiment, the antibody or antibody fragment separated according to the present disclosure does not induce ADCC or ADCP in vitro.

[0320] In one embodiment, the variant Fc region of the isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions, which, when compared to the wild-type Fc region, reduce or eliminate the binding of the variant Fc region to one or more Fc receptors and / or to C1q. In one embodiment, the variant Fc region of the isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions, which, when compared to the wild-type Fc region, reduce or eliminate the ability of the antibody to induce effector function. In a specific embodiment, the one or more amino acid substitutions can reduce the binding affinity of the variant Fc region to one or more Fc receptors and / or to C1q by at least 2 times, at least 5 times, at least 10 times, at least 20 times or even at least 50 times when compared to the wild-type Fc region. In an alternative embodiment, 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 function by at least 2 times, at least 5 times, at least 10 times, at least 20 times or even at least 50 times when compared to the wild-type Fc region.

[0321] In one embodiment, the variant 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 variant Fc region of the antibody according to the present disclosure substantially eliminates the ability of the antibody to induce effector function. In one embodiment, the antibody or antibody fragment according to the present disclosure does not substantially induce effector function. 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 function, which means that the level of induced effector function measured in the absence of the antibody is not significantly higher than background.

[0322] In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human FcγRIIIa, FcγRI, FcγRIla and / or FcγRIlb.

[0323] In one embodiment, an isolated antibody or antibody fragment according to the present disclosure comprises a variant human IgG1 Fc region comprising one or more amino acid substitutions compared to a wild-type human IgG1 Fc region. In one embodiment, the one or more amino acid substitutions reduce or eliminate the binding of the variant Fc region to an Fc receptor and / or to C1q and / or reduce the ability of the antibody to induce effector function when compared to a wild-type Fc region.

[0324] Various Fc modifications are commonly used. For a review, see, for example, Antibodies (2020) 9:64. Silent functions include (according to the EU index numbering) LALA (L234A / L235A), PA-LALA (L234A / L235A / P329A) and PG-LALA (L234A / L235A / P329G) mutations, and AEASS mutations (L234A / L235E / G237A / A330S / P331S).

[0325] In certain embodiments, the present disclosure relates to humanized antibodies specific for CD117, wherein the humanized antibodies have a silent Fc region. In certain embodiments, the present disclosure relates to humanized antibodies specific for CD117, wherein the humanized antibodies or antibody fragments are in AEASS format.

[0326] In certain embodiments, the disclosure relates to humanized antibodies specific to CD117, wherein the humanized antibodies include Fc regions, wherein at least the following modifications are present: L234A, L235E, G237A, A330S, P331S (numbered according to the EU index). In certain embodiments, the disclosure relates to humanized antibodies specific to CD117, wherein the humanized antibodies include Fc regions, wherein the following modifications are present: L234A, L235E, G237A, A330S, P331S (numbered according to the EU index).

[0327] In certain embodiments, the disclosure relates to humanized antibodies specific to CD117, wherein the humanized antibody or antibody fragment is in PA-LALA format. In certain embodiments, the disclosure relates to humanized antibodies specific to CD117, wherein the humanized antibody comprises an Fc region, wherein at least the following modifications are present: L234A, L235A, P329A (numbered according to the EU index). In certain embodiments, the disclosure relates to humanized antibodies specific to CD117, wherein the humanized antibody comprises an Fc region, wherein the following modifications are present: L234A, L235A, P329A (numbered according to the EU index).

[0328] In certain embodiments, the disclosure relates to humanized antibodies specific to CD117, wherein the humanized antibody or antibody fragment is in PG-LALA format. In certain embodiments, the disclosure relates to humanized antibodies specific to CD117, wherein the humanized antibody comprises an Fc region, wherein at least the following modifications are present: L234A, L235A, P329G (numbered according to the EU index). In certain embodiments, the disclosure relates to humanized antibodies specific to CD117, wherein the humanized antibody comprises an Fc region, wherein the following modifications are present: L234A, L235A, P329G (numbered according to the EU index).

[0329] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody comprises:

[0330] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0331] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0332] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0333] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 71, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0334] e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0335] f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0336] g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 121, and a LCDR3 region of SEQ ID NO: 115,

[0337] h) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 123, and a LCDR3 region of SEQ ID NO: 115,

[0338] i) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 125, and a LCDR3 region of SEQ ID NO: 115,

[0339] j) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 127, and a LCDR3 region of SEQ ID NO: 115,

[0340] k) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0341] 1) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0342] m) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9,

[0343] n) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9,

[0344] The Fc region of the humanized antibody is silenced.

[0345] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody comprises:

[0346] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0347] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0348] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0349] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 71, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0350] e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0351] f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0352] g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 121, and a LCDR3 region of SEQ ID NO: 115,

[0353] h) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 123, and a LCDR3 region of SEQ ID NO: 115,

[0354] i) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 125, and a LCDR3 region of SEQ ID NO: 115,

[0355] j) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 127, and a LCDR3 region of SEQ ID NO: 115,

[0356] k) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0357] 1) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0358] m) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9,

[0359] n) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9,

[0360] The Fc region of the humanized antibody comprises modifications L234A, L235E, G237A, A330S and P331S (numbering according to the EU index).

[0361] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody comprises:

[0362] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0363] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0364] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0365] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 71, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0366] e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0367] f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0368] g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 121, and a LCDR3 region of SEQ ID NO: 115,

[0369] h) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 123, and a LCDR3 region of SEQ ID NO: 115,

[0370] i) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 125, and a LCDR3 region of SEQ ID NO: 115,

[0371] j) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 127, and a LCDR3 region of SEQ ID NO: 115,

[0372] k) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0373] 1) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0374] m) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9,

[0375] n) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9,

[0376] The Fc region of the humanized antibody comprises modifications L234A, L235A and P329A (numbering according to the EU index).

[0377] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody comprises:

[0378] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0379] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0380] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0381] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 71, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0382] e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0383] f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 115,

[0384] g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 121, and a LCDR3 region of SEQ ID NO: 115,

[0385] h) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 123, and a LCDR3 region of SEQ ID NO: 115,

[0386] i) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 125, and a LCDR3 region of SEQ ID NO: 115,

[0387] j) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 127, and a LCDR3 region of SEQ ID NO: 115,

[0388] k) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 107, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 113,

[0389] 1) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 95,

[0390] m) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, or

[0391] n) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9,

[0392] The Fc region of the humanized antibody comprises modifications L234A, L235A and P329G (numbering according to the EU index).

[0393] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody comprises:

[0394] a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98,

[0395] b) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 99,

[0396] c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100,

[0397] d) the variable heavy chain of SEQ ID NO: 70 and the variable light chain of SEQ ID NO: 98,

[0398] e) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 105,

[0399] f) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 105,

[0400] g) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 117,

[0401] h) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 118,

[0402] i) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 119,

[0403] j) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 120,

[0404] k) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 100,

[0405] 1) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 97,

[0406] m) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 61, or

[0407] n) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 62,

[0408] The Fc region of the humanized antibody is silenced.

[0409] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody comprises:

[0410] a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98,

[0411] b) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 99,

[0412] c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100,

[0413] d) the variable heavy chain of SEQ ID NO: 70 and the variable light chain of SEQ ID NO: 98,

[0414] e) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 105,

[0415] f) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 105,

[0416] g) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 117,

[0417] h) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 118,

[0418] i) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 119,

[0419] j) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 120,

[0420] k) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 100,

[0421] 1) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 97,

[0422] m) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 61, or

[0423] n) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 62,

[0424] The Fc region of the humanized antibody comprises modifications L234A, L235E, G237A, A330S and P331S (numbering according to the EU index).

[0425] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody comprises:

[0426] a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98,

[0427] b) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 99,

[0428] c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100,

[0429] d) the variable heavy chain of SEQ ID NO: 70 and the variable light chain of SEQ ID NO: 98,

[0430] e) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 105,

[0431] f) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 105,

[0432] g) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 117,

[0433] h) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 118,

[0434] i) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 119,

[0435] j) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 120,

[0436] k) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 100,

[0437] 1) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 97,

[0438] m) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 61, or

[0439] n) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 62,

[0440] The Fc region of the humanized antibody comprises modifications L234A, L235A and P329A (numbering according to the EU index).

[0441] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody comprises:

[0442] a) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 98,

[0443] b) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 99,

[0444] c) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 100,

[0445] d) the variable heavy chain of SEQ ID NO: 70 and the variable light chain of SEQ ID NO: 98,

[0446] e) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 105,

[0447] f) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 105,

[0448] g) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 117,

[0449] h) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 118,

[0450] i) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 119,

[0451] j) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 120,

[0452] k) a variable heavy chain of SEQ ID NO: 106 and a variable light chain of SEQ ID NO: 100,

[0453] 1) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 97,

[0454] m) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 61, or

[0455] n) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 62,

[0456] The Fc region of the humanized antibody comprises modifications L234A, L235A and P329G (numbering according to the EU index).

[0457] Treatment

[0458] Antibodies and antibody fragments disclosed herein, or pharmaceutical compositions containing them, can be used to treat various conditions. For example, such antibodies can be used to ablate endogenous hematopoietic stem cells and progenitor cells (HSPC) in a subject in need thereof. Ablation of endogenous HSPC is the initial step of stem cell replacement therapy. Stem cell replacement therapy generally involves reducing or eliminating endogenous HSPC that is defective in some aspects, and replacing them with alternative HSPC. Alternative HSPC can be autologous, allogeneic or xenogeneic. Endogenous HSPC may be defective due to genetic mutations (e.g., sickle cell anemia or thalassemia) that impair function or expression, due to blood cancer or due to damage from chemotherapy for the treatment of cancer. Endogenous HSPC can also be used in combination with organ transplantation because endogenous HSPC can cause immune attacks of transplantation.

[0459] Anti-CD117 antibodies can also be used to treat 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. As determined by immunohistochemical assays, the expression of CD117 is preferably at a higher level than that of tissue-matched normal control cells.

[0460] Isolated antibodies or antibody fragments according to the present disclosure may also be used in other therapeutic approaches, such as treating inflammatory diseases, autoimmune diseases, urticaria, prurigo nodularis, eosinophilic esophagitis, mastocytosis, hematological malignancies, and potentially other diseases.

[0461] In certain embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure in medicine. In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for treating cancer. In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for treating AML. In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for treating solid cancers. In certain embodiments, the solid cancer is mast cell cancer, testicular stromal cancer, gastrointestinal stromal cancer, melanoma, breast cancer or lung cancer.

[0462] In certain embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for treating diseases associated with the undesirable 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 treating diseases associated with the undesirable presence of CD117-positive cells.

[0463] In one embodiment, the disease to be treated is a proliferative disease. In a specific embodiment, the disease is cancer. Non-limiting examples of cancer include hematological malignancies, such as chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), myelodysplastic syndrome, mastocytosis 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.

[0464] In one embodiment, the present disclosure provides a method for treating a disease.

[0465] In one embodiment, the present disclosure provides a method for treating a disease, comprising administering the antibody or antibody fragment of the present disclosure to a patient.

[0466] In one embodiment, the present disclosure provides a method for treating a disease, comprising administering an antibody or antibody fragment of the present disclosure to a subject in need thereof.

[0467] In one embodiment, the present disclosure provides a method for preventing a disease.

[0468] In one embodiment, the present disclosure provides a method for preventing a disease, comprising administering the antibody or antibody fragment of the present disclosure to a subject.

[0469] 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 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 thereof.

[0470] In one embodiment, the present disclosure provides the use of an isolated antibody or antibody fragment according to the present disclosure for the preparation 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 thereof.

[0471] 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 suffering from a disease, the method comprising administering to the subject a therapeutically effective amount of the antibody or antibody fragment according to the present disclosure.

[0472] 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 therapeutic methods, the isolated antibodies or antibody fragments according to the present disclosure will be formulated, dosed, and administered in a manner consistent with good medical practice.

[0473] Pharmaceutical composition

[0474] 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.

[0475] The antibody is administered in an effective regimen, which means a dose, route of administration, and frequency of administration that achieves the desired purpose, such as a reduction in endogenous HSPCs or cancer cells expressing CD117. In some instances, efficacy can be observed in a single patient relative to historical controls or past experience of the same patient. In other instances, efficacy can be demonstrated in a control population of untreated patients in a treated patient population relative to a control population of untreated patients in a preclinical or clinical trial.

[0476] The pharmaceutical composition may further include at least one other pharmaceutically active compound. The pharmaceutical composition according to the present disclosure may be used for diagnosing, preventing and / or treating diseases associated with undesirable CD117 (particularly human CD117). The pharmaceutical composition according to the present disclosure may be used for diagnosing, preventing and / or treating diseases associated with undesirable CD117 positive cells (particularly human CD117 positive cells). In particular, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure, which is suitable for prevention, treatment and / or diagnosis of mammals (more particularly humans).

[0477] Typically, antibodies or antibody fragments according to the present disclosure can be formulated as pharmaceutical compositions comprising at least one antibody or antibody fragment according to the present disclosure and at least one pharmaceutically acceptable carrier or excipient, and optionally one or more additional pharmaceutically active compounds. Such preparations can be suitable for oral, parenteral, topical or administration by inhalation. Therefore, a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure can be administered parenterally, such as intravenously, intramuscularly or subcutaneously. Alternatively, the antibodies of the present invention can be administered by non-parenteral routes, such as by oral or topical administration. In a preferred embodiment, a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure is administered intravenously or subcutaneously.

[0478] 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 to prevent and / or treat diseases involving target antigens of interest, with or without synergistic effects. Examples of such compounds and their routes of administration, methods, and pharmaceutical preparations or compositions will be clear to the clinician.

[0479] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure, for preventing and / or treating a disease associated with undesirable 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 preventing and / or treating a disease associated with undesirable CD117-positive cells (particularly human CD117-positive cells). In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure used as a medicine. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure, for preventing and / or treating autoimmune diseases and / or inflammatory diseases and / or cancer.

[0480] In one embodiment, the present disclosure provides a method of treating an autoimmune disease and / or inflammatory disease and / or cancer in a subject in need thereof using a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure.

[0481] Further provided is a method for producing 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 a method according to the present disclosure, and (b) formulating the antibody or antibody fragment with at least one pharmaceutically acceptable carrier or excipient, thereby formulating a preparation of the antibody or antibody fragment for in vivo administration. A 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.

[0482] Diagnostic Uses

[0483] In one embodiment, the present disclosure provides the use of antibodies or antibody fragments according to the present disclosure specific for human CD117 for diagnosing diseases. In one embodiment, the present disclosure provides the use of antibodies or antibody fragments according to the present disclosure for detecting CD117 (particularly human CD117). In one embodiment, the present disclosure provides a method for detecting CD117 in a subject or sample, including the step of contacting the subject or sample with antibodies or antibody fragments specific for human CD117 according to the present disclosure. In one embodiment, the present disclosure provides a method for diagnosing a disease in a subject, including the step of contacting the subject or sample with antibodies or antibody fragments according to the present disclosure. Antibodies can also be used to determine the expression level of CD117 in cells from patients. CD117 expression levels can be used as therapeutic biomarkers, for example, for patient stratification.

[0484] Example

[0485] Example 1: Anti-CD117 antibodies of the present invention

[0486] 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 US20200165337A1 (see SEQ ID No. 1 and 5 therein).

[0487] The sequences of the variable chains and CDRs are shown in the following table:

[0488] Table 1:

[0489]

[0490] The humanization of SR-1 is described in WO2007 / 127317 and WO2020 / 112687. WO2007 / 127317 uses a classical humanization method to produce an antibody designated as AMG191 by CDR transplantation. No further engineering was performed on AMG191. In addition, WO2020 / 112687 uses a relatively classical approach to introduce certain modifications in the framework region. Therefore, such antibodies still have disadvantages, such as the appearance of unwanted post-translational modification sites or sites that make humanized antibodies prone to breakage. These sites appear within the CDRs of SR-1, and neither of the two mentioned humanization methods is intended to remove these sites, because it is expected that engineering within the CDRs may affect the properties of the antibody, such as affinity. Through a complex method, the inventors of the present application successfully humanized SR-1 while removing unwanted post-translational modifications and breakage sites. Surprisingly, the antibodies produced also did not impair the affinity and activity of the antibodies. This was achieved by a step-by-step approach, as further described in the following examples.

[0491] Example 2: Humanization of mouse antibody SR-1

[0492] The humanization process relies on the combination of CDR grafting technology with the latest research on antibody structure and an updated database of mature human IgG sequences. Many human framework sequences used as "acceptor" frameworks for target CDR sequences were identified. All acceptor sequences are from mature human IgG. Therefore, it is expected that the humanized sequences will not be immunogenic and retain the canonical structure of the CDR loops.

[0493] Example 2.1: Design of humanized variants of variable heavy chain

[0494] The closest human germline V region identified in Homo sapiens is IGHV1-3*01:

[0495] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO. 16).

[0496] A database of human IgG sequences was searched using the BLAST search algorithm for comparison with the mouse VH domains, and candidate variable domains were selected from the top 200 BLAST results. These were reduced to three candidates based on a combination of framework homology, preservation of key framework residues, and canonical loop structure.

[0497] The three receptor frameworks are:

[0498] >QEP24303:

[0499] QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYGISWVRQAPGQGLEWMGWISTYNGDTNYAQKLRGRVTMTTDTSSTAYMELRSLTSDDTAVYYCARDRLSTGTTFYYWGQGTLVTVSS(SEQ ID NO.17)

[0500] >ADWO8092:

[0501] QVQLVESGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQAPGQRLEWMGEINPDNGHTNYNEKFKSRVTITVDKSASTAYMELSSLRSEDTAVYYCAREADYSYGAFDIWGPGTTVTVSS(SEQ ID NO.18)

[0502] >AKU38660:

[0503] QVQLVQSGAEVRKPGASVKLSCKASGYTFTRYSMHWVRQAPGQGLEWMGIlNPSGGSTSYAQNFQGRVTLTRDTSTSTVYMELSSLRSEDTAVYYCTRDRLRNWFDPWGQGTLVTVSS(SEQ ID NO.19)

[0504] Two additional frameworks were designed based on homology and sequence, which allow for larger sequences to achieve more stable products:

[0505] >IGHV3—23*O3:

[0506] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK(SEQ ID NO.20)

[0507] >IGHV5-51*01:

[0508] EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCAR(SEQ ID NO.21)

[0509] The CDRs of the murine VH of SR-1 were transplanted into the above-mentioned receptor frameworks, and the following humanized variants were selected and synthesized:

[0510] >VH1

[0511] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTMTTDTSTSTAYMELRSLTSDDTAVYYCARERDTRFGNWGQGTLVTVSA(SEQ ID NO.22)

[0512] >VH2

[0513] QVQLVESGAEVKKPGASVKLSCKASGYTFTSYNMHWVRQAPGQRLEWMGVIYSGNGDTSYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVTVSA(SEQ ID NO.23)

[0514] >VH3

[0515] QVQLVQSGAEVRKPGASVKLSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTLTRDTSTSTVYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVTVSA(SEQ ID NO.24)

[0516] >VH4

[0517] EVQLLESGGGLVKPGASLRLSCAASGYTFTSYNIHWVRQAPGQGLEWVSAIYSGNVDTSYSQKFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERDTRFGNWGQGTLVTVSA(SEQ ID NO.25)

[0518] >VH5

[0519] EVQLVQSGAEVKKPGESLKISCKGSGYTFTSYNMHWVRQMPGKGLEWMGVIYSGNADTSYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARERDTRFGNWGQGTLVTVSA(SEQ ID NO.26)

[0520] The SEQ ID No. of the variable heavy chain (including its CDR) is shown in the following table. Table 2:

[0521]

[0522] SEQ ID No.27:SYNIH

[0523] SEQ ID No.28:AIYSGNVDTSYSQKFKG

[0524] SEQ ID No.29:VIYSGNADTSYNQKFKG

[0525] SEQ ID No.30: IYSGNVDT

[0526] SEQ ID No.31: IYSGNADT

[0527] Alignment of the humanized variants with the original mouse SR-1 sequence is shown in Figure 1 The homology of the humanized variants to the original mouse VH sequence is shown in the table below.

[0528] Table 3:

[0529] Same amino acid Common amino acids VH1 83.8％ 92.3％ VH2 82.9％ 90.6％ VH3 83.8％ 90.6％ VH4 72.6％ 84.6％ VH5 76.9％ 88.0％

[0530] Example 2.2: Design of humanized variants of variable light chain

[0531] The closest human germline V region identified in Homo sapiens is IGKV3D-11*02:

[0532] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWH (SEQ ID NO. 32).

[0533] A database of human IgK sequences was searched using the BLAST search algorithm for comparison with the mouse VL domains, and candidate variable domains were selected from the top 200 BLAST results. These were reduced to two candidates based on a combination of framework homology, preservation of key framework residues, and canonical loop structure.

[0534] The two acceptor frameworks are:

[0535] >ADU57829:

[0536] EIVLTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIKS(SEQ ID NO.33)

[0537] >CAB46458:

[0538] DIVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPYTFGQGTKLEIK(SEQ ID NO.34)

[0539] The latter framework (CAB46458) was used in two approaches, one with the framework as shown above (VL2), and another with additional mutations introduced (VL3). Three additional variants were also selected based on homology:

[0540] >IGKV3—15*01

[0541] EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP(SEQ ID NO.35)

[0542] >IGKVl—39*01

[0543] DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP(SEQ ID NO.36)

[0544] >IGKV3D—11*01

[0545] EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWH(SEQ ID NO.37)

[0546] The CDRs of the mouse VL of SR-1 were grafted into the above acceptor framework and the following humanized variants were selected and synthesized:

[0547] >VL1

[0548] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIK(SEQ ID NO.38)

[0549] >VL2

[0550] DIVMTQSPSTLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQNNEDPYTFGQGTKLEIK(SEQ ID NO.39)

[0551] >VL3

[0552] DIVMTQSPSTLSASVGDRVTITCRASESVDIYGSSFMHWYQQKPGQPPKLLIYLASNLESGVPSRFSGSGSGTEFTLTISSLEADDVATYYCQQNNEDPYTFGGGTKLEIK(SEQ ID NO.40)

[0553] >VL4

[0554] EIVMTQSPATLSVSPGERATLSCRASESVDIYGQSFMHWYQQKPGQAPRLLIYLASNLESGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQNDEEPYTFGGGTKLEIK(SEQ ID NO.41)

[0555] >VL5

[0556] DIQMTQSPSSLSASVGDRVTITCRASESVDIYGNAFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANEEPYTFGGGTKLEIK(SEQ ID NO.42)

[0557] >VL6

[0558] EIVLTQSPATLSLSPGERATLSCRASESVDIYGQSFMHWYQQKPGQAPRLLIYLASNLESGlPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQNNENPYTFGGGTKLEIK(SEQ ID NO.43)

[0559] The following table shows the SEQ ID No. of the variable light chain including its CDR.

[0560] Table 4:

[0561]

[0562] SEQ ID No.44:RASESVDIYGSSFMH

[0563] SEQ ID No.45:RASESVDIYGQSFMH

[0564] SEQ ID No.46:RASESVDIYGNAFMH

[0565] SEQ ID No.47:ESVDIYGSSF

[0566] SEQ ID No.48:ESVDIYGQSF

[0567] SEQ ID No.49:ESVDIYGNAF

[0568] SEQ ID No.50:QQNDEEPYT

[0569] SEQ ID No.51:QQANEEPYT

[0570] SEQ ID No.52:QQNNENPYT

[0571] SEQ ID No.53:QQNDEEPYTF

[0572] SEQ ID No.54:QQANEEPYTF

[0573] SEQ ID No.55:QQNNENPYTF

[0574] Alignment of the humanized variants with the original mouse SR-1 sequence is shown in Figure 2 The homology of the humanized variants to the original mouse VL sequence is shown in the following table.

[0575] Table 5:

[0576] Same amino acid Common amino acids VL1 82.0％ 88.3％ VL2 80.2％ 88.3％ VL3 83.8％ 91.9％ VL4 79.3％ 90.1％ VL5 78.4％ 87.4％ VL6 81.1％ 89.2％

[0577] Example 2.3: Expression and purification of humanized variants

[0578] DNA encoding the amino acid sequence of each antibody was synthesized and cloned into a mammalian transient expression plasmid, pETE V2 (fusion antibody, 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 by affinity chromatography using prior art chromatography equipment. The purified antibodies were buffer exchanged into phosphate buffered saline solution. The purity of these antibodies was greater than 95% as judged by reduction and denaturation sodium dodecyl sulfate polyacrylamide gel. Antibody concentration was determined by measuring the absorbance at 280 nm.

[0579] Five humanized variable heavy chains were tested in combination with five humanized variable light chains, resulting in a total of twenty-five (25) combinations. Of these 25 combinations, nine were unproducible. These nine combinations included all combinations containing humanized heavy chain VH4 or humanized light chain VL6. The remaining 16 combinations could be produced in sufficient quantities for further characterization.

[0580] Example 2.4: Affinity determination of humanized variants

[0581] IgG antibody was fixed on the biosensor using a suitable capture surface, and the binding of soluble antigen to fixed antibody was monitored by BLI (Octet). The sensorgram obtained was analyzed using the software provided (Fortebio). As antigen, human recombinant CD117 (c-Kit) (Catalog No. 11996-H08H, Batch No. LC14SE0812) from Sino Biological was used. As a pre-experiment, a series of experiments were carried out to optimize the assay parameters. The following parameters were selected:

[0582] Table 6:

[0583] Antibody loading concentration 0.15ug / ml Antigen stability @25°C, shaking @1000rpm >4 hours Running buffer HBS-P + contains 10uM DTT Combination time 900 seconds Combination time 900 seconds Required Kinetic Screening Protocol Modified Antigen screening range (seven concentrations, 3-fold dilution) 100-0.1372nM Interaction / fit model 1:1

[0584] In the main experiment, IgG was first captured using an anti-human Fc biosensor and a kinetic assay was performed. The mAb-captured biosensors were then immersed in wells containing different concentrations of antigen (binding phase), followed by a dissociation step in running buffer. To allow for a double reference correction, the IgG-captured sensor was immersed in a well containing only buffer and a blank sensor was also immersed in the well containing the antigen. This reference provided a way to compensate for both the natural dissociation of the captured IgG and the nonspecific binding of the antigen to the sensor surface. These steps were performed at 25°C with constant shaking at 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.

[0585] All samples were diluted in freshly prepared running buffer. Antibody variants were immobilized on the surface of a series of biosensors using the capture method described. Antigen was passed over the surface to generate a binding reaction. Binding data for IgG:antigen interactions were collected on the biosensors at 25°C. A dilution series of antigen was used in the binding step to globally fit the results and obtain the best values ​​for ka, kd, and KD. The reaction data for antigen binding to surface-immobilized IgG were fit to a 1:1 binding model. The kinetic parameters are summarized in the table below. HC0 LC0 refers to mouse SR-1.

[0586] Table 7:

[0587]

[0588]

[0589] Compared with the original antibody SR-1 (HC0 LC0), five antibodies showed a dissociation constant KD in the range of 2-fold, namely the combinations VH1 / VL1, VH1 / VL2, VH1 / VL3, VH3 / VL1 and VH5 / VL1. Three antibodies even showed an improved dissociation constant KD compared with the original antibody SR-1, namely the combinations VH1 / VL1, VH1 / VL2 and VH1 / VL3.

[0590] Example 2.5: Biophysical properties of five pre-selected antibodies

[0591] It is accepted that protein aggregation along non-native pathways is caused by protein conformational instability or partial unfolding of the protein. The unfolding of the protein exposes hydrophobic core residues, which then form dimers with other partially unfolded monomers, ultimately becoming nucleation sites for mass aggregation. Therefore, conformational stability indicates that the protein has a tendency to aggregate non-natively. Thermal stability is considered to be a feasible method for monitoring protein conformational stability by determining the melting point (Tm) of the protein.

[0592] Differential Scanning Fluorometry (DSF) allows for the monitoring of conformational stability. Multiple studies have found highly correlated data between Differential Scanning Calorimetry (DSC) and DSF, indicating that DSF can successfully monitor the thermal stability of proteins and ultimately non-native aggregation propensity. DSF uses a fluorescent dye that is quenched in an aqueous environment but fluoresces under hydrophobic conditions. Therefore, upon thermal exposure, the antibody begins to lose conformational stability and unfold, exposing the hydrophobic core residues, which can be measured by an increase in the fluorescence signal. A plot of temperature versus fluorescence (melting temperature curve) can then be used to determine the melting temperature (dF / dT curve) of the protein of interest.

[0593] Many antibody molecules exhibit a two-phase melting curve diagram. Published studies have shown that mAbs display unique unfolding characteristics due to the melting transitions of their individual domains (CH2, CH3, and Fab). The thermal stability of IgG molecules is affected by all of these domains. Reported antibody DSF curves show two clearly distinguishable transitions: the lower temperature represents the unfolding of the CH2 domain (Tm1), and the higher temperature represents the melting of the CH3-Fab domain (Tm2). The Fab melting transition is usually well defined, with its peak being up to 2-3 times larger than the peaks of CH2 or CH3.

[0594] For DSF analysis, 5 μl of Sypro Orange (diluted 1 / 200 in water; Sigma) and 45 ul of a 0.3 mg / ml solution of the antibody to be tested were added to a test tube (Bio-Rad; TLS0831). The tube was sealed with an optical flat cap (Bio-Rad; TCS0803) and heated from 20°C to 95°C in 0.5°C increments in an i-Cycler iQ5 real-time PCR detection system (Bio-Rad). Fluorescence changes in the wells were monitored simultaneously with a charge coupled device (CCD) camera. The wavelengths of excitation and emission were 485 nm and 575 nm, respectively. The temperature midpoint Tm of the protein unfolding transition was calculated using the Bio-Rad iQ5 software.

[0595] 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 loaded independently onto a Superdex 200 increase 10 / 300 GL gel filtration column. 50 μl of sample was injected, and the column flow rate was maintained at 0.75 ml / min. The separation step and equilibration step 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).

[0596] Melting curves for all samples were successfully generated. All antibodies tested showed a single phase transition melting curve. Tm values ​​are summarized in the table below.

[0597] All samples were also successfully analyzed by SEC. The peak quality of SEC analysis was good in all samples. All antibodies eluted as one major peak. According to the column calibration, the major peak corresponds to monomeric IgG.

[0598] Table 8:

[0599] Antibody Tm1[℃] Tm2[℃] %monomer Comment VH0 / VL0 74.0 N / A >98 Good peak quality VH1 / VL1 69.3 N / A >98 Good peak quality VH1 / VL2 67.0 N / A >98 Good peak quality VH1 / VL3 68.3 N / A >98 Good peak quality VH3 / VL1 70.3 N / A >98 Good peak quality VH5 / VL1 68.7 N / A >98 Good peak quality

[0600] Example 2.6: Summary and selection of final candidates for further engineering

[0601] All combinations except those containing VH4 and / or VL6 could be successfully expressed and purified. From SDS-PAGE analysis, all antibodies showed adequate purity levels. Under reducing conditions, both the heavy and light chains of the antibodies were visible and observed at expected molecular weights of approximately 50 kDa and 25 kDa, respectively. Under non-reducing conditions, one major band and several minor bands were observed. Additional bands (impurities) may be the result of non-glycosylated IgG and IgG degradation products (e.g., one [partial] light chain, a combination of two heavy chains and one light chain, a combination of two heavy chains, a combination of two heavy chains and one light chain).

[0602] In kinetic (Octet) analysis, combinations VH1 / LC1, VH1 / LC2, VH1 / LC3, VH3 / LC1 and VH5 / LC1 displayed similar binding properties to the control antibody HCO LCO, showing dissociation constants within 2-fold of that of the control antibody.

[0603] The biophysical properties of all antibodies were as good as those of the original SR-1 antibody.

[0604] For further engineering, the antibody VH1 / LC1 was selected, which had an affinity of 0.79 nM, almost 2-fold higher than the original SR-1 antibody (1.47 nM).

[0605] Example 3: Removal of single sites prone to PTM or fragmentation

[0606] Example 3.1: Design of engineered, humanized variants

[0607] In the next engineering step, the antibody was screened for sites that were prone to Fv glycosylation, deamidation, isomerization, fragmentation or other types of post-translational modification (PTM) sequence hotspots. Several such sites were identified in the CDRs of the antibody. See the table below.

[0608] Table 9:

[0609]

[0610]

[0611] In order to remove these harmful amino acid motifs, variants of antibody VH1 / VL1 were generated. As described below, the method of corresponding changes was rationalized through analysis.

[0612] Sequence- and structure-based descriptors were calculated in silico to identify suitable variants that would maintain the structural integrity and biological activity of the antibody while enhancing feasibility, binding affinity, and solubility. 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 library from the Observed Antibody Space database (http: / / opig.stats.ox.ac.uk / webapps / oas / ). Sequence alignments, antibody numbering, and CDR canonical structures were annotated when the variable domain sequences were aligned with the isotype-specific Hidden Markov Model (HMM) described in NatProtoc (2014) 9:2771-83. Based on Nucleic Acid Res (2017) 45: W17-W23 and Bioinformatics (2014) 30: 2733-40 described in the method, the framework method based on the template, the canonical structure modeling of CDR and the random forest machine learning modeling method of the heavy chain loop H3 were used to establish a three-dimensional model. For VH / VL packaging, the template-based method based on the global similarity and identity at the residue position L44 as described in FEBS J. (2011) 278: 2858-66 was used. In the sequence descriptor, the antibody solubility curve is calculated as a linear combination of different physicochemical properties, such as hydrophobicity, static charge at neutral pH, α-helix and β-chain tendency of each amino acid (Nat Struct Biol (1996) 3: 842-8, J Mol Biol (1994) 238: 693-708). To account for the effects of neighboring amino acids, we used a sliding window approach in which the per-residue solubility descriptors were averaged over a window of seven consecutive amino acids and corrected for the occurrence of polar / nonpolar aggregation-prone patterns (J Mol Biol (2000) 296: 961-8; Methods Mol Biol (2022) 2313: 57-113). Although we did not impose specific target values ​​for local and global solubility during the engineering step, we excluded variants that would lead to the formation of covalent aggregates (cysteine) and solvent-exposed hydrophobic patches, as assessed by the relative solvent accessibility per residue calculated on a 3D model of the paired VH / VL domains (F1000 Res (2016) 5: 189). In addition, variants that affected the CDR conformation and, therefore, the antibody binding mode were also excluded.This was done by assessing the contribution of the engineered sites to any established canonical structure (Nat Protocol (2014) 9:2771-83) and by evaluating the expected binding modes of natural and engineered molecules using per-residue probability scores (Bioinformatics (2013) 29:2285-91). In addition, we excluded / prioritized variants that would result in increased / decreased immunogenicity risk, as assessed by comparing the in silico immunogenicity profiles to a large dataset of natural human lineage and therapeutic antibodies of known immunogenicity (ADA frequency). To this end, we employed a re-adaptation of the NetMHCIIpan neural network algorithm to screen natural and variant antibody sequences for the presence of class II-restricted HLA ligands and putative T-cell epitopes (Nucl Acids Res (2020) 48:W449-W454). We calculated global and local immunogenicity profiles and corrected for the presence of human germline-shared HLA ligands that are likely to be tolerogenic or tolerogenic. Higher priority was given to variants that did not carry neo-epitopes (new HLA binders versus native epitopes) or cross-reactive epitopes (ligands recognized by multiple HLA alleles), whereas lower priority was given to variants with higher epitope content in antibody regions with lower immunogenicity scores in the reference antibody dataset.

[0613] The following table summarizes the new variants.

[0614] Table 10:

[0615] Antibody Removal of... exchange VH1 / VL1.2 Deamidation site in LCDR1 NS=>QS VH1 / VL1.3 Deamidation site in LCDR1 NS=>RS VH1 / VL1.4 Deamidation site in LCDR1 NS=>SS VH1 / VL1.5 Deamidation site in LCDR1 NS=>KS VH1 / VL1.6 Deamidation site in LCDR1 NS=>FS VH1 / VL1.7 Deamidation site in LCDR1 NS=>ES VH1 / VL1.8 Deamidation site in LCDR1 NS=>YS VH1 / VL1.9 Deamidation site in LCDR3 NN=>GN VH1 / VL1.10 Deamidation site in LCDR3 NN=>RN VH1 / VL1.11 Deamidation site in LCDR3 NN=>EN VH1 / VL1.12 Deamidation site in LCDR3 NN=>HN VH1 / VL1.13 Deamidation site in LCDR3 NN=>NK VH1 / VL1.14 Isomerization and fragmentation sites in LCDR3 DP=>PP VH1 / VL1.15 Isomerization and fragmentation sites in LCDR3 DP=>EP VH1.2 / VL1 Deamidation site in HCDR2 NG=>SG

[0616] The sequences of the new VH and VL chains are shown below.

[0617] >VL1.2

[0618] EIVLTQSPSSSLSASVGDRVTITCRASESVDIYGQSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV(SEQ ID NO.56)

[0619] >VL1.3

[0620] EIVLTQSPSSSLSASVGDRVTITCRASESVDIYGRSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV(SEQ ID NO.57)

[0621] >VL1.4

[0622] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGSSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV(SEQ ID NO.58)

[0623] >VL1.5

[0624] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGKSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV(SEQ ID NO.59)

[0625] >VL1.6

[0626] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGFSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV(SEQ ID NO.60)

[0627] >VL1.7

[0628] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV(SEQ ID NO.61)

[0629] >VL1.8

[0630] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGYSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTV(SEQ ID NO.62)

[0631] >VL1.9

[0632] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNEDPYTFGQGTKLEIKRTV(SEQ ID NO.63)

[0633] >VL1.10

[0634] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRNEDPYTFGQGTKLEIKRTV(SEQ ID NO.64)

[0635] >VL1.11

[0636] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQENEDPYTFGQGTKLEIKRTV(SEQ ID NO.65)

[0637] >VL1.12

[0638] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEDPYTFGQGTKLEIKRTV(SEQ ID NO.66)

[0639] >VL1.13

[0640] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEDPYTFGQGTKLEIKRTV(SEQ ID NO.67)

[0641] >VL1.14

[0642] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEPPYTFGQGTKLEIKRTV(SEQ ID NO.68)

[0643] >VL1.15

[0644] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEEPYTFGQGTKLEIKRTV(SEQ ID NO.69)

[0645] >VH1.2

[0646] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGSGDTSYNQKFKGRVTMTTDTSTSTAYMELRSLTSDDTAVYYCARERDTRFGNWGQGTLVTVSA(SEQ ID NO.70)

[0647] The following table shows the SEQ ID Nos. of the variable light chains including their CDRs.

[0648] Table 11:

[0649]

[0650]

[0651] SEQ ID No.71:VIYSGSGDTSYNQKFKG

[0652] SEQ ID No.72:IYSGSGDT

[0653] SEQ ID No.73:RASESVDIYGRSFMH

[0654] SEQ ID No.74:ESVDIYGRSF

[0655] SEQ ID No.75:RASESVDIYGKSFMH

[0656] SEQ ID No.76:ESVDIYGKSF

[0657] <h2 style=";text-align:left;direction:ltr">SEQ ID No.77:RASESVDIYGFSFMH<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0658] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.78:ESVDIYGFSF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0659] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.79:RASESVDIYGESFMH<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0660] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.80:ESVDIYGESF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0661] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.81:RASESVDIYGYSFMH<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0662] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.82:ESVDIYGYSF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0663] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.83:QQGNEDPYT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0664] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.84:QQGNEDPYTF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0665] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.85:QQRNEDPYT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0666] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.86:QQRNEDPYTF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0667] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.87:QQENEDPYT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0668] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.88:QQENEDPYTF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0669] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.89:QQHNEDPYT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0670] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.90:QQHNEDPYTF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0671] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.91:QQNKEDPYT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0672] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.92:QQNKEDPYTF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0673] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.93:QQNNEPPYT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0674] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.94:QQNNEPPYTF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0675] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.95:QQNNEEPYT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0676] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.96:QQNNEEPYTF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0677] <h2 style=";text-align:left;direction:ltr"> Example 3.2: Testing of engineered, humanized variants

[0678] The engineered variants were subjected to the same types of analysis and characterization as the initial humanized variants. The experimental setup is described in Example 2.

[0679] All variants could be expressed and purified, but the yields were insufficient for all variants to perform all assays, particularly differential scanning fluorimetry (DSF). The dissociation constant KD of four antibodies increased more than 2-fold compared to the original SR-1 antibody (VH1 / VL1.3, VH1 / VL1.5, VH1 / LC1.10 and VH1 / VL1.11). Five antibodies showed a dissociation constant KD even lower than that of the original antibody SR-1, namely VH1 / LC1.2, VH1 / LC1.4, VH1 / LC1.7, VH1 / LC1.8 and VH1 / LC1.15. The monomer content of three variants was less than 85% (HC1 / LC1.2, HC1 / LC1.10 and HC1 / LC1.11). In DSF, some antibodies showed slightly lower TM1. For some antibodies, TM2 could also be measured. The results are summarized in the table below.

[0680] Table 12:

[0681] Antibody <![CDATA[K D [nM]]]> Tm1[℃] Tm2[℃] %monomer SR-1 0.9 69.0 74.5 90.2 VH1 / VL1 0.4 69.3 N / A 98.0 VH1 / VL1.2 0.3 N / D N / D 81 VH1 / VL1.3 3.5 70.5 75.0 91.3 VH1 / VL1.4 0.5 70.5 N / A 90.1 VH1 / VL1.5 3.7 69.5 76.0 90.2 VH1 / VL1.6 1.2 N / D N / D 95 VH1 / VL1.7 0.2 69.5 N / A 88 VH1 / VL1.8 0.2 N / D N / D 96.7 VH1 / VL1.9 1.3 68.5 81.5 87.8 VH1 / VL1.10 4 68.0 83.0 81.2 VH1 / VL1.11 2.2 67.0 81.5 81.15 VH1 / VL1.12 1.3 69.5 NA 87.5 VH1 / VL1.13 1.4 69.0 81.5 90.3 VH1 / VL1.14 1.4 N / D N / D 93.2 VH1 / VL1.15 0.4 68.5 80.0 86.7 VH1.2 / VL1 1.3 69.0 N / A 99

[0682] Example 3.3: Summary and selection of candidates for further engineering

[0683] In summary, it was possible to remove unwanted motifs from the sequences of humanized variants of SR-1. This was particularly surprising since the sequence motifs were located within the CDR regions. However, the new variants still retained their ability to bind to the target antigen (with approximately the same affinity) 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 acceptable ranges.

[0684] For further engineering, five antibodies (VH1 / VL1.7, VH1 / VL1.8, VH1 / VL1.13, VH1 / VL1.15, and VH1.2 / VL1) were selected that overall showed the most promising properties for further derivatization.

[0685] Example 4: Removal of multiple sites prone to PTM or fragmentation

[0686] Example 4.1: Further engineering, humanized variant design

[0687] Encouraged by the successful removal of deleterious sequence motifs in the CDRs of humanized variants of SR-1, the inventors next investigated whether it would even be possible to remove multiple such motifs without losing binder affinity or function.

[0688] In addition to the sites engineered in Example 3, the γL motif in the variable light chain was also engineered. The following variants were generated.

[0689] Table 13 ("---" indicates that the site has not changed):

[0690]

[0691] Variable light chains VL1.28 and VL1.29 each contain additional L-to-K and N-to-R mutations, respectively.

[0692] The sequences of the new VH and VL chains are shown below.

[0693] >VL1.16

[0694] EIVLTQSPSSSLSASVGDRVTITCRASESVDIYGYSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEEPYTFGQGTKLEIKRTV(SEQ ID NO.97)

[0695] >VL1.17

[0696] EIVLTQSPSSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEEPYTFGQGTKLEIKRTV(SEQ ID NO.98)

[0697] >VL1.18

[0698] EIVLTQSPSSSLSASVGDRVTITCRASESVDIYGYSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV(SEQ ID NO.99)

[0699] >VL1.19

[0700] EIVLTQSPSSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV(SEQ ID NO.100)

[0701] >VL1.25

[0702] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGQSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV(SEQ ID NO.101)

[0703] >VL1.26

[0704] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGSSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV(SEQ ID NO.102)

[0705] >VL1.27

[0706] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGHSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV(SEQ ID NO.103)

[0707] >VL1.28

[0708] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGSSFMHWYQQKPGKAPKLLIYKASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNKEEPYTFGQGTKLEIKRTV(SEQ ID NO.104)

[0709] >VL1.29

[0710] EIVLTQSPSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNREEPYTFGQGTKLEIKRTV(SEQ ID NO.105)

[0711] >VH1.4

[0712] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGQGDTSYNQKFKGRVTMTTDTSSTAYMELRSLTSDDTAVYYCARERDTRFGNWGQGTLVTVSA(SEQ ID NO.106)

[0713] The following table shows the SEQ ID No. of the variable light chain including its CDR.

[0714] Table 14:

[0715]

[0716]

[0717] SEQ ID No.107:VIYSGQGDTSYNQKFKG

[0718] SEQ ID No.108: IYSGQGDT

[0719] SEQ ID No.109:RASESVDIYGHSFMH

[0720] SEQ ID No.110:ESVDIYGHSF

[0721] SEQ ID No.111: KASNLES

[0722] SEQ ID No.112: KAS

[0723] SEQ ID No.113:QQNKEEPYT

[0724] SEQ ID No.114:QQNKEEPYTF

[0725] SEQ ID No.115:QQNREEPYT

[0726] SEQ ID No.116:QQNREEPYTF

[0727] Example 4.2: Testing of further engineered, humanized variants

[0728] The engineered variants were subjected to the same types of analysis and characterization as the initial humanized variants. The experimental setup is described in Example 2.

[0729] All variants could be expressed and purified in sufficient yields. The dissociation constants K of both antibodies were significantly higher than those of the original SR-1 antibody. DFive of the new antibodies (i.e., VH1 / VL1.16, VH1 / VL1.17, VH1 / VL1.19, VH1.2 / VL1.16, and VH1.2 / VL1.17) showed a dissociation constant K D At least two times lower than the original antibody SR-1. The monomer content of all antibodies was above 92%. In SEC, TM2 could be measured for all antibodies. The table below summarizes the results.

[0730] Table 15:

[0731]

[0732]

[0733] Example 4.3: Summary and selection of candidates for further engineering

[0734] In summary, several undesired motifs could even be removed from the sequences of humanized variants of SR-1. At the same time, many variants had affinities even higher than the original SR-1 antibody, and the biophysical properties of the binders remained within the range of SR.1-.

[0735] Several antibodies appear to be particularly preferred (VH1 / VL1.17, VH1 / VL1.19, VH1 / VL1.27 and VH1.4 / VL1.29), which show the most promising properties. In each of these antibodies, at least two deleterious sequence motifs were removed: the deamidation site in 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 in LCDR3 (DP to EP in all mentioned binders). In addition, in VH1 / VL1.19 and VH1 / VL1.27, the deamidation site in LCDR3 was changed from NN to NK, and in VH1.4 / VL1.29, the deamidation site was changed from NN to NR. Furthermore, in binder VH1.4 / VL1.29, the deamidation site in HCDR2 was removed (NG to QG).

[0736] Also particularly preferred are antibodies having affinities as high as or even 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.

[0737] Example 5: Additional sequence changes

[0738] Example 5.1: Design of further engineered, humanized variants

[0739] Based on the results and experience gained so far, further sequence changes were designed. These mutations were intended to further incorporate additional mutations and also increase hydrophilicity.

[0740] The following variants were generated.

[0741] Table 16 (“---” indicates that the site has not changed)

[0742]

[0743] In addition, binders VH1.4 / VL1.30, VH1.4 / VL1.31, VH1.4 / VL1.32, and VH1.4 / VL1.33 contain L-to-H, L-to-H, L-to-H, and L-to-H mutations, respectively, to increase hydrophilicity.

[0744] The sequences of the new VH and VL chains are shown below.

[0745] >VL1.30

[0746] EIVLTQSPSSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYHASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNREEPYTFGQGTKLEIK(SEQ ID NO.117)

[0747] >VL1.31

[0748] EIVLTQSPSSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYEASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNREEPYTFGQGTKLEIK(SEQ ID NO.118)

[0749] >VL1.32

[0750] EIVLTQSPSSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYSASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNREEPYTFGQGTKLEIK(SEQ ID NO.119)

[0751] >VL1.33

[0752] EIVLTQSPSSSLSASVGDRVTITCRASESVDIYGESFMHWYQQKPGKAPKLLIYQASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNREEPYTFGQGTKLEIK(SEQ ID NO.120)

[0753] The following table shows the SEQ ID No. of the variable light chain including its CDR.

[0754] Table 17:

[0755]

[0756] SEQ ID No.121:HASNLES

[0757] SEQ ID No.122:HAS

[0758] SEQ ID No.123: EASNLES

[0759] SEQ ID No.124: EAS

[0760] SEQ ID No.125:SASNLES

[0761] SEQ ID No.126:SAS

[0762] SEQ ID No.127:QASNLES

[0763] SEQ ID No.128:QAS

[0764] Example 5.2: Testing of further engineered, humanized variants

[0765] The engineered variants were subjected to the same types of analysis and characterization as the initial humanized variants. The experimental setup is described in Example 2.

[0766] All variants could be expressed and purified in adequate yields. The antibodies showed dissociation constants K in the range of the original SR-1 antibody. D Measured affinity. Many antibodies even have increased affinity. The biophysical properties of the antibodies are good.

[0767] Example 5.3: Summary and selection of candidates for further engineering

[0768] In summary, even several unwanted motifs could be removed from the sequences of humanized variants of SR-1. At the same time, the affinities of many variants were 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 SR-1.

[0769] Certain antibodies appear to be particularly preferred (VH1 / VL1.17, VH1 / VL1.19, VH1 / VL1.27, and VH1 / LC1.29), which show the most promising properties. In each of these antibodies, deleterious sequence motifs were removed: deamidation sites in LCDR1 (NS to ES in VH1 / VL1.17, VH1 / VL1.19, and VH1.4 / VL1.29, and NS to HS in VH1 / VL1.27), deamidation sites in LCDR3 (NN to NK in VH1 / VL1.19 and VH1 / VL1.27, and NN to NR in VH1.4 / VL1.29), and isomerization / fragmentation sites in LCDR3 (DP to EP in all of the above binders). In addition, in binder VH1.4 / VL1.29, the deamidation site in HCDR2 was removed (NG to QG).

[0770] Example 6: Summary of Antibody Sequences Disclosed in This Patent

[0771] The following table summarizes the key antibodies produced and tested in the present disclosure. The VH sequence and VL sequence of the antibody are shown. Each VH sequence, VL sequence and CDR sequence can be found in the previous examples.

[0772] Table 18:

[0773]

[0774]

[0775] Example 7: Antibody-mediated inhibition of SCF-dependent proliferation

[0776] SCF-dependent proliferation is a function of CD117. The ability of the humanized antibodies of the present disclosure to inhibit SCF-dependent proliferation was tested compared to the original SR-1 antibody and some other prior art antibodies. In addition to the SR-1 antibody described above, the antibodies in Table 19 were also tested and compared to the antibodies of the present disclosure.

[0777] Table 19:

[0778]

[0779]

[0780] NegCtrl is a human IgG1 control antibody specific for chicken lysozyme. AMG191 and FSI-174 are human IgG1 antibodies specific for human CD117. AMG191 is a humanized derivative of SR-1, but the six CDRs are the same as SR-1, that is, AMG191 still contains key sites in the CDRs, which have been removed in the humanized antibodies disclosed herein. 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. These changes were introduced to make them closer to the human germline sequence, thereby increasing the humanization of the antibody. A change introduced in FSI-174 simultaneously removed the deamidation site in LCDR1. However, the LCDR3 of FSI-174 is the same as the LCDR3 of SR-1, that is, the deamidation hotspot and isomerization / fragmentation hotspot in LCDR3 still exist in FSI-174. The CDRs (according to Kabat) of SR-1, AMG191 and FSI-174 are shown in Table 20.

[0781] Table 20:

[0782]

[0783] SCF-dependent proliferation was measured as follows: TF-1 cells were seeded at a cell concentration of 150,000 cells / ml in sterile leukocyte culture treated plates in RPMI medium + 10% FCS. Cells were treated with 100 ng / ml of SCF and titrations of various CD117 antibodies or isotype control antibodies at different concentrations. After 3 days of incubation, CellTiter-Glo 2.0 assay (Promega) was performed and luminescence was read in a luminometer (Envision, Perkin Elmer) and IC50 was determined. The results are shown in Table 21.

[0784] Table 21:

[0785]

[0786]

[0787] In this SCF-dependent proliferation assay, several antibodies generated had IC50s that were within the range of or even lower than those of the original SR-1 antibody and its known humanized derivatives.

[0788] Example 8: Antibody-mediated inhibition of SCF-dependent phosphorylation

[0789] SCF-dependent phosphorylation is another function of CD117. Antibody-mediated inhibition of SCF-dependent phosphorylation was measured using TF-1 cells expressing CD117. TF-1 cells were seeded at a concentration of 1 million cells / mL in 2 mL of culture medium (RPMI1640 supplemented with GlutaMAX + 10% heat-inactivated FBS) in 6-well plates. Cells were pretreated with 0.005 ug / mL, 0.05 ug / mL, and 0.5 ug / mL of antibody for 30 minutes, and then 100 ng / mL of recombinant human SCF (1 mg / mL) was added within 5 minutes. Recombinant human SCF). The cells were collected, washed with ice-cold PBS, and then resuspended in 500 μL of lysis buffer (lysis buffer from signal transduction 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. Undiluted samples were prepared as described in Thaw and process according to the manufacturer's protocol of Phospho-c-Kit (Tyr719) Sandwich ELISA Kit (Cell Signaling) and Human c-Kit (CD117) ELISA Kit. Absorbance (OD_450nm) was assessed using Envision (Perkin Elmer). The antibodies tested were the same as those in Example 7. The results are shown in Figure 3 as shown in .

[0790] As can be seen, most of the antibodies of the disclosure tested inhibited SCF-dependent phosphorylation at least as strongly as the SR-1 antibody and the humanized derivatives AMG191 and FSI-174.

[0791] Example 9: Inhibition of SCF binding in TF-1 cells

[0792] In this experiment, it was tested whether the antibodies of the present disclosure were able to inhibit the binding of SCF to CD117.

[0793] To this end, 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 test antibody at a fixed concentration of 50 μg / ml. After 15 minutes, a mixture of biotinylated SCF (Acro-Biosystems) and streptavidin PE (Invitrogen) at different concentrations was added. After a further 30 minutes, the cells were washed and live-dead staining was performed by 7AAD (7-aminoactinomycin D) (BioLegend) using 0.5 μg / mL of 7AAD for 10 minutes, and then the cells were analyzed in a flow cytometer. The following antibodies were tested: NegCtrl, SR-1, AMG191, FSI-174, Dufour, Matterhorn, Piz Bernina, Allalinhorn, Piz Zupo, Eiger, Piz Argient, Piz Roseg, Bietschhorn, and Bellavista. Figure 4 as shown in .

[0794] Except for Piz Roseg and Piz Argient, all antibodies disclosed herein effectively inhibited the binding of SCF to CD117. NegCtrl antibody (anti-chicken lysozyme) did not inhibit the binding of SCF to CD117.

[0795] Example 10: Binding of Antibodies to CD117 in TF-1 Cells

[0796] Binding of the disclosed antibodies 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, cells were treated with different concentrations of the antibodies to be tested (0.0001-5 μg / ml). After another 30 minutes, cells were washed and secondary antibodies (IgG (H+L) cross-adsorbed goat anti-human Alexa Fluor 500 antibody) were added to the cells. 488). After 30 min and two additional washing steps, live-dead staining was performed by 7AAD (7-aminoactinomycin D) using 0.5 μg / mL 7AAD for 10 min, and the cells were then analyzed in a flow cytometer. Figure 5 as shown in .

[0797] All antibodies disclosed herein, except Piz Roseg and Piz Argient, strongly bind to CD117 on TF-1 cells. Binding of Piz Roseg and Piz Argient could also be detected, although only at higher antibody concentrations. NegCtrl antibody (anti-chicken lysozyme) did not show any binding to TF-1 cells.

[0798] Example 11: Inhibition of SCF Binding in Hematopoietic Stem Cells

[0799] In this experiment, it was tested whether the antibodies of the present disclosure were able to inhibit the binding of CD117 to SCF in HSPCs (CD34+CD38- cells) and HSCs (CD34+CD38-CD90+CD45RA- cells).

[0800] Experiments were performed in low cytokine medium and high cytokine medium. Low cytokine medium consisted 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). High cytokine medium consisted of the same ingredients plus 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. Results are shown in Table 1. Figure 6 as shown in .

[0801] The same results were obtained for both cell populations tested and for both culture media. Antibodies SR-1 and Allalinhorn both completely inhibited the binding of SCF to CD117 and to the same extent. NegCtrl antibody (anti-chicken lysozyme) did not inhibit the binding of SCF to CD117.

[0802] Example 12: Proliferation Inhibition in HSPCs and HSCs

[0803] SCF-dependent proliferation was also measured in mobilized CD34-positive HSPCs isolated from peripheral blood (CliniMACS Prodigy, Miltenyi Biotech). Cells were seeded in sterile leukocyte culture medium treated plates at a cell concentration of 75,000 cells / ml in complete Stemspan AOF medium containing cytokines (100ng / ml SCF; FLT3, TPO and IL-3), with a transparent bottom. Cells were treated with antibodies of the present disclosure at different concentrations. After incubation for 3 days, CellTiter-Glo 2.0 was measured (Promega), and luminescence was read in a photometer (Envision, Perkin Elmer), and IC50 was determined. The results are shown in Table 22.

[0804] Table 22:

[0805] Antibody IC50[μg / ml] NegCtrl >10 SR-1 0.0623 AMG191 0.0954 FSI-174 0.02532 Dufour 0.09488 Matterhorn 0.07156 Piz Bernina 0.00521 Allalinhorn 0.0313 Piz Zupo 0.1139 Eiger 0.5935 Piz Argient >10 Piz Roseg >10 Bietschhorn 0.08423 Bellavista 0.09599

[0806] The results obtained using CD34-positive HSPCs correlated well with those obtained using TF-1 cells. In this SCF-dependent proliferation assay, the IC50s of several antibodies generated were within the range of or even lower than those of the original SR-1 antibody and its known humanized derivatives.

[0807] For human CD34+HSC (different donors), cells were seeded in complete Stemscpan AOF medium containing cytokines at a cell density of 75,000 cells / ml in sterile leukocyte culture treated plates: 100ng / ml SCF, 100ng / ml TPO, 60ng / ml IL-3 and 100ng / ml FLT-3 ligand. HSC was then treated with various CD117 antibodies or isotype control antibodies at different concentrations, and the plates were incubated for 3 days. After 3 days, CellTiter-Glo2.0 was measured (Promega) and luminescence was read in a photometer (e.g., Envision microplate reader).

[0808] Example 13: HSC depletion experiment

[0809] Pang et al, (Blood (2019) 133:2069-78) adapted a method to deplete human HSCs in mice using various mAbs.

[0810] NBSGW mice (Jackson Laboratories) were injected with 1 Mio of HSPCs. On days 8, 10, 12, and 14 after cell injection, mice were intravenously injected with 25 mg / kg of the antibody Allalinhorn per dose. After 16 weeks, mice were euthanized, and blood, spleen, and bone marrow were analyzed by FACS. Fig.10 This figure shows the in vivo depletion of HSCs after Allalinhorn injection compared to animals receiving an isotype control antibody. HSCs were identified by FACS as live / hCD45+ / CD34+ / CD38- / CD45RA- / CD90+.

[0811] In another experiment, NSG mice (Jackson Laboratories) were sublethally irradiated one day before injection with 1 Mio gene-edited HSPCs carrying the E73K variant of CD117. On days 8, 10, 12, and 14 after cell injection, mice were intravenously injected with 25 mg / kg of the antibody Allalinhorn or 25 mg / kg of an isotype control antibody per dose. After 16 weeks, mice were euthanized, and blood, spleen, and bone marrow were analyzed by FACS.

[0812] Bone marrow results Fig.11 As shown in . CD117+ myeloid cells were identified by FACS as live / hCD45+ / CD33+ / CD117+. To identify CD117+ cells, antibody clone 104D2 was used, 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-).

[0813] Fig.11 Depletion of unedited cells in mice that received the antibody Allaninhorn compared to mice that received an isotype control antibody is shown. In contrast, injection of Allaninhorn resulted in enrichment of E73K variant cells compared to animals that received an isotype control antibody.

[0814] Example 14: Removal of key sites in CDR has a beneficial effect on antibody stability

[0815] The positive effects of removing critical sites were tested by subjecting the antibodies to different stress conditions, such as heat stress and pH stress.

[0816] For heat stress, the antibody solution was incubated at 50°C in the dark for 5 days. For pH stress conditions, the antibody solution was diluted to 1 mg / ml in PBS and the buffer was exchanged with 20 mM citrate buffer pH 3 or 20 mM Tris buffer pH 9. The low pH and high pH samples were then incubated in the dark at room temperature for 1 day, 3 days and 7 days. At the end of the incubation period, the stressed samples were neutralized by diluting 1:2 in 200 mM phosphate buffer at pH 7.4 and stored at -80°C. All samples were then analyzed by high performance size exclusion chromatography (HP-SEC), cation exchange chromatography (CEX), CE-SDS and / or RP-HPLC and peptide mapping to determine chemical and physical degradation. In order to evaluate the effect of forced degradation on the biological activity of the molecule, the stressed antibodies were also measured for binding to CD117.

[0817] Example 15: Antibody-mediated inhibition of HSPC colony formation

[0818] The influence of antibody on HSPC colony formation was tested as follows.HSPC was laid on methylcellulose containing hematopoietic cytokines (MethoCultGF H84435) (500 cells per 35mm culture dish, in duplicate), and in the presence of anti-CD117 antibodies SR-1, Dufour and Allalinhorn, grown for 14 days at 37°C, 5% CO2. Antibodies were added to the culture medium once at the specified final concentration (0.1ug / mL, 1ug / mL and 10ug / mL) during plating. Colonies were manually counted using an inverted microscope (and grid scoring culture dish).

[0819] The results are as follows Figure 7 All three anti-CD117 antibodies tested resulted in a dose-dependent reduction in colonies. Antibody Allalinhorn caused the most dramatic reduction in colony formation.

[0820] Example 16: SCF-dependent phosphorylation of CD117

[0821] TF-1 cells (wild type, knockout and variants E73K, D121K and S123K of CD117) were seeded at a concentration of 1 million cells / mL in 2 mL of culture medium (RPMI1640 supplemented with GlutaMAX + 10% heat-inactivated FBS) in 6-well plates. Before cell collection, cells were treated with 1-20 ng / mL of recombinant human SCF (1 mg / mL of PeproGMP recombinant human SCF) for 5 minutes. The cells were 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 quickly frozen in liquid nitrogen, thawed twice at 37°C, and then frozen at -80°C until further use. Undiluted samples were thawed and processed according to the manufacturer's protocol of the PathScan Phospho-c-Kit (Tyr719) Sandwich ELISA Kit (Cell Signaling). The absorbance (OD_450 nm) was assessed using Envision (Perkin Elmer).

[0822] An exemplary result for variant E73K is shown in Figure 8 As shown. Wild-type TF-1 cells and E73K, D121K and S123K variants of CD117 show SCF-dependent phosphorylation at position Tyr719 of CD117. Phosphorylation levels in knockout cells are at background levels.

[0823] Example 17: Retained signaling of CD117 variants in the presence of blocking antibodies

[0824] 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 culture medium (RPMI1640 supplemented with GlutaMAX + 10% heat-inactivated FBS) in 6-well plates. 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 of PeproGMP recombinant human SCF) was added within 5 minutes. The cells were collected, washed once with ice-cold PBS, and resuspended in 500 μL of lysis buffer (lysis buffer from signaling containing protease inhibitors and phosphatase inhibitors). The cells were then quickly frozen in liquid nitrogen, thawed twice at 37°C, and then frozen at -80°C until further use. 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 Human c-Kit (CD117) ELISA Kit. The absorbance (OD_450nm) was assessed using Envision (Perkin Elmer).

[0825] Exemplary results for variants D121K and S123K are shown in Fig. 9 As shown in . SCF-dependent CD117 phosphorylation in wild-type TF-1 cells was blocked by antibodies Piz Bernina and Allalinhorn, whereas it was not affected in TF-1 cells with the D121K and S123K variants of CD117 phosphorylation. The same was observed for the variants E73K, E73Y and S123F (data not shown).

[0826] Example 18: CD117 variants are resistant to ADC treatment

[0827] In this experiment, DF-1 cells were used. DF-1 cells lack CD117. Cells were transfected with selected CD117 variants (E73Y, D121K and S123K). Transfected cells were treated with ADC (antibodies PizBernina and Allalinhorn, both coupled to teserine) at concentrations of 0.1 μg / ml, 1 μg / ml and 10 μg / ml. After 48 hours, cells were analyzed by FACS.

[0828] The results are as follows Fig.12D121K A5 and D121K A6 represent two different batches of plasmids of the same variant. Both the Piz Bernina-teserine and Allalinhorn-teserine ADCs tested resulted in efficient depletion of wild-type DF-1 cells, but this was not the case for DF-1 cells transfected with the D121K or S123K variants of CD117.

[0829] Example 19: Additional HSC depletion experiments

[0830] Pang et al, (Blood (2019) 133:2069-78) adapted the use of various mAbs to deplete human HSCs in mice.

[0831] NBSGW mice (Jackson Laboratories) were injected with 1Mio HSPCs from 2 different donors carrying E73K, S123K, or D121K variants of CD117. Control mice received unedited electroporated control HSPCs. Mice received 4 mg / kg of the antibody Allalinhorn intravenously at 7, 9, 11, and 12 days after cell injection. After 16 weeks, mice were euthanized and blood, spleen, and bone marrow were analyzed by FACS. Fig.13 Results of depletion of CD117 unedited cells and enrichment of CD117 edited cells are shown in . To identify CD117+ cells, antibody clone 104D2 was used, 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-).

[0832] Fig.13 Depletion of unedited CD34+ progenitor cells (gated as live / hCD45+ / CD34+ / CD38-) in mice receiving the antibody Allalinhorn compared to mice receiving the isotype control antibody is shown. In contrast, injection of Allalinhorn resulted in enrichment of E73K, D121K, and S123K edited variant cells compared to animals receiving the isotype control antibody.

Claims

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

2. The humanized antibody or antibody fragment according to claim 1, wherein 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 replaced by serine, b) the second asparagine in the LCDR3 region (SEQ ID NO: 9) is replaced by lysine, or c) Aspartic acid in the LCDR3 region (SEQ ID NO: 9) is replaced by Glutamic acid.

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

9.

4. The humanized antibody or antibody fragment according to any one of claims 1 to 3, wherein the humanized antibody or antibody fragment comprises: a) a variable heavy chain of SEQ ID NO: 22 and a 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) a variable heavy chain of SEQ ID NO: 22 and a 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.

5. A humanized antibody according to any one of the preceding claims, wherein the antibody is of human IgG1 class, optionally wherein the antibody is a silent IgG1 antibody, such as an antibody with a PA-LALA mutation.

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

7. A humanized antibody or antibody fragment according to any one of the preceding claims, 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 and a variable light chain, wherein the variable heavy chain comprises a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprises a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO:

9.

8. The humanized antibody or antibody fragment of any of the preceding claims, wherein the antibody or antibody fragment binds to the same epitope on CD117 as an antibody or antibody fragment comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain comprises a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and the variable light chain comprises a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO:

9.

9. The humanized antibody or antibody fragment according to any one of the preceding claims, wherein the antibody or antibody fragment is a monoclonal antibody or antibody fragment.

10. A humanized antibody or antibody fragment according to any one of the preceding claims for use in medicine.

11. The humanized antibody or antibody fragment according to any one of the preceding claims, wherein the use in medicine is the treatment of cancer, such as a blood cancer or a solid cancer, or the treatment of an inflammatory disease, an autoimmune disease, urticaria, prurigo nodularis, eosinophilic esophagitis or mastocytosis.

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