Compositions and methods for reducing CD117+ cells

Abstract

To provide antibodies useful for reducing CD117+ cells and treating various hematopoietic diseases, metabolic disorders, cancers (e.g., acute myeloid leukemia (AML)) and autoimmune diseases.SOLUTION: Provided is an isolated anti-CD117 antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising a CDR1 comprising a first specified amino acid sequence, a CDR2 comprising a second specified amino acid sequence, and a CDR3 comprising a third specified amino acid sequence; and a light chain variable region comprising a CDR1 comprising a fourth specified amino acid sequence, a CDR2 comprising a fifth specified amino acid sequence, and a CDR3 comprising a sixth specified amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application is a continuation of U.S. Provisional Application No. 62 / 576,572, filed October 24, 2017; U.S. Provisional Application No. 62 / 596,569, filed December 8, 2017; and U.S. Provisional Application No. 62 / 632,967, filed February 20, 2018. No. 62 / 638,053, filed March 2, 2018. The contents of the prior application are incorporated herein by reference.

[0002] The present invention provides a method for treating, in particular, blood diseases, metabolic disorders, and the like, by administering anti-CD117 antibodies, antibody-drug conjugates (ADCs), and antigen-binding fragments thereof, as well as antibodies or antibody-drug conjugates (ADCs) capable of binding to antigens expressed by hematopoietic cells such as hematopoietic stem cells. The present invention relates to methods for treating patients suffering from a variety of conditions, such as cancer, autoimmune diseases, and the like. [Background technology]

[0003] Despite advances in medical technology, there remains a need to treat hematopoietic conditions, particularly those involving certain blood cell diseases, metabolic disorders, cancer, and autoimmune conditions. While hematopoietic stem cells have significant therapeutic potential, limitations associated with the difficulty of hematopoietic stem cell transplants in reliably engrafting in the host have hindered their clinical use.

[0004] There is currently a need for compositions that specifically target endogenous stem cells and can be used as conditioning agents to promote engraftment of exogenous hematopoietic stem cell grafts, such that the pluripotency and hematopoietic functionality of these cells is preserved in patients after transplantation.

[0005] CD117 (also called c-kit or Stem Cell Factor Receptor (SCFR)) cKIT (pronounced c-kit) is a single transmembrane receptor tyrosine kinase that binds to its ligand, stem cell factor (SCF). SCF induces the homodimerization of cKIT, enhancing its tyrosine kinase activity. activating the PI3-AKT pathway and signaling through both the PI3-AKT and MAPK pathways (Kindblom et al., Am J. Path.1998 152(5):1259).

[0006] CD117 was originally discovered as an oncogene and has been studied in the field of oncology (see, e.g., Stankov et al. (2014) Curr Pharm Des. 20(17):2849-80). An antibody-drug conjugate (KTN0158) targeting CD117 is currently being investigated for the treatment of refractory gastrointestinal stromal tumors (GIST) (see, e.g., "KTN0158, a humanized anti-KIT monoclonal antibody, demonstrates biological activity"). against both normal and malignant canine mast cells” London et al. (2016) Clin Cancer Res DOI: 10.1158 / 1078-0432.CCR-16-2152).

[0007] CD117 is highly expressed on hematopoietic stem cells (HSCs). This expression pattern allows CD117 to be widely expressed. It is a potential target for conditioning across a range of diseases. However, there remains an unmet need for anti-CD117-based therapies that are effective in conditioning patients for transplantation, such as bone marrow transplantation. Summary of the Invention [Problem to be solved by the invention]

[0008] Antibodies and antigen-binding portions thereof that specifically bind to human CD117 (also known as c-kit), and Described herein are compositions and methods for using said antibodies. In particular, the antibodies and fragments described herein are used in anti-CD117 antibody-drug conjugates (ADCs). It is possible. [Means for solving the problem]

[0009] In certain embodiments, the present invention provides compositions and methods for directly treating various disorders of the hematopoietic system, metabolic disorders, cancer, and autoimmune diseases. Additionally, the present invention features methods for conditioning a patient, such as a human patient, prior to undergoing hematopoietic stem cell transplantation therapy to promote hematopoietic stem cell transplant graft survival. The patient may be suffering from one or more blood disorders, such as a hemoglobinopathy or other hematopoietic condition, and the following may be used: Hematopoietic stem cells are capable of differentiating into multiple cell types of the hematopoietic lineage and may be administered to patients to populate or repopulate deficient cell types in the patient. The present invention provides a method for treating, in particular, the hematological disorders described herein by (i) selectively reducing the population of cells expressing CD117 (e.g., abnormal blood cells, cancer cells, or autoimmune cells). (ii) directly treat a disease, such as a metabolic disease, cancer, or autoimmune disease, and / or (iii) deplete the population of endogenous hematopoietic stem cells in said patient, using antibodies and antibody-drug conjugates capable of binding to proteins expressed by hematopoietic cells, such as CD117 (including, for example, GNNK+ CD117, etc.). The present invention features methods for treating patients with anti-cancer drug derivatives (ADCs). CD117 can be expressed by autoimmune lymphocytes, such as cancer cells and T-cells expressing T-cell receptors that cross-react with self-antigens, and the activity of the former may result in the proliferation of cells of the hematopoietic lineage. This allows for the direct treatment of a wide range of related disorders. The latter activity (selectively depleting hematopoietic stem cells) creates a vacancy that can then be filled by exogenous (e.g., The graft is filled by transplanting hematopoietic stem cells (e.g., autologous, allogeneic, or syngeneic). Thus, the present invention relates to, inter alia, the treatment of sickle cell anemia, thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosine deaminase deficiency-severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome. Schwachman-Diamond syndrome, human immunodeficiency virus infection, and acquired immunodeficiency syndrome The present invention provides methods for treating various hematopoietic conditions, such as syndromes, as well as cancer and autoimmune diseases.

[0010] In one embodiment, the present invention provides antibodies, antigen-binding fragments thereof, capable of binding to CD117. A method is provided for reducing the population of CD117+ cells in a human patient by administering an effective amount, wherein the antibody or fragment is conjugated to a cytotoxin (forming an ADC).

[0011] In another aspect, the present invention provides a method of reducing the population of CD117+ cells in a human patient in need of hematopoietic stem cell transplantation by administering to the patient an effective amount of an antibody or antigen-binding fragment thereof (ADC) conjugated to a cytotoxin and capable of binding to CD117 prior to receiving the transplant comprising hematopoietic stem cells.

[0012] In another aspect, the invention features a method of treating a human patient in need of hematopoietic stem cell transplantation, e.g., comprising administering to the human patient a transplant comprising hematopoietic stem cells, wherein the patient is administered an antibody or antigen-binding fragment thereof capable of binding to CD117 (e.g., a cytotoxin-linked antibody or antigen-binding fragment thereof). and (forming ADCs) to reduce the population of CD117+ cells in the patient. The drug is administered in advance in an amount sufficient to cause the

[0013] In a further aspect, the invention features a method of treating, e.g., a human patient in need of a hematopoietic stem cell transplant, comprising: a cytotoxin-conjugated antibody that binds to CD117; The resulting antibody or antigen-binding fragment thereof (forming an ADC) is administered to a human patient. in an amount sufficient to reduce the population of CD117+ cells in the patient, and thereafter administering to said patient a transplant comprising hematopoietic stem cells.

[0014] In any of the above embodiments, the cytotoxin can be, for example, Pseudomonas exotoxin A, de Bouganin, diphtheria toxin, amatoxins such as α-amanitin, saporin, maytansine benzodiazepines, maytansinoids, auristatins, anthracyclines, calicheamicins, irinotecans, SN-38, duocarmycins, pyrrolobenzodiazepines, pyrrolobenzodiazepines The compounds may be benzodiazepine dimers, indolinobenzodiazepines, and indolinobenzodiazepine dimers, or variants thereof.

[0015] In some embodiments of any of the above aspects, the cytotoxin is α-amanitin. , β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanu In one embodiment, the cytotoxin is amanitin, such as amatoxin, amanurinic acid, and proamanuline, or a derivative thereof.

[0016] In some embodiments of any of the above aspects, the cytotoxin is an amatoxin, and the antibody, or antigen-binding fragment thereof, conjugated to the cytotoxin has the formula Ab-ZL-Am where Ab is an antibody or antigen-binding fragment thereof, and L is a linker. , Z is a chemical moiety, and Am is an amatoxin. In some embodiments, the amatoxin-linker conjugate Am-LZ is represented by formula (I): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C , OR D , R C , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl). nyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C1-C6 alkylene), an optionally substituted heteroalkylene (e.g., C1-C6 heteroalkylene), an optionally substituted optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic alkylene; alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene; dipeptide, -(C=O)-, peptide, or combinations thereof; and Z is a reactive group present on L and an antibody that binds to CD117 (such as GNNK+CD117); It is a chemical moiety formed from a coupling reaction between reactive substituents present within the antigen-binding fragment.

[0017] In some embodiments, Am is exactly one R C Contains substituents.

[0018] In some embodiments, the linker L and the chemical substructure Z together form LZ. Considered, [ka] where S is a sulfur atom representing a reactive substituent present in the antibody, or antigen-binding fragment thereof, that binds to CD117 (e.g., derived from the -SH group of a cysteine ​​residue).

[0019] In some embodiments, LZ is [ka] is.

[0020] In some embodiments, the Am-LZ-Ab is one of the following: [ka] Here, X is —S—, —S(O)—, or —SO2—.

[0021] In some embodiments, Am-LZ-Ab is [ka] is.

[0022] In some embodiments, Am-LZ-Ab is [ka] is.

[0023] In some embodiments, Am-LZ-Ab is [ka] is.

[0024] In some embodiments, Am-LZ is represented by formula (IA): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C , OR D , R C , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHRC , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl). nyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C1-C6 alkylene), an optionally substituted heteroalkylene (e.g., C1-C6 heteroalkylene), an optionally substituted optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic alkylene; alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -(C=O)-, peptide, or combinations thereof; It is a linker that is like; Z is a reactive group present on L and an antibody that binds to CD117 (such as GNNK+CD117); a chemical moiety formed from a coupling reaction between a reactive substituent present in the antigen-binding fragment; and where Am is exactly one R C Contains substituents.

[0025] In some embodiments, the linker L and the chemical substructure Z together form LZ. Considered, [ka] is.

[0026] In some embodiments, LZ is [ka] is.

[0027] In some embodiments, Am-LZ is represented by formula (IB): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C , OR D , R C , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl). nyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C1-C6 alkylene), an optionally substituted heteroalkylene (e.g., C1-C6 heteroalkylene), an optionally substituted optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic alkylene; alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -(C=O)-, peptide, or combinations thereof; It is a linker that is like; Z is a reactive group present on L and an antibody that binds to CD117 (such as GNNK+CD117); a chemical moiety formed from a coupling reaction between a reactive substituent present in the antigen-binding fragment; and where Am is exactly one R C Contains substituents.

[0028] In some embodiments, R A and R B together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl group of the formula: [ka] Here, Y is -(C=O)-, -(C=S)-, -(C=NR E) - or - (CR E R E' )-; and R E and R E' are each independently optionally substituted C-C alkylene-R C ,any optionally substituted C1-C6 heteroalkylene-R C , optionally substituted C2-C6 alkenyl Ren-R C, optionally substituted C2-C6 heteroalkenylene-R C , optionally substituted C2-C6 alkynylene-R C , optionally substituted C2-C6 heteroalkynylene-R C , optionally substituted cycloalkylene-R C , optionally substituted heterocycloalkylene N-R C、 optionally substituted arylene-R C、 or optionally substituted heteroarylene-R C , is.

[0029] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B together with the oxygen atoms to which they are attached to form: [ka] R3 is H or R C is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C , OR D , R C , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or RD is; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; and where R C and R D are each as defined above.

[0030] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B together with the oxygen atoms to which they are attached to form: [ka] R3 is H or R C is; R4 and R5 are each independently H, OH, OR C , R C OR D is; R6 and R7 are each H; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; and where R C is as defined above.

[0031] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): where R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; R A and RB together with the oxygen atoms to which they are attached to form: [ka] R3, R4, R6, and R7 are each H; R5 is OR C is; R8 is OH or NH2; R9 is H or OH; and where R C is as defined above.

[0032] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): wherein R1 and R2 are each independently H or OH; R3 is R C is; R4, R6, and R7 are each H; R5 is H, OH, or OC1-C6 alkyl; R8 is OH or NH2; R9 is H or OH; and where R C is as defined above.

[0033] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): wherein R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are independently H, OH, or OR C , or R C is; R8 is OH or NH2; R9 is H or OH; and where R C is as defined above.

[0034] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): wherein R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are each independently H or OH; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; and where R C is as defined above.

[0035] In some embodiments, the linker L and the chemical substructure Z together form LZ. Considered, [ka] is.

[0036] In some embodiments, LZ is [ka] is.

[0037] In some embodiments, Am-LZ is represented by formula (II), formula (IIA), or formula (IIB): [ka] where X is S, SO, or SO2; R1 is H or a compound connected to said antimony group via chemical moiety Z. is a linker covalently attached to the antibody or antigen-binding fragment thereof (Z is the linker and R2 is H or a chemical moiety. a linker covalently attached to the antibody or antigen-binding fragment thereof via a moiety Z; (Z is a group consisting of a reactive substituent present on the linker and an antibody or its antigen-binding fragment.) wherein when R1 is H, R2 is a linker, and when R2 is H, R1 is a linker.

[0038] In some embodiments, the linker is -(CH) 2n -units, where n is an integer between 2 and 6.

[0039] In some embodiments, R1 is a linker and R2 is H, and the linker and The mathematical substructures are grouped together as LZs, [ka] is.

[0040] In some embodiments, Am-LZ-Ab is [ka] is.

[0041] In some embodiments, Am-LZ-Ab is [ka] is.

[0042] In some embodiments of any of the above aspects, the cytotoxin is a maytansinoid selected from the group consisting of DM1 and DM4. In some embodiments, the cytotoxin is an auristatin selected from the group consisting of monomethyl auristatin E and monomethyl auristatin F. In some embodiments, the cytotoxin is an anthracycline selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin.

[0043] In another aspect, the present invention provides a method for increasing a population of CD117+ cells in a human patient by administering an effective amount of an antibody, antigen-binding fragment, or ADC thereof capable of binding to GNNK+CD117. The present invention is characterized by a method for reducing

[0044] Furthermore, the present invention provides a method for reducing the population of CD117+ cells in a human patient in need of hematopoietic stem cell transplantation by administering to the patient an effective amount of an antibody capable of binding to GNNK+CD117, or an antigen-binding fragment thereof, or an ADC, prior to receiving a transplant containing hematopoietic stem cells. The method is characterized.

[0045] In another aspect, the invention features a method of treating a human patient in need of hematopoietic stem cell transplantation, e.g., comprising administering to the human patient a transplant comprising hematopoietic stem cells, wherein the patient has previously been administered an antibody, antigen-binding fragment thereof, or ADC capable of binding to GNNK+CD117 in an amount sufficient to reduce the population of CD117+ cells in the patient.

[0046] In a further aspect, the invention features a method of treating, e.g., a human patient in need of hematopoietic stem cell transplantation, comprising: administering to said patient an antibody, antigen-binding fragment thereof, or ADC capable of binding to GNNK+CD117 in an amount sufficient to reduce the population of CD117+ cells in said patient. to a human patient, and subsequently administering to said patient a transplant comprising hematopoietic stem cells.

[0047] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, or a dual variable immunoglobulin. domain (dual-variable immunoglobulin domain), single-chain Fv molecule (scFv), diabody The antibody may be selected from the group consisting of a antibody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment, an F(ab')2 molecule, and a tandem di-scFv. In some embodiments, the antibody is selected from the group consisting of IgG, IgA, IgM, IgD, and IgE. It has an isotype that is

[0048] In some embodiments of any of the above aspects, the antibody, or antigen-binding fragment thereof, is internalized by hematopoietic cells, such as hematopoietic stem cells, cancer cells, or autoimmune cells after administration to the patient. For example, the antibody, or antigen-binding fragment thereof, or ADC is internalized (e.g., upon binding to cell surface CD117, such as GNNK+ CD117). In some embodiments, the cytotoxin covalently attached to the antibody, or antigen-binding fragment thereof, can be chemically cleaved (e.g., as described herein) and then internalized by hematopoietic stem cells, cancer cells, or autoimmune cells via receptor-mediated endocytosis. may be released intracellularly by enzymatic or non-specific cleavage of the linker described The cytotoxin may then access its intracellular target (such as the mitotic spindle apparatus, nuclear DNA, ribosomal RNA, or topoisomerases, among others) to promote the death of endogenous hematopoietic cells (e.g., endogenous hematopoietic stem cells prior to transplantation therapy), endogenous cancer cells, or endogenous autoimmune cells, among others.

[0049] In some embodiments of any of the above aspects, the antibody, or antigen-binding fragment thereof, or ADC is capable of targeting, inter alia, hematopoietic cells such as hematopoietic stem cells, cancer cells, or autologous cells. In some embodiments, the antibody or antigen-binding fragment thereof, when administered to the patient, can promote necrosis of one or more complement proteins. By recruiting cells such as hematopoietic stem cells, natural killer cells, macrophages, neutrophils, and / or eosinophils, it may promote the death of endogenous hematopoietic stem cells, endogenous cancer cells, or endogenous autoimmune cells, particularly before transplantation therapy.

[0050] In some embodiments of any of the above aspects, the transplant comprising hematopoietic stem cells is administered to a patient in need thereof such that the concentration of the antibody, antigen-binding fragment thereof, or ADC is substantially cleared from the patient's blood. After the dose is adjusted, it is administered to the patient.

[0051] In some embodiments of any of the above aspects, the hematopoietic stem cells or their progeny are functionally expressed after 2 or more days (e.g., about 2 to about 5 days, about 2 to about 7 days, about 2 to about 20 days, about 2 to about 30 days, e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or more) after transplantation of the hematopoietic stem cells into the patient. maintains the possibility.

[0052] In some embodiments of any of the above aspects, the hematopoietic stem cells or their progeny may be localized in hematopoietic tissue (e.g., bone marrow) and / or may re-establish hematopoiesis after transplantation of the hematopoietic stem cells into the patient.

[0053] In some embodiments of any of the above aspects, the hematopoietic stem cells, when transplanted into the patient, produce megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, or the like. cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, A population of cells selected from the group consisting of primary presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes is recovered.

[0054] In some embodiments of any of the above aspects, the method is used to treat one or more disorders, such as by reducing the population of hematopoietic stem cells in a patient prior to hematopoietic stem cell transplantation therapy to provide a niche to which transplanted hematopoietic stem cells can home. After transplantation, the hematopoietic stem cells may establish productive hematopoiesis, thereby replacing cell types that are deficient in the patient or that have been actively damaged or injured, e.g., by chemotherapeutic methods. For example, the patient may be suffering from a stem cell disorder. In some embodiments, the patient is suffering from a hemoglobinopathy disorder, such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome. The patient may be suffering from an immunodeficiency disease, such as a congenital immunodeficiency disorder or an acquired immunodeficiency disorder (e.g., human immunodeficiency virus or acquired immunodeficiency syndrome). In some embodiments, the patient is suffering from a metabolic disorder, such as glycogen storage disease, mucopolysaccharidosis, Gaucher disease, Hurler disease, sphingolipidosis, and metachromatic leukodystrophy. In some embodiments, the patient is suffering from adenosine deaminase deficiency and severe combined immunodeficiency, hyperimmunoglobulin M syndrome. In some embodiments, the patient is suffering from a disorder selected from the group consisting of rheumatoid arthritis, thyroid cancer, thyroid cancers, thyroid cancers, thyroid cancers associated with thyroid cancer ... The patient is suffering from cancer, such as a hematological cancer, or a myeloproliferative disorder. In some embodiments, the patient is suffering from acute myeloid leukemia (AML), acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, or myeloproliferative disorder. In some embodiments, the patient is suffering from lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. , suffer from a myelodysplastic disease such as myelodysplastic syndrome.

[0055] In some embodiments of any of the above aspects, the method is used to reduce the population of CD117+ cancer cells in a patient by administering an antibody, or antigen-binding fragment thereof, or ADC, and / or to reduce the population of endogenous hematopoietic stem cells prior to hematopoietic stem cell transplantation. The administration of antibodies, or antigen-binding fragments thereof, directly treats cancers characterized by CD117+ cells (e.g., leukemias characterized by CD117+ cells) to reduce the number of CD117+ cells present in the tumor. In the latter case, the transplant can then reconstitute a population of cells that was reduced during the process of eradicating the cancer cells. The cancer may be a blood cancer such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.

[0056] In some embodiments of any of the above aspects, the method is used to administer an antibody, antigen-binding fragment thereof, or ADC to reduce the population of CD117+ autoimmune cells. and / or by administration of antibodies, or antigen-binding fragments thereof, or ADCs to reduce the endogenous hematopoietic stem cell population prior to hematopoietic stem cell transplantation. In the latter case, the transplant can in turn reconstitute a population of cells that was reduced during the process of eradicating autoimmune cells, for example. The autoimmune diseases include, for example, scleroderma, multiple sclerosis (MS), human systemic lupus erythematosus (SLE), arthritis recurrence, and the like. Treatment of equine arthritis (RA), inflammatory bowel disease (IBD), psoriasis, type 1 diabetes mellitus (type 1 diabetes), acute disseminated cerebrospinal Myelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid syndrome (APS) , aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Baro's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammation Demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythematosus, autonomic neuropathy, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenia Purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki disease, Lichen planus, Lyme disease, Meniere's disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus-myoclonus syndrome (OMS), optic neuritis, ordothyroid Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and skin It may be myositis, primary biliary cirrhosis, polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-person syndrome, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia ("vulvar vestibulitis"), and Wegener's granulomatosis.

[0057] Thus, in some embodiments of any of the above aspects, the invention features methods of treating a hemoglobinopathy disorder, such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, Wiskott-Aldrich syndrome, etc. In some embodiments, the invention relates to a method of treating a congenital immunodeficiency disorder or an acquired immunodeficiency disorder (e.g., human immunodeficiency virus (HIV)-associated leukemia). The present invention features methods for treating immune deficiency disorders such as HIV-1, HIV-2, and HIV-1-associated leukemia (HIV-1 or HIV-1-associated acquired immune deficiency syndrome). In some embodiments, the present invention provides a method for treating metabolic disorders such as glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidosis, and metachromatic leukodystrophy. In some embodiments, the present invention provides a method for treating adenosine deaminase deficiency and severe combined immunodeficiency, hyperimmunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphoma, and the like. The present invention features a method for treating a disorder selected from the group consisting of histiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major, systemic sclerosis, systemic lupus erythematosus, and juvenile rheumatoid arthritis. In some embodiments, the present invention also features a method for treating an autoimmune disease, such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, and type 1 diabetes. In some embodiments, the invention features a method of treating a cancer or myeloproliferative disorder, such as a hematological cancer. In some embodiments, the invention features a method of treating acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the patient is suffering from a myelodysplastic disorder, such as myelodysplastic syndrome. In these embodiments, the method includes administering to the patient an antibody that binds to CD117 (e.g., GNNK+CD117), or an antigen-binding fragment thereof. or ADC, and / or hematopoietic stem cell transplant according to any of the above aspects and embodiments of the invention. The method may include administering according to the method.

[0058] Similarly, in some embodiments of any of the above aspects, the invention provides methods of directly treating cancer, such as cancer characterized by CD117+ cells (e.g., leukemia characterized by CD117+ cells). In these embodiments, the methods comprise administering an antibody, antigen-binding fragment thereof, or ADC that binds to CD117 (e.g., GNNK+ CD117). The cancer may be a blood cancer such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.

[0059] Further, in embodiments of any of the above aspects, the present invention provides a method for treating, for example, multiple sclerosis (MS), human systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), psoriasis, type 1 diabetes (T1DM), acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, Autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Barrow's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease , cicatricial pemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythematosus, autonomic nervous system Menstrual disorders, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, suppurative Hidradenitis, idiopathic and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy -, interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, Lyme disease, Meniere's disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus-myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-person syndrome, Takayasu's arteritis, temporal arteritis ( (also known as "giant cell arteritis"), ulcerative colitis, uveitis, vasculitis, vitiligo, Methods for treating autoimmune diseases such as vulvar pain ("vulvar vestibulitis") and Wegener's granulomatosis are provided. In these embodiments, the methods comprise administering an antibody, or antigen-binding fragment thereof, or ADC that binds to CD117 (e.g., GNNK+ CD117).

[0060] In another aspect, the present invention provides a method for the treatment of rheumatoid arthritis by contacting a population of CD117+ (e.g., GNNK+CD117+) cells with an effective amount of a conjugate (or ADC) represented by the formula Ab-ZL-Am. The present invention features a method for reducing or eliminating CD117, wherein Ab is an antibody that binds to CD117 or its antigen binding. Am-LZ is a conjugated fragment, Z is a chemical substructure, L is a linker, and Am is an amatoxin. Am-LZ may be represented by formula (IA): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B taken together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R5, R6, and R7 are each independently H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl). nyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C1-C6 alkylene), an optionally substituted heteroalkylene (e.g., C1-C6 heteroalkylene), an optionally substituted optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic alkylene; alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -(C=O)-, peptide, or combinations thereof; and Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an antibody, or antigen-binding fragment thereof; where Am is exactly one R C Contains substituents.

[0061] In some embodiments, the linker L and the chemical substructure Z together form LZ. Considered, [ka] is.

[0062] In some embodiments, LZ is [ka] is.

[0063] In some embodiments, Am-LZ is represented by formula (IB): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C , OR D , R C , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, ORC , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl). nyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C1-C6 alkylene), an optionally substituted heteroalkylene (e.g., C1-C6 heteroalkylene), an optionally substituted optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic alkylene; alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -(C=O)-, peptide, or combinations thereof; It is a linker that is like; Z is a reactive group present on L and an antibody that binds to CD117 (such as GNNK+CD117); a chemical moiety formed from a coupling reaction between a reactive substituent present in the antigen-binding fragment; and where Am is exactly one R C Contains substituents.

[0064] In some embodiments, the linker L and the chemical substructure Z together form LZ. Considered, [ka] is.

[0065] In some embodiments, LZ is [ka] is.

[0066] In some embodiments, Am-LZ-Ab is any of the following: [ka] Here, X is —S—, —SO—, or —SO2—.

[0067] In some embodiments, Am-LZ-Ab is [ka] is.

[0068] In some embodiments, Am-LZ-Ab is [ka] is.

[0069] In some embodiments, Am-LZ-Ab is [ka] is.

[0070] In another aspect, the invention features a conjugate represented by the formula Ab-ZL-Am. wherein Ab is an antibody that binds to CD117 (e.g., GNNK+ CD117), or an antigen-binding fragment thereof, and Am is an amatoxin. In some embodiments, Am-LZ is represented by the above formula (I): , (IA), (IB), (II), (IIA), (IIB), (IV), (IVA), or (IVB).

[0071] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to an amatoxin via a cysteine ​​residue in the Fc domain of the antibody or antigen-binding fragment thereof. In some embodiments, the cysteine ​​residue is introduced into the Fc domain of the antibody or antigen-binding fragment thereof by mutation. For example, the cysteine ​​residue may be selected from the group consisting of Cys118, Cys239, and Cys265.

[0072] In some embodiments of these aspects, the cysteine ​​residue is naturally present in the Fc domain of the antibody, or antigen-binding fragment thereof. For example, the Fc domain can be an IgG Fc domain, such as a human IgG1 Fc domain, and the cysteine ​​residue is naturally present in the Fc domain of the antibody, or antigen-binding fragment thereof. The amino acid residue may be selected from the group consisting of Cys261, Csy321, Cys367, and Cys425.

[0073] In some embodiments of these aspects, R1 is H, OH, or OR A is; R2 is H, OH, or OR Bis; R A and R B When present, together with the oxygen atom to which they are attached, form: [ka] R3, R4, R6, and R7 are each H; R5 is OR C is; R8 is OH or NH2; R9 is H or OH; and X is —S—, —S(O)—, or —SO 2 —.

[0074] In some embodiments, R1 and R2 are each independently H or OH; R3 is R C is; R4, R6, and R7 are each H; R5 is H, OH, or OC1-C6 alkyl; R8 is OH or NH2; R9 is H or OH; and X is —S—, —S(O)—, or —SO 2 —.

[0075] In some embodiments, R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 is OR C or R C is; R5 is H, OH, or OC1-C6 alkyl; R8 is OH or NH2; R9 is H or OH; and X is —S—, —S(O)—, or —SO 2 —.

[0076] In some embodiments, R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are each independently H or OH; R8 is OR C or NHR C is; R9 is H or OH; and X is —S—, —S(O)—, or —SO 2 —.

[0077] In some embodiments of these aspects, the antibody, or antigen-binding fragment thereof, is internalized by CD117+ cells.

[0078] In some embodiments of these aspects, the antibody, or antigen-binding fragment thereof, has a K for CD117 of less than 1 μM, less than 750 nM, less than 500 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 10 pM, less than 1 pM, or less than 0.1 pM. d In some embodiments, the K d is about 0.1 pM to about 1 μM.

[0079] In some embodiments of these aspects, the antibody, or antigen-binding fragment thereof, has a concentration of about 9×10 -2 M -1 s -1 ~Approx. 1×10 2 M -1 s -1 k on It binds to CD117.

[0080] In some embodiments of these aspects, the antibody, or antigen-binding fragment thereof, competitively inhibits CD117 binding to a second antibody, or antigen-binding fragment thereof, or binds to the same epitope as a second antibody, wherein the second antibody, or antigen-binding fragment thereof, has the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence SYWIG (SEQ ID NO: 1); CDR-H2 having the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 2); CDR-H3 having the amino acid sequence HGRGYNGYEGAFDI (SEQ ID NO:3); CDR-L1 having the amino acid sequence RASQGISSALA (SEQ ID NO: 4); CDR-L2 having the amino acid sequence DASSLES (SEQ ID NO: 5); and CDR-L3 having the amino acid sequence CQQFNSYPLT (SEQ ID NO: 6).

[0081] The present invention further provides heavy and light chain CDRs and variable regions set forth in Table 1, Table 6, or Table 8. Isolated antibodies that may be used in the antibody drug conjugates (ADCs) disclosed herein, including The present invention provides an anti-CD117 antibody.

[0082] In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO: The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 7, and the amino acid sequence of SEQ ID NO: 8 In one embodiment, the anti-CD117 antibody or its derivatives comprises a light chain variable region as shown in the sequence The antigen-binding portion of the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 7, and and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:9. wherein the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 10. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 11. The anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:12. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:13. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, The antigen-binding portion of the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 7. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 15. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 15. The portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:16. The anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 17. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 17. the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 7, and the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 18 In one embodiment, the anti-CD117 antibody comprises a light chain variable region shown in the amino acid sequence The antibody, or an antigen-binding portion thereof, may comprise a heavy chain polypeptide as set forth in the amino acid sequence of SEQ ID NO:7. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 20. The original binding portion comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 7, and a sequence In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises: The anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:23. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:24, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:25. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:25. the heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 26, and the amino acid sequence of SEQ ID NO: 27 In one embodiment, the anti-CD117 antibody or The antigen-binding portion thereof comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 28 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 29. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 30. and the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 31. In one embodiment, the anti-CD117 antibody or its antigen-binding In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 32 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 33. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 34. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 35. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 35. In one embodiment, the anti-CD117 antibody comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 36 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 37. The antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 38 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 39. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 40 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 41. In one embodiment, the anti-CD117 antibody or its derivatives comprises a light chain variable region as shown in the sequence The antigen-binding portion of the anti-CD117 antibody comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 32 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 42. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 43. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 44. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 45 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 46. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 47. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 48. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 49. In one embodiment, the anti-CD117 antibody comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 49, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 50. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 51 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 52. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 53 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 54. In one embodiment, the anti-CD117 antibody or its The antigen-binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 55 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 56. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 57. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 58. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 59 and the heavy chain variable region shown in SEQ ID NO: 60 are In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO:61. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 62 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 63. In one embodiment, the anti-CD117 antibody comprises a light chain variable region shown in the amino acid sequence: or an antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 64 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 65. In one embodiment, the anti-CD117 antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 66 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 67. In one embodiment, the anti-CD117 antibody or its anti-CD117 antibody comprises a light chain variable region as shown in The antigen-binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 68 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 69. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 70. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 71. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 72, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 73. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 74. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 76 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 77. In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence: or an antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 78 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 79. In one embodiment, the anti-CD117 antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 80. and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 81. In one embodiment, the anti-CD117 antibody or its antigen The binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 82 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 83. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 84. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 85. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises: The anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 86 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 87. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 8 ... In one embodiment, the anti-CD117 antibody or its derivatives comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:89. The antigen-binding portion of the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 7, and and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 90. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 91. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 91. The anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 92. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 93 ... The antigen-binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 95. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 95. , a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD117 antibody, or an antigen-binding portion thereof, comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO:7. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 97. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 97. The antigen-binding portion comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 143. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 144. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 151 and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 152. In one embodiment, the anti-CD117 antibody or its anti-CD117 antibody comprises a light chain variable region as shown in The original binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 143, and The antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 156. wherein the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 159 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 156. In one embodiment, the anti-CD117 antibody or its antigen-binding The portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 160, and a heavy chain variable region set forth in SEQ ID NO: In one embodiment, the antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 152. and wherein the anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO:98. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 99. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises: In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:99. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 100. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 98 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 101. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 101. The antigen-binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:98 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:102.

[0083] In some embodiments of these aspects, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a dual variable immunoglobulin dopant (BIG) antibody, or a humanized antibody or antigen-binding fragment thereof. Dual-variable immunoglobulin domain (DV), single-chain Fv molecule (scFv), diabody, Triabodies, nanobodies, antibody-like protein scaffolds, Fv fragments, Fab In one embodiment, the antibody is an intact antibody, selected from the group consisting of a fragment, a F(ab')2 molecule, and a tandem dis-scFV.

[0084] In another aspect, the invention features a conjugate (ADC) represented by the formula Ab-ZL-Cy, where Ab is an antibody or antigen-binding fragment thereof that binds to CD117 (e.g., GNNK+ CD117), and Cy is a cytotoxin. In some embodiments of this aspect, the cytotoxin is Pseudomonas exotoxin A, de bouganin, diphtheria toxin, saporin, Maytansine, maytansinoids, auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepines, pyrrolobe The cytotoxin is an indolinobenzodiazepine dimer, an indolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a variant of any of the aforementioned cytotoxins.

[0085] In some embodiments of this aspect, the antibody, antigen-binding fragment thereof, or ADC is internalized by CD117+ cells.

[0086] In some embodiments of this aspect, the antibody, antigen-binding fragment thereof, or ADC has a denaturing activity of less than 1 μM, less than 750 nM, less than 500 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, as measured by Bio-Layer Interferometry (BLI). less than 75 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 10 pM, less than 1 pM, or K<0.1 pM d In some embodiments, the K d is approximately 0.1 pM to about 1 μM.

[0087] In some embodiments of this aspect, the antibody, antigen-binding fragment thereof, or ADC has a cytoplasmic activity of about 9×10 as measured by Bio-Layer Interferometry (BLI). -2 M -1 s -1 ~Approx. 1×10 2 M -1 s -1 k on It binds to CD117.

[0088] In certain embodiments, the anti-CD117 antibody or antigen-binding fragment thereof is When used as part of a conjugate, the anti-CD117 antibody has a particular dissociation rate that is particularly advantageous. For example, in certain embodiments, the anti-CD117 antibody has a dissociation rate of 1×10 as measured by Bio-Layer Interferometry (BLI). -2 ~1×10 -7 , 1×10 -3 ~1×10 -7 , 1×10 -4 ~1×10 -7 , 1×10 -5 ~1×10 -7 , or 1×10 -6 ~1×10 -7 s -1 Off-rate of human CD117 It has a constant (Kdis).

[0089] In some embodiments of this aspect, the antibody, antigen-binding fragment thereof, or ADC competitively inhibits CD117 from binding to a second antibody, or antigen-binding fragment thereof. wherein the second antibody, or antigen-binding fragment thereof, has the following CDRs: a. CDR-H1 having the amino acid sequence SYWIG (SEQ ID NO: 1); b. CDR-H2 having the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 2); c. CDR-H3 having the amino acid sequence HGRGYNGYEGAFDI (SEQ ID NO:3); d. CDR-L1 having the amino acid sequence RASQGISSALA (SEQ ID NO:4); e. CDR-L2 having the amino acid sequence DASSLES (SEQ ID NO: 5); and f. CDR-L3 having the amino acid sequence CQQFNSYPLT (SEQ ID NO: 6).

[0090] In some embodiments of this aspect, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a dual variable immunoglobulin domain antibody, or a humanized antibody or antigen-binding fragment thereof. dual-variable immunoglobulin domain (dual-variable immunoglobulin domain), single-chain Fv molecules (scFv), diabodies, Ribodies, nanobodies, antibody-like protein scaffolds, Fv fragments, Fab fragments In some embodiments, the antibody is selected from the group consisting of IgG, IgA, IgM, IgD, and IgE. It has an isotype.

[0091] In certain embodiments, the methods and compositions include an isolated anti-CD117 antibody or antigen-binding fragment thereof comprising the CDRs set forth in the heavy and light chain amino acid sequences set forth in Table 1, Table 6, or Table 8. In certain embodiments, the methods and compositions include an isolated anti-CD117 antibody or antigen-binding fragment thereof comprising the variable regions set forth in the heavy and light chain amino acid sequences set forth in Table 1. In certain embodiments, the methods and compositions include an IgG1 anti-CD117 antibody or its derivatives comprising variable regions set forth in heavy and light chain amino acid sequences set forth in Table 1, Table 6, or Table 8. antigen-binding fragments.

[0092] In another aspect, the present invention provides a method for treating acute myeloid leukemia (AML) in a human patient. and a method for treating AML, the method comprising administering to the human patient an effective amount of an anti-CD117 ADC such that the AML is treated, wherein the anti-CD117 ADC is conjugated to a cytotoxin. In some embodiments, the cytotoxin is an RNA polymerase inhibitor. In other embodiments, the RNA polymerase inhibitor is an amatoxin. In another embodiment, the anti-CD117 antibody comprises the CDR sequences set forth in the heavy and light chain amino acid sequences of Table 1. In yet another embodiment, the anti-CD117 antibody is an intact antibody. In embodiments, the antibody is an IgG1 or IgG4.

[0093] In another embodiment, the present invention provides a conjugate represented by the formula Ab-ZL-Am. wherein Ab is an antibody or antigen-binding fragment thereof that binds to CD117, L is a linker, Z is a chemical moiety, and Am is an amatoxin, or an antigen-binding fragment thereof comprising the CDRs set forth in the heavy and light chain amino acid sequences set forth in Table 1, Table 6 or Table 8.

[0094] In some embodiments, the conjugate's linker-chemical moiety-amato The oxine moiety (Am-LZ) is represented by formula (IA) [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R5, R6, and R7 are each independently H, OH, OR C , ORD , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted C1-C6 alkylene, an optionally substituted C1-C6 hetero optionally substituted C2-C6 alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -(C=O)-, peptide, or combinations thereof; and, Z is a reactive substituent present on L and a substituent present in the antibody or antigen-binding fragment thereof. is a chemical moiety formed from a coupling reaction between a reactive substituent and where Am is exactly one RC Contains substituents.

[0095] In some embodiments, LZ is [ka] is.

[0096] In some embodiments, Am-LZ is represented by formula (IB): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R5, R6, and R7 are each independently H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R Dis optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted C1-C6 alkylene, an optionally substituted C1-C6 hetero optionally substituted C2-C6 alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -(C=O)-, peptide, or combinations thereof; and, Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an antibody, or antigen-binding fragment thereof; where Am is exactly one R C Contains substituents.

[0097] In some embodiments, LZ is [ka] is.

[0098] In some embodiments, the conjugate's linker-chemical moiety-amato The oxine moiety (Am-LZ) is one of the following: [ka] where X is -S-, -S(O)-, or -SO2-, and Ab is shown to indicate the site of attachment of the antibody.

[0099] In some embodiments, the conjugate's linker-chemical moiety-amato The oxine moiety (Am-LZ) is [ka] where Ab is shown to indicate the site of binding of the antibody.

[0100] In some embodiments, the conjugate's linker-chemical moiety-amato The oxine moiety (Am-LZ) is [ka] where Ab is shown to indicate the site of binding of the antibody.

[0101] In some embodiments, the conjugate's linker-chemical moiety-amato The oxine moiety (Am-LZ) is [ka] where Ab is shown to indicate the site of binding of the antibody.

[0102] In some embodiments, the Am-LZ precursor is [ka] where the maleimide reacts with a thiol group on a cysteine ​​in the antibody.

[0103] In some embodiments, the Am-LZ precursor is [ka] where the maleimide reacts with a thiol group on a cysteine ​​in the antibody.

[0104] In some embodiments, Am-LZ is represented by formula (II), formula (IIA), or formula (IIB): [ka] where X is S, SO, or SO; R1 is H or a linker covalently attached to the antibody or antigen-binding fragment thereof via a chemical moiety Z (where Z is a functional group that binds to a reactive substituent present on the linker and to an antigen-binding fragment of the antibody). or an antigen-binding fragment thereof); and R2 is H or a linker covalently attached to the antibody or antigen-binding fragment thereof via a chemical moiety Z (where Z is a reactive substituent present on the linker and a functional group that binds to the antibody or antigen-binding fragment thereof). or formed from a coupling reaction between a reactive substituent present in the antigen-binding fragment thereof; wherein when R1 is H, R2 is a linker, and when R2 is H, R1 is a linker.

[0105] In some embodiments, LZ is [ka] is.

[0106] In some embodiments, Ab-ZL-Am is [ka] is.

[0107] In some embodiments, Ab-ZL-Am is [ka] is. [Brief explanation of the drawings]

[0108] [Figure 1] Figure 1 shows the Bio-Layer Interferometry binding of the indicated purified IgG (sensor bound) to purified human CD117 ectodomain (R&D Systems #332-SR) at concentrations of 33.3 nM (top trace) and 11 nM (bottom trace) versus time. The purified IgGs correspond to Ab1 (i.e., 001), Ab2 (i.e., 002), Ab3 (i.e., 003), Ab4 (i.e., 004), Ab5 (i.e., 005), Ab6 (i.e., 006), Ab7 (i.e., 007), Ab8 (i.e., 008), Ab9 (i.e., 009), Ab10 (i.e., 010), Ab11 (i.e., 011), Ab12 (i.e., 012), Ab13 (i.e., 013), Ab14 (i.e., 014), Ab15 (i.e., 015), and Ab16 (i.e., 016). [Figure 2] Figures 2A and 2B graphically depict the results of an in vitro cytotoxicity assay, showing Kasumi-1 cell viability (Figure 2A) as measured by luminescence (RLU) with Celltiter Glo, or the number of viable CD34+ CD90+ cells (Figure 2B) in the presence of CD117-ADC or control (y-axis) versus the concentration of CD117-ADC or control (x-axis). [Figure 3]Figures 3A, 3B, 3C, and 3D graphically depict the results of an in vivo cell depletion assay demonstrating that CD117-ADC selectively depletes human HSCs in humanized NSG mice. (A) Schematic of the in vivo mouse model. The percentage of human myeloid cells (B) or T cells (C) present in the peripheral blood of mice treated with CD117-ADC or control was expressed as a percentage of the corresponding cell population before treatment (normalized to baseline). (D) Absolute numbers of CD34+ cells in the bone marrow of mice treated with CD117-ADC or control 21 days after a single ADC administration are shown. [Figure 4] Figures 4A, 4B, 4C, and 4D graphically show the results of an in vivo tumor study demonstrating that CD117-ADC effectively reduces human leukemia cells in NSG mice. (A) Phenotypic analysis of cultured Kasumi-1 cells. (B) Phenotypic analysis of Kasumi-1 cells derived from the bone marrow of tumor-bearing mice at the time of euthanasia. (C) Survival curves of mice treated with CD117-ADC or naked anti-CD117 antibody, or untreated controls, 7 days after tumor injection. (D) Survival curves of mice treated with CD117-ADC or naked anti-CD117 antibody, or untreated controls, 42 days after tumor injection. [Figure 5]Figures 5A, 5B, and 5C graphically show the results of an in vivo cell depletion assay in B6 mice, demonstrating that CD117-ADC selectively depletes HSCs. (A) Schematic of the in vivo depletion assay in the B6 mouse model. (B) Results of a mouse HSC depletion assay following administration of a single dose of anti-CD117-ADC (i.e., "CD117-saporin") compared to controls (i.e., "control" and "isotype-saporin"). Results are expressed as the number of Kit+SCA+CD150+CD48- cells (y-axis) versus a single dose of anti-CD117-ADC (i.e., "CD117-saporin") or control (x-axis). (C) Results of an engraftment assay are expressed as the percentage of donor chimerism (y-axis) versus treatment mode (i.e., "control," "isotype-saporin," and "CD117-saporin"). [Figure 6] Figure 6 graphically depicts the results of a non-human primate pharmacokinetic assay, showing the concentration (ng / mL) of an isotype control antibody (i.e., "wild-type antibody") compared to a CK6 variant antibody with a shorter half-life, versus time on the horizontal axis (i.e., hours after administration; x-axis). [Figure 7]Figures 7A and 7B graphically depict the results of an in vivo depletion assay demonstrating that CD117-ADC effectively depletes human CD34+ cells in humanized NSG mice. (A) Phenotypic analysis of human cells in NSG bone marrow on day 21 when treated with a single dose of (i) wild-type anti-CD117-ADC, (ii) a CK6 variant anti-CD117-ADC with a shorter half-life, or (iii) a control. (B) depicts the results of a depletion assay in humanized NSG mice after administration of a single dose of wild-type anti-CD117-ADC (mg / kg) compared to a CK6 variant anti-CD117-ADC with a shorter half-life (mg / kg) and various controls (i.e., "control," "isotype-ADC (1 mg / kg)," and "anti-CD117 (1 mg / kg)"). The horizontal axis (x-axis) represents the number of CD34+ cells per femur (y-axis) versus a single injection of (i) wild-type anti-CD117-ADC, (ii) a "short half-life" variant anti-CD117-ADC, or (iii) a control. [Figure 8]Figures 8A, 8B, and 8C graphically depict the results of an in vivo depletion assay demonstrating the maintenance of human peripheral lymphocytes 21 days after administration of a single dose of anti-CD117 ADC in a humanized mouse model. (A) shows the percentage of human CD33+ cells (relative to baseline) maintained after 21 days in mice treated with a single dose of various concentrations of (i) wild-type anti-CD117 ADC (mg / kg), (ii) a CK6 variant anti-CD117 ADC with a shorter half-life (mg / kg), or (iii) various controls (i.e., "control," "isotype-ADC (1 mg / kg)," and "anti-CD117 (1 mg / kg)"). (B) shows the percentage of human CD3+ cells (relative to baseline) maintained after 21 days in mice treated with a single dose of various concentrations of (i) wild-type anti-CD117-ADC (mg / kg), (ii) a CK6 variant anti-CD117-ADC with a shorter half-life (mg / kg), or (iii) various controls (i.e., "control," "isotype-ADC (1 mg / kg)," and "anti-CD117 (1 mg / kg)"). (C) shows the percentage of human CD19+ cells (relative to baseline) maintained after 21 days in mice treated with a single dose of various concentrations of (i) wild-type anti-CD117-ADC (mg / kg), (ii) a CK6 variant anti-CD117-ADC with a shorter half-life (mg / kg), or (iii) various controls (i.e., "control," "isotype-ADC (1 mg / kg), and "anti-CD117 (1 mg / kg)"). [Figure 9]Figures 9A, 9B, 9C, and 9D depict Bio-Layer Interferometry measurements of anti-CD117 antibody binding of the indicated purified IgG (sensor-bound) to purified human CD117 ectodomain (R&D Systems #332-SR) at concentrations of 33.3 nM (top trace) and 11 nM (bottom trace) over time for (A) HC-1 / LC-1 (Ab1), (B) HC-77 / LC-77 (Ab77), (C) HC-79 / LC-79 (Ab79), and (D) HC-81 / LC-81 (Ab81). [Figure 10] Figures 10A and 10B depict antibody binding measurements by Bio-Layer Interferometry of the indicated purified IgG (sensor-bound) to purified human CD117 ectodomain (R&D Systems #332-SR) at concentrations of 33.3 nM (top trace) and 11 nM (bottom trace) for (A) HC-85 / LC-85 antibody and (B) HC-1 / LC-1 antibody over time. [Figure 11] Figures 11A, 11B, 11C, and 11D provide the amino acid sequences of the variable heavy (VH) and variable light (VL) chain regions for CK6, Ab85, and Ab249. (A) depicts an alignment of the variable heavy (VH) chain regions of CK6 (SEQ ID NO: 161) and Ab85 (SEQ ID NO: 143). (B) depicts an alignment of the variable light (VL) chain regions of CK6 (SEQ ID NO: 162) and Ab85 (SEQ ID NO: 144). (C) depicts an alignment of the variable heavy (VH) chain regions of CK6 (SEQ ID NO: 161) and Ab249 (SEQ ID NO: 98). (D) depicts the alignment of the variable light (VL) chain regions of CK6 (SEQ ID NO: 162) and Ab249 (SEQ ID NO: 102). [Figure 12]Figures 12A, 12B, and 12C depict the binding by Bio-Layer Interferometry of the indicated purified IgG (sensor-bound) to purified human CD117 ectodomain (R&D Systems #332-SR) at concentrations of 33 nM (top trace) and 11 nM (bottom trace) over time for (A) CK6 antibody, (B) HC-85 / LC-85 (Ab85) antibody, and (C) HC-249 / LC-249 (Ab 249) antibody. [Figure 13] Figures 13A and 13B show the percentage of acidic variants present in the indicated antibodies relative to TO as measured by capillary electrophoresis under the indicated incubation conditions (x-axis) for (A) day 7 (25°C and 50°C) and (B) day 15 (25°C and 50°C). [Figure 14] Figures 14A, 14B, 14C, 14D, and 14E show chromatograms showing the elution profiles of (A) CK6 antibody, (B) HC-77 / LC-77 (Ab77) antibody, (C) HC-79 / LC-79 (Ab79) antibody, (D) HC-81 / LC-81 (Ab81) antibody, and (E) HC-85 / LC-85 (Ab85) antibody after analysis by hydrophobic interaction chromatography (HIC) under the indicated incubation conditions. [Figure 15] Figure 15 graphically depicts the results of an in vitro cytotoxicity assay showing Kasumi-1 cell viability, as measured by luminescence (RLU) in Celltiter Glo, versus the concentration of the indicated anti-CD117 ADC or control. IC50 (M) was determined by a nonlinear regression four-parameter fit in Graphpad Prism. [Figure 16]Figure 16 graphically depicts the results of an in vitro cytotoxicity assay, showing the number of viable CD34+ CD90+ cells versus the concentration of the indicated anti-CD117 ADC or control. IC50 (M) was determined by a nonlinear regression 4-parameter fit in Graphpad Prism. [Figure 17] Figures 17A and 17B graphically depict the results of an in vivo cell depletion assay demonstrating that a 0.3 mg / kg dose of the HC-85 / LC-85 antibody (i.e., Ab85) ADC selectively depletes human HSCs in humanized NSG mice. (A) Shows the percent of human bone marrow cells present in the peripheral blood of mice treated with Ab85-ADC or control. Samples taken on days 0, 7, and 14 are expressed as a percent of that cell population before treatment (normalized to baseline). (B) Shows the absolute number of CD34+ cells in the bone marrow of mice treated with HC-85 / LC-85-ADC (i.e., Ab85-ADC) or control (14 days after a single dose of ADC or control (i.e., PBS)). [Figure 18] Figure 18 graphically depicts the results of an in vitro toxicity assay using human bone marrow CD34+ cells for an anti-CD117 antibody (CK6) site-specifically conjugated to various cytotoxic payloads. The results demonstrate that an anti-CD117 (CK6) antibody conjugated to amanitin depletes human HSCs by more than 90% in humanized mice at 0.3 mg / kg. These results also demonstrate that calicheamicin and amanitin ADCs deplete HSCs equivalently. [Figure 19]Figure 19 graphically depicts the number of hematopoietic stem and progenitor cells in bone marrow. The results demonstrate that a single dose of amanitin-conjugated anti-CD117 (CD117-AM) depletes bone marrow HSCs in cynomolgus monkeys. Male cynomolgus monkeys were intravenously administered a single dose of anti-CD117-AM, isotype-AM (IgG1-AM), or unconjugated anti-CD117 antibody. Seven days after administration, bone marrow HSC numbers were measured by flow cytometry. The percentage of HSCs reduced after treatment with anti-CD117-AM, IgG1-AM, or unconjugated anti-CD117 was calculated compared to the PBS group. [Figure 20] Figure 20 graphically depicts the number of hematopoietic stem and progenitor cells in bone marrow. The results demonstrate that single or divided doses of amanitin-conjugated anti-CD117 modified with a fast half-life Fc (H435A) (HC-85-LC 85) deplete bone marrow HSCs in cynomolgus monkeys. Male cynomolgus monkeys received a single intravenous dose (0.1 or 0.3 mg / kg) or divided intravenous doses (0.3 mg / kg and 0.2 mg / kg Q3D) of the anti-CD117 ADC. Bone marrow HSC counts were measured by flow cytometry 7 days after administration. [Figure 21]Figures 21A, 21B, and 21C graphically depict the results of a survival study using three AML patient-derived xenografts developed from FLT-3+NPM1+ AML samples, demonstrating a significant increase in survival in mouse models treated with 0.3 mg / kg anti-CD117-amanitin or 1 mg / kg anti-CD117-amanitin. (A) Survival curves of AML-PDX developed from FLT-3+NPM1+ AML sample J000106132 after a single intravenous administration of ADC (anti-CD117-AM, isotype-AM), unconjugated anti-CD117 antibody, or PBS (control). (B) Survival curves of AML-PDXs generated from FLT-3+NPM1+ AML sample J000106565 after a single intravenous administration of ADC (anti-CD117-AM, isotype-AM), unconjugated anti-CD117 antibody, or PBS (control). (C) Survival curves of AML-PDXs generated from FLT-3+NPM1+ AML sample J000106134 after a single intravenous administration of ADC (anti-CD117-AM, isotype-AM), unconjugated anti-CD117 antibody, or PBS (control). [Figure 22] Figure 22 shows Bio-Layer Interferometry measurements of the binding of the indicated purified IgG (sensor-bound) to 100 nM purified human CD117 ectodomain (R&D Systems #332-SR) versus time. The purified IgGs correspond to Ab17 (i.e., 017), Ab18 (i.e., 018), Ab19 (i.e., 019), Ab20 (i.e., 020), Ab21 (i.e., 021), Ab22 (i.e., 022), Ab23 (i.e., 023), Ab24 (i.e., 024), Ab25 (i.e., 025), Ab27 (i.e., 027), and Ab28 (i.e., 028). [Figure 23]Figure 23 shows Bio-Layer Interferometry measurements of the binding of the indicated purified IgG (sensor-bound) to 100 nM purified rhesus CD117 ectodomain (R&D Systems #332-SR) versus time. The purified IgGs correspond to Ab17 (i.e., 017), Ab18 (i.e., 018), Ab19 (i.e., 019), Ab20 (i.e., 020), Ab21 (i.e., 021), Ab22 (i.e., 022), Ab23 (i.e., 023), Ab24 (i.e., 024), Ab25 (i.e., 025), Ab27 (i.e., 027), and Ab28 (i.e., 028). DETAILED DESCRIPTION OF THE INVENTION

[0109] [Detailed explanation] Described herein are isolated anti-CD117 human antibodies that bind to human CD117. The antibodies provided herein have many advantageous properties for therapeutic use, including methods for conditioning human patients for stem cell transplantation. For example, in certain embodiments, the antibodies disclosed herein have high affinity and low off-rate for human CD117, as well as the ability to be internalized into cells expressing CD117. Furthermore, certain antibodies provided herein have improved biophysical stability. These characteristics also make the anti-CD117 antibodies disclosed herein advantageous for use in conjugates (antibody-drug conjugates (ADCs)) for delivering cytotoxins to CD117-expressing cells. do.

[0110] The present invention provides anti-CD117 antibodies, particularly isolated human anti-CD117 antibodies that bind to the ectodomain of human CD117. The binding regions of the isolated anti-CD117 antibodies identified herein are described below and in Tables 1, 6, and 8.

[0111] The anti-CD117 antibodies and ADCs described herein may be used in methods for treating a variety of disorders, including, inter alia, cell-type diseases in the hematopoietic lineage, cancer, autoimmune diseases, metabolic disorders, and stem cell disorders. The compositions and methods described herein can be used to treat (i) cancer (ii) directly reducing the population of disease-causing cells, such as the population of immune cells (e.g., leukemia cells) and autoimmune cells (e.g., autoreactive T-cells); and / or (iii) reducing the population of endogenous hematopoietic stem cells, thereby promoting engraftment of transplanted hematopoietic stem cells by providing a niche to which the transplanted cells can home. The above activities can be achieved by ADCs capable of binding to antigens expressed by endogenous disease-causing cells or hematopoietic stem cells. This can be achieved by administering an ADC, antibody, or antigen-binding fragment thereof. When directly treating a disease, this administration can reduce the amount of cells causing the pathology of interest. In cases where a patient is being prepared for hematopoietic stem cell transplantation, this administration selectively reduces the population of endogenous hematopoietic stem cells, thereby creating a vacancy in hematopoietic tissues, such as bone marrow, which can then be filled with transplanted exogenous hematopoietic stem cells. The present invention is based, in part, on the discovery that an ADC, antibody, or antigen-binding fragment thereof capable of binding to CD117 (e.g., GNNK+ D117) can be administered to a patient suffering from cancer or an autoimmune disease to directly reduce the population of cancerous or autoimmune cells, and can also be administered to a patient in need of hematopoietic stem cell transplantation therapy to promote the survival and engraftment of transplanted hematopoietic stem cells.

[0112] The engraftment of hematopoietic stem cell transplants following administration of anti-CD117 ADCs, antibodies, or antigen-binding fragments thereof can be demonstrated by various experimental measurements. For example, the engraftment of transplanted hematopoietic stem cells can be evaluated by assessing the amount of competitive repopulating units (CRUs) present in the bone marrow of patients after administration of an ADC, antibody, or antigen-binding fragment thereof capable of binding to CD117 and subsequent administration of a hematopoietic stem cell transplant. Furthermore, the engraftment of hematopoietic stem cells can be monitored by incorporating a reporter gene, such as an enzyme that catalyzes a chemical reaction that produces a fluorescent, chromogenic, or luminescent product, into a vector transfected into the donor's hematopoietic stem cells, and then monitoring the corresponding signal in the tissue to which the hematopoietic stem cells have homed, such as the bone marrow. Furthermore, the engraftment of hematopoietic stem cells can be monitored by, for example, fluorescence activated cell sorting (FACS) analysis methods known in the art. Hematopoietic stem cell engraftment can also be monitored by assessing the quantity and viability of hematopoietic stem and progenitor cells, as measured by measuring peripheral blood white blood cell counts and / or bone marrow cell recovery by donor cells in bone marrow aspirate samples during the post-transplant period.

[0113] The following sections describe ADCs, antibodies, and other therapeutic agents that can be administered to patients, such as patients suffering from cancer (e.g., acute myeloid leukemia or myelodysplastic syndrome) or autoimmune diseases, or patients requiring hematopoietic stem cell transplantation therapy to promote engraftment of hematopoietic stem cell grafts. or antigen-binding fragments thereof, as well as methods of administering such therapeutic agents to patients (eg, prior to undergoing hematopoietic stem cell transplantation).

[0114] [Definition] As used herein, the term "about" means within 10% of the stated value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

[0115] As used herein, the term "amatoxin" refers to a member of the amatoxin family of peptides produced by Amanita phalloides mushrooms, or a variant or derivative thereof (e.g., a variant or derivative thereof that is capable of inhibiting RNA polymerase II activity). Amatoxins useful in conjunction with the compositions and methods described herein include, but are not limited to, α-amanitin, β-amanitin, γ-amanitin, amanitine, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, or Compounds according to formula (III) include proamanulin and the like. Thus, the amatoxin can be attached to an antibody, or its antigen-binding fragment, for example, via a linker moiety (L). The amatoxins may be conjugated to an antibody or antigen-binding fragment (thereby forming an ADC). Exemplary methods for conjugating amatoxins and linkers useful in such processes are described below. Exemplary linker-containing amatoxins useful for conjugation to antibodies or antigen-binding fragments according to the compositions and methods are also described herein.

[0116] Formula (III) is as follows: [ka] where R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; R A and R Bwhen present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D is; R4 is H, OH, OR D , or R D is; R5 is H, OH, OR D , or R D is; R6 is H, OH, OR D , or R D is; R7 is H, OH, OR D , or R D is; R8 is OH, NH2, or OR D is; R9 is H, OH, or OR D is; X is -S-, -S(O)-, or -SO2-; and R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl). nyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0117] For example, amatoxins useful in conjunction with the compositions and methods described herein include compounds according to formula (IIIA): [ka] where R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D is; R4 is H, OH, OR D , or R D is; R5 is H, OH, OR D , or R D is; R6 is H, OH, OR D , or R D is; R7 is H, OH, OR D , or R D is; R8 is OH, NH2, or OR D is; R9 is H, OH, or OR D is; X is -S-, -S(O)-, or -SO2-; and R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl). nyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0118] Amatoxins useful in conjunction with the compositions and methods described herein also include compounds according to formula (IIIB): [ka] where R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D is; R4 is H, OH, OR D , or R D is; R5 is H, OH, OR D , or R D is; R6 is H, OH, OR D , or R D is; R7 is H, OH, OR D , or R D is; R8 is OH, NH2, or OR D is; R9 is H, OH, or OR D is; X is -S-, -S(O)-, or -SO2-; and R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl). nyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0119] As described herein, amatoxins can be attached to, for example, a hydroxyl group via a linker moiety (L). The antibody or antigen-binding fragment thereof may be conjugated to the antibody or antibody-binding fragment thereof (thereby (to form an ADC). Exemplary methods and linkers for conjugating amatoxins useful in such processes are described below, including in Table 3. Exemplary linker-containing amatoxins useful for conjugation to antibodies or antigen-binding fragments according to the compositions and methods described herein are shown in structural formula (I), (IA), (IB), (II), (IIA), or (IIB), as listed herein.

[0120] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen, and includes monoclonal, genetically engineered, and other modified forms of antibodies, including, but not limited to, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi-, tri-, and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments, etc.).

[0121] The antibodies of the present invention are generally isolated or recombinant. "Isolated," as used herein, refers to a polypeptide (e.g., an antibody) that has been identified and separated and / or recovered from a cell or cell culture that expresses it. Typically, an isolated antibody is prepared by at least one purification step. Thus, an "isolated antibody" is an antibody that has been isolated from a different antibody. For example, an antibody that specifically binds to CD117 is an antibody that is substantially free of other antibodies with the same specificity. The isolated antibody is substantially free of antibodies that specifically bind to antigens other than CD117. .

[0122] Unless otherwise specified, the term "monoclonal antibody" (mAb) refers to a monoclonal antibody specific to a target protein. Intact molecules and antibody fragments (e.g., Fab fragments) capable of specifically binding and F(ab')2 fragments, etc. As used herein, When used herein, Fab and F(ab')2 fragments refer to antibody fragments that lack the Fc fragment of an intact antibody. Examples of these antibody fragments are described herein.

[0123] Generally, an antibody comprises a heavy chain and a light chain, each of which contains an antigen-binding region. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as HCVR or VH). The heavy and light chain variable regions comprise a heavy chain constant region (hereinafter abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability (called complementarity determining regions (CDRs)), interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain binding domains that interact with antigens. The antibody constant region is responsible for the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). It may act as an intermediary between two substances.

[0124] As used herein, "intact" or "full length" antibody An antibody refers to an antibody in which two heavy (H) chain polypeptides and two light (L) chain polypeptides are interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL). The VH and VL regions are composed of a VH domain (CL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions are composed of hypervariable regions (called complementarity determining regions (CDRs)). It can be further subdivided and is interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, and is arranged from the amino terminus to the carboxyl terminus. The heavy and light chain variable regions contain a binding domain that interacts with an antigen. The antibody constant region is a region that allows the immunoglobulin to bind to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q, etc.).

[0125] The terms "Fc," "Fc region," and "Fc domain," as used herein, refer to the IgG Refers to the portion of an IgG antibody that is a crystallizable fragment obtained by papain digestion of the molecule. The Fc region is the C-terminal region of the two heavy chains of an IgG molecule linked by disulfide bonds. The Fc region contains a second constant domain, CH2 (e.g., β-glucan), which does not have antigen-binding activity but contains carbohydrate moieties and binding sites for complement and Fc receptors, including the FcRn receptor. As used herein, the Fc region includes a third constant domain, CH3 (e.g., EU positions 231-340 of IgG1) and a third constant domain, CH4 (e.g., EU positions 341-447 of human IgG1). The Fc includes the lower hinge region (e.g., residues 233-239 of EU position in IgG1). The term "wild-type IgG Fc domain" or "WT IgG Fc domain" may refer to this region in isolation or in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphism has been observed at many positions within the Fc domain, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, and therefore slight differences may exist between the sequences presented in this application and those known in the art. Thus, "wild-type IgG Fc domain" or "WT IgG Fc domain" refers to a naturally occurring IgG Fc region (i.e., any allele). Human IgG1 The sequences of the heavy chains of IgG1, IgG2, IgG3, and IgG4 can be found in many sequence databases, for example, in the Uniprot database (www.uniprot.org) under accession numbers P01857 (IGHG1_human), P01859 (IGHG2_human), P01860 (IGHG3_human), and P01861 (IGHG1_human), respectively. An example of a "WT" Fc region is shown in SEQ ID NO: 183 (heavy chain constant region including the Fc region). (provided).

[0126] As used herein, the term "altered Fc region" or "variant Fc region" refers to a polypeptide that contains one or more amino acid substitutions, deletions, insertions, or modifications introduced anywhere within the Fc region. The term "IgG Fc domain" refers to the IgG Fc domain containing the IgG Fc domain.

[0127] As used herein, the term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. The function can be performed by a fragment of a full-length antibody. The antibody fragment can be, for example, a Fab, F(ab')2, scFv, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, V L , V H , C L and C H Monovalent fragments consisting of one domain (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) V H and C H Fd fragment consisting of one domain (iv) V of a single arm of an antibody L and V H(v) Fv fragment consisting of V domains; H and V L (vi) V domain-containing dAb; H (vii) dAb fragments consisting of the V domain (see, e.g., Ward et al., Nature 341:544-546, 1989); H or V L (viii) isolated complementarity determining regions (CDRs); and (ix) optionally linked by a synthetic linker. Combinations of two or more (e.g., two, three, four, five, or six) isolated CDRs of an Fv fragment. L and V H ) are encoded by separate genes, but they can be joined by a linker using recombinant methods to form a single protein chain (V L and V H The Fv domains can be paired to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al. (See, e.g., et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Antigen-binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments can be screened for utility in the same manner as are intact antibodies. Antigen-binding fragments can be produced by any number of techniques, including recombinant DNA techniques, enzymatic synthesis of intact immunoglobulins, and the like. They can be produced by natural or chemical cleavage, or in some cases by chemical peptide synthesis procedures known in the art.

[0128] As used herein, the term "anti-CD117 antibody" or "antibody that binds to CD117" refers to an antibody that is useful as a diagnostic and / or therapeutic agent that targets CD117. As used herein, the term refers to an antibody that is capable of binding to CD117 with sufficient affinity.

[0129] As used herein, the term "bispecific antibody" refers to a " refers to, for example, a monoclonal antibody capable of binding to at least two different antigens. Often referred to as a human antibody or a humanized antibody. For example, one of the binding specificities may be a hematopoietic stem cell antibody. One antibody can be directed against a cell surface antigen, CD117 (e.g., GNNK+ CD117), and the other can specifically bind to a different hematopoietic stem cell surface antigen or another cell surface protein, such as a receptor or receptor subunit involved in a signaling pathway that specifically enhances cell proliferation.

[0130] As used herein, the term "complementarity determining region" (CDR) refers to the light and heavy chains of an antibody. The term "hypervariable region" refers to a hypervariable region found in both the nucleotide sequence and the nucleotide sequence of an antibody. The more highly conserved portions of variable domains are called framework regions (FRs). The amino acid positions representing hypervariable regions of an antibody may vary depending on the context and the various definitions known in the art. Some positions within variable domains may be considered hybrid hypervariable positions, in that these positions may be considered within a hypervariable region under one set of criteria, while being considered outside a hypervariable region under another set of criteria. One or more of these positions may also be found in an extended hypervariable region. As described herein, The antibodies described herein may contain modifications at these hybrid hypervariable positions. The variable domains of naturally occurring heavy and light chains each adopt a predominantly β-sheet structure and are connected by three CDRs. The CDRs in each chain are arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, with the framework regions grouped together in close proximity and, together with the CDRs from other antibody chains, forming the antibody. (See Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987.) In certain embodiments, immunoglobulin amino acid residues are numbered according to the immunoglobulin amino acid residue numbering system of Kabat et al. (but not limited to any antibody numbering scheme, including, but not limited to, IMGT and Chothia, etc.) unless otherwise specified. may be used).

[0131] As used herein, the terms "conditioning" and "conditioning" refer to the process of preparing a patient to receive a transplant containing hematopoietic stem cells. Such treatment promotes engraftment of the hematopoietic stem cell transplant (e.g., (as inferred from the sustained increase in the amount of viable hematopoietic stem cells in blood samples isolated from patients after conditioning treatment and subsequent hematopoietic stem cell transplantation) According to the methods described herein, a patient may be conditioned for hematopoietic stem cell transplantation therapy by administering to the patient an ADC, antibody, or antigen-binding fragment thereof capable of binding to an antigen expressed by hematopoietic stem cells, such as CD117 (e.g., GNNK+CD117). As described herein, the antibody may be covalently linked to a cytotoxin to form an ADC. Antibodies capable of binding to one or more of the above antigens, administering the antibody-binding fragment, or ADC, to a patient in need of hematopoietic stem cell transplantation therapy. For example, the present invention provides a method for selectively depleting endogenous hematopoietic stem cells, thereby increasing the number of exogenous hematopoietic stem cells. It may promote the engraftment of hematopoietic stem cell grafts by creating voids that are filled by the cell transplant.

[0132] As used herein, the term "conjugate" refers to a compound formed by chemical bonding of a reactive functional group of one molecule, such as an antibody, or antigen-binding fragment thereof, with a suitable reactive functional group of another molecule, such as a cytotoxin described herein. A conjugate may include a linker between the two molecules attached to each other. Examples of linkers that can be used to form conjugates include peptide-containing linkers (e.g., linkers containing naturally occurring or non-naturally occurring amino acids, e.g., D-amino acids). Linkers may be prepared using a variety of strategies, described herein and known in the art. Depending on the reactive moiety therein, the linker may be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012). Such conjugates are referred to herein as "drug-antibody conjugates," "antibody-drug ... They are also referred to interchangeably as "conjugates" and "ADCs."

[0133] As used herein, the term "coupling reaction" refers to a chemical reaction in which two or more substituents that are suitable to react with each other react to form a chemical substructure in which the molecular fragments attached to each substituent are linked (e.g., covalently). In some embodiments, the antibody or antigen-binding fragment thereof (e.g., a cytotoxin known in the art or described herein) may have a reactive group attached to the fragment that is a cytotoxin (e.g., a cytotoxin known in the art or described herein) attached to the antibody or antigen-binding fragment thereof (e.g., a cytotoxin known in the art or described herein, e ... Antibodies, antigen-binding fragments thereof, specific for Examples of suitable reactive substituents include nucleophilic / electrophilic pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / haloalkyl pairs, among others). carbonyl / α,β-unsaturated carbonyl pairs), diene / dienophile pairs (e.g., azido-, among others) Coupling reactions include, but are not limited to, These include thiol alkylation, hydroxyl alkylation, amine alkylation, amine condensation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, among others), aromatic nucleophilic substitution, aromatic electrophilic substitution, and other reaction modes known in the art or described herein.

[0134] As used herein, a "competitive repopulating unit (CRU)" is a unit of measurement for long-term engrafting stem cells that can be detected after in vivo transplantation. This refers to:

[0135] As used herein, "drug-to-antibody ratio" or "DAR" " refers to the number of cytotoxins (e.g., amatoxins) attached to the antibody of an ADC. The DAR of an ADC can range from 1 to 8, although higher loadings are possible depending on the number of attachment sites on the antibody. Thus, in certain embodiments, the ADCs described herein have a DAR of 1, 2, 3, 4, 5, 6, 7, or 8.

[0136] As used herein, the term "donor" refers to a human or animal from which one or more cells have been isolated prior to administering the cells or their progeny to a recipient. The one or more cells may be, for example, a population of hematopoietic stem cells.

[0137] As used herein, the term "diabody" refers to a molecule comprising two polypeptide chains. A bivalent antibody is one in which each polypeptide chain is connected to a V on the same peptide chain. H and VL A linker that is so short (e.g., consisting of five amino acids) that intramolecular association of the domains is not possible V linked by a linker H and V L This configuration includes the domain. This domain pairs with a complementary domain on another polypeptide chain to form a homodimeric structure. Thus, the term "triabody" refers to a trivalent antibody comprising three peptide chains, Each of them is a V within the same peptide chain. H and V L A single V linked by a linker (e.g., a linker consisting of 1–2 amino acids) that is too short to allow intramolecular association of the domains H Domain and one V L Peptides constructed in this way usually require the V domains of adjacent peptide chains to fold into their native structure. H Domain and V L They trimerize so that the domains are spatially close to each other (see, eg, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993).

[0138] As used herein, a "dual variable domain immunoglobulin" ("DVD-Ig") is an immunoglobulin that combines the target-binding variable domains of two monoclonal antibodies via a linker. This refers to a tetravalent, dual-targeting, single-agent antibody (see, e.g., Gu et al., Meth. Enzymol. 502:25-41, 2012).

[0139] As used herein, the term "endogenous" describes a substance such as a molecule, cell, tissue, or organ that is naturally found in a particular organism, such as a human patient (e.g., a hematopoietic stem cell, or a cell of the hematopoietic lineage such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte).

[0140] As used herein, the term "engraftment potential" refers to the ability of a hematopoietic stem cell to engraft. The term "engraftment" is used to refer to the ability of hematopoietic stem cells and hematopoietic progenitor cells to repopulate tissues, regardless of whether such cells are naturally circulating or provided by transplantation. The term encompasses all events surrounding or leading to engraftment, such as tissue homing and colonization of cells into the tissue of interest. Engraftment efficiency or engraftment rate can be assessed or quantified using any clinically accepted parameter known to those skilled in the art, including, for example, assessing competitive repopulating units (CRUs); marker uptake or expression in the stem cell-homed, colonized, or engrafted tissue; or assessing subject progression by disease progression, hematopoietic stem and progenitor cell survival, or recipient survival. Engraftment can also be assessed by measuring the number of white blood cells in peripheral blood after transplantation. Engraftment can also be assessed by measuring the recovery of bone marrow cells by donor cells in bone marrow aspirate samples.

[0141] As used herein, the term "exogenous" describes a substance, such as a molecule, cell, tissue, or organ, that is not naturally found in a particular organism, such as a human patient (e.g., a hematopoietic stem cell, or a cell of the hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte). Exogenous material includes materials that are introduced into or extracted from an organism from an external source. This includes those provided in the cultures that were released.

[0142] As used herein, the term "framework region" or "FW region" includes the amino acid residues adjacent to the CDRs of an antibody, or antigen-binding fragment thereof. Residues in the FW region include, for example, In particular, human antibodies, humanized antibodies, monoclonal antibodies, antibody fragments, Fab fragments It may be present in antibodies, single chain antibody fragments, scFv fragments, antibody domains, and bispecific antibodies.

[0143] As used herein, the term "hematopoietic stem cell" ("HSC") refers to a cell that has the ability to self-renew. , and granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, CD34 refers to immature blood cells that have the ability to differentiate into mature blood cells of various lineages, including, but not limited to, red blood cells, thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells, and T-cells). Such cells express the CD34 + May contain CD34 cells. + CD34+ cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells exhibit the characteristics of stem cells as defined above. In mice, HSCs are thought to comprise a subpopulation of cells that have CD34-, whereas in mice, HSCs are CD34-. Furthermore, HSCs are also referred to as long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). LT-HSCs and ST-HSCs are distinguished based on functional potential and expression of cell surface markers. For example, Human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A, etc.). In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, CD48-, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra, etc.). Negative for lineage markers, whereas ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra, etc.). In addition, ST-HSCs are involved in the maintenance of homeostasis. Under certain conditions, LT-HSCs are less quiescent and more proliferative than LT-HSCs. However, LT-HSCs have a high self-renewal capacity (i.e., they survive throughout adulthood and are continuously transferred through successive recipients). In contrast to ST-HSCs, which are capable of transplantation, ST-HSCs have limited self-renewal capacity (i.e., they survive for only a limited period of time and cannot be serially transplanted). Either of these HSCs can be used in the methods described herein. ST-HSCs are particularly useful because they are highly proliferative and therefore can give rise to differentiated progeny more quickly.

[0144] As used herein, the term "functional potential of hematopoietic stem cells" refers to 1) pluripotency (which includes, but is not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), 2) pluripotency (which includes, but is not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), 3) pluripotency (which includes, but is not limited to, erythrocytes, erythrocytes, thromb ... 1) self-renewal (which refers to the ability to differentiate into multiple different blood lineages, including cells that grow in the mother, such as dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells)); 1) refers to the functional properties of hematopoietic stem cells, including (i) the ability of hematopoietic stem cells to give rise to daughter cells of equivalent potential to the original stem cell, and further, this ability can occur repeatedly throughout an individual's lifetime without being exhausted, and (ii) the ability of hematopoietic stem cells or their progeny to home to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis upon reintroduction into a transplant recipient.

[0145] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or by gene rearrangement or somatic mutation in vivo). However, as used herein, the term "human antibody" is intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Human antibodies may be produced in human cells (e.g., by recombinant expression) or by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. When a human antibody is a single-chain antibody, it may contain a linker peptide not found in native human antibodies. For example, an Fv may contain a linker peptide, such as two to about eight glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region. Such linker peptides are considered to be of human origin. Human antibodies may be made by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes (e.g., See, for example, PCT Publication Nos. WO 1998 / 24893; WO 1992 / 01047; WO 1996 / 34096; WO 1996 / 33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598.

[0146] As used herein, a patient "in need" of a hematopoietic stem cell transplant is one or more hematopoietic stem cell transplant recipients. This includes patients with defects or deficiencies in liquid cell types, as well as patients with stem cell disorders, autoimmune diseases, cancer, or other conditions described herein. Hematopoietic stem cells generally exhibit: 1) pluripotency, thus capable of differentiating into multiple different blood lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells); 2) self-renewal, thus capable of giving rise to daughter cells with potential equivalent to that of the parent cell; and 3) the ability to be reintroduced into a transplant recipient, home to the hematopoietic stem cell niche, and re-establish productive and sustained hematopoiesis. Thus, patients with defects or deficiencies in one or more cell types of the hematopoietic lineage and administering hematopoietic stem cells to a patient in vivo to reconstitute a defective or missing cell population. For example, the patient may be suffering from cancer, and the deficiency may be caused by administering a chemotherapeutic or other agent that selectively or non-specifically reduces a cancerous cell population. Additionally or alternatively, the patient may be suffering from a hemoglobinopathy (e.g., a non-malignant hemoglobinopathy), such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome. The subject may be suffering from adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome. The subject may have or be affected by an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune disease. Additionally or alternatively, the subject may have or be affected by a malignant tumor, such as neuroblastoma or a blood cancer. For example, the subject may have leukemia, lymphoma, or myeloma. In some embodiments, the subject has acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the subject has myelodysplastic syndrome. In some embodiments, the subject has an autoimmune disease such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, type 1 diabetes, or another autoimmune condition described herein. In some embodiments, the subject is in need of chimeric antigen receptor T-cell (CART) therapy. In some embodiments, the subject has or is affected by a metabolic storage disorder. The subject has a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidosis, metachromatic leukodystrophy, or any other disease or disorder that can benefit from the treatments and therapies disclosed herein, including but not limited to, severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytic leukemia, Hematopoietic stem cell transplantation therapy is a treatment for a variety of conditions, including bone marrow disease, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis, as well as those described in "Bone Marrow Transplantation for Non-Malignant Disease," ASH Education Book, 1:319-338 (2000) (the disclosure of which relates to conditions that may be treated by hematopoietic stem cell transplantation therapy, and which is incorporated herein by reference in its entirety). Additionally or alternatively, a patient "in need of" a hematopoietic stem cell transplant may be suffering from or affected by one of the aforementioned conditions. In patients with or without the disease (thrombocytes, megakaryocytes, and The expression of one or more endogenous cell types within the hematopoietic lineage, such as platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes. One of skill in the art can determine whether the levels of one or more of the above cell types or other blood cell types are decreased relative to other healthy subjects, for example, by flow cytometry and fluorescence activated cell sorting (FACS) methods, among other procedures known in the art. It can be easily determined.

[0147] As used herein, the term "recipient" refers to a patient receiving a transplant, such as a transplant comprising a population of hematopoietic stem cells. The transplanted cells administered to the recipient can be, for example, autologous, syngeneic, or allogeneic cells.

[0148] As used herein, the term "sample" refers to a specimen (e.g., a sample) taken from a subject. , blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or dermis), pancreatic juice, chorionic villus samples, and cells).

[0149] As used herein, the term "scFv" refers to a single chain Fv antibody in which the variable domain of the heavy chain and the variable domain of the light chain from an antibody are combined to form a single chain. The variable region of an antibody light chain (V) is separated by a linker. L ) (e.g., CDR-L1, CDR-L2, and / or CDR-L3) and the variable region of the antibody heavy chain (V H ) (e.g., CDR-H1, CDR-H2, and / or The V of the scFv fragment contains a single polypeptide chain containing the CDR-H3 or CDR-H4. L Area and V H region The linker connecting the scFv fragments may be a peptide linker composed of proteinogenic amino acids. Alternative linkers may be used to increase the resistance of the scFv fragment to proteolysis (e.g., linkers containing D-amino acids) or to increase the solubility of the scFv fragment (e.g., polyethylene glycol-containing linkers or repeating linkers). hydrophilic linkers, such as polypeptides containing glycine and serine residues, To improve physical stability (e.g., by forming intramolecular or intermolecular disulfide bonds), a linker containing a cysteine ​​residue that reduces the immunogenicity of the scFv fragment, or For example, linkers containing glycosylation sites may be used to enhance the binding of scFv molecules to the corresponding antibody. Those skilled in the art will also appreciate that the variable regions of the scFv molecules described herein may be modified to vary in amino acid sequence from the antibody molecule from which they are derived. For example, nucleotide or amino acid substitutions resulting in conservative substitutions or changes at amino acid residues may be made (e.g., in CDR and / or framework residues) to preserve or enhance the potency of the scFv (its ability to bind to an antigen recognized by the corresponding antibody).

[0150] As used herein, the terms "subject" and "patient" refer to an organism, such as a human, receiving treatment for a particular disease or condition as described herein. For example, a patient, such as a human patient, may receive treatment prior to hematopoietic stem cell transplantation therapy to promote engraftment of exogenous hematopoietic stem cells.

[0151] As used herein, the phrase "substantially cleared from the blood" refers to the time after administration of a therapeutic agent (e.g., an anti-CD117 antibody, or antigen-binding fragment thereof) to a patient. and the concentration of the therapeutic agent in a blood sample isolated from the patient is such that the therapeutic agent is not detectable by conventional methods (e.g., the therapeutic agent is The term "therapeutic agent" refers to a therapeutic agent that is not detectable above the noise threshold of the device or assay used to detect the therapeutic agent. ELISA-based detection assays known in the art or described herein Further assays that can be used to detect antibodies or antibody fragments include immunoprecipitation and immunoblot assays, among others, known in the art.

[0152] As used herein, the phrase "stem cell disorder" refers to a disorder that is caused by conditioning a target tissue of a subject and / or by removing an endogenous stem cell population in the target tissue (e.g., removing an endogenous hematopoietic stem cell population or hematopoietic progenitor population from bone marrow tissue of a subject). by removing a cell population) and / or engrafting stem cells into a target tissue of interest. The term "hematopoietic stem cell transplantation" broadly refers to any disease, disorder, or condition that can be treated or cured by transplanting or transplanting stem cells. For example, type 1 diabetes has been shown to be cured by hematopoietic stem cell transplantation. and can benefit from conditioning in accordance with the compositions and methods described herein. Additional disorders that may be treated using the compositions and methods described herein include, but are not limited to, sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome. Additional diseases that may be treated using the methods of patient conditioning and / or hematopoietic stem cell transplantation described herein include inherited blood disorders (e.g., sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome). red cell anemia) and autoimmune diseases such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease Additional diseases that may be treated using the conditioning and / or transplantation methods described herein include malignant tumors (e.g., neuroblastoma), or hematological cancers (e.g., For example, the cancer may be acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma Additional diseases treatable using the conditioning and / or transplantation methods described herein include myelodysplastic syndromes. In some embodiments, the subject has or is affected by a metabolic storage disorder. For example, the subject may have a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidosis, metachromatic leukodystrophy, or any other disease or disorder that can benefit from the treatments and therapies disclosed herein (including, but not limited to, severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis), as well as those described in "Bone Marrow Transplantation for Non-Malignant disease," ASH Education Book, 1:319-338 (2000), the disclosure of which relates to conditions that may be treated by hematopoietic stem cell transplantation therapy and which is incorporated herein by reference in its entirety.

[0153] As used herein, the term "transfection" includes electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, and Commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as recombinant DNA technology. The term "technology" refers to any of a wide variety of techniques commonly used in the medical field.

[0154] As used herein, the term "treating" or "treatment" refers to reducing the severity and / or frequency of disease symptoms, eliminating disease symptoms and / or the underlying cause of said symptoms, reducing the frequency or likelihood of disease symptoms and / or their underlying cause, and ameliorating or repairing damage caused directly or indirectly by the disease. Beneficial or desired clinical outcomes include, but are not limited to, the improvement or repair of a patient's condition following antibody conditioning therapy and subsequent hematopoietic stem cell transplantation therapy as described herein.

[0010] Further beneficial results include enhanced engraftment of exogenous hematopoietic cells in a patient in need of hematopoietic stem cell transplantation after administering a conditioning regimen and subsequent exogenous hematopoietic stem cell graft to said patient. Beneficial results of the therapies described herein also include enhanced engraftment of one or more hematopoietic lineages after administering a conditioning regimen and subsequent hematopoietic stem cell transplantation regimen. cells (e.g., megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells) cells, myeloblasts, basophils, neutrophils, eosinophils, microglial cells, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, or This includes an increase in the number or relative concentration of cells (e.g., B lymphocytes). Further beneficial results include the ability to differentiate cancer cells (e.g., CD117+ leukemia cells) or autoimmune cells. The effects of preventing a disorder may include reducing the amount of a disease-causing cell population, such as a population of CD117+ autoimmune lymphocytes (e.g., CD117+ T-cells that express a T-cell receptor that cross-reacts with an autoantigen). To the extent that the methods of the invention are directed to preventing a disorder, it is understood that the term "preventing" does not require that the disease state be completely averted. Rather, as used herein, the term preventing refers to the ability of one of skill in the art to identify a population susceptible to a disease, administer a compound of the invention prior to the onset of the disease, and the like. The term does not imply that the disease state is completely avoided.

[0155] As used herein, the terms "variant" and "derivative" are used interchangeably and refer to naturally occurring, synthetic, and semi-synthetic analogs of the compounds, peptides, proteins, or other substances described herein. Variants or derivatives of the compounds, peptides, proteins, or other substances described herein may retain or improve the biological activity of the original substance.

[0156] As used herein, the term "vector" includes plasmids, DNA vectors, and nucleic acid vectors, such as nucleotides, RNA vectors, viruses, or other suitable replicons. The expression vectors described herein may contain polynucleotide sequences and additional sequence elements used, for example, to express proteins and / or integrate these polynucleotide sequences into the genome of mammalian cells. Particular vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that increase the translation efficiency of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements may include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to ensure efficient transcription of genes harbored on the expression vector. The expression vectors described herein may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.

[0157] As used herein, the term "acyl" refers to -C(=O)R, where R is hydrogen ("aldehyde"), alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl, as defined herein. Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, and acryloyl.

[0158] As used herein, the term "alkyl" refers to an alkyl group having, for example, 1 to 20 carbon atoms in the chain. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and the like. Examples include ethyl, isopentyl, tert-pentyl, hexyl, and isohexyl.

[0159] As used herein, the term "alkylene" refers to a straight or branched chain divalent alkyl group. The divalent positions may be on the same atom or on different atoms within the alkyl chain. Examples of alkylene include methylene, ethylene, propylene, isopropylene, and the like.

[0160] As used herein, the term "heteroalkyl" refers to a heteroalkyl group having, for example, 1 to 20 alkyl groups in the chain. It refers to a straight or branched chain alkyl group having carbon atoms and further containing one or more heteroatoms (such as oxygen, nitrogen, or sulfur, among others) in the chain.

[0161] As used herein, the term "heteroalkylene" refers to a straight-chain or branched-chain divalent heteroalkyl group. The divalent positions can be on the same atom or on different atoms within the heteroalkyl chain. The divalent positions can be at one or more heteroatoms.

[0162] As used herein, the term "alkenyl" refers to an alkyl group having, for example, 2 to 20 carbon atoms in the chain. It refers to a straight or branched chain alkenyl group having 1 to 3 carbon atoms. Examples of alkenyl groups include vinyl, propenyl, isopropenyl, butenyl, tert-butylenyl, hexenyl, etc. do.

[0163] As used herein, the term "alkenylene" refers to a straight or branched chain divalent alkenyl group. The divalent positions may be on the same atom or different atoms within the alkenyl chain. Examples of alkenylene include ethenylene, propenylene, isopropenylene, butenylene, etc.

[0164] As used herein, the term "heteroalkenyl" refers to a heteroaryl group having, for example, 2 to 20 alkyl groups in the chain. "Alkenyl" refers to a straight- or branched-chain alkenyl group having 1 to 10 carbon atoms and further containing one or more heteroatoms (such as oxygen, nitrogen, or sulfur, among others) in the chain.

[0165] As used herein, the term "heteroalkenylene" refers to a straight or branched chain divalent heteroalkenyl group. The divalent positions can be on the same atom or on different atoms within the heteroalkenyl chain. The divalent positions can be one or more heteroatoms. do.

[0166] As used herein, the term "alkynyl" refers to an alkynyl group having, for example, 2 to 20 carbon atoms in the chain. "alkynyl" refers to a straight or branched chain alkynyl group having 1 to 4 carbon atoms. Examples of alkynyl groups include propargyl, butynyl, pentynyl, hexynyl, and the like.

[0167] As used herein, the term "alkynylene" refers to a straight or branched chain divalent alkynyl group. The divalent positions can be on the same atom or on different atoms within the alkynyl chain.

[0168] As used herein, the term "heteroalkynyl" refers to a heteroalkynyl group having, for example, 2 to 20 alkyl groups in the chain. "alkynyl" refers to a straight- or branched-chain alkynyl group having 1 to 10 carbon atoms and further containing one or more heteroatoms (such as oxygen, nitrogen, or sulfur, among others) in the chain.

[0169] As used herein, the term "heteroalkynylene" refers to a straight or branched chain divalent heteroalkynyl group. The divalent positions can be on the same atom or on different atoms within the heteroalkynyl chain. The divalent positions can be one or more heteroatoms. do.

[0170] As used herein, the term "cycloalkyl" refers to a monocyclic or fused, bridged, or spiro polycyclic ring structure that is saturated and has, for example, from 3 to 12 carbon ring atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and bicyclo[3.1.0]hexane. Examples include:

[0171] As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl group. The divalent positions may be on the same atom or different atoms within the ring structure. Examples of cycloalkylene include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and the like.

[0172] As used herein, the term "heterocyloalkyl" refers to a saturated and having 3 to 12 ring atoms per ring structure selected, for example, from carbon atoms and heteroatoms (e.g., selected from nitrogen, oxygen, and sulfur, among others). Heterocyclo refers to a heterocyclic, monocyclic, or fused, bridged, or spiro polycyclic ring system. The ring system may contain one or more oxo groups, for example, on carbon, nitrogen, or sulfur ring members. Examples of alkyl include dihydropyridyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahy- droxyphenyl, tetrahydropyranyl ... Examples include, but are not limited to, hydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinuclidinyl, and morpholinyl.

[0173] As used herein, the term "heterocycloalkylene" refers to a divalent heterocycloalkyl group. The divalent positions may be on the same atom or on different atoms within the ring structure.

[0174] As used herein, the term "aryl" refers to an aryl group containing, for example, 6 to 19 carbon atoms. Aryl groups include, but are not limited to, phenyl, fluorenyl, naphthyl, etc. The divalent position may be one or more heterocyclic groups. It can be an atom.

[0175] As used herein, the term "arylene" refers to a divalent aryl group. The divalent positions may be on the same atom or on different atoms.

[0176] As used herein, the term "heteroaryl" refers to a monocyclic heteroaromatic, or a bicyclic or tricyclic fused-ring heteroaromatic group in which one or more ring atoms is a heteroatom. Heteroaryl groups include pyridyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolidinyl, quinazolinyl, phthalazinyl, quinoxalinyl, cinnolinyl, napthyridinyl, pyrido[3,4-b]pyridyl, Examples include pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl, and benzoquinolyl.

[0177] As used herein, the term "heteroarylene" refers to a divalent heteroaryl group. The divalent positions may be on the same atom or on different atoms. The divalent positions may be at one or more heteroatoms.

[0178] Unless otherwise constrained by the definition of the individual substituents, the aforementioned chemical moieties, e.g., "alkyl," "alkylene," "heteroalkyl," "heteroalkylene," "alkenyl," "alkenylene," "heteroalkenyl," "heteroalkenylene," "alkynyl," "alkynylene," "heteroalkynyl," "heteroalkynylene," "Cycloalkyl," "cycloalkylene," "heterocycloalkyl," "heterocycloalkylene," "aryl," "arylene," "heteroaryl," and "heteroarylene" groups and the like may be optionally substituted with 1 to 5 substituents selected from the group consisting of, for example, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, alkylcycloalkyl, alkylheterocycloalkyl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, nitro, and the like. Exemplary substituents include, but are not limited to, -X, -R, -OH, -OR, -SH, -SR, NH, -NHR, -N(R)2, -N + (R)3, -CX3, -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO2, -N3, -NC(=O)H, -NC(=O)R, -C(=O)H, -C(=O)R, -C(=O)NH2, -C(=O)N(R)2, -SO3-, -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NH 2, -S(=O)2N(R)2, -S(=O)R, -OP(=O)(OH) 2, -OP(=O)(OR)2, -P(=O)(OR)2, -PO3, -PO3H2, -C(=O)X, -C(=S)R, -CO2H, -CO2R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NH 2,-C(=O)N(R)2, -C(=S)NH 2, -C(=S)N(R)2, -C(=NH)NH 2, and —C(═NR)N(R)2; where each X is independently selected at each occurrence from F, Cl, Br, and I; and each R is alkyl, aryl, heterocycloalkyl, or heteroaryl, protecting groups, and prodrug moieties, independently in each occurrence. Wherever a group is described as being "optionally substituted," that group may be substituted, independently in each occurrence, with one or more of the above substituents. The substitution may include ring closure with adjacent substituents (e.g., ring closure of adjacent functional substituents) to form, for example, lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals, aminals, and hemiaminals (which are formed by ring closure, e.g., to provide a protecting group).

[0179] It should be understood that the naming convention for a given radical may include either a mono-radical or a di-radical, depending on the context. For example, if a substituent is a 2-membered group to the rest of the molecule, If two bonding positions are required, the substituent is understood to be a di-radical. For example, substituents identified as alkyl, which require two points of attachment, include di-radicals such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc. Other radical naming conventions are described in the above It is clearly indicated that the dialkyl is a di-radical such as "alkylene," "alkenylene," "arylene," "heterocycloalkylene," etc.

[0180] The substituent is shown as a di-radical (i.e., the positions of both bonds to the rest of the molecule are In all cases where a group has a substituent, the substituents may be bonded in any directional configuration unless otherwise indicated. It should be understood that the various

[0181] Anti-CD117 antibody The present invention is based in part on the discovery of novel anti-CD117 antibodies and antigen-binding portions thereof that are useful for therapeutic purposes. The present invention is based on the use of antibodies capable of binding to CD117, such as GNNK+ CD117, or antigen-binding fragments thereof, as the sole therapeutic agent or as an ADC, for example, as follows: (i) CD117+ cells, based in part on the discovery that CD117+ cells can be used for the purposes of (ii) to treat cancers (e.g., acute myeloid leukemia or myelodysplastic syndromes) and autoimmune diseases characterized by the presence of CD117, and (iii) to promote the engraftment of transplanted hematopoietic stem cells in patients requiring transplantation therapy. These therapeutic activities are mediated by, for example, the binding of CD117 expressed on the surface of cells such as cancer cells, autoimmune cells, or hematopoietic stem cells (e.g., GNNK+ CD117) to anti-CD117 antibodies. The effect of hematopoietic stem cells is due to the binding of the antigen-binding fragment thereof to the target cells and subsequent induction of cell death. The depletion of endogenous hematopoietic stem cells provides a niche to which transplanted hematopoietic stem cells can home, and subsequently establish productive hematopoiesis. In this manner, transplanted hematopoietic stem cells can successfully engraft in patients, such as human patients suffering from stem cell disorders described herein.

[0182] Human CD117 (also known as c-Kit, mRNA NCBI reference sequence: NM_000222.2, protein NCBI reference sequence: Antibodies and antigen-binding fragments capable of binding to GNNK+CD117 (reference sequence: NP_000213.1) (including antibodies and antigen-binding fragments capable of binding to GNNK+CD117) may be used in combination with the compositions and methods described herein to condition patients for hematopoietic stem cell transplantation therapy. Polymorphisms affecting this region or the extracellular domain are currently not well understood for indications other than oncology. At least four isoforms of CD117 have been identified, and additional isoforms may be expressed in tumor cells. Two of these are localized in the intracellular domain of the protein, and two in the outer juxtamembrane region. Two extracellular isoforms, GNNK+ and GNNK-, are characterized by the presence (GNNK+) or absence (GNNK-) of a four amino acid sequence. These isoforms have been reported to have the same affinity for the ligand (SCF), but ligand binding to the GNNK- isoform is not observed in cells. It has been reported that GNNK+ isoform increases uptake and degradation in the cytosol. The GNNK+ isoform may be used as an immunogen to generate antibodies capable of binding to CD117, as antibodies generated against this isoform include GNNK+ and GNNK- proteins.

[0183] The present invention provides novel anti-CD117 antibodies whose heavy and light chain amino acid sequences are provided in Tables 1, 6, 8, and 16. Accordingly, the present invention also includes anti-CD117 antibody drugs comprising the binding regions (heavy and light chain CDRs or variable regions) set forth in SEQ ID NOs: 7 to 168. In one embodiment, the anti-CD117 antibody, or an antigen-binding portion thereof, is a conjugate. The anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:8. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:9. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:10. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:10. The antigen-binding portion of the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 7. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 11. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 12. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 12. The portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:13. The anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 14. the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 7, and the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 15 In one embodiment, the anti-CD117 antibody comprises a light chain variable region shown in the amino acid sequence The antibody, or an antigen-binding portion thereof, may comprise a heavy chain polypeptide as set forth in the amino acid sequence of SEQ ID NO:7. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 17. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 17. The original binding portion comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 7, and a sequence In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 19. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises: In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:20. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO:21, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:22. In one embodiment, the anti-CD117 antibody or its derivatives comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:22. The antigen-binding portion of the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 7, and and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 23. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises the amino acid sequence of SEQ ID NO: 24. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 25. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 26 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 27. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 28. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:29. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:29. In one embodiment, the anti-CD117 antibody comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 30, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 31. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 32 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 33. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 34 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 35. In one embodiment, the anti-CD117 antibody or its The antigen-binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 36 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 37. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 38. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 39. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, The heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 40, and the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 41. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 32. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 43 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 44. In one embodiment, the anti-CD117 antibody comprises a light chain variable region shown in the amino acid sequence: or an antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 45 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 46. In one embodiment, the anti-CD117 antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 47 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 48. In one embodiment, the anti-CD117 antibody or its anti-CD117 antibody comprises a light chain variable region as shown in The antigen-binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 49 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 50. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 51. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 52. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 53, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 54. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 55. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 57 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 58. In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence: or an antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 59 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 60. In one embodiment, the anti-CD117 antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 61. and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 50. In one embodiment, the anti-CD117 antibody or its antigen The binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 62 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 63. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 64. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 65. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises: In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 66 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 67. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 68. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 69, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 70. In one embodiment, the anti-CD117 antibody or and an antigen-binding portion thereof comprising a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 72 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 73. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 74. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 75. In one embodiment, the anti-CD117 antibody, or its antigen-binding domain, In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 76 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 77. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 78. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 79. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 79. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 80 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 81. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 82. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 83, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 84. a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 84, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 85; In one embodiment, the anti-CD117 antibody or The antigen-binding portion thereof comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 86 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 87. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 88. In one embodiment, the anti-CD117 antibody, or the antigen-binding portion thereof, comprises a light chain variable region comprising The fragment comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 89. In one embodiment, the fragment The anti-CD117 antibody, or antigen-binding portion thereof, may be selected from the group consisting of SEQ ID NO: 7, and the heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 90 In one embodiment, the anti-CD117 antibody or its derivatives comprises a light chain variable region as shown in the sequence The antigen-binding portion of the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 7, and and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 91. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 92. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 93. The anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:94. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:95. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, The antigen-binding portion of the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 7. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:96.

[0184] In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO: In one embodiment, the anti-CD117 antibody or its derivatives comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:97. The antigen-binding portion of the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 7, and In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 143 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 144. In one embodiment, the anti-CD117 antibody or its antigen-binding The portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 151, and a heavy chain variable region set forth in SEQ ID NO: 152. In one embodiment, the antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 152. The anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 143 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 156. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 160 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 152. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: In one embodiment, the anti-CD117 antibody comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 98 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 99. The antibody, or an antigen-binding portion thereof, may comprise a heavy chain polypeptide as set forth in the amino acid sequence of SEQ ID NO:7. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 100. In one embodiment, the anti-CD117 antibody or its The antigen-binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 98 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 101. wherein the anti-CD117 antibody, or antigen-binding portion thereof, is an amino acid sequence of SEQ ID NO:98. and the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 102. The light chain variable region comprises a light chain variable region.

[0185] In one embodiment, the antibody is an intact antibody comprising the heavy and light chain variable regions set forth in Table 1. In one embodiment, the anti-CD117 antibody is designed to have a short half-life. It is being manipulated in this way.

[0186] (SEQ ID NO: 7) [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13]

[0187] The nucleic acid sequences corresponding to the heavy and light chain regions of certain of the sequences above are shown in Table 2.

[0188] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15]

[0189] As described below, human antibody scFV phage display library screening was performed to identify novel anti-CD117 antibodies and fragments thereof with therapeutic applications. In this screen, antibodies 85 (Ab85), 86 (Ab86), 87 (Ab87), 88 (Ab88), and 89 (Ab89), among others, were identified.

[0190] The heavy chain variable region (VH) amino acid sequence of Ab85 is shown below as SEQ ID NO: 143. The VH CDR amino acid sequences of Ab85 are underlined and shown below: NYWIG (VH CDR1; SEQ ID NO: 145); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 146); and HGRGYEGYEGAFDI (VH CDR3; SEQ ID NO: 147).

[0191] VH sequence of Ab85 [ka] (SEQ ID NO: 143)

[0192] The amino acid sequence of the light chain variable region (VL) of Ab85 is set forth below as SEQ ID NO: 144. The VL CDR amino acid sequences of Ab85 are underlined and shown below: RSSQGIRSDLG (VL CDR1; SEQ ID NO: 148); DASNLET (VL CDR2; SEQ ID NO: 149); and QQANGFPLT (VL CDR3; SEQ ID NO: 150).

[0193] VL sequence of Ab85 [ka] (SEQ ID NO: 144)

[0194] Antibody HC-86 / LC-86(Ab86) The heavy chain variable region (VH) amino acid sequence of Ab86 is shown below as SEQ ID NO: 151. The VH CDR amino acid sequences of Ab86 are underlined and shown below: NYWIG (VH CDR1; SEQ ID NO: 145); IIYPGDSDIRYSPSLQG (VH CDR2; SEQ ID NO: 153); and HGRGYNGYEGAFDI (VH CDR3; SEQ ID NO: 3).

[0195] VH sequence of Ab86 [ka] (SEQ ID NO: 151)

[0196] The amino acid sequence of the light chain variable region (VL) of Ab86 is shown below as SEQ ID NO: 152. The VL CDR amino acid sequences of Ab86 are underlined and shown below: RASQGIGDSLA (VL CDR1; SEQ ID NO: 154); DASNLET (VL CDR2; SEQ ID NO: 149); and QQLNGYPIT (VL CDR3; SEQ ID NO: 155).

[0197] VL sequence of Ab86 [ka] (SEQ ID NO: 152)

[0198] Antibody HC-87 / LC-87(Ab87) The heavy chain variable region (VH) amino acid sequence of Ab87 is shown below as SEQ ID NO: 143. The VH CDR amino acid sequences of Ab87 are underlined and shown below: NYWIG (VH CDR1; SEQ ID NO: 145); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 146); and HGRGYEGYEGAFDI (VH CDR3; SEQ ID NO: 147).

[0199] VH sequence of Ab87 [ka] (SEQ ID NO: 143)

[0200] The amino acid sequence of the light chain variable region (VL) of Ab87 is set forth below as SEQ ID NO: 156. The VL CDR amino acid sequences of Ab87 are underlined and shown below: RASQGIRNDLG (VL CDR1; SEQ ID NO: 157); DASSLES (VL CDR2; SEQ ID NO: 5); and QQLNGYPIT (VL CDR3; SEQ ID NO: 155).

[0201] VL sequence of Ab87 [ka] (SEQ ID NO: 156)

[0202] Antibody HC-88 / LC-88(Ab88) The heavy chain variable region (VH) amino acid sequence of Ab88 is shown below as SEQ ID NO: 158. The VH CDR amino acid sequences of Ab88 are underlined and shown below: NYWIG (VH CDR1; SEQ ID NO: 145); IIYPGDSLTRYSPSFQG (VH CDR2; SEQ ID NO: 159); and HGRGYNGYEGAFDI (VH CDR3; SEQ ID NO: 3).

[0203] Ab88 VH sequence [ka] (SEQ ID NO: 158)

[0204] The amino acid sequence of the light chain variable region (VL) of Ab88 is shown below as SEQ ID NO: 156. The VL CDR amino acid sequences of Ab88 are underlined and shown below: RASQGIRNDLG (VL CDR1; SEQ ID NO: 157); DASSLES (VL CDR2; SEQ ID NO: 5); and QQLNGYPIT (VL CDR3; SEQ ID NO: 155).

[0205] Ab88 VL sequence [ka] (SEQ ID NO: 156)

[0206] Antibody HC-89 / LC-89(Ab89) The heavy chain variable region (VH) amino acid sequence of Ab89 is shown below as SEQ ID NO: 160. The VH CDR amino acid sequences of Ab89 are underlined and shown below: NYWIG (VH CDR1; SEQ ID NO: 145); IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 2); and HGRGYNGYEGAFDI (VH CDR3; SEQ ID NO: 3).

[0207] VH sequence of Ab89 [ka] (SEQ ID NO: 160)

[0208] The amino acid sequence of the light chain variable region (VL) of Ab89 is set forth below as SEQ ID NO: 152. The VL CDR amino acid sequences of Ab89 are underlined and shown below: RASQGIGDSLA (VL CDR1; SEQ ID NO: 154); DASNLET (VL CDR2; SEQ ID NO: 149); and QQLNGYPIT (VL CDR3; SEQ ID NO: 155).

[0209] VL sequence of Ab89 [ka] (SEQ ID NO: 152)

[0210] Antibody HC-249 / LC-249(Ab249) The heavy chain variable region (VH) amino acid sequence of Ab249 is shown below as SEQ ID NO:98. The VH CDR amino acid sequence of Ab249 is underlined and shown below: TSWIG (VH CDR1; sequence IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 2); and HGLGYNGYEGAFDI (VH CDR3; SEQ ID NO: 187).

[0211] VH sequence of Ab249 [ka] (SEQ ID NO: 98)

[0212] The light chain variable region (VL) amino acid sequence of Ab249 is set forth below as SEQ ID NO: 102. The VL CDR amino acid sequence of Ab249 is underlined and shown below: RASQGIGSALA (VL CDR1; SEQ ID NO: 188); DASNLET (VL CDR2; SEQ ID NO: 149); and QQLNGYPLT (VL CDR3; SEQ ID NO: 189).

[0213] VL sequence of Ab249 [ka] (SEQ ID NO: 102)

[0214] Both human antibodies Ab85 and Ab249 are derived from the antagonist anti-CD117 antibody CK6. Comparisons of the amino acid sequences of the variable regions of Ab85 and Ab249 with those of CK6 are shown in Figures 11A to 11D (where the CDRs are also designated). Both antibodies have improved properties (e.g., improved binding properties) compared to CK6.

[0215] CK6 identifies potential deamidation sites in the CDR3 domain of the heavy chain variable region. For future production of the antibody, it would be advantageous to remove the asparagine, but the location of the asparagine is a major issue. However, in the Ab85 heavy chain CDR3, the antibody (Ab85 heavy chain The CDRs of the α- and β-chain ... The potential deamidation sites were successfully removed while maintaining the potency of Ab85. Furthermore, Ab85 exhibited an improved off-rate compared to its parent.

[0216] Thus, in certain embodiments, the anti-CD117 antibody comprises a heavy chain comprising the CDR set (CDR1, CDR2 and CDR3) set forth in SEQ ID NOs: 145, 146 and 147, and a CDR set forth in the sequence and expressing CD117, comprising a light chain comprising a set of CDRs set forth in SEQ ID NOs. (SEQ ID NOs.): 148, 149, and 150. The antibody was taken up into the cells and measured by BLI at 5 x 10 -4 s -1 The following k off Has speed.

[0217] As described in Jain et al. (2017) PNAS 114 (5) 944-949, antibody activity is therapeutic. Key to their development as therapeutics, but often overlooked, is the "developability" of antibodies for industrial production purposes. It is difficult to identify antibodies that can achieve both therapeutic and good biophysical properties. In fact, the biophysical properties of antibodies are essential for antibodies to be used for therapeutic purposes. Biophysical evaluation of Ab 85 revealed that it is a particularly stable For example, the Ab85 antibody population maintains low levels of acid variants even when left at higher temperatures (see Figures 13A and 13B). This was also true in the context of other CK6-derived antibodies. Thus, in certain embodiments, Therefore, the compositions included in the present invention were stored at 25°C for 7 days and then analyzed by capillary electrophoresis. wherein the antibody is an IgG antibody that specifically binds to CD117 and comprises a heavy chain comprising the CDR set (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: (SEQ ID NOs): 145, 146, and 147, and a light chain comprising the CDR set forth in SEQ ID NOs: (SEQ ID NOs): 148, 149, and 150.

[0218] The anti-CD117 antibodies or binding fragments described herein also include those disclosed in the art. As known, the properties of the antibody and / or fragment may be modified (e.g., to increase half-life). These may also include modifications and / or mutations (e.g., that increase or decrease ADCC) that increase or decrease ADCC.

[0219] In one embodiment, the anti-CD117 antibody, or binding fragment thereof, comprises a variant Fc region. wherein the variant Fc region has at least one amino acid sequence different from that of the wild-type Fc region. The Fc region contains amino acid modifications that result in the molecule having altered affinity for FcγR. The specific amino acid positions are known from crystallographic studies of direct contact with FcγR. Specifically, amino acids 234 to 239 (hinge region), amino acids 265 to 269 (B / C loop), amino acids 297 to 299 (C' / E loop), and amino acids 327 to 332 (F / G loop) (see Sondermann et al., 2000 Nature, 406: 267-273). For example, amino acids 234 and 235 of the Fc region These amino acid substitutions have been identified as reducing the affinity of IgG antibodies for binding to Fc receptors, particularly Fc gamma receptors (FcγRs). In certain embodiments, the anti-CD117 antibodies described herein comprise an Fc region containing amino acid substitutions at L234 and / or L235 (e.g., L234A and L235A) (EU index). Thus, the anti-CD117 antibodies described herein may comprise a variant Fc region in which at least one residue that directly contacts FcγRs has been altered based on structural and crystallographic analysis. In certain embodiments, the Fc region of the anti-CD117 antibodies may be modified. The Fc region (or a fragment thereof containing the Fc region) is described in Kabat et al., Sequences of Proteins of "EU index as in Kabat" or "EU index" refers to the amino acid substitution at amino acid 265 according to the EU index as in Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991), which is expressly incorporated herein by reference. Unless otherwise specified, the numbering refers to the human IgG1 EU antibody and the Fc amino acid positions are designated herein as Used for reference.

[0220] In one embodiment, the Fc region comprises a D265A mutation. contains the D265C mutation.

[0221] In some embodiments, the Fc region of the anti-CD117 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index as in Kabat. In some embodiments, the Fc region comprises a L234A mutation. The Fc region of the IgG1 gene (or fragment thereof) is amino acid 235 according to the EU index as per Kabat. In one embodiment, the Fc region comprises an amino acid substitution at L235A. In one embodiment, the Fc region comprises the L234A and L235A mutations. In a further embodiment, the Fc region comprises the D265C, L234A, and L235A mutations.

[0222] In certain embodiments, the variant IgG Fc domain comprises one or more amino acid substitutions. The Fc binding interaction comprises one or more amino acid substitutions that result in reduced or abolished binding affinity for Fc gamma R and / or C1q compared to a wild-type Fc domain that does not contain the Fc gamma R and / or C1q. , various effector functions and downstream signaling events (e.g., but not limited to, antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)). Thus, in certain embodiments, antibodies comprising an altered Fc region (e.g., , L234A, L235A, and D265C mutations) substantially reduce or eliminate effector function.

[0223] Affinity for the Fc region can be measured using a variety of techniques known in the art, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; radioimmunoassay (RIA)), or surface plasmon resonance assays or other mechanism-based kinetic assays (e.g., BIACORE TM Analysis or Octet TM analysis (forteBIO)), as well as other methods (e.g., indirect binding assays, Measurements can be made using competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). These and other methods utilize labels present on one or more components of interest. The antibody may be labeled with a marker and / or may utilize various detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which is incorporated herein by reference in its entirety. An example of a competitive binding assay is the radioimmunoassay, which involves incubating a labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen and detecting antibody bound to the labeled antigen. The affinity and binding off-rate of the antibody of interest for a particular antigen may be determined from the data by Scatchard plot analysis. Competition with a second antibody may also be measured using radioimmunoassays, in which the antigen is conjugated to a labeled compound in the presence of increasing amounts of an unlabeled second antibody. Incubate with the conjugated antibody of interest.

[0224] In certain embodiments, the anti-CD117 antibodies described herein comprise the amino acid sequence of L235A, L235A, and D265C. (EU index) containing the Fc region. The antibodies of the present invention can be further modified to form antibodies as described, for example, in (Dall'Acqua et al. (2006) J Biol Chem 281: 23514-24), (Zalevsky et al. (2010) Nat Biotechnol 28: 157-9), (Hinton et al. (2004) J Biol Chem 279: 6213-6), (Hinton et al. (2006) J Immunol 176: 346-56), (Shields et al. (2001) J Biol Chem 276: 6591-604), (Petkova et al. (2006) Int Immunol 18: 1759-69), (Datta-Mannan et al. (2007) Drug Metab Dispos 35: 86-94), (Vaccaro et al. (2005) Nat Biotechnol 23: 1283-8), (Yeung et al. (2010) Cancer Res 70: 3269-77) and (Kim et al. (1999) Eur J Immunol 29: 2819-25), and may involve positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434, and 435. Exemplary mutations that may be made alone or in combination include T250Q, M252Y, 1253A, S254T, T256E, P2571, T307A, D376V, E380A, M428L, H433K, N434S, N434A , N434H, N434F, H435A and H435R mutations.

[0225] Thus, in one embodiment, the Fc region comprises a mutation that results in a reduced half-life. Antibodies with short half-lives are advantageous in certain instances where the antibody is expected to function as a short-lived therapeutic agent, such as in the conditioning techniques described herein, where the antibody is administered followed by HSCs. Ideally, unlike endogenous stem cells, these cells would generally express CD117. Before administering to HSCs that express, but are not the target of, the anti-CD117 antibody, the antibody should be substantially cleared. In one embodiment, the Fc region comprises a mutation at position 435 (EU index according to Kabat). In one embodiment, the mutation is an H435A mutation. .

[0226] In certain embodiments, the anti-CD117 antibodies described herein are administered for up to 24 hours, up to 22 hours, or up to 8 p.m. The antibody has a half-life of 1 hour or less, 18 hours or less, 16 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, or 11 hours or less. In some embodiments, the half-life of the antibody is 11 to 24 hours, 12 to 22 hours, 10 to 20 hours, 8 to 18 hours, or 14 to 24 hours.

[0227] Anti-CD117 antibodies that can be used in conjunction with the methods of conditioning a patient described herein include, for example, the SR-1 antibody, identified as ATCC Accession No. 10716 (BA7.3C.9). and antibodies produced and released from the CD117 antibody deposit, which are described, for example, in U.S. Pat. No. 5,489,516, the disclosure of which is incorporated herein by reference as it relates to anti-CD117 antibodies. , which is incorporated by reference.

[0228] In certain embodiments, the anti-CD117 antibodies described herein comprise an Fc region comprising L235A, L235A, D265C, and H435A (EU index).

[0229] Additional anti-CD117 antibodies that can be used in combination with the methods of conditioning a patient described herein include the anti-CD117 antibodies described in U.S. Pat. No. 7,915,391 (which describes, e.g., the humanized SR-1 antibody); U.S. Pat. No. 5,808,002 (which describes, e.g., the anti-CD117 A3C6E2 antibody), as well as the anti-CD117 antibodies described in, e.g., WO 2015 / 050959 (which describes anti-CD117 antibodies that bind to an epitope including Pro317, Asn320, Glu329, Val331, Asp332, Lus358, Glue360, Glue376, His378, and / or Thr380 of human CD117); and US 2012 / 0288506 (also published as U.S. Pat. No. 8,552,157), which describes, for example, the anti-CD117 antibody CK6 having the following CDR sequences: CDR-H1 having the amino acid sequence SYWIG (SEQ ID NO: 1); CDR-H2 having the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 2); CDR-H3 having the amino acid sequence HGRGYNGYEGAFDI (SEQ ID NO:3); CDR-L1 having the amino acid sequence RASQGISSALA (SEQ ID NO: 4); CDR-L2 having the amino acid sequence DASSLES (SEQ ID NO: 5); and CDR-L3 having the amino acid sequence CQQFNSYPLT (SEQ ID NO: 6) ) are mentioned.

[0230] The heavy chain variable region amino acid sequence of CK6 is provided in SEQ ID NO: 161: [ka] (SEQ ID NO: 161; CDRs are underlined and in bold).

[0231] The light chain variable region amino acid sequence of CK6 is provided in SEQ ID NO: 162: [ka] (SEQ ID NO: 162; CDRs are underlined and in bold).

[0232] Additional anti-CD117 antibodies that can be used in combination with the compositions and methods described herein include: The antibodies and their antigen-binding fragments include clones 9P3, NEG024, NEG027, NEG085, and NEG086. and those described in US 2015 / 0320880, such as 20376.

[0233] The disclosure of each of the foregoing publications is incorporated herein by reference as it relates to anti-CD117 antibodies. Antibodies and antigen-binding fragments that can be used in conjunction with the compositions and methods described herein include the antibodies and antigen-binding fragments described above, as well as humanized variants of the non-human antibodies and antigen-binding fragments described above, and antibodies (e.g., competitive CD117 (When assessed by binding assay) antibodies or antigen-binding fragments that bind to the same epitopes as those described above.

[0234] Exemplary antigen-binding fragments of the antibody include, among others, dual-variable immunoglobulin domain, single-chain Fv molecules (scFv), diabodies, and the like. These include di-, triabodies, nanobodies, antibody-like protein scaffolds, Fv fragments, Fab fragments, F(ab')2 molecules, and tandem di-scFv.

[0235] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, the anti-CD117 antibodies described herein may be produced using In a further embodiment, isolated nucleic acids are provided that encode the VL and / or VH amino acid sequences of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors comprising such nucleic acids are provided. In further embodiments, a host cell is provided that comprises such nucleic acid. In some such embodiments, the host cell comprises (e.g., For example, it has been transformed with: (1) a vector containing a nucleic acid encoding an amino acid sequence containing the VL of the antibody and an amino acid sequence containing the VH of the antibody, or (2) an amino acid sequence containing the VL of the antibody. A first vector containing a nucleic acid encoding a sequence and a second vector containing a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic organism, e.g., a mammalian cell, e.g., a mammalian cell. Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method of producing an anti-CLL-1 antibody is provided, comprising: culturing a host cell comprising nucleic acid encoding an antibody as provided above under conditions suitable for expression of said antibody, and optionally culturing said host cell (or host cell culture medium). and recovering the antibody from the culture medium.

[0236] To recombinantly produce an anti-CD117 antibody, nucleic acid encoding the antibody (e.g., a nucleic acid as described above) is isolated and further cloned and / or expressed in a host cell. The nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to the genes encoding the heavy and light chains of the antibody). can be done.

[0237] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, particularly where glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, including a description of expressing antibody fragments in E. coli). (It has been described that, after expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0238] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be beneficial. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney cell lines (see, e.g., Graham et al., 293 or 293 cells as described in [Illegible], J. Gen Virol. 36:59 (1977); Baby Ham Star kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver human liver cells (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include chicken Chinese hamster ovary (CHO) cells (DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines (e.g., Y0, NS0, and Sp2 / 0). For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0239] In one embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, is Amino acids that are at least 95%, 96%, 97% or 99% identical to the SEQ ID No. Alternatively, the anti-CD117 antibody, or an antigen-binding fragment thereof, may comprise a variable region having the following sequence: The compounds have framework regions of the variable regions described herein that have amino acid sequences that are at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos. disclosed herein. and CDRs comprising the SEQ ID Nos. disclosed herein.

[0240] In one embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, is In another embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region and a heavy chain constant region having the amino acid sequence disclosed herein. In yet another embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, comprises a light chain variable region and a light chain constant region having an amino acid sequence according to any of the amino acids disclosed herein. The antibody comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region having the amino acid sequence.

[0241] Methods for identifying anti-CD117 antibodies Antibodies, or antibody fragments, for molecules capable of binding to CD117 (e.g., GNNK+CD117) Methods for high-throughput screening of libraries, as described herein, can be used to identify and affinity mature antibodies useful for treating cancer, autoimmune diseases, and conditioning patients (e.g., human patients) in need of hematopoietic stem cell therapy. Such methods include in vitro display techniques known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display, among others. The use of phage display to isolate ligands that bind to biologically relevant molecules is reviewed, for example, in Felici et al., Biotechnol. Annual Rev. 1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4:1-20, 1998 (the disclosures of each of which are incorporated herein by reference as they relate to in vitro display techniques). As described in Kay, Perspective. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, 1996 (the disclosures of each of which are incorporated herein by reference as they relate to the discovery of antigen-binding molecules), randomized combinatorial peptide libraries have been constructed to select polypeptides that bind to cell surface antigens. Phage display of functional molecules (e.g., multimeric proteins) is , which have been successfully implemented (e.g., EP 0349578; EP 4527839; and EP 0589877, as well as See also Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992, which (The disclosures of each of these publications are incorporated herein by reference as they relate to the use of in vitro display techniques to discover antigen-binding molecules.) Additionally, functional antibody fragments (e.g., Fab fragments and scFv fragments) have been expressed in in vitro display formats (see, e.g., McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, each of which discloses the use of in vitro display techniques to discover antigen-binding molecules. (These technologies are incorporated herein by reference as they relate to in vitro display platforms for discovering synthetic molecules.) Among other things, these technologies can be used to detect hematopoietic stem cell transplantation. Identify antibodies that bind to CD117 (e.g., GNNK+CD117), which may in turn be used to deplete endogenous hematopoietic stem cells in patients (e.g., human patients) requiring transplantation therapy. and may improve antibody affinity.

[0242] In addition to in vitro display techniques, computational modeling techniques can be used to design and characterize antibodies or antibody fragments in silico that bind to CD117 (e.g., GNNK+ CD117). For example, using computational modeling techniques, one skilled in the art can screen a library of antibodies or antibody fragments in silico for molecules capable of binding to a specific epitope (e.g., an extracellular epitope of the antigen). Antibodies, or antigen-binding fragments thereof, identified by these computational techniques can be used in the therapeutic methods described herein (e.g., the cancer and autoimmune diseases described herein). These methods may be used in combination with other methods for treating a variety of conditions, including but not limited to the ...

[0243] Further techniques can be used to bind to CD117 (e.g., GNNK+CD117) on the surface of cells (e.g., cancer cells, autoimmune cells, or hematopoietic stem cells) and induce cell death, e.g., by receptor-mediated endocytosis. For example, the in vitro display techniques described above may be adapted to screen for antibodies or antigen-binding fragments thereof that bind to CD117 (e.g., GNNK+ CD117) on the surface of cancer cells, autoimmune cells, or hematopoietic stem cells and are subsequently internalized by the cells. Phage display represents one technique that can be used in conjunction with this screening paradigm. Antibodies or antigen-binding fragments thereof that bind to CD117 (e.g., GNNK+ CD117) and are subsequently internalized by cancer cells, autoimmune cells, or hematopoietic stem cells may be identified. To identify antibodies or fragments thereof containing randomized amino acid cassettes (e.g., in one or more or all of the CDRs or equivalent regions, or the antibody or antibody fragment), one of skill in the art may adapt phage display technology, e.g., as described in Williams et al., Leukemia 19:1432-1438, 2005, the disclosure of which is incorporated herein by reference in its entirety. For example, mutagenesis methods known in the art may be used to identify scFv fragments, Fab fragments, dimers, and other antibodies or fragments thereof containing randomized amino acid cassettes (e.g., in one or more or all of the CDRs or equivalent regions, or the antibody or antibody fragment). Antibodies, such as abodies and triabodies, antibody fragments, and 10 Fn3 domain, encoding The framework regions, hinges, Fc domains, and other regions of the antibodies or antibody fragments may be engineered to be non-immunogenic in humans, for example, by having sequences that are the same as human germline antibodies or that exhibit only minor variations relative to human germline antibodies.

[0244] Using phage display techniques described herein or known in the art, a phage library containing randomized antibodies or antibody fragments covalently attached to phage particles may be incubated with a CD117 (e.g., GNNK+ CD117) antigen, e.g., to initially generate phage encoding antibodies or fragments thereof that exhibit nonspecific protein binding and antibodies or fragments thereof that bind to the Fc domain. To remove phages encoding the target gene, the phage library is first incubated with a blocking agent (e.g., milk protein, bovine serum albumin, and / or IgG). The method is carried out by bating the phage library with a population of hematopoietic stem cells and then incubating the phage library with a population of target cells (e.g., cancer cells). In some cases, the cells are incubated with other cells (e.g., autoimmune cells, or hematopoietic stem cells). The antibody is a CD117-specific antibody, or an antigen-binding fragment thereof (e.g., GNNK+ CD117-specific The incubation may be performed for a period of time sufficient for the antibody, or antigen-binding fragment thereof, to bind to the cell surface CD117 (e.g., cell surface GNNK+CD117) antigen and subsequently be internalized by cancer cells, autoimmune cells, or hematopoietic stem cells (e.g., 30 minutes to 6 hours at 4°C, 1 hour at 4°C, etc.). Phage containing antibodies or fragments thereof that do not exhibit sufficient affinity to bind to one or more of these antigens and that do not exhibit internalization by cancer cells, autoimmune cells, or hematopoietic stem cells are then incubated with the cells (e.g., in cold (4°C) 0.1 M glycine buffer). The antibodies or fragments thereof can be removed by washing with a buffer (pH 2.8). Phages that are bound to the antibodies or fragments thereof or that have been internalized by cancer cells, autoimmune cells, or hematopoietic stem cells can be identified, for example, by lysing the cells and recovering the internalized phage from the cell culture medium. The phage can then be amplified in bacterial cells, for example, by incubating the bacterial cells with the recovered phage in 2xYT medium using methods known in the art. The phage recovered from this culture medium may then be identified, for example, by determining the nucleic acid sequence of the gene(s) encoding the antibody or fragment thereof that was inserted into the genome of the phage. The encoded antibody or fragment thereof may then be synthesized chemically (e.g., for antibody fragments such as scFv fragments) or by polymerization (e.g., by ELISA). Antibodies can be prepared de novo by recombinant expression (e.g., of full-length antibodies).

[0245] The ability of the prepared antibodies or fragments thereof to be internalized within cells may be assessed using, for example, radionuclide internalization assays known in the art. For example, antibodies or fragments thereof identified using in vitro display techniques described herein or known in the art may be fused with a radioisotope (e.g., 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu, 177 Lu, 77 As, 72 As, 86 Y,90 Y, 89 Zr, 212 Bi, 213 Bi, or 225 Ac) to make it functional. For example, 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 Radioactive halogens such as At, can be converted into electrophilic halogens. Beads, such as polystyrene beads containing reagents (e.g., Iodination Beads, Thermo Fisher Scientific, Inc., Cambridge, MA), can be used to incorporate the antibody or its fragment. The radiolabeled antibody or fragment thereof may be incubated with cancer cells, autoimmune cells, or hematopoietic stem cells for a time sufficient for cellular uptake (e.g., 30 minutes to 6 hours at 4°C, 1 hour at 4°C, etc.). The cells are then washed (e.g., Antibodies or fragments thereof that have not been internalized can be removed using cold (4°C) 0.1 M glycine buffer (pH 2.8). Internalized antibodies or fragments thereof may be identified by detecting radiation (e.g., gamma rays) emitted from the resulting cancer cells, autoimmune cells, or hematopoietic stem cells, while comparing this radiation (e.g., gamma rays) with that emitted from the recovered wash buffer.

[0246] Antibody-drug conjugates (ADCs) cytotoxin The anti-CD117 antibodies and antigen-binding fragments thereof described herein are cytotoxin-conjugated. In some embodiments, the cytotoxic molecule may be conjugated to an antibody, or antigen-binding fragment thereof, that is internalized as disclosed herein, such that after the antibody, or fragment thereof, is internalized, the cytotoxin may access its intracellular target and mediate the death of the hematopoietic cell. Any number of cytotoxins, e.g., 1, 2, 3, 4, 5, 6, 7, or 8, can be coupled to an anti-CD117 antibody. It can be conjugated.

[0247] Suitable cytotoxins for use with the compositions and methods described herein include those known in the art, among others, DNA-intercalating agents (e.g., anthracyclines, cyclohexyltransferases ... icrin), drugs that can disrupt the spindle apparatus (e.g., vinca alkaloids, maytansine, maytansinoids, and their derivatives), RNA polymerase inhibitors (e.g., amatoxins such as α-amanitin, and their derivatives), and drugs that can disrupt protein biosynthesis. Agents that can inhibit rRNA N-glycosidase activity (e.g., saporin and ricin A chain) Drugs) are included.

[0248] In some embodiments, the cytotoxin is a microtubule binding agent (e.g., maytansine or Maytansinoids), amatoxins, Pseudomonas exotoxin A, debouganin, diphtheria toxin, saporin, auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycins, pyrrolobenzodiazepines, pyrrolobenzodiazepine diamines The compound may be an indolinobenzodiazepine, an indolinobenzodiazepine dimer, or a variant thereof, or any other cytotoxic compound described herein or known in the art.

[0249] Additional cytotoxins suitable for use with the compositions and methods described herein include, but are not limited to, 5-ethynyluracil, abiraterone, acytoxin, cytotoxins ... Acylfulvene, adecypenol, adozelesin, Aldesleukin, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitor, antarelix, anti-dorsalizing morphogenetic protein-1, antiandrogen, prostate cancer, antiestrogen, antineoplastic agent, antisense oligonucleotide, aphidicolin glycinate, apoptosis gene modulator, apoptosis Stress regulator, apurinic acid, asulacrine, atametha atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III III.) derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorins, benzoylstaurosporine, β-lactam derivatives, β-alethine, β-betaclamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistratene A, bizelesin, breflate, bleomycin A2, bleomycin B2, bropirimine, budotitane, buthionine sulfoximine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives (e.g., 10-hydroxycamptothecin), capecitabine, carboxamido-amino-triazoles, carboxyamidotriazoles, carzelesin, casein kinase inhibitors, castanospermine, cecropin B, cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicapros cicaprost, cis-porphyrin, cladribine, clomiphene and its analogs, clotrimazole, collismycin A , Collismycin B, Combretastatin A4, Combretastatin analogues body, conagenin, crambecidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine ocfosfate ocfosfate, cytolytic factors, cytostatin, dacliximab, decitabine, dehydrodidemnin B, 2'-deoxycoformycin (DCF), deslorelin, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, di Demnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin, di Phenyl spiromustine, discodermolide, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, Elemene, emitefur, epothilones, epithilones, epristeride, estramustine and its analogs, etoposide, etoposide 4'-phosphate (also called etopophos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, fretinoids, Flezelastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors , gemcitabine, glutathione inhibitors, hepsulfam, homoharringtonine (HHT), hypericin, ibandron Ibandronic acid, idoxifene, idramantone, Ilmofosine, Ilomastat, Imidazoacridone, Imi Imiquimod, immune stimulating peptides, iobenguane, iododoxol Iododoxorubicin, ipomeanol, irinotecan, iroplact, irsogladine, isobengazole, jasp Jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol, lonidamine, losoxantrone, loxoribine, lurtotecan, lutetium texaphyrin texaphyrin, lysofylline, masoprocol, maspin, matrix metalloproteinases enzyme inhibitors, menogaril, merbarone, meterelin , methioninase, metoclopramide, MIF inhibitors, mifepristone, miltefosine, mirimostim, mithracin, mitoguazone, mitolactol, mitomycin and its analogs, mitonafide, mitoxantrone, mofarotene, molgramostim, mycaperoxide B, milia Myriaporone, N-acetyldinaline, N-substituted benzamide, Nafarelin, Nagrestip, Napavin, Nafterpi naphterpin, nartograstim, nedaplatin, nemol Nemorubicin, neridronic acid, nilutamide, nisamycin, nitrullyn, octreotide, oxenone, onapristone, ondansetron, oracin, ormaplatin, oxaliplatin, oxaunomycin unomycin, paclitaxel and its analogs, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, pentasulfate sodium, pentostatin, pentrozole, perflu perflubron, perfosfamide, phenazinomycin, picibanil, pirarubicin, piritrexim, podophyllotoxin, porfiromycin, Purine nucleoside phosphorylase inhibitors, raltitrexed, lysozyme Rhizoxin, rogletimide, rohitukine, rubiginone B1, ruboxyl, safingol, saintopin, sarcophytol A, sargramostim, sobuzoxa sobuzoxane, sonermin, sparfosic acid, spicamycin D, spiromustine, stipiamide, sulphonamide Finosin (sulfinosine), tallimustine (tallimustine), tegafur (tegafur), temozolomide (temozolomide), teniposide (teniposide), thaliblastine (thaliblastine), These include thiocoraline, tirapazamine, topotecan, topsentin, triciribine, trimetrexate, veramine, vinorelbine, vinxaltine, vorozole, zeniplatin, and zilascorb.

[0250] The anti-CD117 antibodies and antigen-binding fragments thereof described herein are microtubule-binding agents. As used herein, the term "microtubule binding agent" refers to a compound that acts by disrupting the microtubule network, which is essential for mitotic and interphase cellular functions in cells. Examples of microtubule binding agents include, but are not limited to, maytansin, maytansinoids, and their derivatives (the present invention). such as those described herein or known in the art), vinblastine, vinbratamine, Vinca alkaloids such as vincristine sulfate, vincristine, vincristine sulfate, vindesine, and vinorelbine; taxanes such as docetaxel and paclitaxel; macrolides such as discodermolide, colchicine, and epothilones, and their derivatives (such as epothilone B or its derivatives).

[0251] Maytansinoids In some embodiments, the microtubule-binding agent is maytansine, a maytansinoid, or a maytansinoid analog. Maytansinoids are mitotic inhibitors that act by binding to microtubules and inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, specific microtubule-binding agents have been isolated. Organisms have also been found to produce maytansinoids (e.g., maytansinol and C-3 maytansinol esters) (U.S. Patent No. 4,151,042). Synthetic maytansinol, as well as its derivatives and analogs, are described in, for example, U.S. Patents 4,137,230; 4,248,870; 4,256,746; and 4,371,533. Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates. The drug moiety of maytansinoids is derived from: (i) fermentation or chemical modification or derivatization of the fermentation product; (ii) are relatively accessible to preparation via non-disulfide linkers (iii) are amenable to derivatization with functional groups suitable for conjugation to antibodies; and (iv) are stable in plasma. and (iv) it is effective against a variety of tumor cell lines.

[0252] Examples of suitable maytansinoids include esters of maytansinol, synthetic maytansinol, and maytansinol analogues and derivatives. Included herein, like maytansinoids, maytansinol, and maytansinol analogues and derivatives, is any cytotoxin that inhibits microtubule formation and is highly toxic to mammalian cells.

[0253] Examples of suitable maytansinol esters include those with modified aromatic rings and those with modifications at other positions. Such suitable maytansinoids are described in U.S. Patents 4,137,230; 4,151,042; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; and 7,473,796, each of which is incorporated by reference herein as relating to maytansinoids and derivatives thereof.

[0254] In some embodiments, the immunoconjugates of the invention comprise a thiol-containing maytansinoid (DM1) (formerly known as N 2 '-Deacetyl-N 2 DM1 is represented by structural formula (VII): [ka] .

[0255] In another embodiment, the conjugate of the invention comprises a thiol-containing maytansinoid N 2 '-Deacetyl-N 2 '(4-methyl-4-mercapto-1-oxopentyl)-maytansine (e.g., DM4) is utilized as a cytotoxic drug. DM4 is represented by structural formula (V): [ka] .

[0256] Another maytansinoid containing a side chain containing a sterically hindered thiol bond is N 2 '-Deacetyl-N- 2 '(4-mercapto-1-oxopentyl)-maytansine (referred to as DM3), which is represented by structural formula (VI): [ka] .

[0257] Each of the maytansinoids taught in U.S. Patent Nos. 5,208,020 and 7,276,497 may also be used in the conjugates of the present invention, the entire disclosures of which are incorporated herein by reference in this regard.

[0258] Many positions on the maytansinoid can serve as sites for chemical attachment of a linking moiety, for example, the C-3 position, which bears a hydroxyl group, or the C-4 position, which is modified with hydroxymethyl. A modified C-14 position, a hydroxy-modified C-15 position, and a C-20 position bearing a hydroxy group are all expected to be useful. In some embodiments, the C-3 position is chemically linked to a linking moiety. In some specific embodiments, the C-3 position of maytansinol serves as the site of attachment to the linking moiety. Many linking groups are known in the art for preparing antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Patent Nos. 5,208,020, 6,441,163, and European Patent No. 0425235 B1; Chari et al., Cancer Research 52:127-131 (1992); and US 2005 / 0169933 A1, the disclosures of which are expressly incorporated herein by reference. Additional linking groups are described and exemplified herein.

[0259] The present invention also includes various isomers and mixtures of maytansinoids and conjugates. Certain compounds and conjugates of the present invention may exist in various stereoisomeric, enantiomeric, and diastereomeric forms. Such antibody-maytansinoid conjugates may be used in combination with other compounds. Producing adjugates is described in U.S. Patents 5,208,020, 5,416,064, 6,333,410, 6,441,163, 6,716,821, and 7,368,565, each of which is incorporated herein in its entirety. (included).

[0260] The therapeutically effective number of maytansinoid molecules bound per antibody molecule can be determined spectrophotometrically by measuring the ratio of absorbance at 252 nm and 280 nm. One molecule of toxin / antibody can enhance cytotoxicity over antibody alone, but antibody molecules alone can enhance cytotoxicity. Conjugation of an average of 3-4 maytansinoid molecules per antibody or antigen-binding fragment thereof can enhance cytotoxicity to target cells without adversely affecting antibody function or solubility. The average number of maytansinoid molecules per antibody or antigen-binding fragment thereof can be, for example, 1-10. Or it may be 2 to 5.

[0261] Anthracyclines In other embodiments, the antibodies and antigen-binding fragments thereof described herein may be conjugated to a cytotoxin that is an anthracycline molecule. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Studies have shown that anthracyclines can kill cells by a number of different mechanisms, including: 1) the drug molecule intercalates into the cell's DNA, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) the drug generates free radicals, which then react with cellular macromolecules and cause cell damage; or 3) the drug molecule interacts with the cell membrane [see, e.g., C. Peterson et al., "Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia" in Anthracycline Antibiotics In Cancer Therapy; See N.R. Bachur, "Free Radical Damage," id. at pp. 97-102. Due to potential cytotoxicity, the risk of leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma Anthracyclines are used to treat a number of cancers, including in Anthracycline: Current Status And New Developments See p. 11. Commonly used anthracyclines include doxorubicin, epirubicin, idarubicin, and daunomycin.

[0262] Doxorubicin (ADRIAMYCINO), an anthracycline analogue, is an intercalating It is thought that doxorubicin interacts with DNA via cleavage and inhibits the progress of the enzyme topoisomerase II, which unwinds DNA for transcription. Doxorubicin stabilizes the topoisomerase II complex after it has broken the DNA strand for replication, preventing the DNA double helix from reclosing, thereby halting the replication process. Doxorubicin and daunorubicin are the prototypic cytotoxic natural product anthracycline chemotherapy agents (Sessa et al., 2004). al., (2007) Cardiovasc. Toxicol. 7:75-79).

[0263] Pyrrolobenzodiazepines (PBDs) In other embodiments, the anti-CD117 antibodies, or antigen-binding fragments thereof, described herein , may be conjugated to a cytotoxin that is a pyrrolobenzodiazepine (PBD) or that contains a PBD. PBDs are natural products produced by certain actinomycetes and have the sequence PBD cytotoxins include, but are not limited to, anthramycin, dimeric PBDs, and the cytotoxins described in, e.g., Hartley, JA (2011). "The development of pyrrolobenzodiazepines as antitumor agents." Expert Opin. Inv. Drug, 20(6), 733-744; and Antonow, D.; Thurston, DE (2011) "Synthesis of DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepines (PBDs)." Chem. Rev. 111: 2815-2864.

[0264] Calicheamicin In other embodiments, the antibodies and antigen-binding fragments thereof described herein may be conjugated to a cytotoxin that is a calicheamicin molecule. The calicheamicin family of antibiotics is capable of generating double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,296 (all to American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, those disclosed in Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the above-referenced U.S. patents to American Cyanamid. Examples include:

[0265] Auristatin The anti-CD117 antibodies and antigen-binding fragments thereof described herein are auristatins. They may be conjugated to certain cytotoxins (U.S. Patent Nos. 5,635,483; 5,780,588). Auristatins are antimitotic agents that interfere with microtubule dynamics, GTP hydrolysis, nuclear division, and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anticancer (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). (U.S. Patent Nos. 5,635,483; 5,780,588). Auristatin drug moieties may be attached to the N-(amino) or C-(carboxy) termini of peptide drug moieties. The hydroxyl terminus may be linked to an antibody (WO02 / 088172).

[0266] Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF, which are disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, March 28, 2004. (the disclosure of which is incorporated herein by reference in its entirety).

[0267] An exemplary auristatin embodiment is MMAE, where the wavy line indicates the antibody-linker the position of covalent attachment of the -conjugate (as described herein -LZ-Ab) to the linker Indicates the location. [ka]

[0268] Another exemplary auristatin embodiment is MMAF, where the wavy line represents an antibody-linker conjugate (as described herein) as disclosed in US 2005 / 0238649. The position of the covalent bond to the linker (-LZ-Ab) is indicated. [ka]

[0269] Auristatins can be prepared according to the following method: U.S. Pat. No. 5,635,483 Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, GR, et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin Trans. 15:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784.

[0270] Amatoxin In some embodiments, the cytotoxin of the antibody-drug conjugate is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is an amatoxin or is its derivative.

[0271] In some embodiments, the cytotoxin is α-amanitin, β-amanitin, γ-amanitin, or Amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, and The structure of various naturally occurring amatoxins is represented by Formula III and is disclosed, for example, in Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459.

[0272] In some embodiments, the cytotoxin is amanitin. For example, an antibody or antigen-binding fragment described herein can be conjugated to a conjugate represented by the formula Ab-ZL-Am. where Ab is an antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an amatoxin. Many positions on an amatoxin or its derivatives can be linked to a linking moiety L to form a bond. In some embodiments, Am-LZ is represented by formula (I): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C , ORD , R C , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl). nyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C1-C6 alkylene), an optionally substituted heteroalkylene (e.g., C1-C6 heteroalkylene), an optionally substituted optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic alkylene; alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene; dipeptide, -(C=O)-, peptide, or combinations thereof; and Z is a reactive group present on L and an antibody that binds to CD117 (such as GNNK+CD117); It is a chemical moiety formed from a coupling reaction between reactive substituents present within the antigen-binding fragment.

[0273] In some embodiments, Am is exactly one R C Contains substituents.

[0274] In some embodiments, the linker is -(CH) 2n -units, where n is an integer from 2 to 6. In some embodiments, the linker is -((CH) n In some embodiments, the LZ comprises: [ka] where S is a sulfur atom representing a reactive substituent present in the antibody, or antigen-binding fragment thereof, that binds to CD117 (e.g., derived from the -SH group of a cysteine ​​residue).

[0275] In some embodiments, LZ is [ka] is.

[0276] In some embodiments, the conjugate has the formula IV, IVA, or IVB: is represented by: [ka] Here, X is -S-, -S(O)-, or -SO2-.

[0277] In some embodiments, Am-LZ-Ab is [ka] is.

[0278] In some embodiments, Am-LZ-Ab is [ka] is.

[0279] In some embodiments, Am-LZ-Ab is [ka] is.

[0280] In some embodiments, Am is represented by formula (IA): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R Bwhen present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C , OR D , R C , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl). nyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C1-C6 alkylene), an optionally substituted heteroalkylene (e.g., C1-C6 heteroalkylene), an optionally substituted optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic alkylene; alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -(C=O)-, peptide, or combinations thereof; It is a linker that is like; Z is a reactive group present on L and an antibody that binds to CD117 (such as GNNK+CD117); a chemical moiety formed from a coupling reaction between a reactive substituent present in the antigen-binding fragment; and where Am is exactly one R C Contains substituents.

[0281] In some embodiments, the linker is -((CH) n In some embodiments, the LZ comprises: [ka] is.

[0282] In some embodiments, LZ is [ka] is.

[0283] In some embodiments, Am-LZ is represented by formula (IB): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C , OR D , R C , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl). nyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C1-C6 alkylene), an optionally substituted heteroalkylene (e.g., C1-C6 heteroalkylene), an optionally substituted optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic alkylene; alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -(C=O)-, peptide, or combinations thereof; It is a linker that is like; Z is a reactive group present on L and an antibody that binds to CD117 (such as GNNK+CD117); a chemical moiety formed from a coupling reaction between a reactive substituent present in the antigen-binding fragment; and where Am is exactly one R C Contains substituents.

[0284] In some embodiments, the linker L and the chemical substructure Z together form LZ. Considered, [ka] is.

[0285] In some embodiments, LZ is [ka] is.

[0286] In some embodiments, R A and R B together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl group of the formula: [ka] Here, Y is -(C=O)-, -(C=S)-, -(C=NR E) - or - (CR E R E' )-; and R E and R E' are each independently optionally substituted C-C alkylene-R C ,any optionally substituted C1-C6 heteroalkylene-R C , optionally substituted C2-C6 alkenyl Ren-R C , optionally substituted C2-C6 heteroalkenylene-R C , optionally substituted C2-C6 alkynylene-R C, optionally substituted C2-C6 heteroalkynylene-R C , optionally substituted cycloalkylene-R C , optionally substituted heterocycloalkylene N-R C、 optionally substituted arylene-R C、 or optionally substituted heteroarylene-R C , is.

[0287] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached, form: [ka] R3 is H or R C is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C , OR D , R C , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , or NHRC is; R9 is H or OH; X is —S—, —S(O)—, or —SO2—; and where R C and R D are each as defined above.

[0288] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached, form: [ka] R3 is H or R C is; R4 and R5 are each independently H, OH, OR C , R C OR D is; R6 and R7 are each H; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; X is —S—, —S(O)—, or —SO2—; and where R C is as defined above.

[0289] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): where R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; R A and R B When present, together with the oxygen atom to which they are attached, form: [ka] R3, R4, R6, and R7 are each H; R5 is OR C is; R8 is OH or NH2; R9 is H or OH; X is —S—, —S(O)—, or —SO2—; and where R C is as defined above. Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2016 / 0002298, the disclosure of which is incorporated herein by reference in its entirety.

[0290] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): wherein R1 and R2 are each independently H or OH; R3 is R C is; R4, R6, and R7 are each H; R5 is H, OH, or OC1-C6 alkyl; R8 is OH or NH2; R9 is H or OH; and where R C is as defined above. Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated herein by reference in its entirety.

[0291] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): wherein R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are independently H, OH, or OR C , or R C is; R8 is OH or NH2; R9 is H or OH; and where R C is as defined above. Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0292] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): wherein R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are each independently H or OH; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; and where R C Such amatoxin conjugates are described, for example, in U.S. Patent Nos. 9,233,173 and 9,399,681, and in U.S. Patent No. 2016 / 0089450. No. 6,219,999, the disclosure of which is incorporated herein by reference in its entirety.

[0293] Additional amatoxins that can be used for conjugation to antibodies, or antigen-binding fragments thereof, in accordance with the compositions and methods described herein are described, for example, in WO 2016 / 142049; WO 2016 / 071856; and WO 2017 / 046658, the disclosures of each of which are incorporated by reference herein in their entireties.

[0294] In some embodiments, Am-LZ is represented by formula (II), formula (IIA), or formula (IIB): [ka] where X is S, SO, or SO2; R1 is H or a compound connected to said antimony group via the chemical moiety Z. a linker covalently attached to the antibody or antigen-binding fragment thereof, and Z is the linker and R2 is formed from a coupling reaction between a reactive substituent present on the hydroxyl group and a reactive substituent present in the antibody or antigen-binding fragment thereof; and R2 is H or a chemical moiety. a linker covalently attached to the antibody or antigen-binding fragment thereof via a moiety Z; Z is a group that represents a reactive substituent present on the linker and a group that represents an antibody or an antigen-binding fragment thereof. wherein when R1 is H, R2 is a linker, and when R2 is H, R1 is a linker.

[0295] In some embodiments, the linker is -(CH) n -units, where n is an integer from 2 to 6. In some embodiments, R1 is the linker and R2 is H and the linker and chemical moiety together are considered LZ; [ka] is.

[0296] In some embodiments, Am-LZ-Ab is: [ka] is.

[0297] In some embodiments, Am-LZ-Ab is: [ka] is.

[0298] In some embodiments, the cytotoxin is α-amanitin. In some embodiments, the α-amanitin is a compound of formula III The α-amanitin is attached to the anti-CD117 antibody via a linker L. The linker L may be attached at any one of several possible positions of the α-amanitin of formula III (e.g., R 1 -R 9 α-amanitin linker of formula I, IA, IB, II, IIA, IIB, IV, IVA, or IVB In some embodiments, the linker is at position R 1 In some embodiments, the linker is attached at position R 2 In some embodiments, the linker is attached at position R 3 In some embodiments, the linker is attached at position R 4 In some embodiments, the linker is attached at position R 5 In some embodiments, the linker is attached at position R 6 In some embodiments, the linker is attached at position R 7 In some embodiments, the linker is attached at position R 8 In some embodiments, the linker is attached at position R 9In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker is -((C=O)(CH2) n -unit, where n is an integer from 1 to 6.

[0299] In some embodiments, the linker is -(CH) n - includes units, where n is 2 In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH) n -is In some embodiments, the linker L and chemical moiety Z are taken together as LZ. And [ka] is.

[0300] In some embodiments, the cytotoxin is β-amanitin. In some embodiments, the β-amanitin is a compound of Formula I. Manitin is a compound of formula III. In some embodiments, the β-amanitin of formula III is , which is linked to the anti-CD117 antibody via a linker L. The linker L is a β-flaxseed compound of Formula III Any one of several possible positions of the chin (e.g., R 1 -R 9) and a group of formula I In some embodiments, the linker is at position R. 1 In some embodiments, the linker is attached at position R 2 In some embodiments, the linker is attached at position R 3 In some embodiments, the linker is attached at position R 4 In some embodiments, the linker is attached at position R 5 In some embodiments, the linker is attached at position R 6 In some embodiments, the linker is attached at position R 7 In some embodiments, the linker is attached at position R 8 In some embodiments, the linker is attached at position R 9 In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. The linker is -((C=O)(CH) n -unit, where n is an integer from 1 to 6.

[0301] In some embodiments, the linker is -(CH) n - includes units, where n is 2 In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH)n -is In some embodiments, the linker L and chemical moiety Z are taken together as LZ. And [ka] is.

[0302] In some embodiments, the cytotoxin is gamma-amanitin. wherein the γ-amanitin is a compound of formula III. In some embodiments, γ-amanitin is attached to the anti-CD117 antibody via a linker L. The linker L may be attached at any one of several possible positions of the γ-amanitin of formula III (e.g., R 1 -R 9 a γ-amanitin-linker conjugate of formula I, IA, IB, II, IIA, or IIB; In some embodiments, the linker is provided at position R 1 In some embodiments, the linker is attached at position R 2 In some embodiments, the linker is attached at position R 3 In some embodiments, the linker is attached at position R 4 In some embodiments, the linker is attached at position R 5 In some embodiments, the linker is attached at position R 6 In some embodiments, the linker is attached at position R 7 In some embodiments, the linker is attached at position R 8 In some embodiments, the linker is attached at position R 9In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-Ala. In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker is -((C=O)(CH) n -units, where n is 1 to 6 is an integer.

[0303] In some embodiments, the linker is -(CH) n - includes units, where n is 2 In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH) n -is In some embodiments, the linker L and chemical moiety Z are taken together as LZ. And [ka] is.

[0304] In some embodiments, the cytotoxin is ε-amanitin. In some embodiments, the ε-amanitin is a compound of formula III The ε-amanitin is attached to the anti-CD117 antibody via a linker L. The linker L may be attached at any one of several possible positions of the ε-amanitin of formula III (e.g., R 1 -R 9ε-amanitin-linker conjugate of formula I, IA, IB, II, IIA, or IIB In some embodiments, the linker is provided at position R 1 In some embodiments, the linker is attached at position R 2 In some embodiments, the linker is attached at position R 3 In some embodiments, the linker is attached at position R 4 In some embodiments, the linker is attached at position R 5 In some embodiments, the linker is attached at position R 6 In some embodiments, the linker is attached at position R 7 In some embodiments, the linker is attached at position R 8 In some embodiments, the linker is attached at position R 9 In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises Val-Ala and Val-Cit. In some embodiments, the linker comprises a dipeptide selected from the group consisting of para- In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker is -((C=O)(CH) n -units, where n is 1 to 6 is an integer.

[0305] In some embodiments, the linker is -(CH) n - includes units, where n is 2 In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) nIn some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH) n -is In some embodiments, the linker L and chemical moiety Z are taken together as LZ. And [ka] is.

[0306] In some embodiments, the cytotoxin is amanin. In some embodiments, the amanin is a compound of Formula III. In some embodiments, the amanin of Formula III is attached to the anti-CD117 antibody via a linker L. The linker L may be attached to any one of several possible positions of the amanin of Formula III (e.g., R 1 -R 9 and a group represented by formula I, IA, or IB Amanine-linker conjugates of I, IIA, or IIB may be provided. In some embodiments, the linker is at position R 1 In some embodiments, the linker is attached at position R 2 In some embodiments, the linker is attached at position R 3 In some embodiments, the linker is attached at position R 4 In some embodiments, the linker is attached at position R 5 In some embodiments, the linker is attached at position R 6 In some embodiments, the linker is attached at position R 7 In some embodiments, the linker is attached at position R 8 In some embodiments, the linker is attached at position R 9In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises the moiety PAB-Ala-Val. Carr is -((C=O)(CH2) n -unit, where n is an integer from 1 to 6.

[0307] In some embodiments, the linker is -(CH) n - includes units, where n is 2 In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH) n -is In some embodiments, the linker L and chemical moiety Z are taken together as LZ. And [ka] is.

[0308] In some embodiments, the cytotoxin is amaninamide. In some embodiments, the amaninamide is a compound of Formula III. In some embodiments, the amaninamide of Formula III is attached to an anti-CD117 antibody via a linker L. The linker L is Any one of several possible positions of the amaninamide of formula III (e.g., R 1 -R 9 amaninamide-linker conjugate of formula I, IA, IB, II, IIA, or IIB In some embodiments, the linker may provide a linker at position R 1 In some embodiments, the linker is attached at position R 2 In some embodiments, the linker is attached at position R 3 In some embodiments, the linker is attached at position R 4 In some embodiments, the linker is attached at position R 5 In some embodiments, the linker is attached at position R 6 In some embodiments, the linker is attached at position R 7 In some embodiments, the linker is attached at position R 8 In some embodiments, the linker is attached at position R 9 In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker is -((C=O)(CH2) n -unit, where n is an integer from 1 to 6.

[0309] In some embodiments, the linker is -(CH) n - includes units, where n is 2 In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH) n -is In some embodiments, the linker L and chemical moiety Z are taken together as LZ. And [ka] is.

[0310] In some embodiments, the cytotoxin is amanuline. , the amanuline is a compound of Formula III. In some embodiments, the amanuline of Formula III is attached to the anti-CD117 antibody via a linker L. The linker L may be attached at any one of several possible positions on the amanuline of Formula III (e.g., R 1 -R 9 ) and a group of formula I amanulin-linker conjugates of IA, IB, II, IIA, or IIB can be provided. In some embodiments, the linker is at position R 1 In some embodiments, the linker is attached at position R 2 In some embodiments, the linker is attached at position R 3 In some embodiments, the linker is attached at position R 4 In some embodiments, the linker is attached at position R 5 In some embodiments, the linker is attached at position R 6 In some embodiments, the linker is attached at position R 7 In some embodiments, the linker is attached at position R 8 In some embodiments, the linker is attached at position R 9 In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. , the linker is -((C=O)(CH2) n -unit, where n is an integer from 1 to 6.

[0311] In some embodiments, the linker is -(CH) n - includes units, where n is 2 In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH) n -is In some embodiments, the linker L and chemical moiety Z are taken together as LZ. And [ka] is.

[0312] In some embodiments, the cytotoxin is amanurinic acid. In some embodiments, the amanurinic acid is a compound of Formula III. In some embodiments, the amanurinic acid of Formula III is attached to the anti-CD117 antibody via a linker L. The linker L may be attached to any one of several possible positions of the amanurinic acid of Formula III (e.g., R 1 -R 9 or any of the following) to provide an amanurinic acid-linker conjugate of formula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker is located at position R 1 In some embodiments, the linker is attached at position R 2 In some embodiments, the linker is attached at position R 3 In some embodiments, the linker is attached at position R 4 In some embodiments, the linker is attached at position R 5In some embodiments, the linker is attached at position R 6 In some embodiments, the linker is attached at position R 7 In some embodiments, the linker is attached at position R 8 In some embodiments, the linker is attached at position R 9 In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group. In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker comprises the moiety PAB-Cit-Val. In the form, the linker is -((C=O)(CH) n -unit, where n is an integer from 1 to 6. be.

[0313] In some embodiments, the linker is -(CH) n - includes units, where n is 2 In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH) n -is In some embodiments, the linker L and chemical moiety Z are taken together as LZ. And [ka] is.

[0314] In some embodiments, the cytotoxin is proamanulin. In some embodiments, the proamanulin is a compound of Formula III. In some embodiments, the proamanulin of Formula III is attached to the anti-CD117 antibody via a linker L. The linker L is Any one of several possible positions of the proamanulin of formula III (e.g., R 1 -R 9 a proamanulin-linker conjugate of formula I, IA, IB, II, IIA, or IIB In some embodiments, the linker may provide a linker at position R 1 In some embodiments, the linker is attached at position R 2 In some embodiments, the linker is attached at position R 3 In some embodiments, the linker is attached at position R 4 In some embodiments, the linker is attached at position R 5 In some embodiments, the linker is attached at position R 6 In some embodiments, the linker is attached at position R 7 In some embodiments, the linker is attached at position R 8 In some embodiments, the linker is attached at position R 9 In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker is -((C=O)(CH2) n -unit, where n is an integer from 1 to 6.

[0315] In some embodiments, the linker is -(CH) n - includes units, where n is 2 In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH) n -is In some embodiments, the linker L and chemical moiety Z are taken together as LZ. And [ka] is.

[0316] The synthesis of amatoxin is described in U.S. Pat. No. 9,676,702. is incorporated herein by reference.

[0317] Antibodies or antigen-binding fragments for use with the compositions and methods described herein can be conjugated to an amatoxin, such as α-amanitin or a variant thereof, using conjugation techniques known in the art or described herein. For example, an antibody or antigen-binding fragment thereof that recognizes and binds to CD117 (such as GNNK+ CD117) can be conjugated to an amatoxin, such as α-amanitin or a variant thereof, as described in US 2015 / 0218220, the disclosure of which is incorporated herein by reference. and covalent linkers that can be used to covalently conjugate amatoxins such as cyclohexylamine or variants thereof, and are incorporated herein by reference.

[0318] Exemplary antibody-drug conjugates useful in conjunction with the methods described herein may be formed by reaction of an antibody or antigen-binding fragment thereof with an amatoxin conjugated to a linker that includes a substituent suitable for reaction with a reactive residue on the antibody or antigen-binding fragment thereof. Amatoxins conjugated to linkers containing substituents suitable for reaction with reactive residues on an antibody, or antigen-binding fragment thereof, as described herein include, but are not limited to, 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin; 7'C-(4-(6-(maleimido)hexanoyl)piperidin-1-yl)-amatoxin; 7'C-(4-(6-(6-(maleimido)hexanoamido)hexanoyl)piperazin-1-yl)-amatoxin; 7'C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperazin-1-yl)-amatoxin; 7'C-(4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)-amatoxin; 7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7'C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7'C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amatoxin;7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexane 7'C-(4-(2-(3-carboxypropanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(4-(maleimido)butanamido)ethyl) Piperidin-1-yl)-amatoxine; 7'C-(4-(2-(maleimido)acetyl)piperazine-1-yl 7'C-(4-(3-(maleimido)propanoyl)piperazin-1-yl)-amatoxin;7'C-(4-(4-(maleimido)butanoyl)piperazin-1-yl)-amatoxin;7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl) Piperidin-1-yl)-amatoxin; 7'C-(3-((6-(maleimido)hexanamido)methyl)piperidin-1-yl)-amatoxin 7'C-(3-((6-(6-(maleimido)hexanamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7'C-(3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrolidin-1-yl)-amatoxin;7'C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7'C-(4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7'C-(4-(2-(4-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin 7'C-(4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)-amatoxine;7'C-(4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazin-1-yl)-amatoxine;7'C-((4-(6-(maleimido)hexanamido)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(6-(maleimido)hexanoyl)piperazin-1-yl)methyl)-amatoxine;(R)-7'C-((3-((6-(maleimido)hexanamido)methyl) (S)-7'C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin 7'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl) 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarbo 7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((3-((4-((maleimido)methyl)silica 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxine 7'C-((4-(2-(3-carboxypropanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(2-(3-carboxypropanamido)ethyl)piperazin-1-yl)methyl)-amatoxin ;7'C-((4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(6-(4-((maleimido)methyl)cyclohexanecarboxamide) 7'C-((4-(2-(maleimido)acetyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(3-(maleimido)propanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(4-(maleimido)butanoyl)piperazin-1-yl)methyl)-amatoxine 7'C-((4-(2-(2-(maleimido)acetamido)ethyl)-amatoxin; 7'C-((4-(2-(4-(maleimido)butanamido)-4-methyl)piperidin-1-yl)methyl)-amatoxine; 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((3-((6-(maleimido)hexanamido)methyl)azetidin-1-yl)methyl)-amatoxine 7'C-((3-(2-(6-(maleimido)hexanamido)ethyl)azeti 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amatoxine;7'C-((3-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)azetidin-1-yl)methyl)-amatoxine 7'C-((3-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxine;7'C-(((2-(6-(maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxine 7'C-(((4-(6-(maleimido)-N-methylhexanamido)butyl(methyl)amino)methyl)-amatoxin;7'C-(((4-(6-(maleimido)-N-methylhexanamido)butyl(methyl)amino)methyl)-amatoxin 7'C-((2-(2-(6-(maleimido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxine;7'C-((2-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxine;7'C-((4-(6-(6-(2-(aminooxy)acetamido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxine 7'C-((4-(1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-oyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(2-(2-(aminooxy)acetamido)acetyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(3-(2-(aminooxy)acetamido)propanoyl)piperazin-1-yl)methyl)-amatoxin 7'C-((4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)- 7'C-((4-(2-(2-(2-(aminooxy)acetamido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxine 7'C-((4-(20-(aminooxy)-4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaicosyl)piperidin-1-yl)methyl)-amatoxin;7'C-(((2-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxin;7'C-(((4-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)butyl)(methyl)amino)methyl)-amatoxin;7'C-((3-((6-(4-((maleimido)methyl)cyclohexanamido) 7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(2-Bromoacetonitrile) 7'C-((4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(3-(pyridin-2-yldisulfanyl)propan ... 6'O-(6-(6-(maleimido)hexanamido)hexyl)-amatoxin; 6'O-(5-(4-((maleimido)hexanamido)hexyl)-amatoxin 6'O-(2-((6-(maleimido)hexyl)oxy)-2-oxoethyl)-amatoxine;6'O-((6-(maleimido)methyl)cyclohexanecarboxamido)pentyl)-amatoxine;6'O-(2-((6-(maleimido)hexyl)oxy)-2-oxoethyl)-amatoxine;6'O-((6-(maleimido) 6'O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexyl)carbamoyl)-amatoxine;6'O-(6-(2-bromoacetamido)hexyl)-amatoxine;7'C-(4-(6-(azido)hexanamido)piperidin-1-yl)- Amatoxin;7'C-(4-(hex-5-ynoylamino)piperidin-1-yl)-amatoxin;7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;7'C-(4-(2-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;7'C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)-amatoxin 6'O-(6-(6-(11,12-didehydro-5,6-dihydro-dibenz[b,f]azocin-5-yl)-6-oxohexanamido)hexyl)-amatoxin;6'O-(6-(hex-5-ynoyl)-amatoxin; ... 6'O-(6-(2-(aminooxy)acetylamido)hexyl)-amatoxin; 6'O-((6-aminooxy)hexyl)-amatoxin; and 6'O-(6-(2-iodoacetamido)hexyl)-amatoxin. The above linkers are, in particular, those described herein. These compositions and methods are useful in conjunction with other compositions and methods described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0319] Additional cytotoxins that can be conjugated to antibodies or antigen-binding fragments thereof that recognize and bind to CD117 (such as GNNK+ CD117) for use in directly treating cancer, autoimmune conditions, or for conditioning patients (e.g., human patients) in need of hematopoietic stem cell transplantation therapy include, but are not limited to, 5-ethynyluracil, abiraterone, acylfulvene, adecypenol, adozelesin, aldesleukin, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrile, amifostine ... amsacrine, anagrelide, anastrozole, androgen andrographolide, an angiogenesis inhibitor; antarelix, anti-dorsalizing morphogenetic protein-1, an anti-androgen; Prostate cancer, antiestrogens, antineoplastic agents (antineoplastons), antisense oligonucleotides, aphidicolin glycinate, apoptosis genes Apoptosis modulator, apurinic acid, aspartate Asulacrine, Atamestane, Atrimustine, Axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorins, benzoylstaurosporine, beta-lactam derivatives, beta-alethine, beta-lactam lamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistratene A, bizelesin, breflate, bleomycin A2, bleomycin B2, bropirimine, budotitane, buthionine sulfonate buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives (e.g., 10-hydroxy- camptothecin, capecitabine, carboxamido-amino-triazole, carboxyamidotriazole, carzelesin, casein kinase inhibitor, castanospermine, cecropin B, cetrorelix, chlorins, chloroquinoxaline sulfonamide sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene and its analogs, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogs, conagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin Cin A derivatives, curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine ocfosfate, cytolytic factors, cytostatin, dacliximab, decitabine, dehydrodidemnin B, 2'-deoxycoformycin (DCF), deslorelin, dexifosfamide, dexrazoxane, dexverapamil, Diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, diazacytidine dioxamycin, diphenyl spiromustine, Discodermolide, docosanol, dolasetron, doxifluridine, droloxifene, dronabi Dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, Eflornithine, elemene, emitefur, epothine epothilones, epithilones, epristeride, e Stramustine and its analogs, etoposide, etoposide 4'-phosphate (also called etopophos), exemestane, fadrozole, fazarabine, fenretin fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin Gadolinium texaphyrin, gallium nitrate, galocitabine, ganirel Ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, homoharringtonine (HHT), Hypericin, Ibandronic acid, Idoxifene, Idramantone, Ilmofosine, Ilomastat, Imidazoacridone, Imiquimod, Immunostimulating peptides, Iobenguane, Iododoxorubicin, Ipomeanol, Irinotecan, Iropract, Irsogladine, Isobenga Isobengazole, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol, lonidamine, losoxantrone, loxoribine, lurtotecan, lutetium texaphyrin texaphyrin, lysofylline, masoprocol, maspin, Matrix metalloproteinase inhibitors, menogaril, merbarone, meterelin, methioninase, metoclopramide , MIF inhibitors, mifepristone, miltefosine, mirimostim, mithracin, mitoguazone, mitolactol, mitomycin and its analogs, mitonafide, mitoxantrone, mofarotene, molgramostim, mycaperoxide B, myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagrestip, napavin, naphterpin, nartograstim, neda Nedaplatin, nemorubicin, neridronic acid, nilutamide, nisamycin, nitrullyn, octreotide, oxenone, onapristone, ondansetron, oracin, ormaplatin, oxaliplatin, oxaunomycin, paclitaxel and its analogs, palauamine, palmitoylrhizoxin, pamidronic acid , panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, pentosan polysulfate sodium, pentostatin, pentro Pentrozole, perflubron, perfosfamide, phenazinomycin, picibanil, pirarubicin, piritrexim, podophyllotoxin, porf Porfiromycin, purine nucleoside phosphorylase inhibitors, raltitrexed, rhizoxin, rogletimide, rohitukine, rubiginone B1, ruboxyl, safingol, saintopin, sarcophytol A, sargramostim, sobuzoxane, sonermin, sparfosic acid, spicamycin D, spiromustine, stipirimidazole ... Stipiamide, sulfinosine, tallimustine, Tegafur, temozolomide, teniposide, talib Thaliblastine, thiocoraline, tirapazamine , topotecan, topsentin, triciribine, trimetrexate, veramine, vinorelbine, vinxalci These include vinxaltine, vorozole, zeniplatin, and zilascorb.

[0320] Linkers for chemical conjugation A variety of linkers can be used to conjugate the antibodies or antigen-binding fragments described herein (e.g., antibodies, or antigen-binding fragments thereof) that recognize and bind to CD117 (such as GNNK+ CD117) to cytotoxic molecules.

[0321] As used herein, the term "linker" refers to a bivalent chemical moiety comprising a covalent bond or chain of atoms that covalently attaches an antibody or fragment thereof (Ab) to a drug moiety (D) to form an antibody-drug conjugate (ADC; Ab-ZLD, where D is a cytotoxin) of the invention. A suitable linker has two reactive ends, one for conjugation to an antibody, The other is for conjugation to a cytotoxin. The reactive end of the linker that conjugates with an antibody (reactive moiety, Z) is typically a cysteine ​​thiol on the antibody. A moiety that can be conjugated to the antibody via a thiol or lysine amine group, and therefore typically contains a thiol-reactive group (e.g., a methyl group) such as a double bond. imide, etc.), or leaving groups such as chloro, bromo, iodo, or R-sulfanyl groups, or a group reactive with amines, such as a carboxyl group; whereas the reactive end of the linker that conjugates with the antibody is typically a site that can be conjugated to a cytotoxin via amide bond formation with a basic amine or carboxyl group on the cytotoxin, and is therefore typically a carboxyl or basic amine group. When the term "linker" is used to describe a conjugated form of a linker, one or both of the reactive ends is either absent (e.g., converted to a reactive moiety Z, a chemical moiety Z) or in the process of reacting (e.g., remaining only as a carboxylic acid carbonyl) since bonds have been formed between the linker and / or the cytotoxin, and between the linker and / or the antibody or antigen-binding fragment thereof. Such conjugation reactions are further described herein below.

[0322] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the Drug unit from the antibody in the intracellular environment. In yet other embodiments, the linker unit is not cleavable, and the Drug is released, for example, by degradation of the antibody. Linkers useful in the ADCs of the invention are preferably cleavable under intracellular conditions. It is extracellularly stable, prevents the ADC molecule from aggregating, and keeps the ADC soluble in aqueous media and in a monomeric form. Prior to being transported or delivered intracellularly, the ADC: Preferably, the linker is stable and remains intact, i.e., the antibody remains linked to the drug moiety. The linker is stable outside the target cell and may be cleaved at an effective rate inside the cell. An effective linker: (i) binds to the specific (ii) maintaining the desired binding properties; (ii) enabling the conjugate or drug moiety to be delivered intracellularly; and (iii) delivering or transporting the conjugate to its target site. and (iv) maintains the cytotoxic, cytotoxic, or cytostatic effect of the cytotoxic moiety. Measured by standard analytical techniques such as mass spectrometry, HPLC, and LC / MS separation / analysis techniques. The covalent bond between the antibody and the drug moiety can be achieved by the linker having two reactive functional groups. (i.e., bivalent in the sense of reactivity). Two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups, are required. Bivalent linker reagents useful for linking molecules are known, and methods for obtaining these conjugates have been described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, pp. 234-242).

[0323] Linkers include, for example, those that can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem, 20:571-582, 2012, the disclosure of which is directed to linkers suitable for covalent conjugation). (which is incorporated herein by reference as relevant).

[0324] Linkers hydrolyzable under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, and the like (e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661, the disclosures of each of which are incorporated herein by reference in their entirety for their disclosures regarding linkers suitable for covalent conjugation). Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but are unstable below pH 5.5 or 5.0, which is approximately the pH of the lysosome.

[0325] Examples of linkers that can be cleaved under reducing conditions include disulfides. For example, disulfide linkers can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), SPDB, and SMPT. Various disulfide linkers are known in the art (e.g., See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U.S. Press, 1987). See also U.S. Pat. No. 4,880,935, the disclosures of each of which are incorporated herein by reference in their entirety for purposes of linkers suitable for covalent conjugation.

[0326] Additional linkers suitable for the synthesis of the drug-antibody conjugates described herein include , p-aminobenzyl alcohol (PABC), p-aminobenzyl (PAB), 6-maleimidohexa phosphate, pH-sensitive carbonate, and Jain et al., Pharm. Res. 32:3526-3540, 2015 (this disclosure) and other reagents described in the above, which are incorporated herein by reference in their entirety.

[0327] In some embodiments, the linker includes the aforementioned PAB or PABC (para-aminobenzaldehyde). and self-immolative groups such as benzoyloxycarbonyl, which are described, for example, in Carl et al., J. Med. Chem. (1981) 24:479-480; Chakravarty et al (1983) J. Med. Chem. 26:638-644; US 6214345; US20030130189; US20030096743; US6759509; US20040052793; US6218519; US6835807; US6268488; US20040018194; WO98 / 13059; US20040052793; US6677435; US5621002; US20040121940; Other methods for making this process are disclosed in WO2004 / 032828. Such chemical moieties ("self-immolative linkers") include methylene carbamates and heteroaryl groups, such as aminothiazoles, aminoimidazoles, and aminopyrimidines. Linkers containing such heterocyclic self-immolative groups are described, for example, in U.S. Patent Applications 20160303254 and 20150079114, and U.S. Patent No. 7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237; US 2005 / 0256030; de Groot et al (2001) J. Org. Chem. 66:8815-8830; and US 7223837.

[0328] Linkers that are susceptible to enzymatic hydrolysis include, for example, intracellular peptidase or protease enzymes (such as lysosomal or endosomal proteases). The linker may be a peptide-containing linker that is cleaved by a suitable cleavage site, including but not limited to, a peptide-containing linker. One advantage of using intracellular proteolytic release of therapeutic agents is In some embodiments, the therapeutic agent is generally attenuated when conjugated, and the serum stability of the conjugate is generally high. In some embodiments, the peptidyl linker is at least two amino acids in length or at least three amino acids in length. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides, or peptides. Examples of suitable peptides include peptides containing amino acids such as valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine. The amino acid residues comprising the amino acid linker component include naturally occurring amino acids, as well as minor amino acids and non-naturally occurring amino acid analogs (e.g., silyl groups). Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include The amino acids include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine. In some embodiments, the linker includes a dipeptide such as Val-Cit, Ala-Val, or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit. Linkers containing covalent bonds are disclosed, for example, in U.S. Pat. No. 6,214,345 (which disclosure also provides covalent bonds). (The entire contents of which are incorporated herein by reference as they relate to linkers suitable for indirect conjugation. In some embodiments, the linker includes a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the dipeptide is used in combination with a self-immolative linker.

[0329] Linkers suitable for use herein include C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroaryl, and the like. and combinations thereof, each of which Each may be optionally substituted. Non-limiting examples of such groups include (CH2) n , (CH2CH2O) n , and -(C=O)(CH2) n -unit, where n is an integer from 1 to 6 and is independently selected for each occurrence.

[0330] In some embodiments, the linker is selected from the group consisting of hydrazine, disulfide, thioether, dipeptide, p-aminobenzyl (PAB) group, heterocyclic self-immolative group, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocycloalkyl, optionally optionally substituted aryl, optionally substituted heteroaryl, acyl, -(C=O)-, or Ha-(CH2CH2O) n - groups, where n is an integer from 1 to 6. Some skilled in the art will recognize that one or more of the listed groups may be a divalent (diradical) species (e.g., C1-C6 alkyl). Recognize that the term may exist in the form of a stencil, ...

[0331] In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the p-aminobenzyl group is positioned between the cytotoxic drug and the protease cleavage site in the linker. In some embodiments, the p-aminobenzyl group is part of a p-aminobenzyloxycarbonyl unit. In some embodiments, the p-aminobenzyl group is part of a p-aminobenzylamide unit.

[0332] In some embodiments, the linker comprises PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0333] In some embodiments, the linker is a peptide, an oligosaccharide, -(CH2) n -, -(CH2CH2O) n -, PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB. The present invention includes a combination of one or more of the following:

[0334] In some embodiments, the linker is -(C=O)(CH) n -unit, where n is an integer from 1 to 6.

[0335] In some embodiments, the linker is -(CH) n -unit, where n is an integer from 2 to 6.

[0336] Linkers that can be used to conjugate an antibody, or an antigen-binding fragment thereof, to a cytotoxic drug include those that are covalently attached to the cytotoxic drug at one end of the linker and contain a chemical moiety formed at the other end of the linker through a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in an antibody or antigen-binding fragment thereof that binds to CD117 (such as GNNK+ CD117). Reactive substituents that may be present in an antibody or antigen-binding fragment thereof that binds to CD117 (such as GNNK+ CD117) include, but are not limited to, hydroxyl moieties of serine, threonine, and tyrosine residues, amino moieties of lysine residues, carboxyl moieties of aspartic acid and glutamic acid residues, and thiol moieties of cysteine ​​residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of unnatural amino acids.

[0337] Examples of linkers useful in the synthesis of drug-antibody conjugates include antibody or antigen-binding fragments. Linkers suitable for reaction with nucleophilic substituents (e.g., amine and thiol moieties) present in the fragment include, but are not limited to, Michael acceptors (e.g., maleimides), activated esters, electron-deficient carbonyl compounds, and aldehydes, among others. For example, linkers suitable for the synthesis of drug-antibody conjugates include, but are not limited to, Among these are succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, as described, for example, in Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference for its disclosure regarding linkers for chemical conjugation. Additional linkers include non-cleavable maleimidocaproyl linkers, which are particularly useful for conjugating microtubule-disrupting agents (e.g., auristatins), and are described in Doronina et al., Bioconjugate Chem. 17:14-24, 2006, the disclosure of which is incorporated herein by reference for its disclosure regarding linkers for chemical conjugation.

[0338] Any one or more of the chemical groups, moieties and features disclosed herein may be combined in a variety of ways. It will be recognized by one of skill in the art that linkers useful for conjugating antibodies and cytotoxins as disclosed herein can be formed in combination. Additional linkers useful in conjunction with the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0339] Linkers useful in the antibody-drug combinations described herein include, but are not limited to: Examples include, but are not limited to, linkers containing chemical moieties formed by coupling reactions such as those shown in Table 3 below. The wavy lines indicate the points of attachment to the antibody or antigen-binding fragment and the cytotoxic molecule, respectively.

[0340] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0341] One skilled in the art will recognize that the reactive substituent Z attached to the linker and the reactive substituent on the antibody or antigen-binding fragment thereof participates in a covalent coupling reaction to generate the chemical moiety Z, and recognizes the reactive substituent Z as such. Antibody-drug conjugates useful in conjunction with the methods described herein include antibodies or an antigen-binding fragment thereof, and the linker or cytotoxin-linker conjugate may comprise a reactive substituent, such as Z, suitable for reaction with a reactive substituent on an antibody or antigen-binding fragment thereof to form a chemical moiety, Z, as described herein. .

[0342] As shown in Table 3, examples of suitable reactive substituents on the linker and antibody, or antigen-binding fragment thereof, include nucleophilic / electrophilic pairs (e.g., thiol / haloalkyl pairs, amino / amino groups, etc.) Coupling reactions between reactive substituents to form chemical substructures Z include, for example, azide / alkyne pairs or diene / α,β-unsaturated carbonyl pairs, diene / dienophile pairs (e.g., azide / alkyne pairs or diene / α,β-unsaturated carbonyl pairs, among others). Reactions include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine or hydroxylamine condensation, hydrazine formation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder, among others), and the like. Cycloaddition, [3+2] Huisgen cycloaddition), aromatic nucleophilic substitution, aromatic electrophilic substitution, and Other reaction formats known in the art or described herein include those described herein. Preferably, the linker comprises an electrophilic functional group for reaction with a nucleophilic functional group on the antibody or antigen-binding fragment thereof.

[0343] As disclosed herein, reactive substituents that may be present in an antibody, or antigen-binding fragment thereof, include, but are not limited to, (i) an N-terminal amine group, (ii) a side chain amine group ( (iii) side chain thiol groups (e.g., lysine), and (iv) sugar hydroxyl or amino groups (where the antibody is glycosylated). As disclosed herein, reactive substituents that may be present in an antibody, or antigen-binding fragment thereof, include, but are not limited to, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine ​​residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., phenylalanine), and phenylalanine residues of unnatural amino acids. Examples include fluoroheteroaryl, haloalkyl, and haloheteroalkyl moieties. In some embodiments, as disclosed herein, reactive substituents present in an antibody, or antigen-binding fragment thereof, include amine or thiol moieties. Certain antibodies have reducible interchain disulfides (i.e., cysteine ​​bridges). Antibodies can be conjugated to linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine ​​bridge thus theoretically forms two reactive thiol nucleophiles. Additional nucleophiles can be introduced into antibodies by reaction of lysines with 2-iminothiolane (Traut's reagent), converting amines to thiols. Reactive thiol groups can be introduced into an antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing a mutant antibody containing one or more cysteine ​​amino acid residues not originally present). US Patent No. 7,521,541 describes the introduction of reactive cysteine ​​amino acids. The present application teaches the use of engineered antibodies.

[0344] In some embodiments, the reactive moiety Z attached to the linker is present on an antibody. The nucleophilic group is a nucleophilic group that is reactive with electrophilic groups present on the antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of the nucleophilic group may react with an electrophilic group on the antibody to form a covalent bond with the antibody. Useful nucleophilic groups include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0345] In some embodiments, Z is a reactive group present in an antibody or antigen-binding fragment thereof. Reactions between reactive nucleophilic substituents (e.g., amine and thiol moieties) and reactive electrophilic substituents Z For example, Z may be a Michael acceptor (e.g., maleimide). , activated esters, electron-deficient carbonyl compounds, or aldehydes.

[0346] In some embodiments, the ADC has the formula I, IA, IB, II, IIA, or IIB via a linker and chemical moiety Z. In some embodiments, the linker is a hydrazine , a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker is In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker is -((C=O)(CH) n -Yu In some embodiments, the linker -PAB-Cit-Val-((C=O)(CH2) n -It is.

[0347] In some embodiments, the linker is -(CH) n - includes units, where n is 2 In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH) n -is In some embodiments, the linker is -(CH2) n In some embodiments, the linker is -((CH) n -, where n is 6.

[0348] In some embodiments, the chemical substructure Z is selected from Table 3. In this embodiment, the chemical substructure Z is [ka] That is, where S is a sulfur atom representing a reactive substituent present in the antibody, or antigen-binding fragment thereof, that binds to CD117 (eg, resulting from the -SH group of a cysteine ​​residue).

[0349] In some embodiments, the linker L and chemical substructure Z are taken together as LZ And [ka] is.

[0350] One skilled in the art would recognize the structure of the linker-reactive substituent as including a maleimi...

Claims

1. 1. An isolated anti-CD117 antibody, or an antigen-binding fragment thereof, comprising: A heavy chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 186, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 187; and a light chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 188, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 149, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:

189.

2. 2. The anti-CD117 antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

98.

3. 3. The anti-CD117 antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

102.

4. The anti-CD117 antibody, or antigen-binding fragment thereof, of any one of claims 1 to 3, wherein the antibody is an IgG.

5. 5. The anti-CD117 antibody, or antigen-binding fragment thereof, of claim 4, wherein the IgG is IgG1 or IgG4.

6. 6. The anti-CD117 antibody, or antigen-binding fragment thereof, according to any one of claims 1 to 5, wherein said antibody, or antigen-binding fragment thereof, comprises an Fc region, and said Fc region comprises at least one amino acid substitution selected from the group consisting of D265C, H435A, L234A, and L235A (numbering according to the EU index).

7. 7. The anti-CD117 antibody, or antigen-binding fragment thereof, of claim 6, wherein the Fc region comprises the amino acid substitutions D265C, H435A, L234A, and L235A (numbering according to the EU index).

8. 8. The anti-CD117 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO:

185.

9. 9. The anti-CD117 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:

179.

10. The anti-CD117 antibody of any one of claims 1 to 9, wherein said antibody is an intact antibody.

11. A pharmaceutical composition comprising the antibody, or antigen-binding portion thereof, of any one of claims 1 to 10 and a pharma- ceutically acceptable carrier.

12. 11. An antibody drug conjugate (ADC) comprising the anti-CD117 antibody, or an antigen-binding portion thereof, of any one of claims 1 to 10, conjugated to a cytotoxin via a linker.

13. 13. The ADC of claim 12, wherein the cytotoxin is selected from the group consisting of DNA intercalators, agents capable of disrupting the mitotic spindle apparatus, RNA polymerase inhibitors, agents capable of disrupting protein biosynthesis, amatoxins, Pseudomonas exotoxin A, de bouganin, diphtheria toxin, saporin, maytansine, maytansinoids, auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycins, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolinobenzodiazepines, and indolinobenzodiazepine dimers.

14. 14. The ADC of claim 13, wherein the amatoxin is selected from the group consisting of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, and proamanuline.

15. An antibody drug conjugate (ADC) represented by the formula Ab-ZL-Am, where Ab is an antibody that binds to human CD117, or an antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an amatoxin, wherein the antibody, or antigen-binding fragment thereof, comprises: a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 186, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 187; and a light chain variable region comprising a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 188, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 149, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 189, wherein Am-LZ is represented by formula (I): 【Chemistry 1】 Here, R 1 H, OH, OR A OR C is; R 2 H, OH, OR B OR C is; R A and R B (when present), together with the oxygen atom to which they are attached, form an optionally substituted 5-membered heterocycloalkyl group; R 3 is H, R C , or R D is; R 4 , R 5 , R 6 , and R 7 are independently H, OH, or OR C , OR D , R C , or R D is; R 8 OH, NH 2 , OR C , OR D , N.H.R. C , or NR C R D is; R 9 H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO 2 -is; R C is -LZ; R D is an optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 Heteroalkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 Heteroalkenyl, optionally substituted C 2 -C 6 Alkynyl, optionally substituted C 2 -C 6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is optionally substituted C 1 -C 6 Alkylene, optionally substituted C 1 -C 6 Heteroalkylene, optionally substituted C 2 -C 6 Alkenylene, optionally substituted C 2 -C 6 Heteroalkenylene, optionally substituted C 2 -C 6 Alkynylene, optionally substituted C 2 -C 6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -(C=O)-, peptide, or combinations thereof; and Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in the antibody or antigen-binding fragment, where Am is exactly one R C Contains substituents.

16. The ADC of claim 15, wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

98.

17. 17. The ADC of claim 15 or 16, wherein the antibody, or antigen-binding fragment thereof, comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

102.

18. The ADC of any one of claims 15 to 17, wherein the antibody is an IgG.

19. 19. The ADC of claim 18, wherein the IgG is IgG1 or IgG4.

20. The ADC of any one of claims 15 to 19, wherein the antibody, or antigen-binding fragment thereof, comprises an Fc region, and the Fc region comprises at least one amino acid substitution selected from the group consisting of D265C, H435A, L234A, and L235A (numbering according to the EU index).

21. 21. The ADC of claim 20, wherein the Fc region comprises the amino acid substitutions D265C, H435A, L234A, and L235A (numbering according to the EU index).

22. The ADC of any one of claims 15 to 21, wherein the antibody, or antigen-binding fragment thereof, comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:

185.

23. The ADC of any one of claims 15 to 22, wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:

179.

24. The ADC of any one of claims 15 to 23, wherein the antibody is an intact antibody.

25. The ADC of any one of claims 15 to 24, wherein Am-LZ is represented by formula (IB): 【Chemistry 2】

26. 26. The ADC of any one of claims 15 to 25, wherein: R 1 is H or OH; R 2 is H or OH; R 3 is H; R 4 , R 5 , R 6 , and R 7 are independently H, OH, or OR C , or R C is; R 8 OH, NH 2 , OR C , or NHR C is; R 9 is H or OH; and X is -S- or -S(O)-.

27. 27. The ADC of any one of claims 15 to 26, wherein: L is optionally substituted C 1 -C 6 Alkylene, optionally substituted C 1 -C 6 It is a heteroalkylene, an optionally substituted arylene, an optionally substituted heteroarylene, a dipeptide, -(C=O)-, a peptide, or a combination thereof.

28. 27. The ADC of claim 26, wherein: R 1 is OH; R 2 is OH; R 3 is H; R 4 , R 6 , and R 7 are H, respectively; R 5 H, OH, OR C , or R C is; R 8 NH 2 or NHR C is; R 9 is OH; and X is -S- or -S(O)-.

29. 29. The ADC of claim 28, wherein: L is optionally substituted C 1 -C 6 Alkylene, optionally substituted C 1 -C 6 It is a heteroalkylene, an optionally substituted arylene, an optionally substituted heteroarylene, a dipeptide, -(C=O)-, a peptide, or a combination thereof.

30. 30. The ADC of claim 29, wherein L is optionally substituted C 1 -C 6 It is alkylene.

31. The ADC of any one of claims 15 to 30, wherein the LZ is as follows: 【Chemistry 3】

32. The ADC of any one of claims 15 to 29, wherein the LZ is as follows: 【Chemistry 4】

33. 30. The ADC of any one of claims 15 to 29, wherein the antibody, or antigen-binding fragment thereof, is conjugated to the amatoxin via a cysteine ​​residue in the Fc domain of the antibody, or antigen-binding fragment thereof.

34. The ADC of any one of claims 15 to 25, represented by any one of the following formulas: 【Chemistry 5】 where X is S, SO, or SO 2 It is.

35. An antibody drug conjugate (ADC) of the formula Ab-ZL-Am, where Ab is an antibody that binds to human CD117, or an antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an amatoxin, wherein the antibody, or antigen-binding fragment thereof, comprises: a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 186, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 187; and a light chain variable region comprising a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 188, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 149, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 189, wherein Am-LZ is represented by formula (II): 【Chemistry 6】 Where: X is S, SO, or SO 2 is; R 1 is Ab-Z-, where Z is formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the antibody or antigen-binding fragment; and R 2 is H.

36. The ADC of claim 35, wherein Ab-ZL-Am is: 【Chemistry 7】

37. The ADC of claim 35, wherein X is S.

38. 38. The ADC of claim 37, wherein L is -(CH 2 ) n -unit, and n is an integer from 2 to 6.

39. The ADC of claim 38, wherein n is 6.

40. 40. The ADC of claim 39, wherein the LZ is: 【Chemistry 8】

41. 41. The ADC of any one of claims 12 to 40 for use in reducing the population of CD117+ cells in a human patient with a stem cell disorder.

42. 41. An ADC according to any one of claims 12 to 40 for use in reducing the population of CD117+ cells in a human patient in need of hematopoietic stem cell transplantation, wherein an effective amount of said ADC is administered to said human patient prior to receiving a transplant comprising hematopoietic stem cells.

43. The ADC of any one of claims 12 to 40 for use in a method comprising: administering the ADC to a human patient in need thereof in an amount sufficient to reduce the population of CD117+ cells in the patient; and then administering to the human patient a transplant comprising hematopoietic stem cells.

44. The ADC of any one of claims 41-43, wherein the human patient has a disorder selected from the group consisting of a hemoglobinopathy, a myelodysplastic syndrome, an immunodeficiency, cancer, an autoimmune disease, or a metabolic disorder.

45. 45. The ADC of claim 44, wherein the cancer is a hematological cancer.

46. 46. ​​The ADC of claim 45, wherein the hematological cancer is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, or multiple myeloma.

47. 45. The ADC of claim 44, wherein the hemoglobinopathy is selected from the group consisting of sickle cell anemia and thalassemia.

48. A pharmaceutical composition comprising the ADC of any one of claims 12 to 40 and a pharma- ceutically acceptable carrier.

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