Conditioning methods for gene therapy

Abstract

To provide a method for hematopoietic stem cell gene therapy.SOLUTION: A method of administering genetically modified stem cells to a human subject in need thereof, comprises: a) administering to the human subject an antibody-drug conjugate (ADC) that binds to a cell surface molecule expressed on hematopoietic stem cells (HSC) and / or immune cells, thereby depleting HSCs and / or immune cells from the human subject; and b) administering to the human subject a transplant comprising a population of genetically modified stem cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application is a continuation of U.S. Provisional Application No. 62 / 838,278, filed April 24, 2019, and U.S. Provisional Application No. 62 / 838,278, filed December 6, 2019. Priority is claimed to U.S. Provisional Application No. 62 / 944,925, filed on 2004 / 05 / 10. The contents of each priority application are as follows: , which is incorporated by reference into this application.

[0002] [Sequence table] This application includes all of the following documents that have been submitted electronically in ASCII format and are incorporated herein by reference in their entirety: The ASCII copy contains the Sequence Listing, which is incorporated by reference. It was created on April 23, 2000, is named M103034_2170WO_SL.txt, and is 341,152 bytes in size. be. [Background technology]

[0003] Hematopoietic stem cells (HSCs) are a major source of hematopoietic stem cells for treating various diseases. More recently, HSC-based therapies have included stem cell genetic modification. This involves the use of powerful gene editing techniques that allow for the targeted modification of HSCs. , among others, certain blood cell disorders (e.g., sickle cell disease), metabolic disorders (e.g., mucopolysaccharide), Delivered to patients suffering from conditions such as diabetes, cancer, and autoimmune conditions (e.g., chronic granulomatous disease). For many patients, HSC-based therapies remain It is the only curative treatment available.

[0004] Hematopoietic stem cell transplant (HSCT) is a procedure that involves determining the target organ before engraftment. It requires conditioning of tissue (e.g., bone marrow tissue). Current target unsqueezed conditioning methods (including, for example, irradiation [e.g., total body irradiation [ total body irradiation (TBI), and DNA alkylating / denaturing agents Highly beneficial for multiple organ systems, hematopoietic and non-hematopoietic cells, and the hematopoietic microenvironment Such harsh conditioning regimens are toxic to the host's immune cells and It effectively damages niche cells and adversely affects multiple organ systems, often resulting in death. This can lead to life-threatening complications. For example, recent advances in gene editing have led to the development of new drugs that can target sickle cell While it has become possible to develop genetically modified stem cells for diseases, it is difficult to develop stem cells that can be used to treat harsh conditions. Current treatments, including steroid regimens, have proven unsuccessful and have led to a decline in patients may develop life-threatening or long-term complications such as cancer (e.g., secondary malignancies) and infertility. Thus, HSCs have great therapeutic potential, but such control is difficult to achieve. limitations have hindered its clinical use.

[0005] Currently, in post-transplant patients, the multipotency and hematopoietic functionality of the HSCs, as well as Genetically modified HSC grafts, such that the corrected or altered genes of the HSCs are preserved in the Methods to promote engraftment are needed.

[0006] [Field] The present disclosure relates to conjugates using antibody drug conjugates (ADCs). In conjunction with conditioning methods, various pathologies (e.g., hematological disorders, metabolic disorders, among others) Genetically modified stem cells for treating patients suffering from various diseases, including cancer and autoimmune diseases. and the use of cells, wherein the ADC is a therapeutic agent for hematopoietic stem cells and / or immune cells. It can bind to a molecule on a cell (eg, CD117 or CD45). Summary of the Invention [Problem to be solved by the invention]

[0007] Described herein is a method for providing stem cell gene therapy, wherein the method comprises administering an antibody to the Conditions involving the use of antibody-drug conjugates (ADCs) administering the genetically modified stem cells, along with a screening method, to a subject in need thereof. The antibodies of the ADCs described in this application target specific populations of endogenous hematopoietic stem cells. and / or immune cells prior to transplantation of targeted and genetically modified stem cells. , is reduced from said subject. [Means for solving the problem]

[0008] In some aspects, the present disclosure provides methods for administering genetically modified stem cells to humans in need thereof. 1. A method of administering to an elephant, said method comprising: a) Binding to cell surface molecules expressed on hematopoietic stem cells (HSCs) and / or immune cells administering to the human subject an antibody-drug conjugate (ADC) comprising the antibody-drug conjugate (ADC) Depleting HSCs and / or immune cells from a human subject; and b) administering to said human subject a transplant comprising a population of genetically modified stem cells; In some embodiments, the ADC is administered to the HSCs to be depleted and / or to the immune system. The present invention provides a method for detecting a leukocyte-associated leukocyte antigen (LEA) that binds to a cell surface molecule expressed on an immune cell.

[0009] In some aspects, the present disclosure provides methods for treating human subjects using genetically modified cells. a method for administering a genetically modified stem cell population to a patient in need thereof, the method comprising administering to the patient a genetically modified stem cell population; wherein the human subject is administering to a human subject a compound selected from the group consisting of a compound having a phenotype on hematopoietic stem cells (HSCs), Antibody-drug conjugates that bind to cell surface molecules expressed on immune cells and / or immune cells. The patient has received conditioning treatment, including anticoagulant drug conjugates (ADCs).

[0010] In some embodiments, the genetically modified stem cells are autologous stem cells. In some embodiments, the genetically modified stem cells are allogeneic stem cells.

[0011] In some embodiments, the genetically modified stem cells are HSCs.

[0012] In some embodiments, the genetically modified stem cells are CD34+ HSCs.

[0013] In some embodiments, the subject has a disease such as cancer, a hemoglobinopathy disorder, or a myelodysplastic disorder. have one or more of the following conditions:

[0014] In some embodiments, the hemoglobinopathy disorder is sickle cell anemia, thalassemia, A, Fanconi anemia, aplastic anemia, or Wiskott-Aldrich syndrome It is selected from one or more of the following.

[0015] In some embodiments, the immunodeficiency disorder is a congenital immunodeficiency or an acquired immunodeficiency. It is a disease.

[0016] In some embodiments, the acquired immunodeficiency disease is a human immunodeficiency virus or an acquired immunodeficiency disease. AIDS (immunodeficiency syndrome).

[0017] In some embodiments, the metabolic disorder is a glycogen storage disease, a mucopolysaccharidosis, a gossypii disease, or a steroid drug. Sphingolimodysplasia, Hurler's disease, sphingolipidosis, globoid cell leukodystrophy Select one or more of the following: leukodystrophy, metachromatic leukodystrophy, or leukodystrophy will be done.

[0018] In some embodiments, the cancer is leukemia, lymphoma, multiple myeloma, or neuroblastoma. tumors, and

[0019] In some embodiments, the cancer is a hematological cancer (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, or multiple myeloma. (It can be).

[0020] In some embodiments, the subject has adenosine deaminase deficiency, severe combined immune deficiency, and immunodeficiency syndrome, hyperimmunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, Osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major, systemic sclerosis, systemic erythema Systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis The person suffers from one or more of the following disorders:

[0021] In some embodiments, the subject is suffering from an autoimmune disorder. In some embodiments, the autoimmune disorder is multiple sclerosis, human systemic lupus erythematosus, rheumatoid arthritis, or the like. Treatment of gout, inflammatory bowel disease, psoriasis, type 1 diabetes (type 1 diabetes), acute disseminated encephalomyelitis, adisothiasis Alopecia universalis, ankylosing spondylitis, antiphospholipid syndrome, aplastic anemia, autoimmune autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune Autoimmune oophoritis, Baro's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, Chronic fatigue immune deficiency syndrome, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial neuropathy Pemphigus, celiac 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 syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, idiopathic Pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, leprosy Meniere's disease, mixed connective tissue disease, myasthenia gravis, neuromyotonia, opsocron Ronus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, Pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, Polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica, primary agammaglobulinemia Primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, Lucoidosis, scleroderma, Sjögren's syndrome, stiff-person syndrome, Takayasu's artery inflammation, temporal arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia, chronic granulomatous disease, or Wegener's granulomatosis.

[0022] In some embodiments, the population of stem cells is genetically engineered to alter a target gene. In some embodiments, the target gene is genetically modified. Targlobin, gamma-globin, adenosine deaminase, arylsulfatase A , WASp gene, phagocyte NADPH oxidase, galactosylceramidase (galatosyla cer amidase), beta-galactosidase, beta-hexosaminidase, alpha-L-iduronate idase, ATM serine / threonine kinase, ribosome maturation protein SBDS, or CCR5 , and is selected from one or more of the following.

[0023] In some embodiments, the graft comprising the population of genetically modified stem cells is a gene-edited In some embodiments, the gene editing system The system is the CRISPR / Cas system.

[0024] In some embodiments, the ADC is selected from the group consisting of CD2, CD5, CD7, CDwl2, CD13, CD15, CD19, CD2 1, CD22, CD29, CD30, CD33, CD34, CD36, CD38, CD40, CD41, CD42a, CD42b, CD42c, CD 42d, CD43, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD48, CD49b, CD49d, CD49e, CD49 f, CD50, CD53, CD55, CD64a, CD68, CD71, CD72, CD73, CD81, CD82, CD85A, CD85K, CD 90, CD99, CD104, CD105, CD109, CD110, CD111, CD112, CD114, CD115, CD117, CD123, CD124, CD126, CD127, CD130, CD131, CD133, CD135, CD137, CD138, CD151, CD157, CD1 62, CD164, CD168, CD172a, CD173, CD174, CD175, CD175s, CD176, CD183, CD191, CD20 0, CD201, CD205, CD217, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD235a, CD235b, CD236, CD236R, CD238, CD240, CD242, CD243, CD277, CD292, CDw293, CD295, CD298, CD309, CD318, CD324, CD325, CD338, CD344, CD 349, or CD350, or an antibody or its anti-body that binds to one or more cell surface molecules selected from The original-binding fragment, including.

[0025] In some embodiments, the ADC comprises an antibody or antigen-binding fragment thereof that binds to CD117. segment, including

[0026] In some embodiments, the ADC is administered to a subject to reduce the population of CD117+ cells. Administer in an amount sufficient to

[0027] In some embodiments, the CD117 is GNNK+ CD117.

[0028] In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof is (a) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 31; a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 32, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (S CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 34, set forth in SEQ ID NO: 35 and the amino acid sequence set forth in SEQ ID NO: 36. and a CDR3 domain comprising: or an antigen-binding fragment thereof, comprising: CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 22, CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 23 domain, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 23. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24; and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 24. a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 26; A light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in (SEQ ID NO): 26. In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof comprises: CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 41, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 42, and an amino acid sequence set forth in SEQ ID NO: 43; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 44; a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 45; a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 46; and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the anti-CD117 antibody or its antigen comprises a light chain variable region comprising a - the binding fragment comprises: the amino acid sequence set forth in SEQ ID NO: 51 a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 52; and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 53. and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 53. and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 54, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 55, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 56. A light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in 56. wherein the anti-CD117 antibody or antigen-binding fragment thereof comprises: SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 61, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 62, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 63; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 64; CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 65; CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 66; domain, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 66. In some embodiments, the anti-CD117 antibody or its antigen-binding fragment comprises a light chain variable region comprising: The fragment comprises: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 71; a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 72, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 73; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in (SEQ ID NO): 73; and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 74; NO): 75, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 76 a light chain variable region comprising a CDR3 domain comprising the amino acid sequence The anti-CD117 antibody or antigen-binding fragment thereof comprises: SEQ ID NO:81 a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 82; a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 83; and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 84. a heavy chain variable region comprising a domain; and an amino acid sequence set forth in SEQ ID NO: 84. a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 85; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof comprises: comprising: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:11, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 12, and 13): a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 15, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the anti-CD117 antibody comprises a light chain variable region comprising a CDR3 domain comprising a CDR4 domain. The antibody or antigen-binding fragment thereof includes: an antibody set forth in SEQ ID NO: 91; CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 92 an R2 domain, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 93; a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 94; and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 94. a main CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 95, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 96; a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof comprises: : CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 101, SEQ ID NO: and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 102, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 103. a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 104, an amino acid sequence set forth in SEQ ID NO: 105, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 106; In some embodiments, the anti-CD117 antibody or and antigen-binding fragments thereof include: the amino acid set forth in SEQ ID NO: 245 CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 246 2 domain, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 247. a heavy chain variable region comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248; a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 249; and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 250. a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 250 .

[0029] In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof has the sequence CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 127, 28, and the CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 129. a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: (SEQ ID NO: 1); a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 130, a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 131, CDR2 domain comprising the amino acid sequence, and the amino acid sequence set forth in SEQ ID NO: 132 and a light chain variable region comprising a CDR3 domain comprising:

[0030] In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof is (a) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 133; a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 134, and a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:135; and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 136, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 137, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 138. In some embodiments, the anti-CD1 light chain variable region comprises a CDR3 domain comprising the amino acid sequence The antibody 17 or antigen-binding fragment thereof has the amino acid sequence set forth in SEQ ID NO:139. a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 140; a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 140; and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 141. a heavy chain variable region; and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 142. a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 143, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 144; a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 144; include.

[0031] In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof is a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 29, and A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 30. The anti-CD117 antibody or antigen-binding fragment thereof comprises: SEQ ID NO: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19, and an amino acid sequence set forth in SEQ ID NO: 20; In some embodiments, the anti-CD117 antibody or its anti-CD117 antibody comprises a light chain variable region comprising the amino acid sequence The original-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 39. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof has the following characteristics: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 49, and the sequence In some embodiments, a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 50. The anti-CD117 antibody or antigen-binding fragment thereof comprises: SEQ ID NO: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 59, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 60 In some embodiments, the anti-CD117 antibody or its light chain variable region comprises the amino acid sequence The antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:69. and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70. In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof comprises: A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 79, and a A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 80. The anti-CD117 antibody or antigen-binding fragment thereof comprises: SEQ ID NO: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 In some embodiments, the anti-CD117 antibody or its light chain variable region comprises the amino acid sequence The antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:89. and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 90. In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof comprises: A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 99, and a A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 100. wherein the anti-CD117 antibody or antigen-binding fragment thereof comprises: SEQ ID NO: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 243, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 244 A light chain variable region comprising the amino acid sequence described.

[0032] In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof is 1×10 as measured by bio-layer interferometry (BLI) -2 From 1 x10 -3 , 1×10 -3 From 1×10 -4 , 1×10 -5 From 1×10 -6 , 1×10 -6 From 1×10 -7 , or 1 × 10 -7 From 1×10 -8 , the dissociation rate (K OFF )

[0033] In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof is a biocompatible antibody. Approximately 100 nM or less as measured by bio-layer interferometry (BLI). Bottom, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less , about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nM or less, about 2 K of nM or less, about 1 nM or less D and binds to CD117.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof is human. In some embodiments, the antibody or antigen-binding fragment thereof is an intact antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG. In some embodiments, the antibody or antigen-binding fragment thereof is IgG1 or I. In some embodiments, the antibody or antigen-binding fragment thereof is monoclonal antibody. It is a clonal antibody.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof has the sequence identified in SEQ ID NO: a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 122, and / or ):121, and a light chain constant region comprising the amino acid sequence set forth in

[0036] In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence D265C, H435A , L234A, and L235A (numbering according to the EU index) In some embodiments, the Fc region comprises at least one amino acid substitution. Contains the amino acid substitutions D265C, L234A, and L235A (numbering according to the EU index).

[0037] In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof has the sequence a light chain comprising the amino acid sequence set forth in SEQ ID NO: 109, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 110 , SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, and and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 114. nothing.

[0038] In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof is a light chain comprising the amino acid sequence set forth in SEQ ID NO: 115, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 120; .

[0039] In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof is a light chain comprising the amino acid sequence set forth in SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, and SEQ ID NO: 278, and a heavy chain comprising an amino acid sequence selected from the group consisting of:

[0040] In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof comprises the HC-CDR 1, HC-CDR2, and HC-CDR3 or Ab55, Ab54, Ab56, Ab57, Ab58, Ab61, Ab66, Ab67 , Ab68, Ab69, Ab85, Ab86, Ab87, Ab88, Ab89, Ab77, Ab79, Ab81, Ab85, or Ab2 49, a heavy chain comprising a variable region sequence derived from the heavy chain variable region of LC-CDR1, LC-CDR2, and LC-CDR3 or Ab55, Ab54, Ab56, Ab57, Ab58, Ab61, Ab66, Ab67, Ab68, Ab69 , Ab85, Ab86, Ab87, Ab88, Ab89, Ab77, Ab79, Ab81, Ab85, or Ab249 light chain In some embodiments, the antibody comprises a light chain comprising a variable region sequence derived from a variable region of the antibody. - the CD117 antibody or antigen-binding fragment thereof comprises HC-CDR1, HC-CDR2, and HC-CDR3 or , SEQ ID NOs: 147, 164, 166, 168, 170, 172, 174, 176, 178, 180, 183, 18 5, 187, 189, 191, 193, 195, 197, 199, 201, 202, 204, 206, 208, 210, 212, 214, 21 6, 218, 220, 222, 224, 226, 238, or 243, derived from the heavy chain variable region amino acid sequence a heavy chain comprising a variable region corresponding to LC-CDR1, LC-CDR2, and LC-CDR3, or SEQ ID NO: 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 240, 241, a light chain comprising a variable region derived from the light chain variable region amino acid sequence of 242, or 244; include.

[0041] In some embodiments, the ADC has the formula Ab-(ZL-Cy) n is represented by, where: Ab is the antibody or antigen-binding fragment thereof; L is a linker; Z is a combination of a reactive substituent on L and a reactive substituent on the antibody or antigen-binding fragment thereof. is a chemical substructure formed by a coupling reaction between a substituent Z′ and Cytotoxins include amatoxin, Pseudomonas exotoxin A, debouganin, diphtheria toxin, and sa Porin, maytansine, maytansinoid, pyrrolobenzodiazepine, pyrrolobenzodiazepine Indolinobenzodiazepine dimer, indolinobenzodiazepine, indolinobenzodiazepine dimer, indolinobenzodiazepine Dolinobenzodiazepine pseudodimers, calicheamicins, auristatins, and anthraquinones a cytotoxin selected from the group consisting of: iucrin; and n is an integer from about 1 to about 20 (which represents the average number of cytotoxins per antibody). ). In some embodiments, n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, It is an integer of about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20. In some embodiments, n is 2.

[0042] In some embodiments, the cytotoxin is an amatoxin.

[0043] In some embodiments, the ADC has formula (I): [ka] where: Q is -S-, -S(O)-, or -SO2-; R1 is H, OH, OR A OR D , is; R2 is H, OH, OR B OR D , is; R A and R B When present, together with the oxygen atom to which they are attached, forming 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 D , or NR C R D , is; R9 is H, OH, OR C , or OR D , is; R C is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkene C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkynyl C1-C6 alkyl, aryl, heteroaryl, or a combination thereof. alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkyl C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or Each of the groups "a" and "b" is an alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl, Cycloalkyl, alkaryl, alkyl, heteroaryl, amino, ammonium, Cyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urei Carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy aryl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydro independently selected in each occurrence from the group consisting of hydroxy, mercapto, and nitro , optionally substituted with 1 to 5 substituents; R D is -LZ-Ab, where the ADC of formula (I) contains exactly one R D Contains substitutions; L is one or more of hydrazines, disulfides, thioethers, amino acids, and up to 10 Peptides consisting of amino acids, p-aminobenzyl (PAB) groups, and heterocyclic self-immolative e) groups such as C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl , C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocycloalkynyl alkyl, aryl, heteroaryl, -(C=O)- group, -C(O)NH- group, -OC(O)NH- group, where p is an integer from 1 to 6 -(CH2CH2O)p- group; wherein each of C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, The cycloalkyl, aryl, or heteroaryl is an alkyl, alkenyl, alkynyl, or heteroaryl. aryl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl and heteroaryl, Amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkane Coxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl , sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, thiazolinone each selected from the group consisting of methyl, cyano, hydroxy, mercapto, and nitro; 1 to 5 (e.g., 1, 2, 3, 4, or 5) substituents, each independently selected in each occurrence; may be optionally substituted with; and Each of C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkene C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocyclo The alkyl, aryl, or heteroaryl group optionally contains an alkyl group selected from O, S, and N. It may be interrupted by one or more heteroatoms.

[0044] In some embodiments, the ADC of Formula (I) is represented by Formula (Ia): [ka] where Q, R1-R9, R A , R B , R C , R D Each of , L, and Z is as previously defined for formula (I). As it was justified.

[0045] In some embodiments, R1 is OR A R2 is OR B and R A and R B teeth , together with the oxygen atom to which they are attached, form: [ka] where: Y is -(C=O)-, -(C=S)-, -(C=NH)-, -(CH)2-, or CR E R E’ and R E and R E’ are H, C1-C6 alkylene-R D , C1-C6 heteroalkylene-R D , C2 -C6 alkenylene-R D , C2-C6 heteroalkenylene-R D , C2-C6 alkynylene-R D , C2-C6 Haitai Alkynylene-R D , cycloalkylene-R D , heterocycloalkylene-R D , Arylene-R D , and heteroarylene-R D, wherein said C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynyl C2-C6 heteroalkynylene, cycloalkylene, heterocycloalkylene, aryl Each of the heteroarylene, heteroarylene, or heteroarylene is an alkyl, alkenyl, alkynyl, cycloalkenyl, or heteroarylene. alkyl, heterocycloalkyl, alkaryl, alkyl-heteroaryl, amino, aryl ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl Nyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl aryl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl , cyano, hydroxy, mercapto, and nitro, in each case 1 to 5 (e.g., 1, 2, 3, 4, or 5) independently selected substituents, optionally may be automatically replaced.

[0046] In some embodiments, Y is C═O, as represented by the formula: [ka] .

[0047] In some embodiments, the linker is one or more of a peptide, an oligosaccharide, -(CH2) p -, -(CH2CH2O) p -, -(C=O)-, -(C=O)(CH2) p -, 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-P AB, or Ala-PAB, where p is 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). It is an integer.

[0048] In some embodiments, the linker is PAB-Ala-Val-propioline, represented by the formula: Nil, including: [ka] .

[0049] In some embodiments, the linker is PAB-Cit-Val-propiothioate, represented by the formula: Nil, including: [ka] .

[0050] In some embodiments, the linker-antibody conjugates are joined together as LZ-Ab. and has the following structure: [ka] , where S is a reactive substituent present in an antibody or antigen-binding fragment thereof. is a sulfur atom.

[0051] In some embodiments, the LZ-Ab has the following structure: [ka] where S is a reactive substituent present in an antibody or antigen-binding fragment thereof. is a sulfur atom.

[0052] In some embodiments, the ADC of Formula (Ia) is selected from the group consisting of: [ka] . [Brief explanation of the drawings]

[0053] [Figure 1] Figures 1A and 1B graphically show the results of an in vitro cytotoxicity assay demonstrating the viability of Kasumi-1 cells, as measured by luminescence (RLU) using Celltiter Glo, in the presence of varying concentrations of the indicated anti-CD117 ADCs or controls. The results are shown graphically for Ab54 (Figure 1A), Ab55 (Figure 1A), Ab56 (Figure 1A), Ab57 (Figure 1A), Ab58 (Figure 1A), Ab61 (Figure 1A), Ab66 (Figure 1B), Ab67 (Figure 1B), Ab68 (Figure 1B), and Ab69 (Figure 1B). [Figure 2] FIG. 2 graphically depicts the quantification of the area under the injury curve for the in vitro cytotoxicity assays depicted in FIGS. 1A and 1B. [Figure 3] Figures 3-6 show that CD117-ADCs are effective against primary human and non-human primate (NHP) CD34+ cells and selective for HSCs. Figure 3 illustrates the results of an in vitro cytotoxicity assay using human and non-human primate (NHP) HSCs, which shows that CD117 ADCs efficiently deplete HSCs and progenitor cells compared to human isotype and NHP isotype controls. [Figure 4] Figure 4 depicts the results of a single dose of CD117-ADC administered to rhesus primates, analyzed using flow cytometry, showing that lymphocytes are preserved while HSCs are significantly depleted. [Figure 5] The present data demonstrated that a single dose of CD117-ADC significantly reduced colony-forming cells in the bone marrow 7 days after administration (Figure 5). [Figure 6]FIG. 6 illustrates the rapid clearance of CD117-ADC, allowing for the administration of grafts by injection within days of administration. [Figure 7] Figures 7-11 show that CD117-ADC is sufficient to enable autologous genetically modified transplantation therapy in rhesus primates. Figure 7A illustrates a treatment scheme for gene-tagged autologous transplantation in CD117 ADC-conditioned primates (administered in a single dose). Figure 7B illustrates a treatment scheme for administering multiple doses of busulfan. [Figure 8] FIG. 8 shows the results of neutrophil counts (103 / μl) versus days post-transplantation on the horizontal axis following administration of a single dose of CD117 ADC. [Figure 9] FIG. 9 shows the results of platelet counts (105 / μl) versus days post-transplantation on the horizontal axis following administration of a single dose of CD117 ADC. [Figure 10] FIG. 10 shows the results of lymphocyte counts (10 3 / μl) versus days post-transplantation on the horizontal axis when a single dose of CD117 ADC was administered. [Figure 11] Figure 11 shows the results of an assay demonstrating peripheral granulocyte β-globin vector copy number (VCN) versus days posttransplant. The data show that peripheral granulocyte vector copy number is stable over time and is comparable to previous data using busulfan conditioning. The shaded boxes indicate the range of VCN with busulfan conditioning. [Figure 12] FIG. 12 illustrates the results of an assay showing levels of aspartate aminotransferase (AST) (U / L) versus days after transplantation on the horizontal axis. DETAILED DESCRIPTION OF THE INVENTION

[0054] Described herein are methods for providing stem cell gene therapy, wherein the methods comprise: The graft containing the genetically modified stem cell (e.g., hematopoietic stem cell) population is delivered to a recipient in need thereof. administering the genetically modified stem cells to a subject, The method involves administering to a subject who has received the method, in particular to a subject who has received the method, a method for the treatment of endogenous hematopoietic stem cells and / or immune cells. Targeting and depleting certain populations from said subjects (conditioning); This involves administering an antibody drug conjugate (ADC). As described herein, the genetically modified stem cells can be used to modify defective genes (e.g., The correction of a genetic mutation (a gene that causes a genetic disease) may be administered to a subject in need of treatment. The present disclosure provides a method for the combined use of stem cell gene therapy and a conditioning method that enhances engraftment. and thereby enabling gene modification.

[0055] For example, autologous stem cells from patients with sickle cell anemia can be genetically modified ex vivo. , correcting a defective gene (e.g., a mutation in the beta-globin gene - HBB gene), and administering to said patient. Genetically modifying stem cells (i.e., gene editing) Various methods are known and available in the art, e.g., zinc zinc finger nucleases (e.g., US 9,834,787), transcriptional activation transcription-activator-like effector nuclease eases (TALENs), viral-mediated gene editing, or CRI SPR / Cas system (e.g., US 2019 / 0010495 A1), etc. Modified stem cells are known in the art (see, e.g., Yong et al. al. “Recent challenges and advances in genetically-engineered cell therapy” J. Pharm. Investig. 48(2):199-208, 2018, and references cited in this application, (see, e.g., the entirety of which is incorporated by reference into this application), any of which may be used in the present method. It is sometimes used in this context.

[0056] As used in this disclosure, ADCs target specific molecules (e.g., For example, CD2, CD5, CD7, CDwl2, CD13, CD15, CD19, CD21, CD22, CD29, CD30, CD33, CD34, CD36, CD38, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD47, CD48, CD49b, CD49d, CD49e, CD49f, CD50, CD53, CD55, CD64a, CD68, CD71, CD72, CD73, CD81, CD82, CD85A, CD85K, CD90, CD99, CD104, CD105, CD1 09, CD110, CD111, CD112, CD114, CD115, CD117, CD123, CD124, CD126, CD127, CD130, CD131, CD133, CD134, CD135, CD137, CD138, CD151, CD157, CD162, CD164, CD168, CD 172a, CD173, CD174, CD175, CD175s, CD176, CD183, CD191, CD200, CD201, CD205, CD2 17, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD235a, CD235b, CD236, CD236R, CD238, CD240, CD242, CD243, CD252, CD277, CD292, CDw293, CD295, CD298, CD309, CD318, CD324, CD325, CD338, CD344, CD349, or CD3 50, and others). For example, a single Described in this application is an ADC comprising an isolated anti-CD117 human antibody. ADCs comprising isolated anti-CD45 human antibodies are also described in this application. have many features that make them advantageous for therapy, e.g., genetically modified and conditioning a human patient for transplantation of stem cells. For example, the antibodies disclosed in this application cross-react with and internalize rhesus monkey CD117. Both of these features allow them to bind cytotoxins to CD117. It may also be advantageous to use it in conjugates for delivery to expressing cells.

[0057] The antibodies described in this application include antagonistic antibodies and neutral antibodies. Specifically, the anti-CD117 antibodies antibody 54 (Ab54), antibody 55 (Ab55), antibody 56 (Ab56), and antibody 57 (Ab57) are included. 6), Antibody 57 (Ab57), Antibody 58 (Ab58), Antibody 61 (Ab61), Antibody 66 (Ab66), Antibody 67 (Ab67), Antibody 68 (Ab68), and antibody 69 (Ab69), which are antibodies against human CD117, respectively, are provided herein. Human anti-CD117 antibodies that specifically bind to the ectodomain. Ab54, Ab55, Ab56, Ab57, Ab The binding regions of Ab58, Ab61, Ab66, Ab67, Ab68, and Ab69 are described below (e.g., in Table 9). The anti-CD117 antibodies disclosed in the present application are also known as anti-CD117 antibody drug conjugates (ADCs; In the application, it may be included in a compound (also referred to as a conjugate).

[0058] ADCs described in this application (e.g., anti-CD117 or anti-CD45 antibody drug conjugates (ADCs)) Genetically modified stem cells in combination with conditioning methods including These include, among others, diseases of blood lineage cell types, cancer, autoimmune diseases, metabolic disorders, and stem cell disorders. The ADC compositions and methods described herein may be used in methods for treating various conditions, including cancers, liver damage, and other conditions. The method enhances transplantation by providing a niche to which transplanted cells can home. The endogenous hematopoietic stem cell population is then increased to enhance the engraftment of the genetically modified hematopoietic stem cells. This activity is due to the binding of hematopoietic stem cell-expressed antigens (e.g., CD117 or CD45). by administering an ADC, antibody, or antigen-binding fragment thereof that can bind to the This administration selectively depletes the endogenous hematopoietic stem cell population and This creates a vacancy in the hematopoietic tissue (e.g., bone marrow), which is then filled by These defects can be filled by transplanted, genetically modified hematopoietic stem cells. The selective reduction is also referred to as "conditioning." The method can be used to, for example, reduce CD117 [e.g., , GNNK+ CD117, etc.], or CD45, The synthetic fragment may be administered to a patient as a conditioning agent. For example, ADCs, antibodies, or antigens thereof that bind to CD117 or CD45. - administering the binding fragment to a patient suffering from cancer (e.g., leukemia) or an autoimmune disease They may be administered to patients to directly reduce the population of cancerous or autoimmune cells. and to promote the survival and engraftment of transplanted genetically modified hematopoietic stem cells. It may also be administered to patients who require hematopoietic stem cell gene therapy, which may improve post-transplant survival. Ensure that the corrected or altered gene is preserved in the patient.

[0059] For example, genetically modified hematopoietic stem cells by administering anti-CD117 or anti-CD45 ADCs. Bone graft survival can be demonstrated in a variety of experimental measures, e.g. The engraftment of the transplanted genetically modified hematopoietic stem cells is then enhanced by administering the ADC described in this application. Competitive repopulation in the patient's bone marrow after administering a graft of genetically modified hematopoietic stem cells By assessing the amount of competitive repopulating units (CRU), In addition, reporter genes (e.g., fluorescent, luminescent, or luminescent) can be used. By incorporating a gene (such as an enzyme that catalyzes a chemical reaction that produces a product) into a vector, The engraftment of genetically modified hematopoietic stem cells may be monitored (see above). The grafts containing the altered hematopoietic stem cells were transfected with the vector. ), and subsequently, the tissue to which the transplanted hematopoietic stem cells have homed (e.g., bone marrow, etc.) The corresponding signal is monitored. Known fluorescence activated cell sorting (FACS) analysis methods By assessing the quantity and survival of hematopoietic stem and progenitor cells, as measured by the Therefore, the engraftment of hematopoietic stem cells may be monitored. and / or bone marrow aspirate samples to assess bone marrow cell recovery by donor cells. Engraftment may also be measured by measuring in vitro the rate of engraftment. Engraftment may also be measured by detecting the presence of a gene sequence, e.g., sickle cell In the treatment of HBV, engraftment is measured by detecting the presence of the corrected HBB gene sequence. This may be determined.

[0060] The following sections discuss how to enhance the engraftment of genetically modified hematopoietic stem cell grafts. patients (e.g., patients suffering from cancer or autoimmune diseases, or patients undergoing hematopoietic stem cell transplantation therapy) ADCs, antibodies, or their antigen-binding fragments, which may be administered to patients in need of treatment and providing such therapeutic agents to patients. (e.g., administering conditioning and genetically modified HSCs) to provide.

[0061] A.Definition As used in this application, the term "about" refers to a value 10% above or below the stated value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

[0062] As used in this application, the term "antibody" refers to an antibody that specifically binds to or specifically binds to a particular antigen. It refers to an immunoglobulin molecule that is immunologically reactive with a specific antigen. Examples of antibodies include, but are not limited to, monoclonal antibodies, multispecific antibodies (e.g., specific antibodies), genetically engineered antibodies, and other modified forms of antibodies antibodies (for example, but not limited to, chimeric antibodies, humanized antibodies, heteroconjugates, specific antibodies [e.g., bi-, tri- and quad-specific antibodies, diabodies, tribodies, abodies, and tetrabodies], and antibody fragments [i.e., antigen-binding fragments of antibodies fragments, such as Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments, (These are included as long as they exhibit the desired antigen-binding activity).

[0063] The term "monoclonal antibody" (mAb) refers to a molecule that specifically binds to a target protein. The antibody fragments can be prepared from intact molecules as well as antibody fragments (e.g., Fab and F(ab')2 fragments). A monoclonal antibody is a single antibody fragment. refers to an antibody obtained from a clone by any means available or known in the art. The present disclosure is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be produced using hybridoma, recombinant, and phage display technologies, or using a wide variety of techniques known in the art, including using combinations thereof. As used herein, Fab and F(ab')2 fragments can be prepared by inducing These antibody fragments lack the Fc fragment of a normal antibody. Examples of such methods are described in this application.

[0064] The antibodies of the present disclosure are generally isolated or recombinant. As used herein, refers to the identification and isolation from a cell or cell culture that expresses it and The term "isolated antibody" broadly refers to a polypeptide (e.g., antibody) that has been isolated and / or recovered. Typically, an isolated antibody is An "isolated antibody" is prepared by at least one purification step. For example, an antibody specific for CD117 is an antibody that is substantially free of other antibodies having antigen specificity other than CD117. The isolated antibody specifically binds to an antigen other than CD117 and is substantially free of antibodies that specifically bind to an antigen other than CD117. Not at all.

[0065] As used in this application, the term "antigen-binding fragment" refers to a fragment that is specific for a target antigen. Antigen-binding refers to a fragment or portion of an antibody that retains its ability to specifically bind to an antigen. The function can be performed by fragments of a full-length antibody. Antibody fragments include, for example, For example, Fab, F(ab')2, scFv, diabody, triabody, affibody, nanobody, It may be an aptamer, or a domain antibody. Examples of binding fragments encompassed by the term include, but are not limited to: (i) Fab fragments, V L , V H , C L and C H A monovalent fragment consisting of one domain (ii) F(ab')2 fragment, two fragments linked by a disulfide bridge at the hinge region (iii) a bivalent fragment containing a Fab fragment of V H and C H Fd flag 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 dAb fragments consisting of domains (e.g., Ward et al., See Nature 341:544-546, 1989); (vii) V H or V L dAb consisting of domains; (viii ) isolated complementarity determining regions (CDRs); and (ix) optionally linked by a synthetic linker. A combination of two or more (e.g., two, three, four, five, or six) isolated CDRs may be Furthermore, the two domains of the Fv fragment (V L and V H ) are coded for by separate genes However, by using recombinant methods, they can be linked by a linker and As a result, a single protein chain (V L and V H (regions pair to form monovalent molecules) (known as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988. These antibody fragments can be obtained using conventional techniques known to those skilled in the art. and the fragments can be used for their utility in the same manner as intact antibodies. Antigen-binding fragments can be screened using recombinant DNA techniques, immunoassays, and other methods. by enzymatic or chemical cleavage of intact immunoglobulins, or in some cases It can be produced by chemical peptide synthesis techniques known in the art.

[0066] As used in this application, the term "anti-CD117 antibody" or "antibody that binds to CD117" refers to an antibody that binds to CD117. The present invention provides a method for the preparation of a CD117-targeting antibody, the method comprising: It refers to an antibody that can bind to CD117 with sufficient affinity. There are two major markers of human CD117. The amino acid sequences of the isoforms are SEQ ID NO: 145 (isoform 1) and SEQ ID NO: The sequence is shown in SEQ ID NO: 146 (isoform 2).

[0067] As used in this application, the term "anti-CD45 antibody" or "antibody that binds to CD45" refers to The antibodies are useful as diagnostic and / or therapeutic agents in targeting CD45. The term "antibody" refers to an antibody that is capable of binding to CD45 with sufficient affinity so as to be useful.

[0068] As used in this application, the terms "anti-CD2 antibody" or "antibody that binds to CD2" or "anti-CD "CD2 ADC" or "CD2-binding ADC" refers to an ADC that binds to CD2, where CD2 is found on the cell surface of cells such as T cells. In this case, the term refers to an antibody or ADC that specifically binds to human CD2.

[0069] As used in this application, the terms "anti-CD5 antibody" or "antibody that binds to CD5" or "anti-CD "CD5 ADC" or "CD5-binding ADC" refers to an ADC that binds to CD5, which is found on the cell surface of cells such as T cells. In this case, the term refers to an antibody or ADC that specifically binds to human CD5.

[0070] As used in this application, the term "anti-CD137 antibody" or "antibody that binds to CD137" refers to The antibody is useful as a diagnostic and / or therapeutic agent by targeting CD137. refers to an antibody that can bind to CD137 with sufficient affinity so as to be useful in .

[0071] As used in this application, the term "bispecific antibody" means At least two different antigens, which may be on the same or different antigens, or two different antigens Antibodies (e.g., monoclonal, often human) capable of binding to different epitopes For example, one of the binding specificities may be a hematopoietic stem cell surface antigen ( For example, epitopes on CD117 [e.g., CD117, such as GNNK+ CD117], or CD45 The target can be directed to one group and the other to a signaling pathway that specifically enhances cell proliferation. Various hematopoietic stem cell surface antigens or other antigens, such as receptors or receptor subunits, It can specifically bind to epitopes on cell surface proteins. In an embodiment, the binding specificity is determined by unique, non-overlapping enzymes on the same target antigen. Antibodies may be directed against a specific antigen (i.e., a biparatopic antibody).

[0072] As used in this application, the term "complementarity determining region" (CDR) refers to the light and heavy chain variable regions of an antibody. The more highly conserved regions of the variable domains are referred to as hypervariable regions found in both the ribosomal and ribosomal domains. The amino acid positions that represent the hypervariable regions of an antibody are referred to as framework regions (FR). and may vary depending on various definitions known in the art. Some positions in may be considered hybrid hypervariable positions, and these positions are may be considered to be within a hypervariable region under one set of criteria, while being One or more of these positions may also be considered to be outside the hypervariable regions. , may be found in the extended hypervariable region. The antibodies described in this application may contain these Hybrid hypervariable positions may contain modifications. Native heavy and light chain variable domains Each of these has a predominantly β-sheet structure and is connected by three CDRs (the CDRs are β-sheets). (connecting and sometimes forming part of the nucleotide structure, forming loops), The CDRs in each chain are arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and are separated by a framework region The CDRs are held together in close proximity by the CDRs from the other antibody chains and act to target the antibody. contributes to the formation of the ATP-binding site (Kabat et al., Sequences of Proteins of Immunological (See, for example, "Interest," National Institute of Health, Bethesda, MD., 1987). In certain embodiments, the numbering of amino acid residues in immunoglobulins is Ka unless otherwise specified. The immunoglobulin amino acid residue numbering system of Bat et al. is used (but is not limited to this). Any antibody numbering scheme may be used, including but not limited to IMGT and Chothia. (Sometimes this happens).

[0073] As used in this application, the terms "conditioning" and "conditioning" are used interchangeably. "Giving" refers to the ability of a person to receive a graft (e.g., a graft containing genetically modified hematopoietic stem cells [HSCs]). This refers to the process or processes by which a patient is prepared for Such techniques can enhance hematopoietic stem cell graft survival (e.g., conditioning). in blood samples isolated from patients after the screening procedure and subsequent hematopoietic stem cell transplantation. (This is inferred from the sustained increase in the amount of viable hematopoietic stem cells.) According to the method described, molecules (e.g., antigens) expressed by hematopoietic stem cells and / or immune cells ( For example, administering to a patient an ADC capable of binding to CD117 (e.g., GNNK+ CD117) or CD45 and conditioning the patient in preparation for transplantation of genetically modified HSCs. As described in this application, the antibody may be used as a drug-antibody conjugate ( covalently attached to a cytotoxin to form an antibody-drug conjugate (ADC) The conjugate may be conjugated to one or more of the HSC-expressed antigens disclosed in the present application. The ADC, antibody, or antigen-binding fragment thereof may be used in hematopoietic stem cell transplantation therapy. by administering it to patients in need of hematopoietic stem cell transplant (HSCT) therapy. For example, endogenous HSCs are selectively depleted, thereby allowing the transplantation of genetically modified HSCs. This may promote HSC graft survival by creating a void that can be filled by .

[0074] As used in this application, the terms "conjugate," "antibody drug conjugate," or A "drug-antibody conjugate" or "ADC" is an antibody or its fragment linked to a cytotoxin. ADC refers to a compound that combines a reactive functional group on an antibody, or an antigen-binding fragment thereof, with a compound of the present invention. by chemical conjugation with an appropriate reactive functional group on another molecule, such as the cytotoxins described in A conjugate is formed between two molecules that are bound to each other (e.g., an antibody and a cytotoxin). ) may contain a linker. Linkers that can be used to form conjugates Examples of linkers include peptide-containing linkers (e.g., naturally occurring or non-naturally occurring amino acids). Linkers include linkers containing D-amino acids. Using a variety of strategies, which are described and known in the art, Depending on the reactive moiety therein, the linker may be prepared by, for example, enzymatic cleavage. Hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide It may be cleaved by amide reduction, nucleophilic cleavage, or organometallic cleavage (e.g., Leriche (See, e.g., et al., Bioorg. Med. Chem., 20:571-582, 2012). As noted above, the term " "Conjugate" (when referring to a compound) is used in this application as a "drug conjugate"; Also referred to interchangeably as "drug antibody conjugate," "antibody drug conjugate," or "ADC" To say.

[0075] As used in this application, the term "coupling reaction" refers to the process of combining two compounds suitable to react with each other. One or more substituents react to form molecular fragments attached to each substituent (e.g., Coupling reaction refers to a chemical reaction that forms chemical substructures that bond together (covalently). In response, a cytotoxin (e.g., a cytotoxin known in the art or described in this application) may be used. The reactive substituent attached to a fragment may be a nucleotide that binds to an antibody or antigen-binding fragment thereof (e.g., For example, CD117 [e.g., GNNK+ CD117] known in the art or described in this application.

[0023] A specific anti-CD117 antibody, an antibody, an antigen-binding fragment thereof, or a specific anti-CD117 antibody, which binds to Reactions involving the reaction of a suitable reactive substituent attached to a fragment include those involving the reaction of a suitable reactive substituent attached to a fragment. Examples of groups include nucleophilic / electrophilic pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pair, or thiol / α,β-unsaturated carbonyl pair, etc.), diene / dienophile pair (e.g., azide / alkyne pairs, among others). Coupling reactions include: Examples include, but are not limited to, thiol alkylation, hydroxyl alkylation, and amine alkylation. Alkylation, amine condensation, amidation, esterification, disulfide formation, cycloaddition (e.g. These include, in particular, [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, aromatic nucleophilic substitution, aromatic aromatic electrophilic substitution, and other reaction modes known in the art or described in this application. Examples include:

[0076] As used in this application, "CRU (competitive repopulating unit)" refers to a "it"))" is a unit of measurement for long-term engrafting stem cells that can be detected after in vivo transplantation. This refers to the following.

[0077] As used in this application, the term "diabody" refers to a bivalent polypeptide comprising two polypeptide chains. refers to an antibody, wherein each polypeptide chain is a V on the same peptide chain H and V L Domain A linker that is so short that intramolecular association is not possible (e.g., a linker consisting of five amino acids) V connected by a H and V L This configuration allows each domain The domain pairs with a complementary domain on another polypeptide chain to form a homodimeric structure Thus, the term "triabody" refers to a trivalent antibody containing three peptide chains, Each V in the same peptide chain H and V L It is too short to allow intramolecular association of the domains. a linker (e.g., a linker consisting of 1 to 2 amino acids) V H Domain and one V L The peptide thus constructed contains the domain. To fold into the native structure, the V H Domain and V L Do They trimerize so that the domains are spatially close to each other (e.g., Holliger et al., Proc (See Natl. Acad. Sci. USA 90:6444-48, 1993).

[0078] As used in this application, "drug-to-antibody ratio" or "DAR" means refers to the number of drugs (e.g., amatoxins) attached to the antibody of the conjugate. The DAR of an ADC ranges from 1 to 8, although higher loadings are possible depending on the number of binding sites. In certain embodiments, the conjugate may be 1, 2, 3, 4, 5, 6, 7, or has a DAR of 8.

[0079] As used in this application, the term "endogenous" refers to a molecule that is naturally present in a particular organism, such as a human patient. The molecules, cells, tissues, or organs found in the mbocyte, platelet, red blood cell, mast cell, myeloblast, basophil , neutrophils, eosinophils, microglial cells, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages cytotoxicity, such as phages, dendritic cells, natural killer cells, T-lymphocytes, or B-lymphocytes Describes substances such as blood lineage cells.

[0080] As used in this application, the term "engraftment potential" refers to the ability of hematopoietic stem cells to engraft. and hematopoietic progenitor cells, and refers to the ability of such cells to repopulate tissues, The term refers to a cell that is present in the blood, whether circulating in the blood or provided by transplantation. The mechanisms surrounding engraftment, such as cell homing and cell colonization within tissues, The engraftment efficiency or engraftment rate is defined as any of the events that lead to engraftment, as known to those skilled in the art. Any clinically accepted parameter can be used to assess or quantify, for example, competitive To evaluate the competitive repopulating unit (CRU); Marker uptake in the ingested, colonized, or engrafted tissue or progression of the disease, hematopoietic stem and progenitor cell production, and assessing the progress of the subject by prolonging the life of the recipient or the recipient's life. Engraftment can also be assessed by measuring the number of white blood cells in the peripheral blood after transplantation. Engraftment is measured by measuring the recovery rate of bone marrow cells from donor cells in bone marrow aspirate samples. It can also be evaluated by

[0081] As used in this application, the term "exogenous" refers to a substance that is naturally present in a particular organism, such as a human patient. molecules, cells, tissues, or organs not found in the human genome (e.g., genetically modified hematopoietic stem cells, or Megakaryocyte, thrombocyte, platelet, red blood cell, mast cell, myeloblast oblast), basophils, neutrophils, eosinophils, microglial cells, granulocytes, monocytes, osteoclasts, anti lymphocytes, macrophages, dendritic cells, natural killer cells, T-lymphocytes, or B- In some embodiments, the present invention describes substances such as cells of the hematopoietic lineage, such as lymphocytes. A substance that is exogenous to a recipient organism (e.g., a recipient patient) is a substance that is exogenous to the recipient organism (e.g., a recipient patient). It may be naturally present in the donor organism (e.g., donor subject) from which it originates. For example, allogeneic cell grafts are exogenous to the recipient but are transplanted from the donor. In some embodiments, the autologous cell graft comprises cells that are native to the exogenous to the recipient (e.g., to correct a mutation present in the recipient) (by correcting) the gene sequence, and therefore, such autologous cell grafts Exogenous materials include substances that are introduced into a living organism from an external source. or cultures extracted therefrom include those provided.

[0082] The terms "Fc", "Fc region", and "Fc domain" as used in this application refer to an IgG antibody. (relating to crystallizable fragments obtained by papain digestion of IgG molecules) The Fc region refers to the C-terminal tails of the two heavy chains of an IgG molecule that are linked by disulfide bonds. The terminal half contains a glycosylated portion that does not have antigen binding activity, and a complement and Fc receptors, including the FcRn receptor (see below). The Fc region comprises a second constant domain CH2 (e.g., EU positions 231 to 340 of IgG1) and a third constant domain CH3 (e.g., EU positions 231 to 340 of IgG1). The antibody used in this application comprises the constant domain CH3 (e.g., residues 341 to 447 of EU position of human IgG1). When the Fc region or domain is a “lower hinge region” ( For example, residues at EU positions 233-239 of IgG1.

[0083] Fc refers to this region in isolation, or to an antibody, antibody fragment, or Fc fusion protein. This region may be referred to as "the region in the context of the EU region." It has been observed at many positions within the Fc domain, including positions 270, 272, 312, 315, 356, and 358. Therefore, there is a significant difference between the sequences presented in this application and those known in the art. Minor differences may exist. Therefore, the term "wild-type IgG Fc domain" or "WT IgG Fc" is used. "c domain" refers to any naturally occurring IgG Fc region (i.e., any allele). 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), the accession numbers are P01857(IGHG1_human), P01859(IGHG2_human), P01860(IGHG3_human) and P01861(IGHG1_hu An example of a "WT" Fc region is SEQ ID NO: 122 (heavy chain constant domain containing the Fc region). Provided in the area).

[0084] As used in this application, the term "altered Fc region" or "variant Fc region" refers to an Fc region IgG containing one or more amino acid substitutions, deletions, insertions or modifications introduced anywhere within the region In certain embodiments, the variant IgG Fc domain comprises one or more amino acids. Binding to Fc gamma R and / or C1q compared to a wild-type Fc domain not containing the amino acid substitution It contains one or more amino acid substitutions that result in reduced or no affinity. Fc binding interactions mediate various effector functions and downstream signaling events (antibody-dependent). Antibody dependent cell-mediated cytotoxicity (ADCC) and These include complement dependent cytotoxicity (CDC), Therefore, in certain embodiments, the variant Fc domain is essential for An antibody (e.g., an antibody, fusion protein, or conjugate) containing one or more amino acids does not contain any amino acid substitutions, deletions, insertions, or modifications (e.g., amino acids naturally occurring at the corresponding positions in the Fc region). the unmodified Fc region, containing the amino acid residues present in the Fc region, has otherwise the same amino acid sequence. at least one or more Fc ligands (e.g., Fc gamma R) compared to a corresponding antibody having ) may change their binding affinity.

[0085] Variant Fc domains are defined according to the amino acid modifications that compose them. For all amino acid substitutions discussed in this application, the numbering is always according to Kabat. Thus, for example, D265C corresponds to the EU index relative to the parent Fc domain. An Fc variant in which the aspartic acid (D) at position 265 is replaced with a cysteine ​​(C). Note that the order of presentation is arbitrary. Similarly, for example, D265C / L234A / L235A is the parent F For the c domain, substitutions were made at EU positions 265 (D to C), 234 (L to A), and 235 (L to A). The variant also defines an Fc variant having a mutated EU amino acid position. They may also be designated according to their final amino acid composition. For example, the L234A / L235A mutation is designated as "L ALA is sometimes called "ALA." Another example is the E233P.L234V.L235A.delG236 (deletion of 236) mutation. is sometimes referred to as "EPLVLAdelG." As yet another example, the I253A.H310A.H435A mutation is It is sometimes referred to as "IHH." Note that the order in which the substitutions are listed is arbitrary.

[0086] The term "Fc gamma receptor" or "Fc gamma R" as used in this application refers to an IgG antibody. A family of proteins that bind to the Fc region of the body and are encoded by the Fc gamma R gene In humans, this family is not limited to However, Fc gamma RI (CD64) (e.g., the isoforms Fc gamma RIa, Fc gamma RIb, and Fc gammaRIc etc.); Fc gammaRII (CD32) (e.g., the isoform Fc gammaRIIa [e.g., , allotypes H131 and R131], Fc gamma RIIb [e.g., Fc gamma RIIb-1 and Fc gamma RIIb Ib-2], and Fc gamma RIIc, etc.); and Fc gamma RIII (CD16) (e.g., isoforms Fc gamma RIIIa [e.g., allotypes V158 and F158] and Fc gamma RIIIb [e.g., allotypes Fc gamma RIIIb-NA1 and Fc gamma RIIIb-NA2], and any undiscovered human Fc gamma Fc gamma R or an isoform or allotype of Fc gamma R. Fc gamma R can be any living organisms (e.g., but not limited to, humans, mice, rats, rabbits, and monkeys) Mouse Fc gamma R may be derived from, but is not limited to, Fc gamma R. Fc gamma RII (CD64), Fc gamma RII (CD32), Fc gamma RIII (CD16), and Fc gamma RIII-2 (CD16-2 ), as well as any undiscovered mouse Fc gamma R or Fc gamma R isoforms or alloforms. Type, including.

[0087] The term "effector function" as used in this application refers to the interaction of an Fc domain with an Fc receptor. The effector function is limited to the following: These include, but are not limited to, ADCC, ADCP, and CDC. "Fc-cells" express one or more Fc receptors and mediate one or more effector functions. Effector cells are cells of the immune system that act as immune cells. Effector cells include, but are not limited to: However, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, and platelets ), B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and gastrointestinal and cytotoxic T cells, as well as any living organism (e.g., but not limited to, The genotypes are derived from a variety of animals (humans, mice, rats, rabbits, and monkeys, but not from humans).

[0088] As used in this application, the terms "silent" and "silenced" Or "silencing" refers to the Fc region of the same antibody containing an unmodified Fc region. Binding to Fc gamma receptors (FcγR) is reduced compared to binding to Fc γ receptors (FcγR) Also refers to antibodies with modified Fc regions as described in this application (e.g., as measured by BLI). For example, when the Fc region is modified, the FcγR binding of the antibody is increased compared to the FcγR binding of the same antibody comprising an unmodified Fc region. A reduction in binding to γR of at least 70%, at least 80%, at least 90%, or at least 95% In some embodiments, the Fc subunit is at least 98%, at least 99%, or 100%. Fc silenced antibodies do not exhibit detectable binding to FcγR. Binding of an antibody having the formula (I) to FcγR can be determined by various techniques known in the art (e.g., limited Although not intended to be a method for determining the activity of a protein, equilibrium methods [e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Ratha Naswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; Radioimmunoassay RIA], or kinetic-based surface plasmon resonance assay or other mechanisms. Assay [e.g., BIACORE TM Parse or Octet TM analysis (forteBIO)], as well as other methods [e.g. For example, indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel Electrophoresis and chromatography (e.g., gel filtration) can be used to measure These and other methods utilize labels present on one or more of the components being evaluated. and / or various detection methods (e.g., but not limited to, Chromatic, fluorescent, luminescent, or isotopic labels may be used. Regarding affinity and kinetics, see Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lip Pincott-Raven, Philadelphia (1999) (which focuses on antibody-immunogen interactions) An example of a competitive binding assay is a method in which a labeled antigen is bound to an antibody of interest. in the presence of increasing amounts of unlabeled antigen; and detecting the antibody bound to the labeled antigen. From the data, Scatchard plot analysis was performed to identify the target antibody against a specific antigen. The binding affinity and binding off-rate of the antibody may be determined. Alternatively, radioimmunoassays may be used to measure the antigen. The antibody of interest conjugated to a compound and increasing amounts of an unlabeled second antibody are added. The mixture is incubated in the presence of

[0089] As used in this application, the term "same antibody with an unmodified Fc region" refers to the same antibody as listed Lacking amino acid substitutions (e.g., D265C, H435A, L234A, and / or L235A), but otherwise refers to an antibody having the same amino acid sequence as the Fc-modified antibody to which it is being compared.

[0090] The term “antibody dependent cell-mediated cytotoxicity” "Anti-Dysplastic Cell Cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which a polypeptide containing an Fc domain The peptide (e.g., antibody) targets certain cytotoxic cells (e.g., primarily NK cells, neutrophils, and It binds to Fc receptors (FcR) present on cells (e.g., macrophages) and damages these cells. The harmful effector cells bind specifically to the antigen-bearing "target cells." and subsequently kills the target cells with its cytotoxin (Hogarth et al., N (Article review Drug Discovery 2012, 11:313). In addition to antibodies and their fragments, Other polypeptides containing an Fc domain that have the ability to specifically bind to antigen-bearing target cells peptides (e.g., Fc fusion proteins and Fc conjugate proteins) are used for cell-mediated cellular transport. It is contemplated that this will result in cell damage.

[0091] For simplicity, cell-mediated cytotoxicity resulting from the activity of polypeptides containing an Fc domain The ADCC activity of any particular polypeptide of the present disclosure is also referred to as ADCC activity in the present application. The ability of a target cell to mediate lysis can be assayed to assess ADCC activity. To achieve this, a polypeptide of interest (e.g., an antibody) is transfected into a target cell along with immune effector cells. The target cells are then added to the target cells, resulting in cytolysis of the target cells. Specifically, the label (e.g., radioactive substrate, fluorescent dye, or natural intracellular protein) is extracted from lysed cells. The effector useful for such assays is -cells include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells A specific example of an in vitro ADCC assay is described in Bruggemann et al., J. Exp. Med. 166:1351 (198 7); Wilkinson et al., J. Immunol. Methods 258:183 (2001); Patel et al., J. Immun ol. Methods 184:29 (1995). Alternatively, or in addition, ADCC of the antibody of interest may be performed. Activity can be measured in vivo (e.g., Clynes et al., Proc. Natl. Acad. Sci. USA 95:652 (1999) 8) may be evaluated in animal models.

[0092] The terms "full-length antibody" and "intact antibody" are used in this application to refer to a substantially intact antibody. are used interchangeably to refer to various forms of antibodies, and antibody fragments as defined in this application. Thus, for IgG antibodies, the intact antibody consists of variable regions, constant regions, and two heavy chains each containing a variable region and a constant region, and More specifically, intact IgG comprises a light chain variable region (VL) and a light It contains two light chains, each containing a chain constant region (CL), and a heavy chain variable region (VH) and three heavy chains It contains two heavy chains, each containing a constant region (CH1, CH2, and CH3). CH2 and CH3 are the Fc regions of the heavy chain. In certain embodiments, the ADCs used in the methods described herein represent stem Binds to antigens expressed on the surface of cells (e.g., human CD117 [hCD117] or human CD45 [hCD45]) and intact antibodies.

[0093] As used in this application, the term "framework region" or "FW region" refers to a region of an antibody or its antigen-binding fragment. The residues in the FW region include those adjacent to the CDRs of the fragment. Humanized antibodies, monoclonal antibodies, antibody fragments, Fab fragments, single-chain antibody fragments fragments, scFv fragments, antibody domains, and bispecific antibodies. This may happen.

[0094] In addition, "conservative sequence modifications" of the sequences set forth in the SEQ ID NOs described in this application are also included. "sequence modifications" (i.e., modifications of the nucleotide sequence and amino acid sequence) Thus, an antigen of an antibody encoded by the nucleotide sequence or an antibody comprising the amino acid sequence Modifications of the nucleotide and amino acid sequences that do not abolish binding to the Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, and These include the addition and deletion of nucleotides and amino acids. Modifications may be made by conventional techniques known in the art, such as mutagenesis and PCR-mediated mutagenesis. may be introduced into the SEQ ID NOs described in this application. Conservative sequence modifications include Conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, are examples. Families of amino acid residues having similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, histidine, acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycolic acid), Synthin, Asparagine, Glutamine, Serine, Threonine, Tyrosine, Cysteine, Triptophan tophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline , phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoforms leucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine, Therefore, preferred examples of the amino acid sequence of the predicted amino acid sequence in the anti-CD117 antibody include amino acids having the amino acid sequence of the predicted amino acid sequence (e.g., cysteine). A non-essential amino acid residue that is present in an antibody is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of nucleotides and amino acids that do not disrupt intact bonds are well known. These methods are well known in the art (e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Koba yashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. A cad. Sci. USA 94:412-417 (1997)).

[0095] As used in this application, the term "half-life" refers to the time period during which a compound is dissolved in the body in a subject, e.g., a human subject. This refers to the time it takes for the plasma concentration of an antibody drug to decrease by half or 50%. A 50% decrease in reflects the amount of drug in circulation.

[0096] As used in this application, the term "stem cell" refers to a multipotent stem cell (e.g., a stem cell that is capable of producing The term refers to totipotent or pluripotent stem cells. This may also occur.

[0097] As used in this application, the term "hematopoietic stem cell" ("HSC") refers to a stem cell that has the ability to self-renew and and granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes , red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, blood platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, cytoplasmic including, but not limited to, bone cells, and lymphocytes (e.g., NK cells, B cells, and T cells). These immature blood cells have the ability to differentiate into mature blood cells, including those with various lineages. These cells express CD34 + May contain CD34 cells. + The cells express the CD34 cell surface marker. In humans, CD34+ cells have the properties of stem cells defined above. In mice, HSCs are thought to comprise a subpopulation of CD34- cells. C also refers to Long Term Renewable HSC (LT-HSC) and Short Term Renewable HSC (ST-HSC). LT-HSC and ST-HSC are distinguished based on their functional potential and expression of cell surface markers. For example, human HS C denotes CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (CD2, CD3, CD4, CD7, CD8, CD1 0, negative for mature lineage markers including CD11B, CD19, CD20, CD56, CD235A, etc. In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, CD 48- and lin- (mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra, etc.) ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, and Slamfl / CD150+ and lin- (constituents including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra, etc.) In addition, ST-HSCs are expressed under homeostatic conditions. They are less quiescent and more proliferative than LT-HSCs. However, LT-HSCs have a higher self-renewal capacity. (i.e., LT-HSCs survive throughout adulthood and can be serially transplanted through successive generations of recipients.) ST-HSCs have limited self-renewal capacity (i.e., they can be regenerated). (They survive only for the period of time they are transplanted and cannot be transplanted serially.) ST-HSCs can be used in the methods described in

[1999] . Therefore, they are particularly useful as they can give rise to differentiated progeny more quickly.

[0098] As used in this application, the terms "genetically modified stem cells" or "genetically modified HS" are used interchangeably. C" has undergone gene editing to modify a target gene in the genome of said cell. or a cell in which an exogenous gene or exogenous gene sequence has been modified to be expressed in said cell. It refers to a cell or multiple cells.

[0099] As used in this application, the term "target gene" refers to a gene sequence or refers to a portion of a gene sequence. In some embodiments, the target gene sequence is In some embodiments, the target gene sequence is a non- In some embodiments, the target A mutant gene sequence is a non-wild type gene sequence (e.g., a mutation) that causes a disorder or disease. In some embodiments, modifying a target gene can include, for example, 1) A gene editing system is used to correct one or more mutations in the target gene sequence. using the gene (e.g., by codon-specific editing; or by editing the entire gene or or by replacing a part of the gene), thereby restoring the function of the gene ( e.g., to produce a functional protein or a functional regulatory sequence), 2) a functional gene sequence Insertion of a gene (e.g., a wild-type or functional variant sequence) into the genome of a stem cell or 3) inserting a functional gene (e.g., a wild-type or functional barrier disrupting target gene sequences (e.g., silencing) Various gene modifications and / or insertions for gene sequences are available. Gene editing systems are known in the art. Examples include the gene encoding the gene that causes sickle cell disease. One or more mutations in the HBB gene that cause the mutations can be repaired by site-specific editing of the mutations or by the gene This can be corrected by replacing the whole or part of the gene. For example, a mutant HBB gene can be silenced by gene editing methods, and functional HBB genes can be In some embodiments, the target gene may be inserted into the genome of the stem cell. The gene is a wild-type gene (e.g., CCR5) that is replaced with a variant sequence. or may be edited to encode a variant sequence (the variant The target sequence provides therapeutic benefit [e.g., CCR5(delta)32 for HIV treatment]. ).

[0100] As used in this application, the term "functional potential of hematopoietic stem cells" refers to: 1) pluripotency (multi-potency) motility) (which includes, but is not limited to, granulocytes [e.g., promyelocytes, neutrophils, eosinophils, basophils], red blood cells [e.g., reticulocytes, erythrocytes], platelets (thrombocytes) [e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets)], monocytes [e.g., monocytes, macrophages], dendritic cells, microglia, osteoclasts, and lymphocytes [e.g. (refers to the ability to differentiate into multiple different blood lineages, including NK cells, B-cells, and T-cells) 2) self-renewal (the ability of hematopoietic stem cells to give rise to daughter cells with potential equivalent to that of the parent cell) Furthermore, this ability can be repeated throughout an individual's life without wearing off. and 3) hematopoietic stem cells or their progeny, when reintroduced into the transplant recipient, produce hematopoietic stem cells. hematopoietic stem cells, including the ability to home to cell niches and re-establish productive and sustained hematopoiesis. Refers to the functional properties of a cell.

[0101] As used in this application, the term "human antibody" refers to an antibody derived from human germline immunoglobulin sequences. A human antibody is intended to include antibodies having variable and constant regions derived from a human. Amino acid residues not encoded by germline immunoglobulin sequences (e.g., in vitro Mutations introduced by random or site-directed mutagenesis in vivo or may contain mutations introduced by gene rearrangement or somatic mutations. As used in this application, the term "human antibody" includes antibodies derived from the germline of another mammalian species, such as a mouse. It is not intended to include antibodies in which CDR sequences derived from a human have been grafted onto human framework sequences. Human antibodies can be produced (e.g., by recombinant expression) or functionally reconstituted in human cells. Non-human animals capable of expressing engineered human immunoglobulin (e.g., heavy and / or light chain) genes. Alternatively, the human antibody may be produced by prokaryotic or eukaryotic cells. In some cases, 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. a linker peptide connecting the heavy chain variable region and the light chain variable region. Such linker peptides are considered to be of human origin. Phage Display Method Using an Antibody Library Derived from Human Immunoglobulin Sequences Human antibodies may be produced by a variety of methods known in the art, including by immunohistochemistry. , which are unable to express functional endogenous immunoglobulins but contain human immunoglobulin genes They can also be produced using transgenic mice capable of expressing the gene. (See, e.g., 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; See Nos. 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. thing).

[0102] A "humanized" antibody refers to an antibody that contains minimal sequence derived from non-human immunoglobulin. Thus, a "humanized" form of a non-human (e.g., murine) antibody can be obtained by removing the sequence derived from the non-human antibody. A chimeric antibody is one that contains minimally all or substantially all of the FW region of a human immunoglobulin G. Typically, a humanized antibody contains at least one typical F / W region of the purine sequence. Typically, the antibody comprises substantially all of two variable domains, including all or substantially all of the CDR regions. The FR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions correspond to those of a human immunoglobulin. Humanized antibodies also contain at least one portion of the immunoglobulin constant region (Fc). and typically contains at least a portion of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art and are described, for example, in Riechman et al. n et al., Nature 332:323-7, 1988; U.S. Patents 5,530,101; 5,585,089; 5,693,761; 5,69 3,762; and 6,180,370 to Queen et al.; EP239400; PCT Publication WO 91 / 09967; U.S. Patent 5 ,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci 91:969-973; and U.S. Pat. No. 5,565,332.

[0103] As used in this application, a patient "in need of" a transplant of genetically modified hematopoietic stem cells is Examples include patients who exhibit defects or deficiencies in one or more blood cell types, as well as those with stem cell disorders. Patients with rheumatoid arthritis, autoimmune diseases, cancer, or other conditions described in this application. In some embodiments, a patient "in need" of a genetically modified hematopoietic stem cell transplant is disorders (e.g., sickle cell anemia, thalassemia, Wiskott-Aldrich syndrome, Patients with a defective gene that causes adenosine deaminase deficiency include Hematopoietic stem cells are generally classified into the following categories: 1) multipotency, and therefore, there is no limit to However, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, red blood cells, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes) cytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia Multiple different types of cells, including lymphocytes (e.g., NK cells, B-cells, and T-cells), are involved in the differentiation of the inflammatory bowel disease. 2) self-renewal, and therefore have the same potential as the parent cell. and 3) when reintroduced into the transplant recipient, they can induce hematopoiesis. They exhibit the ability to home to stem cell niches and re-establish productive and sustained hematopoiesis. and are defective or incapable of producing hematopoietic cells (e.g., due to a mutated gene) in one or more cell types of the hematopoietic lineage. to modify the target gene [e.g., mutant gene] in a patient with a deficiency. In vivo (by artificial means) to reconstitute the defective or missing cell population. Additionally or alternatively, the patient may have sickle cell anemia. , thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome Hemoglobinopathy (e.g., non-malignant hemoglobinopathy) The subject has adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV / AIDS, , metachromatic leukodystrophy, Diamond-Blackfan anemia, and Shwachman- The subject may be suffering from Diamond syndrome, an inherited blood disorder. Having or being affected by an autoimmune disease (e.g., sickle cell anemia) Additionally or alternatively, the subject has a malignant tumor, such as neuroblastoma or a blood cancer. For example, the subject may have or be affected by leukemia, lymphoma, or other cancers. In some embodiments, the subject may have acute myeloma or myeloma. Myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple bone marrow leukemia 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 a disease such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, have an autoimmune disease such as type 1 diabetes, or another autoimmune condition described in this application. 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 is suffering from glycogen storage disease, mucopolysaccharidosis, Gaucher disease, Hurler disease. a metabolic disorder selected from the group consisting of: erythropoietin, erythrolipidosis, and metachromatic leukodystrophy; or any other disease or disorder that can benefit from the treatments and therapies disclosed in this application. Harms (including but not limited to severe combined immunodeficiency syndrome, Wiskott-Aldrich syndrome) group, hyperimmunoglobulin M 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 "Bone Marrow Tra" "Insplantation for Non-Malignant Disease", ASH Education Book, 1:319-338(2000) The disclosure relates to conditions that may be treated by prescribing hematopoietic stem cell transplant therapy. and the entire contents of which are incorporated by reference into this application) may suffer from or be affected by a disorder.

[0104] As used in this application, a "neutral antibody" is a neutral antibody that binds to a receptor ligand. individual or specific targets, including binding to or interaction with an enzyme's substrate, etc. (e.g., CD117 or CD45) In one embodiment, a neutral antibody is an antibody or antigen-binding fragment thereof that is unable to bind to a neutralizing antibody. The anti-CD117 antibody, or a fragment thereof, does not substantially inhibit SCF-dependent cell proliferation, and It is an anti-CD117 antibody that does not cross-block the binding of CD117 to other antibodies. An example of an antibody is Ab67 (or an antibody having the binding region of Ab67). Anti-CD117 antibodies inhibit SCF-dependent proliferation and inhibit SCF binding to CD117. An example of an antagonist antibody is Ab55 (or Ab55 binding). (Antibody having a specific region).

[0105] As used in this application, the term "recipient" refers to a recipient of a genetically modified hematopoietic stem cell population. The term "transplant" refers to a patient receiving a transplant, such as a transplant containing a group of cells. The transplanted cells administered to the recipient are For example, they may be autologous, syngeneic, or allogeneic cells.

[0106] As used in this application, the term "donor" refers to a person who provides cells or subsequently refers to a human or animal from which one or more cells have been isolated prior to administration of the cells. The cells may be, for example, a population of hematopoietic stem cells.

[0107] As used in this application, the term "autologous" refers to a tissue that is transplanted into a subject and originates from the same subject. In certain instances, the autologous cell or cells are returned to the subject. Before transplantation, the cells are genetically modified.

[0108] As used in this application, the term "allogeneic" refers to cells of the same species (when compared to a refers to cells that are genetically distinct from the cells (i.e., derived from a different human subject). For example, cells from one human subject may be transplanted into a different human subject. is intended to refer to the source of the cells as compared to the recipient, and does not include genetic It is not intended to refer to the state of the cells with respect to the modification.

[0109] As used in this application, the term "sample" refers to a specimen (e.g., blood) taken from a subject. fluids, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissues (e.g., placenta or dermis), pancreatic juice, chorionic villus samples, and cells).

[0110] As used in this application, the term "scFv" refers to a fragment of an antibody comprising the variable domain of a heavy chain and a variable domain of a light chain from an antibody. The term "scFv fragment" refers to a single-chain Fv antibody in which the variable domain is bound to form a single chain. is the variable region (V) of an antibody light chain 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 C The scFv fragment V contains a single polypeptide chain containing the nucleotides (e.g., DR-H3). L Area and V H Combine Regions The linker used is a peptide linker made up of amino acids that make up proteins. Alternative linkers may be used to increase the resistance of scFv fragments to proteolysis. To increase the solubility of the scFv fragment (e.g., linkers containing D-amino acids), To increase the hydrophilic linkers, such as polypeptides containing sucrine and serine residues, To improve the chemical stability (e.g., by forming intramolecular or intermolecular disulfide bonds), linkers containing stearate residues), or to reduce the immunogenicity of the scFv fragment For example, a linker containing a glycosylation site may be used. The variable regions of the scFv molecules that are produced are modified so that their amino acid sequences vary from the antibody molecules from which they are derived. Those skilled in the art will also understand that the activity of scFv can be modified in the same way as described above. In order to preserve or enhance the ability of the corresponding antibody to bind to the antigen recognized by the corresponding antibody, Nucleotide or amino acid substitutions resulting in conservative substitutions or changes in the CDRs and / or or backbone residues).

[0111] The terms "specific binding" or "specifically binding" as used in this application In this case, the antibody (or ADC) specifically recognizes and binds to the protein rather than broadly. The ability of an antibody to recognize and bind to a specific protein structure (epitope). If the antibody is specific for the topope "A," then the antibody is added to the reaction containing the labeled "A" and the antibody. In the presence of a molecule containing vertope A (or free, unlabeled A), labeled A binds to the antibody. For example, if the antibody is labeled, the amount of the corresponding unlabeled antibody is reduced. If a specific antibody can competitively dissociate it from its target, "Specifically binds." In one embodiment, the antibody specifically binds to a target (e.g., CD117). specifically binds to the target (if the antibody binds to the target -4 M or less, 10 -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less (less than 10 -12 means a number that is less than Taste, for example, 10 -13 )K D (This is the case when the above-mentioned K D An inclusive range of values, e.g., 1 0 -8 M to 10 -12 M, 10 -9 M to 10 -12 M or 10 -10 M to 10 -12 M, are also included in this application. In embodiments, the terms "specific binding to CD117" or "specific binding to CD117" as used in this application "Specifically binds" refers to the binding of antibodies or CD117 to the target protein as measured by surface plasmon resonance. 1.0×10 -7 The dissociation constant (K DIn one embodiment, , K. D (M) according to standard Bio-Layer Interferometry (BLI) In one embodiment, K off (1 / s) was measured using standard Bio-Layer Interferometry (Bio-Layer However, the antibody is a sequence-related antibody. It is understood that one can specifically bind to more than one antigen. For example, In some embodiments, antibodies can be used to identify human and non-human (e.g., murine or non-human) antibodies, e.g., CD117 or CD45. It may bind specifically to both human and nonhuman primate orthologs.

[0112] As used in this application, the terms "subject" and "patient" refer to a particular subject as described in this application. A living organism, such as a human, receiving treatment for a specific disease or condition. For example, a human patient Such patients receive genetically modified hematopoietic stem cells (which can be autologous or allogeneic). Treatment may be given before hematopoietic stem cell transplantation to promote engraftment of the tumor.

[0113] As used in this application, the phrase "substantially cleared from the blood" refers to a therapeutic agent (e.g., an anti-CD117 antibody or antigen-binding fragment thereof) to the patient. wherein the concentration of the therapeutic agent in a blood sample isolated from the patient is If the therapeutic agent is such that it cannot be detected by conventional methods (e.g., The therapeutic agent is detected above the noise threshold of the device or assay used to detect the therapeutic agent. Various techniques known in the art (e.g. , ELISA-based detection assays known in the art or described in this application, etc. ) may be used to detect antibodies or antibody fragments. Further assays that can be used to detect the fragment include, inter alia, those known in the art. These include immunoprecipitation and immunoblot assays.

[0114] As used in this application, the phrase "stem cell disorder" refers to a condition that conditions a target tissue of a subject. by selectively targeting and / or depleting the endogenous stem cell population in the target tissue. by (e.g., by retrieving endogenous hematopoietic stem cell populations or hematopoietic progenitor cells from the subject's bone marrow tissue) population) and genetically modified stem cells into the target tissue of interest. Any disease, disorder, or condition that can be treated or cured by engrafting or transplanting a The term "condition" broadly refers to any condition that may be treated using the compositions and methods described in this application. These include, but are not limited to, sickle cell anemia, thalassemia, Fanconi anemia, regenerative Aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV / AIDS, metachromatic leukodystrophy Trophy, Diamond-Blackfan anemia, and Shwachman-Diamond disease The patient conditioning and genetically modified constructs described in this application include Further diseases that can be treated using the method of blood stem cell transplantation include inherited blood disorders ( For example, sickle cell anemia) and scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease The conditioning and transplantation methods described in this application can be used to treat autoimmune diseases. Further diseases that can be treated with steroids include malignant tumors (e.g., neuroblastoma), or hematological cancers (e.g., leukemia, leukemia, and leukemia). For example, the cancer may be acute myeloid leukemia, lymphoma, or myeloma. Hemophilia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, and Diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma Using the conditioning and / or transplantation methods described in this application, Further diseases that can be treated include myelodysplastic syndromes. The subject has or is affected by a metabolic storage disorder. The subjects are glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipid a metabolic disorder selected from the group consisting of leukodystrophy, metachromatic leukodystrophy, and leukodystrophy; and any other disease or disorder that can benefit from therapy, including but not limited to: However, severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M ( IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta , storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis), and "B one Marrow Transplantation for Non-Malignant disease", ASH Education Book, 1:319 -338(2000) (This disclosure relates to the potential for treatment by hematopoietic stem cell transplantation therapy). and which relate to certain disease states, and which are incorporated by reference in their entirety into this application. may be suffering from or affected by a disease or disorder.

[0115] As used in this application, the term "transfection" refers to electroporation. , lipofection, calcium phosphate precipitation, DEAE-dextran transfection Generally, exogenous DNA is introduced into prokaryotic or eukaryotic host cells, such as yeast. This refers to any of a wide variety of techniques that may be used.

[0116] As used in this application, the term "treat" or "treatment" refers to the treatment of a disease, including the severity and / or severity of the disease symptoms. or reducing the frequency, eliminating disease symptoms and / or the underlying causes of said symptoms. , reducing the frequency or likelihood of disease symptoms and / or their underlying causes, and ameliorating or repairing damage caused directly or indirectly by lengthening, reduction of morbidity, and / or alternative therapeutic modalities any improvement in any outcome of the disease, including reduction in side effects that are a by-product of the treatment; However, as will be readily understood in the art, complete eradication of the disease is desirable. However, it is not a requirement for therapeutic intervention. Beneficial or desired clinical results include but are not limited to: Although not intended to be limiting, the ADC conditioning regimen and subsequent Enhanced hematopoietic cell engraftment in patients after transplantation of genetically modified hematopoietic stem cells Further beneficial outcomes include improved outcomes for patients requiring hematopoietic stem cell transplantation. administering a conditioning regimen followed by a hematopoietic stem cell transplant to said patient. After the treatment, the number of hematopoietic stem cells increases or the relative concentration increases. A beneficial result of the therapy described in this application also includes conditioning therapy and subsequent After hematopoietic stem cell transplantation, one or more types of cells of the hematopoietic lineage (megakaryocytes, platelets (thrombocytes)) , platelets, red blood cells, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microfilariae granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, Increased or delayed cell counts (e.g., neural killer cells, T-lymphocytes, or B-lymphocytes) A further beneficial effect may be an increase in the concentration of ATP in cancer cells. (e.g., CD117+ leukemia cells) or autoimmune cells (e.g., T-cells that cross-react with autoantigens) populations of CD117+ autoimmune lymphocytes (e.g., CD117+ T-cells expressing the receptor) that cause disease Further beneficial results may include a reduction in the amount of cells that cause As described in this application, the present invention aims to modify a target gene that causes a disease. As a result, the presence or detection of a functional protein expressed in the patient. Insofar as the methods of the present disclosure are directed to preventing a disorder, the term "preventing" is not intended to be limiting. It is understood that "does not require that the disease state be completely prevented. Rather, As used in the application, the term prevent refers to identifying a population susceptible to a disease and preventing the disease. The term "anticoagulant" refers to a compound of the present disclosure administered to a patient before the onset of the disease. However, this does not mean that the disease state will be completely avoided.

[0117] As used in this application, the terms "variant" and "derivative" are used interchangeably, and naturally occurring sequences of the compounds, peptides, proteins, or other substrates described in this application. The term "compounds" refers to synthetic and semi-synthetic analogues of the compounds described in this application. A variant or derivative of a compound, peptide, protein, or other substance is a derivative of the biological activity of the original substance. The therapeutic activity may be maintained or improved.

[0118] As used in this application, the term "vector" includes plasmids, DNA vectors, This includes nucleic acid vectors, such as vectors, RNA vectors, viruses, or other suitable replicons. The expression vectors described in the application can be used to express polynucleotide sequences as well as proteins, e.g. and / or incorporating these polynucleotide sequences into the genome of mammalian cells. The antibodies and polypeptides of the present disclosure may contain additional sequence elements that are used to embed the polypeptide. Particular vectors that can be used to express antibody fragments include those containing the gene Plasmids containing regulatory sequences, such as promoter regions and enhancers, that direct transcription are included. Other useful vectors for expressing antibodies and antibody fragments include those enhance the translation rate of the gene or to improve the stability or nuclear export of mRNA resulting from gene transcription These sequence elements include, for example, polynucleotide sequences that improve expression 5' and 3' untranslated regions and polynucleotides that allow efficient transcription of genes carried on the vector The expression vectors described in this application may also include a denylation signal site. In addition, a polynucleotide encoding a marker for selecting cells containing such a vector may be used. Examples of suitable markers include ampicillin, chloramphenicol, Resistance to antibiotics such as benzodiazepine, kanamycin, and nourseothricin Genes that encode sex include:

[0119] The term "acyl" as used herein refers to -C(=O)R, as defined herein. R is hydrogen ("aldehyde"), C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkini C3-C7 carbocyclyl, C6-C 20 Aryl, 5-10 membered heteroaryl, or 5-10 membered heteroaryl Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, and the like. acryloyl, and acryloyl.

[0120] The term "C1-C 12 "Alkyl" means a straight or refers to branched saturated hydrocarbons. Typical C1-C 12 The alkyl group is not limited to However, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl are On the other hand, the branch C1-C 12 Alkyl includes, but is not limited to, -isopropyl. -propyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl Examples include C1-C 12 The alkyl group may be unsubstituted or substituted. be.

[0121] The term "alkenyl" as used in this application means an alkyl group having at least one site of unsaturation, i.e., carbon. Carbon, sp 2 C2-C containing normal, secondary, or tertiary carbon atoms with double bonds 12 Refers to hydrocarbons. Examples include, but are not limited to: ethylene or vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, - 2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl The alkenyl group may be unsubstituted or substituted. do.

[0122] As used in this application, "alkynyl" refers to an alkyl group having at least one site of unsaturation, i.e., carbon-carbon. C2-C containing normal, secondary, or tertiary carbon atoms with an oxygen, sp triple bond 12 hydrocarbons Examples include, but are not limited to, acetylene and propargyl. An alkynyl group can be unsubstituted or substituted.

[0123] As used in this application, "aryl" refers to a C-C 20 It refers to a carbocyclic aromatic group. Examples of aryl groups are Examples include, but are not limited to, phenyl, naphthyl, and anthracenyl. The aryl group can be unsubstituted or substituted.

[0124] As used in this application, "arylalkyl" refers to an alkyl group having an alkyl group containing at least one carbon atom (typically a terminal or sp 3 carbon Acyclic alkyl groups in which one of the hydrogen atoms bonded to the alkyl group (atom) is replaced by an aryl radical Typical arylalkyl groups include, but are not limited to, , benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2- Naphthylethan-1-yl, 2-naphthylethan-1-yl, naphthobenzyl, 2-naphthophenyl The arylalkyl group may contain 6 to 20 carbon atoms. For example, the alkyl moiety of an arylalkyl group (e.g., alkanyl, alkenyl or The alkyl group (e.g., alkynyl group) has 1 to 6 carbon atoms, and the aryl moiety has 5 to 6 carbon atoms. The alkaryl group may be unsubstituted or substituted. There is.

[0125] As used in this application, "cycloalkyl" refers to a saturated carbocyclic radical, which is a monocyclic Cycloalkyl groups may be monocyclic or bicyclic. Examples of monocyclic rings include rings having 7 to 12 carbon atoms, and bicyclic rings include rings having 7 to 12 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclopentyl. hexyl, cycloheptyl, and cyclooctyl. may not be replaced or may be substituted.

[0126] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic radical, which is a monocyclic The cycloalkenyl group may be a monocyclic group having 3 to 6 carbon atoms. Examples include rings having 1 to 12 carbon atoms, or bicyclic rings having 7 to 12 carbon atoms. Examples of cycloalkenyl groups include 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, and the like. 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl. Cycloalkenyl groups are unsubstituted , or may be substituted.

[0127] As used in this application, "heteroaralkyl" refers to a heteroaralkyl group containing a carbon atom (typically a terminal or sp 3 carbon Acyclic, in which one of the hydrogen atoms bonded to the heteroaryl group is replaced by a heteroaryl radical It refers to an alkyl radical. Typical heteroarylalkyl groups include, but are not limited to: Although not limited to the above, examples include 2-benzimidazolylmethyl and 2-furylethyl. The heteroarylalkyl group contains 6 to 20 carbon atoms, for example, heteroarylalkyl. The alkyl moiety of the group (including alkanyl, alkenyl, or alkynyl groups) may be one or more and heteroaryl moieties are 5 to 14 carbon atoms and and 1 to 3 heteroatoms selected from N, O, P, and S. The heteroaryl portion of the alkyl group is a monocyclic alkyl group having from 3 to 7 ring members (2 to 6 carbon atoms). It may be a ring or may have 7 to 10 ring members (4 to 9 carbon atoms and 1 or more of N, O, P, and S). Bicyclic rings may be bicyclic rings with 1 to 3 heteroatoms selected from the group consisting of: , [5,5], [5,6], or [6,6] systems).

[0128] As used in this application, "heteroaryl" and "heterocycloalkyl" are each defined as an aromatic refers to an aromatic or non-aromatic ring system in which one or more ring atoms are heteroatoms, e.g., nitrogen The heteroaryl or heterocycloalkyl radicals are hydrogen, oxygen, and sulfur. It contains from 1 to 20 carbon atoms and from 1 to 3 heteroatoms selected from N, O, P, and S. Heteroaryl or heterocycloalkyl has 3 to 7 ring members (2 to 6 carbon atoms and N , 1 to 3 heteroatoms selected from O, P, and S), or has 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 substituents selected from N, O, P, and S) It may be a bicyclic ring with a heteroatom (e.g., bicyclic [4,5], [5,5], [5,6], or [6 ,6] system). Heteroaryl and heterocycloalkyl are unsubstituted or It may have been replaced.

[0129] Heteroaryl and heterocycloalkyl groups are described in Paquette, Leo A.; "Principles of M "Modern Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968), especially 1, 3, 4, 6, Chapters 7 and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" ( John Wiley & Sons, New York, 1950 to present), especially volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.

[0130] Examples of heteroaryl groups include, but are not limited to, pyridyl, thiazolinone ... Zolyl, tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyridinyl Imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indole benzoyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, isoxazolyl , pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-isoindolyl Ndazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalyl nyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, Phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl , phenothiazinyl, furazanil, phenoxazinyl, isochromanyl, chromanyl, i Midazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, benzotriazolyl , benzisoxazolyl, and isatinoyl.

[0131] Examples of heterocycloalkyl include, but are not limited to, dihydropyridyl. Lysyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, piperidinyl , 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydro tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroiso Quinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinunyl Examples include clidinyl, and morpholinyl.

[0132] By way of example and not limitation, carbon-bonded heteroaryls and heterocycloalkyls include 2, 3, 4, 5, or 6 position of lysine, 3, 4, 5, or 6 position of pyridazine, 2 position of pyrimidine , 4, 5, or 6-position of pyrazine, 2, 3, 5, or 6-position of pyrazine, furan, tetrahydrofuran , thiofuran, thiophene, pyrrole or tetrahydropyrrole, 2-, 3-, 4- or 5-position, 2-, 4-, or 5-position of oxazole, imidazole, or thiazole, iso-oxazole the 3-, 4-, or 5-position of a thiazole, pyrazole, or isothiazole; the 2-position of an aziridine; or 3-position, 2-, 3-, or 4-position of azetidine, 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of quinoline or at the 1, 3, 4, 5, 6, 7, or 8 position of the isoquinoline. Examples of carbon-bonded heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, and 5-pyridyl. , 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2- Pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrimidinyl Razinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl Examples include:

[0133] By way of example and not limitation, nitrogen-linked heteroaryls and heterocycloalkyls include Diridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole , imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyra Zoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indole The 1st position of the azole, the 2nd position of the isoindole or isoindoline, the 4th position of the morpholine, and the carboxyl group The bond is typically at the 9-position of a beta-carboline or beta-benzol. Heterocylic rings include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, and 1-imidazolyl. Examples include aryl, 1-pyrazolyl, and 1-piperidinyl.

[0134] As used in this application and as described above, alkyl, alkenyl, alkynyl, aryl, and aryl are Applies to any of arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, etc. "Substituted" means that one or more hydrogen atoms are each independently replaced with a substituent. Unless otherwise constrained by the definition of the individual substituents, the aforementioned chemical Examples of such moieties include "alkyl," "alkylene," "heteroalkyl," and "heteroalkynyl." "Alkenyl", "alkenylene", "heteroalkenyl", "heteroalkenylene", "Alkynyl", "alkynylene", "heteroalkynyl", "heteroalkynylene", "cycloalkynyl" "Alkyl", "cycloalkylene", "heterocycloalkyl", "heterocycloalkylene", " The "aryl", "arylene", "heteroaryl", and "heteroarylene" groups are optionally Exemplary substituents include, but are not limited to, -X, -R, -O H, -OR, -SH, -SR, NH2, -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 F, Cl, Br, and I. and each R is independently selected in each occurrence from C-C 12 Alkyl, C6- C 20 Aryl, C3-C 14 Heterocycloalkyl or heteroaryl, protecting groups and prodrugs The group is independently selected in each instance from the group consisting of "optionally substituted" and "substituted" moieties. In any case where it is stated that "the It may be independently substituted with one or more of the above substituents.

[0135] The naming convention for a given radical may include naming it as either a mono-radical or a di-radical, depending on the context. For example, it is important to understand that a substituent may have two or more substituents attached to the rest of the molecule. If two bonding positions are required, the substituent is understood to be a di-radical. For example, substituents that are identified as alkyls requiring two bond positions include -CH2-, -C Di-radicals such as -H2CH2-, -CH2CH(CH3)CH2-, etc. Other radical naming conventions are The radical may be "alkylene," "alkenylene," "arylene," or "heterocycloalkane." It clearly shows that it is a di-radical such as "chiren".

[0136] The substituent is shown as a di-radical (i.e., the positions of both bonds to the rest of the molecule are Whenever a group has a substituent, said substituents may be attached in any orientation unless otherwise specified. It should be understood that this is possible.

[0137] "Isomerism" means two compounds that have the same molecular formula but differ in the arrangement of bonds of their atoms or their The spatial arrangement of atoms is called an isomer. Stereoisomers that are not mirror images of each other are called "diastereomers", and Stereoisomers that are mirror images and cannot be superimposed on each other are called "enantiomers." and are sometimes called "optical isomers."

[0138] A carbon atom bonded to four non-identical substituents is called a "chiral center." means a compound having at least one chiral center. The compounds may be presented as individual diastereomers or as "diastereomeric mixtures." It may exist as a mixture of diastereomers called a "diastereomeric mixture." When chiral centers are present, stereoisomers are distinguished by the absolute configuration (R or S) of the chiral centers. Absolute configuration refers to the spatial arrangement of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are arranged according to the Cahn, Ingold and Prelog arrangement rules. Therefore, they are ranked. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; erra ta 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116). A mixture containing equal amounts of individual enantiomeric forms of opposite chirality. The substance is called a "racemic mixture."

[0139] The compounds disclosed and claimed herein may contain one or more asymmetric centers. and that different diastereomers and / or enantiomers of each compound exist. In this specification and claims, any reference to a compound is Unless otherwise specified, all enantiomers, diastereomers and mixtures thereof are included. Furthermore, in this specification and claims, any compound All references refer to individual enantiomers as well as to the enantiomers listed above unless otherwise specified. This means that the structure of a compound is not limited to a specific enzyme, and includes any mixture, racemic or otherwise, of the compound. When depicted as an antiomer, the disclosure of this application is limited to that particular enantiomer. It should be understood that the enantiomers, optical isomers, and the like of each structural formula of the present disclosure are not intended to be limiting. , and diastereomers are contemplated in this application. Although the formulas represent specific isomers in some cases for convenience, the present disclosure does not include geometric isomers. , including all isomers such as optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc. It is understood that not all isomers have the same level of activity. Compounds according to the present disclosure may exist in different tautomeric forms. Unless otherwise specified, all tautomeric forms are intended to be included. When depicted as a tautomer, the disclosure of this application is not limited to that particular tautomer. should be understood.

[0140] The compounds of any formula described in this application may be used in conjunction with the compounds themselves, as well as with the compounds themselves, if applicable. For example, salts include those formed by combining an anion and a positive ion on a compound of the present disclosure, and solvates thereof. Suitable anions include: , chloride, bromide, iodide, sulfate, bisulfate, sulfamate fermate), nitrate, phosphate, citrate, methanesulfonate, trifluoroacetic acid Salt, glutamate, glucuronate, glutarate, malate, maleate, succinate Citrate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalene sulfonate The term "pharmaceutically acceptable" includes phosphates and acetates (e.g., trifluoroacetates). "Anion" refers to a suitable anion for forming a pharmaceutically acceptable salt. A salt is formed between a cation and a negatively charged group (e.g., carboxylate) on a compound of the present disclosure. Suitable cations include sodium ions, potassium ions, ions, magnesium ions, calcium ions, and tetramethylammonium ions Some suitable substituted ammonium cations include ammonium ions. Examples of ions include those derived from: ethylamine, diethylamine amine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, Ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzene amino acids such as thiamin, choline, meglumine, and tromethamine, lysine, and arginine Acids. The compounds of the present disclosure also include salts containing a quaternary nitrogen atom.

[0141] Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: Examples include: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, and nitrous acid. Acid, phosphoric and phosphorous. Examples of suitable organic anions include, but are not limited to: Although not specifically mentioned, some of the compounds are derived from the following organic acids: 2-acetoxybenzoates, Benzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid Cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoside Propionic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxy Sinaphthalene carboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, Pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid , salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluene sulfone Examples of suitable polymeric organic anions include, but are not limited to: , and those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0142] Additionally, the compounds of the present disclosure (e.g., salts of the compounds) may be in hydrated or non-hydrated forms. It may exist in the form of a hydrate (water) or as a solvate with other solvent molecules. Typical examples include monohydrates, dihydrates, etc. Non-limiting examples of solvates include: Examples of solvates include ethanol solvates and acetone solvates. Solvent-added forms containing either stoichiometric or non-stoichiometric amounts of solvent Some compounds trap a fixed molar ratio of solvent molecules in the crystalline solid phase. When the solvent is water, it tends to form a solvate. When the solvent is an alcohol, the solvate formed is a hydrate; and when the solvent is an alcohol, the solvate formed is a hydrate. A hydrate is a compound formed by combining one molecule of a substance with one or more molecules of water. Hydrates are formed by the reaction of water with the water molecule in the form of H2O. , monohydrate, dihydrate, trihydrate, etc.

[0143] Furthermore, the compounds represented by the formulas disclosed in this application or salts thereof may have crystalline polymorphism. Any crystalline form, mixture of crystalline forms, or anhydrous or hydrated forms thereof may be used in the present invention. Please note that this is within the scope of the disclosure.

[0144] As used in this application, the term "amatoxin" refers to the compound found in Amanita phalloides mushrooms. Amatoxin family member peptide or variant thereof produced by or derivatives thereof (e.g., variants thereof capable of inhibiting RNA polymerase II activity) Suitable amatoxins and derivatives thereof are further described below in this application. As described herein, amatoxins may be, for example, a linker moiety (L): may be conjugated to an antibody, or antigen-binding fragment thereof, via Thus, a conjugate (i.e., an ADC) is formed. Methods of conjugation, and linkers useful in such processes, are described below. and known in the art.

[0145] B. Stem Cell Gene Therapy Methods The disclosed methods involve administering a genetically modified gene to a patient suffering from a condition resulting from a defective gene (e.g., a mutation). Broadly, the method involves administering a population of modified stem cells to living cells (e.g., stem cells). In particular, this application relates to stem cell gene therapy, which involves modifying the genome of a living cell (e.g., a human cell) for therapeutic purposes. As described in, the therapeutic effect may be achieved by correcting the defective gene. For example, hematopoietic stem cells (HSCs) can be transplanted into patients suffering from disorders caused by defective genes (e.g., The cells were extracted from patients with sickle cell anemia (who have the HBB gene) and CD34-expressing cells (CD34+ The isolated cells are purified by selecting for the desired cell type, as known in the art. The genome may be treated ex vivo using methods known in the art, e.g., to remove defects. By editing a target gene to make it functional, Such modified stem cells may then be returned to the patient. The transplanted stem cells colonize the patient's bone marrow and mature into normally functioning proteins; and The cells to be produced are replicated and produced, thereby solving the problem.

[0146] Isolating stem cells from a source and further processing the cells ex vivo (e.g., expanding Methods for (breeding, propagating, and modifying genomes) are known in the art, and In some embodiments, the stem cells are available to the mammal to which they are administered. In some embodiments, the stem cells are allogeneic, whereas the stem cells are allogeneic. In relation to mammals, it is the self.

[0147] In some embodiments, the stem cells are isolated from bone marrow. In some embodiments, the stem cells are isolated from peripheral blood (e.g., mobilized peripheral blood). In some embodiments, the mobilized peripheral blood is isolated from a subject to which G-CSF has been administered. In embodiments, the mobilized peripheral blood is treated with a mobilization agent other than G-CSF (e.g., Plerixa). For [Plerixafor 登録商標 (AMD3100)]) from subjects administered the In embodiments, the stem cells are isolated from umbilical cord blood.

[0148] In some embodiments, the isolated stem cells comprise or consist of CD34+ cells. In some embodiments, the cells are substantially free of CD34- cells. In some embodiments, the cells comprise or consist of CD34+ / CD90+ stem cells. In some embodiments, the cells comprise or consist of CD34+ / CD90- cells. The cells are a population comprising one or more cell types described above or in this application.

[0149] In some embodiments, any one or more of the known genetically modified stem cells are used in the method. For example, Strimvelis 登録商標 (encoding the human ADA cDNA sequence) Autologous CD34+ enrichment containing CD34+ cells transduced with retroviral vectors (cell fractions).

[0150] The genetically modified HSCs described in this application can be used in genetically modified stem cell therapy, or It may be used in stem cell gene therapy (in which cells are transfected before being introduced into the patient) In vitro, cells can be gene-edited (e.g., by CRISPR / Cas systems or by viral Thus, the methods described in this application are The genetically modified stem cells are then used to generate the stem cells, because they contain the modified or corrected genes. and / or contain exogenous genes, and therefore may be used in gene therapy methods. In addition, all or most of the progeny derived from the genetically modified stem cells described in this application are The genetically modified stem cells are then transformed into progeny that contain the altered or corrected gene. Thus, the modified hematopoietic cells can be used in methods of gene therapy, e.g. Conditions such as, but not limited to, genetic disorders, cancer, and certain viral infections It may be used to treat afflicted mammalian subjects (eg, human subjects).

[0151] As described herein, genetically modified stem cells can be used to treat ADCs and The patient is conditioned using the method and administered (i.e., transplanted) to achieve engraftment. Ensure or improve

[0152] 1. Methods for modifying stem cell genomes Various methods for editing the genome of a cell (e.g., a stem cell) are available (e.g., as described in this application). Stem cells that can be manipulated include those known in the art (see, for example, the references cited above). Stem cells derived from an individual's isolated stem cells or stem cell lineages established from said isolated stem cells. These stem cells have one or more nucleic acid mutations. using any suitable genetic engineering method known in the art and described herein, The genome of said stem cells is edited or altered. In certain embodiments, the genome of said stem cells is Genetic modification of the stem cells may correct nucleic acid mutations in the stem cells. In some embodiments, the genetic modification of the genome of the stem cells involves introducing an exogenous gene into the stem cells. In certain embodiments, nucleic acid manipulation reagents (e.g., gene editing systems) may be introduced. In some embodiments, genetic material (a component of the stem cell) is introduced into the stem cell. exogenous genes to be expressed), and / or nucleic acid manipulation reagents (e.g., The components of the editing system are delivered using viral vectors (e.g., retroviral vectors). Lus, adenovirus, AAV, helper-dependent adenovirus system, hybrid Adenovirus system, herpes simplex, pox virus, lentil virus viruses, Epstein-Barr virus), and non-viral systems (e.g., physical Various systems [intact DNA, DNA bombardment, electroporation, hybridization] hydrodynamic, ultrasound, magnetfection] ), and chemical systems (cationic lipids, various cationic polymers, and In some embodiments, the virus may be a lipid polymer. The vector is a lentivirus. Subsequently, the stem cells are transformed with nucleic acid manipulation reagents. Such reagents correct nucleic acid mutations in the target cells, resulting in the formation of engineered stem cells. The target polynucleotide sequence is expressed in a target cell (e.g., a stem cell) and / or a target It works by allowing the DNA sequence (DNA sequence) to be efficiently and accurately modified. and typically a nucleic acid-guide that recognizes a nucleic acid sequence in the target cell. Endonuclease (nucleic acid-guided endonuclease) (e.g., RNases such as Cas9) A-guide endonuclease, or DNA-guide endonuclease) -Directly modifying polypeptides.

[0153] Site-directed modifying polypeptides for use in the compositions and methods disclosed in the present application The polypeptide itself, or a related molecule, is a specific nucleic acid sequence or a series of similar in that it recognizes and targets the target sequence In some embodiments, the site-directed modifying polypeptide ( or related molecules) may contain conserved bases or recognizes sequences that contain motifs and have similar sequences.

[0154] In certain embodiments, the site-directed modifying polypeptide is a polypeptide that is capable of modifying its target sequence. Modifying a polynucleotide at a specific position (or positions) outside the sequence (i.e., modification site) The modification site(s) to be modified by the specific site-directed modifying polypeptide. The site of the mutation is also generally specific to a particular sequence or series of similar sequences. In some embodiments, the site-directed modifying polypeptide comprises a Modification of sequences with similar sequences, including conserved bases or motifs, which may be degenerate In another embodiment, the site-directed modifying polypeptide is Modifying a sequence within a specific position relative to the sequence, e.g., site-directed modification The polypeptide may be a specific number of nucleic acids upstream or downstream from the target sequence. The sequence in may be modified.

[0155] As used in this application, the term "modification" refers to a site-directed modification of a polypeptide. "Modification" or "alteration" refers to the modification of at least one nucleotide at the modification site. Any insertion, deletion, substitution, or chemical modification of the leucine or adjacent to the target site. This means that the expression of the gene corresponding to at least one of the modified sites is altered. Nucleotide substitutions are similar to those in the domains of cytidine deminase or adenine deminase. This may be the result of recruitment of base-editing domains (e.g., See, for example, Eid et al. (2018) Biochem J475(11):1955-1964, which is incorporated herein by reference in its entirety. incorporated into the application).

[0156] The alteration of expression of a gene adjacent to the target site is determined by the promoter of said gene. Recruitment of transcriptional activation or repression domains to the region; The gene expression of neighboring genes is altered without changing the DNA sequence. DNA or histone transcription factors to alter histone and / or chromosome structure Covalently modifying proteins, epigenetic modification domains mediate recruitment The term "modification" or "alteration" n)" are site-directed nucleic acid sequences that allow for the detection of specific nucleic acid sequences (e.g., disease-associated sequences). The polypeptide may be conjugated to a target molecule, such as a polypeptide that specifically modifies the target gene, or a related molecule (e.g., gRNA). and recruiting a detectable label to the target site.

[0157] In some embodiments, the site-directed modifying polypeptide is a nuclease or a variant thereof, and a drug comprising said nuclease or a variant thereof. As used herein, a "nuclease" refers to an enzyme that binds phosphodiesterase (PNP) to the backbone of a polynucleotide chain. It refers to an enzyme that cleaves diester bonds. The enzyme may have endonuclease and / or exonuclease activity. Exonucleases cleave nucleotides one by one from the end of a polynucleotide chain. Endonucleases break down the phosphodiester bonds at both ends of a polynucleotide chain. By cleaving the phosphodiester bonds within the polynucleotide chain, The nuclease cleaves the RNA polynucleotide chain (i.e., ribonucleotide chain). enzymes that cleave DNA polynucleotide chains (i.e., deoxyribonucleases) and / or DNA polynucleotide chains (i.e., deoxyribonucleases) This may happen.

[0158] Nucleases cleave polynucleotide chains, creating cleavage sites. When used herein, the term "cleave" refers to the breaking of a phosphodiester bond in the backbone of a polynucleotide chain. Nuclease cleavage as disclosed in this application refers to single-strand cleavage or In some embodiments, the double-strand break in DNA can be a 2 This is achieved through cleavage by two nucleases, where each nuclease cleaves the DNA. The cleavage by the nuclease can be performed at blunt ends or staggered ends. This can result in a catastrophic event.

[0159] Non-limiting examples of nucleases suitable for the compositions and methods disclosed in the present application include: Meganucleases such as taming endonucleases; Type IIS endonucleases ( Restriction endonucleases, such as FokI; zinc finger nucleases (zinc finger nuclease; transcription activator-like effector nuclease tactic-like effector nucleases (TALENs), and nucleic acid-guided nucleases (nucleic acid cid-guided nucleases) (e.g., RNA-guided endonucleases, DNA-guided endonucleases) nucleases, or DNA / RNA-guided endonucleases).

[0160] As used in this application, a "meganuclease" refers to a nuclease that can bind to a target molecule greater than 12 base pairs in length. Meganuclease refers to an endonuclease that binds to DNA at the same sequence as the heterodimer. As a result, the meganucleotides bind to double-stranded DNA. Homing endonucleases (e.g., , LAGLIDADG (SEQ ID NO: 321) family of endonucleases [this amino

[0033] or variants thereof).

[0161] As used in this application, "zinc finger nuclease" or "ZFN" refers to an enzyme. xonucleases or endonucleases (e.g., restriction endonucleases or meganucleases) Zinc finger DNA binding domains fused to nuclease domains derived from nucleases (e.g., nucleases) This refers to a chimeric protein containing a zinc finger DNA-binding domain. The ATP-binding protein binds to zinc ions, which act to stabilize the specific structure.

[0162] As used in this application, "transcription activator-like effector nuclease" or "TA" "LEN" refers to an exonuclease or endonuclease (e.g., a restriction endonuclease) Multiple TAL domains fused to a nuclease domain from a nuclease (e.g., a nuclease or meganuclease) This refers to a chimeric protein that contains a DNA-binding domain and a main repeat. The in-repeat sequence is found in the Proteobacteria Xanthomonas TAL domain lipids may be derived from the TALE family of proteins from the genus TAL. The sequence is a hypervariable repeat variable diresidue (RVD). The RVD is a sequence of 33-34 amino acids, with the 12th and 13th amino acids of The TAL domain repeats confer specificity to target sequence binding. To generate variant TALENs with sequence specificity, rational or experimental means are used. (e.g., Boch et al. (2009) Science326(5959):1509-151 2 and Moscou and Bogdanove (2009) Science 326(5959):1501, which are The entire contents of which are incorporated by reference into this application. Two DNA target sequences are required, flanked by a suitable spacer region, where each DNA target In some embodiments, the TALEN is a TALEN monomer. Compact TALEN (which expresses homing endonucleases in any direction) (e.g., I-TevI, MmeI, EndA, End1, I-BasI, I-TevII, I-TevIII, I-TwoI, MspI, One or more TAL domain lipids fused to any part of the TAL domains (MvaI, NucA, and NucM). (Refers to endonucleases that contain a DNA-binding domain and have a compact TALENs do not require dimerization for DNA processing activity (hence, single This is advantageous in that it requires only one target site.

[0163] As used herein, a "nucleic acid-guided nuclease" refers to a nucleic acid-guided nuclease. The guide nucleic acid (i.e., guide RNA or gRNA, guide DNA or gDNA, or guide Complementarity (full or partial) between the target sequence and the target DNA / RNA hybrid It refers to a nuclease that targets a specific target sequence based on the guide RNA. The binding between the nuclease and the target sequence serves to concentrate the nuclease in the vicinity of the target sequence. Suitable nucleic acid-guided nucleases for the compositions and methods disclosed herein include, but are not limited to: Typical examples include naturally occurring clusters from prokaryotes (e.g., bacteria, archaea). Clustered Regularly Interspaced Short Palindromic Repeats (Clustered R typically Interspaced Short Palindromic Repeats (CRISPR)-associated(Cas) CRISPR sequences found in prokaryotes include: , a sequence derived from a fragment of a polynucleotide from an invading virus, and Recognize similar viruses during subsequent infections and cleave viral polynucleotides CRISPR-associated (Cas) polypeptides that function as RNA-guided nucleases to cleave As used in this application, "CRIS" refers to a sequence that cleaves a viral polynucleotide via a nucleic acid sequence. "PR-associated polypeptides" or "Cas polypeptides" refer to polypeptides that are present in naturally occurring CRISPR systems. Refers to a naturally occurring polypeptide found within the vicinity of a CRISPR sequence. The polypeptide functions as an RNA-guided nuclease.

[0164] There are at least two classes of naturally occurring CRISPR systems: class 1 and class 2. Generally, the nucleic acid-guided nucleases of the compositions and methods disclosed herein are Class 2 CRISPR systems are Cas polypeptides or variants thereof (Class 2 CRISPR systems are nuclear Class 1 CRIs contain a single polypeptide with acid-guided nuclease activity, whereas class 1 CRIs contain a single polypeptide with acid-guided nuclease activity. The SPR system hypothesizes that a protein complex is required for nuclease activity. There are at least three known types of Class 2 CRISPR systems: Type II, Type V, and Type VI, among which there are multiple subtypes (subtype II -A, II-B, II-C, VA, VB, VC, VI-A, VI-B, and VI-C, and other undefined or assumed sub-series. In general, Type II and Type VB systems require a In contrast, types VA and VI require tracrRNA in addition to RNA for activity. All known type II and type V RNA-guided nucleotides require only cr-RNA. While all known type VI RNA-guided ATPases target double-stranded DNA, The nuclease targets single-stranded RNA. The cytoplasmic nuclease is referred to as Cas9 in this application and in the literature. The nucleic acid-guided nuclease of the compositions and methods disclosed in the application is a type II Cas9 protein. Type V Cas proteins or their variants function as RNA-guided nucleases. The polypeptide is cleaved by trac In this application and in the literature, the RNA-guided CRISPR system of type VA is The nuclease is called Cpf1; it is the RNA-guided nuclease of the type VB CRISPR system. The RNA-guided nuclease of the type VC CRISPR system is called Cas12C or C2C3. The RNA-guided nuclease of type VIA CRISPR systems is called C2C2 or Cas13A1. The RNA-guided nuclease of the type VIB CRISPR system is called Cas13B; The RNA-guided nuclease in this CRISPR system is called Cas13A2. In some embodiments, the nucleic acid-guided nucleases of the compositions and methods disclosed herein are Type V A Cpf1 protein or its variants that function as nucleic acid-guiding nucleases. Naturally occurring Cas polypeptides and variants thereof are known in the art. Examples include, but are not limited to, Streptococcus pyogenesCas9, Staphylococcus ccus aureus Cas9, Streptococcus thermophilus Cas9, Francisella novicida Cpf1, and Shmakov et al. (2017) Nat Rev Microbiol15(3):169-182; Makarova et al. (2015) N at Rev Microbiol 13(11):722-736; and U.S. Pat. No. 9,790,490 (each of which is a translation of a separate document). Class 2, Type V CRIS PR nucleases include Cas12 and any subtype of Cas12, such as Cas12a and Cas12b. , Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, and Cas12i. The type VI CRISPR nucleases (including Cas13) of the two genes were used to cleave the RNA target sequence. It may be used for this purpose.

[0165] The nucleic acid-guided nucleases of the compositions and methods disclosed herein are naturally occurring A nucleic acid-guided nuclease (e.g., S. pyogenes Cas9) or a variant thereof Variant nucleic acid-guided nucleases may be engineered. or have properties that alter or modify, for example, the activity of one or more nuclease domains. Heterologous domains (e.g., transcription activation domains, epigenetic modification domains) that confer fusing the nucleic acid-guided nuclease to a detectable label; amino acid substitutions that modify the stability of the nuclease or that modify the specificity of the nuclease; They may be naturally occurring variants, including deletions or additions.

[0166] In some embodiments, the nucleic acid-guided nuclease is capable of targeting the target protein in the intracellular microenvironment. Contains one or more mutations to improve specificity and / or stability for the target site For example, if the protein is Cas9 (e.g., SpCas9) or a modified Cas9, the Rec2 domain It may be beneficial to delete any or all (inclusive) of residues N175 to R307 of the nucleotide sequence. Smaller or lower molecular weight forms of nucleases have been found to be more effective. In some embodiments, the nuclease is a naturally occurring form of a nuclease. For example, the protein may be a Cas9 or modified If the Cas9 is a mutated one, mutations at C80 or C574 (or their homologs, In modified proteins with indels, this can be beneficial. Preferred substitutions include C80A, C80L, C80I, C80V, C80K, C574E, C574D, C574N, and C 574Q (in any combination). To reduce intracellular protein binding and / or increase target site specificity In addition, or alternatively, substitutions may be included to reduce the off-target toxicity of the composition. Substitutions may be included to reduce the relevance.

[0167] The nucleic acid-guided nuclease may comprise a guide nucleic acid (e.g., a guide RNA (gRNA), a guide DNA (gDNA) or a guide RNA (gRNA)). A (gDNA) through interactions with the nucleic acid guide The nuclease binds to the guide nucleic acid through non-covalent interactions and in this way The polynucleotide-targeting nucleic acid is a nucleic acid having a sequence corresponding to the target sequence. The complex has target specificity by including a nucleotide sequence complementary to The complex is then bound to or fused to a nucleic acid-guided nuclease. or otherwise conjugated to a domain or label. In other words, the nucleic acid-guided nuclease has site-specific activity. The enzyme binds a protein-binding segment of a polynucleotide-targeting guide nucleic acid For interaction, target polynucleotide sequences (e.g., in chromosomal nucleic acids) target sequences in extrachromosomal nucleic acids, e.g., episomal nucleic acids , minicircle; target sequence in mitochondrial nucleic acid; chloroplast nucleic acid The target sequence is guided by the target sequence in the plasmid.

[0168] Thus, the guide nucleic acid comprises a "polynucleotide-targeting segment" and The "segment" includes two segments: a "polypeptide-binding segment" and a "polypeptide-binding segment." A segment / section / region of a molecule (e.g., a continuous stretch of nucleotides in RNA) A segment may also be defined as a region of a molecule, such that the segment may comprise multiple regions of the molecule. , may refer to a region / section of a complex. For example, in some cases, The polypeptide-binding segment of the nucleic acid target (described below) is and thus the polypeptide-binding segment comprises a region of the nucleic acid molecule. In other cases, the polypeptide-binding segment (see below) of the DNA-targeting nucleic acid may be It comprises two separate molecules hybridized along complementary regions.

[0169] The polynucleotide-targeting segment (or "polynucleotide-targeting segment") A "guiding sequence" or "guide sequence" is a sequence that aligns (completely or a nucleotide sequence that is (partially) complementary to the target DNA sequence (e.g., the complementary sequence of the target DNA sequence) The polypeptide-binding segment (or "polypeptide-binding sequence") ) interacts with nucleic acid-guided nucleases. The site-specific cleavage or modification of the target DNA by the enzyme can be achieved by (i) cleaving the polynucleoside of the nucleic acid. (ii) base pair complementarity between the targeting sequence and the target DNA; and A short motif (protospacer adjacent motif) in the target DNA The location of the protein occurs at a location determined by both the protein and the protein motif (called the protein-protein motif (PAM)).

[0170] The protospacer adjacent motifs can be of different lengths and can be targeted to However, the PAM generally has a distance of 100 nm from the target sequence. From the target sequence, about 1 to about 10 nucleotides (e.g., from the target sequence, about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides, etc. The PAM may be 5' or 3' of the target sequence. The PAM is a consensus sequence of about 3-4 nucleotides, but in certain embodiments, is 2 nucleotides in length. , 3, 4, 5, 6, 7, 8, 9 or more nucleotides. Methods for identifying preferred PAM sequences or consensus sequences for ATPases are known in the art. Known in, and including but not limited to, Karvelis et al. (2015) Gen PAM depletion assay (PAM depletion assay) as described in [SEQ ID NO: 1]. y), or as disclosed in Pattanayak et al. (2013) Nat Biotechnol 31(9):839-43, and assays for detecting chromatin-binding proteins (each of which is incorporated by reference in its entirety).

[0171] The polynucleotide-targeting sequence (i.e., guide sequence) is a sequence that targets a desired target. The guide sequence is a nucleotide sequence that hybridizes directly to the target sequence. The target sequence can be modified to be fully or partially complementary to the target sequence. In embodiments, the guide sequence is from about 8 nucleotides to about 30 nucleotides or more. For example, the guide sequence may be about 8, about 9, about 10, about 11, about 12, About 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about It may be 26, about 27, about 28, about 29, about 30, or more nucleotides. In this embodiment, the guide sequence is about 10 to about 26 nucleotides in length, or about In certain embodiments, the guide sequence is about 30 nucleotides in length. In some embodiments, a suitable alignment algorithm is between a guide sequence and its corresponding target sequence when optimally aligned using The degree of complementarity is about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84% ,approximately 85%,approximately 86%,approximately 87%,approximately 88%,approximately 89%,approximately 90%,approximately 91%,approximately 92%,approximately 93%,approximately 94%,approximately 95%, In certain embodiments, the guide alignment is about 96%, about 97%, about 98%, about 99%, or more. The sequence may be, for example, but not limited to, mFold (Zuker and Stiegler (1981) Nucleic Acids Res. 9:133-148) and RNAfold (Gruber et al. (2008) Cell 106(1):23- Any suitable polynucleotide known in the art, such as Predictable using a polynucleotide folding algorithm It does not take the secondary structure.

[0172] Also, in some embodiments, the guide nucleic acid may be composed of two separate nucleic acid molecules (hereinafter, "active "targeter-nucleic acid" and "targeter-nucleic acid" and In the application, this is referred to as a "dual-molecular guide nucleic acid" or "two-molecular guide nucleic acid." The subject guide nucleic acid is a single nucleic acid molecule (single polynucleotide), and Herein, we refer to these as "single-molecule guide nucleic acids," "single-guide nucleic acids," or "sgNAs." "Guide Nucleic Acid" or "gNA" is a generic term that includes both dual-molecule guide nucleic acids and single-molecule guide nucleic acids. In those embodiments where the guide nucleic acid is RNA, the gRNA refers to both the guide nucleic acid (i.e., sgNA) and the guide nucleic acid (i.e., sgNA). A can be a double-guide RNA or a single-guide RNA. In those embodiments in which the nucleic acid is DNA, the gDNA may be a dual-guide DNA or a single-guide DNA. This may be the case.

[0173] Exemplary bi-molecular guide nucleic acids include crRNA-like ("CRISPR RNA") or "targeter RNA" ("TAR"). "crRNA" or "crRNA repeat" molecules, and the corresponding tracrRNA-like ("trans-acting CRISPR RNA," or "activating CRISPR RNA") crR includes a "activator-RNA" or "tracrRNA" or "tracrRNA" molecule. The NA-like molecule (targeter RNA) binds to the polynucleotide-targeting segment of the guide RNA. a dsRNA duplex (dsRNA) of the polypeptide-binding segment of the guide RNA; A stretch of nucleotides that forms one half of a duplex (a "duplex-forming segment"). lex-forming segment) (in this application, CRISPR repeat sequence) This includes both the

[0174] The term "activator-nucleic acid" or "activator-NA" is used in the present application to refer to a dual-molecular guide nucleic acid. The term "targeter nucleic acid" or "targeting nucleic acid" is used to refer to a tracrRNA-like molecule. "Getter-NA" is used in this application to refer to a crRNA-like molecule of a dual-molecular guide nucleic acid. The term "duplex-forming segment" is used in this application. , used to refer to the nucleotide extension of an activator-NA or targeter-NA molecule and hybridizes to the nucleotide stretch of the corresponding activator-NA or targeter-NA molecule. By activating the dsRNA, it contributes to the formation of dsRNA duplexes. The factor-NA contains a duplex-forming segment complementary to the duplex-forming segment of the corresponding target-NA. Thus, the activator-NA contains a duplex-forming segment, while The targeter-NA is a duplex-forming segment and a DNA-targeting segment of the guide nucleic acid. Thus, the subject dual-molecular guide nucleic acid contains both the corresponding It may contain an activator-NA and a targeter-NA pair.

[0175] The activator-NA has a region that is sufficiently complementary to hybridize with the activator-NA. A CRISPR repeat sequence comprising a nucleotide sequence comprising a region (the polypeptide of the guide nucleic acid) In various embodiments, the CRISPR repeat comprises a fragment of the CRISPR fragment (the other half of the CRISPR fragment). The target sequence may contain from about 8 nucleotides to about 30 nucleotides or more. For example, the CRISPR repeat sequence can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, About 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about It may be 28, about 29, about 30, or more nucleotides. When optimally aligned using a preferred alignment algorithm, CRISPR Complementarity between a repeat sequence and the antirepeat region of its corresponding tracr sequence The degree of sexuality is approximately 50%, approximately 60%, approximately 70%, approximately 75%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%. ,approximately 86%,approximately 87%,approximately 88%,approximately 89%,approximately 90%,approximately 91%,approximately 92%,approximately 93%,approximately 94%,approximately 95%,approximately 96%, It may be about 97%, about 98%, about 99%, or more.

[0176] The corresponding tracrRNA-like molecule (i.e., activator-NA) is a polypeptide of the guide nucleic acid. -a stretch of nucleotides that forms the other half of the double-stranded duplex of the binding segment (duplex-forming segment). In other words, the stretch of nucleotides in the crRNA-like molecule ( i.e., CRISPR repeat sequences) are stretches of nucleotides (i.e., anti-ribonucleotides) in tracrRNA-like molecules. and hybridizes to the antirepeat sequence, The crRNA-like molecule forms a double-stranded duplex of the polypeptide-binding domain of the guide nucleic acid. provides a single-stranded DNA-targeting segment. Thus, the crRNA-like molecule and The tracrRNA-like molecules (as a matched pair) hybridize to form the guide nucleic acid. The exact sequence of a given crRNA or tracrRNA molecule is crucial for determining the identity of the CRISPR system and its RNA molecules. The target double-molecule guide RNA is a gene that can be used in combination with any corresponding crRNA and tracrRNA. May contain pairs of A.

[0177] trans-activating-like CRISPR RNA or tracrRNA-like The molecule (also referred to in this application as "activator-NA") is referred to in this application as an antirepeater. The CRISPR repeat sequence of the crRNA is sufficiently complementary to hybridize with the CRISPR repeat sequence, called the at region. In some embodiments, the nucleotide sequence comprises a region having a tracrRNA-like sequence. The gene may further comprise a region with secondary structure (e.g., a stem-loop) or a corresponding crR. In certain embodiments, the CRISPR repeat forms a secondary structure when hybridized with the NA. The region of the tracrRNA-like molecule that is fully or partially complementary to the target sequence is The 5' end of the tracrRNA-like molecule and the 3' end of the tracrRNA-like molecule contain secondary structure. The region is generally found adjacent to an antirepeat sequence, e.g., a nexus It contains several hairpin structures, such as the nexus hairpin. The hairpin shares the motif UNANNC, which is found in many nexus hairpins in tracrRNA. They often contain conserved nucleotide sequences within the bases of the hairpin stem. The 3' end of acrRNA often contains terminal hairpins, the structure and number of which vary. , often GC-rich, Rho-independent transcription termination hairpins (followed by a string of U's at the 3' end) For example, Briner et al. (2014) Molecular Cell 56:333-339, Briner and Barr angou (2016) Cold Spring Harb Protoc; doi: 10.1101 / pdb.top090902, and US publication no. See, e.g., US Pat. No. 2017 / 0275648, each of which is incorporated herein by reference in its entirety. (included).

[0178] In various embodiments, the CRISPR repeat sequence is fully or partially complementary to the CRISPR repeat sequence. The antirepeat region of a tracrRNA-like molecule can range from about 8 nucleotides to about 30 nucleotides. nucleotides or more. For example, tracrRNA-like anti-repeat sequences The base pair region between the sequence and the CRISPR repeat sequence is about 8, about 9, about 10, about 11, about 12, or about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26 , about 27, about 28, about 29, about 30, or more nucleotides. In embodiments, a CRISPR repeat sequence and its corresponding tracrRNA-like antirepeat at) The degree of complementarity between the sequences is optimally determined using a suitable alignment algorithm. When aligned, approximately 50%, approximately 60%, approximately 70%, approximately 75%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, Approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 9 5%, about 96%, about 97%, about 98%, about 99%, or more.

[0179] In various embodiments, the entire tracrRNA-like molecule comprises from about 60 nucleotides to more than about 140 nucleotides. For example, the tracrRNA-like molecule may be about 60, about 6 5, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125 In certain embodiments, the sequence is about 130, about 135, about 140, or more nucleotides. The tracrRNA-like molecule may, for example, have a length of about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 1 7, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, and It is about 80 to about 100 nucleotides in length, such as about 100 nucleotides.

[0180] The subject single-molecule guide nucleic acid (i.e., sgNA) comprises two mutually complementary nucleotides (target activator-NA and activator-NA) (which contain intervening nucleotides ["linker covalently linked by a "linker nucleotide" or "linker nucleotide") and hybridized The double-stranded nucleic acid duplex of the protein-binding segment is formed, thus forming a stem-loop The targeter-NA and the activator-NA are combined to form a target-NA. A may be covalently linked via the 3' end of A and the 5' end of the activator-NA; or The targeter-NA and the activator-NA are attached to the 5' end and front end of the targeter-NA. The activator may be covalently bound via the 3' end of the NA.

[0181] The linker of the single-molecule DNA-targeting nucleic acid may be from about 3 nucleotides to about 100 nucleotides. For example, the linker may have a length of from about 3 nucleotides (nt) to from about 90 nt, from about 3 nt to about 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60 nt, from about 3 nt to about from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, from about 3 nt to about 30 nt, from about 3 nt to about 20 nt, or from about 3 nt to about 50 nt. The length of the nucleic acid sequence may be, for example, but not limited to, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 10 nt. About 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, It may have about 19, about 20, or more nucleotides. The linker of the single-molecule DNA-targeting nucleic acid is 4 nt.

[0182] Exemplary single-molecule DNA-targeting nucleic acids hybridize to a specific target sequence. Two nucleotides hybridize to form a double-stranded duplex along a guide sequence that The complementary extension of the nucleotides is included.

[0183] Suitable naturally occurring related crRNAs (and, in some embodiments, tracrRNAs) Pairs are known for most Cas proteins (Cas proteins have known anti- tracrRN by searching for the antirepeat-coding sequence or its variants. A - Cas nucleic acid-guide nucleic acid to identify the coding sequence (hence, tracrRNA sequence) Sequencing the flanking sequences of the cleavage protein Identifying nucleic acid-guided nuclease activity by analyzing (Already discovered or determined as naturally occurring Cas proteins) The antirepeat region of the tracrRNA is one half of the ds protein-binding duplex. The complementary repeat sequences comprising half of the ds protein-binding duplex are referred to as CRISPR repeats. The CRISPR repeats utilized by known CRISPR nucleic acid-guided nucleases are called and antirepeat sequences are known in the art, e.g., The online CRISPR database can be found at crispr.i2bc.paris-saclay.fr / crispr / You can attach it.

[0184] Single guide nucleic acid or dual-guide nucleic acid leic acid) may be synthesized chemically or by in vitro transcription. The assay for measuring sequence-specific binding between a nuclease and a guide nucleic acid comprises: Any of the nucleic acids known in the art and including, but not limited to, expressed nucleic acids - guide nucleic acids a cleavage enzyme and a guide nucleic acid (which is tagged with a detectable label [e.g., biotin]) and the identification of nucleoprotein complexes via detectable labels [e.g., streptomycin-containing nucleoprotein complexes]. Pull-down detection assay using avidin beads In vitro binding assays between the guide nucleic acid and the nucleic acid are also possible. a control guide nucleic acid having a sequence or structure unrelated to the nucleic acid-guide nucleic acid; Negative control for non-specific binding of nuclease to nucleic acids It may be used as a control.

[0185] In certain embodiments, the site-directed modification of the compositions and methods disclosed herein is The polypeptides include nuclease variants that function as nickases. wherein the nuclease has a higher affinity for double-stranded nucleic acid molecules than a wild-type nuclease. can cleave only a single strand of the (i.e., nuclease-dead) contains a mutation that renders the nuclease

[0186] Nucleases such as nucleic acid-guided nucleases that function as nickases are In some of these embodiments, the nuclease domain contains only one functional nuclease domain. The nuclease domain is a protein that has been engineered to reduce or eliminate the nuclease activity of that particular domain. It is further modified to

[0187] In other embodiments, the nuclease (e.g., RNA-guided nuclease) is completely Lacking nuclease activity, and in this application referred to as nuclease-dead In some of these embodiments, all of the nucleases in the nuclease The domain is mutated to eliminate all nuclease activity of the polypeptide. The methods described in U.S. Publication No. 2014 / 0068797 and U.S. Patent Application Publication No. 2014 / 0068797 may be used to prepare a soluble cellulose membrane comprising: No. 9,790,490 (each of which is incorporated by reference in its entirety). embedded in one or more nuclease domains of a site-directed nuclease, Mutations may be introduced.

[0188] Any mutation in the nuclease domain that reduces or eliminates nuclease activity Using nucleic acid-guided nucleases or nuclease-deck nucleases with nickase activity Nuclease-dead nucleic acid-guided nucleases can be created. Variants are known in the art and include, but are not limited to, the RuvC domain. D10A mutation in the HNH domain of S. pyogenes Cas9 or H840A mutation in the HNH domain of S. pyogenes Cas9 or Streptococcus pyogenes (S. pyogenes) Cas9. Mutations at similar positions in other nucleic acid-guided nucleases are also observed. Mutations can be introduced to create nickase- or nuclease-dead proteins. Other mutations in the nuclease domain of S. pyogenes Cas9 may be involved. Positions include G12, G17, E762, N854, N863, H982, H983, and D986. Nuclease- or nuclease-dead proteins, which may lead to nucleic acid-guiding nucleases. Other mutations within the nuclease domain of the nuclease include those in Francisella novicida (Franc Isella novicida Cpf1 protein, D917A, E1006A, E1028A, D1227A, D1255A, N1257A , D917A, E1006A, E1028A, D1227A, D1255A, and N1257A, or Francisella novicida (Francisella novicida) Cpf1 protein. Mutations at similar positions in another nucleic acid-guided nuclease when can be.

[0189] The site-directed modifying polypeptide comprising a nuclease-dead domain may further comprise: It may contain a domain capable of modifying a polynucleotide. Non-limiting examples of modifying domains that may be fused to a modified domain include These include, but are not limited to, transcriptional activation or repression domains, base editing domains, and epigenetic domains. In other embodiments, the nucleotide sequence may be a nuclease-dead domain. The site-directed modifying polypeptide containing the domain further detects the presence of the target sequence. It contains a detectable label which may aid in detection.

[0190] Epigenetic modification domains that may be fused to nuclease-dead domains Insin acts by covalently modifying DNA or histone proteins, altering the DNA sequence itself. alters histone and / or chromosome structure without altering gene expression site-directed Non-limiting examples of epigenetic modifications that can be induced by polypeptides that modify Examples include the following modifications in histone residues and their reverse reactions: sumoylation methylation of arginine or lysine residues, acetylation or ubiquitination of lysine residues, Phosphorylation of serine and / or threonine residues; and the following modifications of DNA and their reverse reactions: Methylation or hydroxymethylation of cystosine residues, thus epigenetic modification Non-limiting examples of domains include histone acetyltransferase domains. , histone deacetylase domain, histone methyltransferase domain, histone histone demethylase domains, DNA methyltransferase These include the methylase domain, the methylase domain, and the DNA demethylase domain. It can be obtained.

[0191] In some embodiments, the site-directed polypeptide comprises a transcriptional regulatory element, and / or Through interactions with transcriptional regulatory proteins such as transcription factors or RNA polymerases, It contains a transcription activation domain that activates transcription of at least one adjacent gene. Transcriptional activation domains are known in the art and include, but are not limited to: However, the VP16 activation domain is one example.

[0192] In other embodiments, the site-directed polypeptide comprises a transcriptional repression domain, which , transcriptional control elements, and / or transcriptional regulatory proteins such as transcription factors or RNA polymerases may interact with the protein, decreasing the transcription of at least one adjacent gene. Suitable transcription repression domains are known in the art, and and, but not limited to, IκB and KRAB domains.

[0193] In yet another embodiment, site-directed modifications containing nuclease-dead domains are The polypeptide may further comprise a polypeptide that detects the presence of a target sequence, which may be a sequence associated with a disease. Detectable labels include those that can aid in detection. Detectable labels are those that can be visualized. The detectable label may be, as a fusion protein, Nucleic acid-guided nucleases may be fused to (e.g., fluorescent proteins) or visual conjugated to a nuclease polypeptide, detectable optically or by other means The nucleic acid-guanine derivatives disclosed in this application may be small molecules containing fusion proteins. The detectable label that may be fused to the endonuclease may be any detectable Protein domains, such as, but not limited to, fluorescent proteins, or specific Examples include protein domains that can be detected using antibodies. - Non-limiting examples include green fluorescent proteins (e.g., GFP, EGFP, ZsGreen1) and yellow fluorescent Proteins (e.g., YFP, EYFP, ZsYellow1). Non-limiting examples include: 3 H and 35 Radiolabels, such as S.

[0194] Nucleic acid-guided nucleases bind to their guide nucleic acids. delivered as part of a delivery system into the cell as a nucleoprotein complex containing Alternatively, the nucleic acid-guide nuclease and the guide nucleic acid may be provided separately. In certain embodiments, the guide RNA is delivered as an RNA molecule into the target cell. The guide RNA may be transcribed in vitro or In another embodiment, the nucleotide sequence encoding the guide RNA may be chemically synthesized. In some of these embodiments, the guide RNA is introduced into the cell. The nucleotide sequence encoding the operably linked to a promoter, and this promoter is a native promoter. or may be heterologous to the nucleotide sequence encoding said RNA. be.

[0195] In certain embodiments, the site-directed polypeptide comprises at least one nuclear localization sequence. The nucleic acid sequence may further include an amino acid sequence such as a nuclear localization sequence (NLS). The nuclear localization sequence directs the site-directed polypeptide to be transported into the nucleus of the cell. Proteins transported into the nucleus generally have amino acid sequences at lysine and arginine residues. The best-binding protein, importin / karypherin protein ( It binds to one or more proteins within the nuclear pore complex, such as ATPases (proteins). A well-understood pathway involves the binding of short peptide sequences to the importin-α protein. These nuclear localization sequences often contain stretches of basic amino acids and are important Given that there are two such binding sites on ATP, at least 10 amino acids Two basic sequences separated by a may constitute a bipartite NLS. The second well-defined pathway for nuclear import involves binding to the importin-β1 protein. Proteins that contain the sequence RKKRRQRRR (e.g., HIV-TAT and HIV-REV proteins) RQARRNRRRRWR (SEQ ID NO: 323) and (which bind to importin-β1, respectively). Other nuclear localization sequences are known in the art. It is known that the nucleus is involved in the nucleus formation in the cortex (e.g., Lange et al., J. Biol. Chem. (2007) 282:5101-510 5). The NLS may be a naturally occurring NLS of the site-directed polypeptide, or a heterologous NLS. As used in this application, "heterologous" when referring to a sequence may be an NLS. "logos" refers to a sequence derived from a foreign species, or, if derived from the same species, , intentional human intervention, in composition and / or genetic loci, that alter their innate A sequence substantially modified from the form of the site-directed nuclear localization of a polypeptide. Non-limiting examples of NLS sequences that may be used to enhance transcription include the SV40 large T- In certain embodiments, the NLSs include those of large T-antigen and c-Myc. contains the amino acid sequence PKKKRKV (SEQ ID NO: 324).

[0196] A site-directed polypeptide may contain one, such as two, three, four, five, six, or more NLS sequences. Each of the multiple NLSs may be unique in sequence. Alternatively, multiple identical NLS sequences may be used. or at the amino-terminal (N-terminal) end, the carboxy-terminal (C-terminal) end of said polypeptide In certain embodiments, the anterior pos ... The site-directed polypeptide comprises four NLS sequences at its N-terminal end. In some embodiments, the site-directed polypeptide comprises a C-terminal In yet another embodiment, the site-directed polypeptide comprises two NLS sequences at the ends. , contains four NLS sequences at its N-terminal end and two NLS sequences at its C-terminal end.

[0197] In certain embodiments, the site-directed polypeptide is a cell-penetrating peptide. The CPP contains a cell penetrating peptide (CPP), which penetrates the cell membrane and binds to the Induces absorption of proteins or peptides, allowing them to self-assemble into membrane-spanning holes or some positively charged residues (which are negatively charged outside the phospholipid) interacting with the membrane, bending said membrane, which in turn activates internalization; Generally, a CPP has the general shape and tendency of: Exemplary penetrating peptides include, but are not limited to, However, transportan, PEP1, MPG, p-VEC, MAP, CADY, polyR, HIV-TAT, HIV-REV, Penetratin, R6W3, P22N, DPV3, DPV6, K-FGF, and C105Y, and van d en Berg and Dowdy (2011) Current Opinion in Biotechnology 22:888-893 and Farkha (2014) Peptides 57:78-94, respectively. , which is incorporated by reference in its entirety into this application).

[0198] In conjunction with, or as an alternative to, an NLS, the site-directed polypeptide may be Detectable labels (e.g., fluorescent proteins) or purification tags described in the application. The purification tag may contain additional heterologous amino acid sequences to form a fusion protein. The protein or fused protein is extracted from a mixture (e.g., a biological sample, a culture medium) Non-limiting examples of purification tags include: These include biotin, myc, maltose binding protein (MBP), and and glutathione-S-transferase (GST). do.

[0199] The compositions and methods disclosed herein can be used to identify double-stranded breaks to be repaired using sequence specific through the introduction of heterologous end-joining (e.g., error-prone non-homologous end-joining) [gous end-joining (NHEJ)], microhomology-mediated end joining [microhomology-media ted end joining (MMEJ), or alternative end-joining (alt-EJ), Genome editing (via other pathways) may be used to introduce mutations at specific genomic locations. Because the degradation process is error-prone, the double-strand break is repaired, resulting in target This results in the target sequence being modified. Alternatively, the donor template polynucleotide However, during the process of repairing the introduced double-strand break, The exogenous donor sequence is introduced into the target gene or is replaced by the target sequence. Thus, the compositions and methods may include flanking homologous ends. These embodiments may further comprise a donor template polynucleotide. In some cases, the donor template polynucleotide is a polynucleotide described elsewhere in this application. the donor is linked to the site-directed polypeptide via a linker The template polynucleotide is linked to the site-directed polynucleotide via a cleavable linker. binds to the polypeptide).

[0200] In some embodiments, the donor sequence modifies the original target sequence, resulting in The newly incorporated donor sequence is not recognized by the nucleic acid-guided nuclease and is not cleaved. The donor sequence has substantial similarity to the sequences adjacent to the target sequence. The DNA fragment contains adjacent sequences with sequence identity and can be repaired through homology-directed repair. The nucleic acid-guided nuclease enhances the incorporation of the double-stranded In certain embodiments, the donor polynucleotide generates staggered breaks. The nucleotides may be flanked by compatible overhangs, which allow repair of the double-strand break. It is possible that the donor sequence is incorporated via a non-homologous repair process during It becomes Noh.

[0201] The nucleic acid manipulation reagents of the present disclosure are introduced into the stem cells using any suitable delivery method. Delivery may be via in vitro, ex vivo, or in vivo administration. Exemplary methods for introducing reagents for manipulation include, but are not limited to, transfection. These include transfection, electroporation, and virus-based methods. In some embodiments, the stem cells are cultured in the subject prior to introducing gene editing components. The stem cells are genetically modified ex vivo and then returned to the subject (e.g., transplanted). In one embodiment, the subject is the same subject from which the cells are isolated. In another embodiment, the subject is different from the subject from which the cells are isolated. In embodiments, autologous stem / progenitor cells are modified ex vivo and returned to the subject. In another embodiment, heterologous stem / progenitor cells are modified ex vivo and administered to the subject. return.

[0202] Genetically modifying stem cells can be achieved by using gRNA molecules, Cas9 molecules, and optionally delivering a donor template nucleic acid to the stem cell. In one embodiment, the gRNA molecule, the Cas9 molecule, or both, and optionally The template nucleic acid may be a viral vector (e.g., an AAV vector or a lentivirus vector). vectors [e.g., integration-deficient lentiviruses] In another embodiment, the gRNA molecule and the Cas9 molecule are delivered by an intracellular vector (IDLV). is delivered as a ribonucleoprotein complex of the gRNA molecule / Cas9 molecule. In this method, the gRNA molecule and the Cas9 molecule are delivered as RNA. It may include at least one exon of a target gene for gene replacement therapy. In certain embodiments, the template nucleic acid is associated with a disease or risk of a disease. The template nucleic acid does not contain a mutation. In certain embodiments, the template nucleic acid may comprise a splicing sequence. In another embodiment, the template nucleic acid comprises a poly(A) donor or acceptor. Contains a denylation signal.

[0203] In some embodiments, the reagent taken up by one or more cells is a transfection reagent. Examples of transfection reagents include polymer-based (e.g. , DEAE dextran) transfection reagents and cationic liposome-mediated transfection Electroporation methods can be used to introduce nucleic acid manipulation reagents. It may also promote drug uptake. By applying an external field, the membrane potential in the cell induces a change in membrane potential, and the net value of the membrane potential (the sum of the difference between the applied potential and the resting potential) is greater than the threshold. When the concentration of the ions in the membrane increases, a transient permeation structure is created within the membrane, and electroporation is achieved. See Gehl et al., Acta Physiol. Scand. 177:437-447 (2003).

[0204] Also, nucleic acid manipulation reagents can be delivered into the stem cells by viral transduction. Suitable viral delivery systems include, but are not limited to, adeno-associated Viral (adeno-associated virus (AAV)), retroviral, and lentiviral delivery systems Such viral delivery systems include those in which the stem cells are transfected. This is particularly useful when the virus is resistant to viral delivery. The method of using the system further includes a step of preparing a viral vector encoding a nucleic acid manipulation reagent. and packaging the vector into a viral particle. .

[0205] Other methods for delivering nucleic acid reagents include, but are not limited to, lipofection. , nucleofection, microinjection, biolistics , virosome, liposome, immunoliposome, polycation or lipid: nucleus Acid conjugates, naked DNA, artificial virions, nanoparticles, and drugs Agent-enhanced uptake of nucleic acids. See also Iwoehner et al., Nucleic Acids Res. 42:1341-1353 (2014), which For all purposes, and particularly for all teachings relating to reagent delivery systems, the entire disclosure of which is incorporated herein by reference. In some embodiments, non-viral vector delivery systems are used. Introduction by stem can be achieved by DNA plasmids, RNA (e.g., vectors described in this application), or the like. transcripts), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. .

[0206] Stem cells can be genetically modified to target a gene of interest (e.g., The target position may be modified, for example, by By repairing (e.g., correcting or modifying) one or more mutations in the gene In a specific embodiment, the mutation may be repaired by homology-directed repair. Using homology-directed repair to correct the mutant allele, and restores the wild-type state. In one embodiment, the mutation in the gene is corrected. By knocking a polynucleotide into the target gene, wild-type gene activity is restored. In one embodiment, the stem cells may be modified by transfection with a polynucleotide. Knocking in the leutide restores wild-type gene activity.

[0207] In certain embodiments, stem cells are modified to knock out the activity of a target gene. Altering the target location by: This can be achieved by: (1) knocking out the gene; (a) knocking out the gene; Insertion or deletion of one or more nucleotides adjacent to or within the initial coding region or (b) a deletion (e.g., an NHEJ-mediated insertion or deletion), or (b) at least one of the genes (2) deletion of a genomic sequence containing a portion of the Cas9 gene (e.g., NHEJ-mediated deletion), or (3) enzymatically inactive Cas9. Enzymatically inactive Cas9 (eiCas9) molecules or eiCas9-fusion proteins (e.g., transcription factors) By targeting the promoter region of the gene via a gene fusion to a repressor That is, knocking down the gene.

[0208] 2. Indications for treatment As described in this application, transplantation therapy of genetically modified HSCs can be performed by targeting a gene. Prescribed to a subject in need of treatment for engraftment of one or more modified blood cell types. Hematopoietic stem cells generally exhibit pluripotency, and therefore may be used in a variety of ways, including, but not limited to, Granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, red blood cells), platelets (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets) , monocytic cells (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and phospholipids. Differentiation into multiple different blood lineages, including lymphocytes (e.g., NK cells, B cells, and T cells) Hematopoietic stem cells also have the ability to self-renew, and therefore have the same potential as their parent cells. can give rise to daughter cells that can be reintroduced into the transplant recipient and become hematopoietic stem cells. They are characterized by their ability to home to niches and re-establish productive and sustained hematopoiesis.

[0209] Therefore, the compositions and methods described herein can be used to treat non-malignant hemoglobinopathies (e.g., Sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiscott anemia Hemoglobinopathies selected from the group consisting of: Additionally or alternatively, the compositions and methods described herein may be used to treat congenital It may also or alternatively be used to treat immune deficiencies, such as immunodeficiency. The compositions and methods described in this application may be used to treat acquired immune deficiency diseases (e.g., HIV and AIDS). The present invention may be used to treat acquired immunodeficiency disorders (acquired immunodeficiency disorders selected from the group consisting of: The compositions and methods described herein are useful in treating metabolic disorders (e.g., glycogen storage diseases, mucopolysaccharidoses, glycoprotein disorders, a disease selected from the group consisting of leukodystrophy, ... It may be used to treat metabolic disorders (including

[0210] In some embodiments, the methods are used to treat a group of disorders that affect hemoglobin. This disorder causes red blood cells to turn into a sickle shape. Atypical hemoglobin (hemoglobin) that may cause the hemoglobin to deform into a crescent shape or The disorder is characterized by a low number of red blood cells (anemia), Symptoms include repeated infections and periodic pain. Mutations in the HBB gene cause sickle cell disease. Various mutations in the HBB gene result in different types of beta-globin. Certain HBB gene mutations result in an abnormal form of hemoglobin known as hemoglobin S (HbS). Other mutations in the HBB gene also produce hemoglobin C (H HBB gene mutations result in abnormal forms of beta-globin, such as hemoglobin B (HbC) and hemoglobin E (HbE). The abnormality can also result in abnormally low levels of beta-globin, a condition known as beta-thalassemia. In people with sickle cell disease, the beta-globin subunit in hemoglobin At least one is replaced by hemoglobin S. This is a common form of sickle cell disease. In sickle cell anemia, both beta-globin subunits in hemoglobin In other types of sickle cell disease, only a small amount of hemoglobin is One beta-globin subunit is replaced by hemoglobin S. The two beta-globin subunits are different abnormal variants of hemoglobin C. For example, people with sickle cell-hemoglobin C (HbSC) disease have beta- Instead of globin, it has a hemoglobin molecule with hemoglobin S and hemoglobin C. The combination of hemoglobin S and beta-thalassemia-causing mutations results in Globin S-beta thalassemia (HbSBetaThal) disease. Using known gene editing methods. and any one or more of the mutations that cause sickle cell disease, for use in the method. Additionally or alternatively, a functional HBB gene may be engineered into stem cells prior to transplantation. It may be introduced into stem cells for the purpose of

[0211] In some embodiments, the methods are used to treat blood disorders that decrease the production of hemoglobin. It may treat beta thalassemia (also called Beta Thal), a condition that is harmful to the body. In subjects with thalassemia, low levels of hemoglobin cause many The patient also develops a lack of red blood cells (anemia) and pale skin. Beta-thalassemia can cause weakness, fatigue, and more serious complications. People with beta-thalassemia are at increased risk of developing abnormal blood clots. Therefore, it is divided into two types: thalassemia major (Cooley's anemia) (also known as thalassemia major) and thalassemia intermedia. Mutations in the HBB gene cause beta-thalassemia. The BB gene gives instructions for making beta-globin. Mutations in the HBB gene Some of the substances that prevent the production of beta-globin are , beta zero (B 0 ) thalassemia. Mutations in other HBB genes cause some beta- Globin is produced, but in reduced amounts, i.e., beta-plus (B + ) · Thalassemia People with both types are known as thalassemia major and thalassemia intermedia. In some embodiments, a rare form of beta-thalassemia is , delta- or gamma-globin (HBG1 and HBG2, UniProt P69891 and P69892, respectively). Using known gene editing methods, it is possible to modify the beta Any one or more of the mutations that cause thalassemia for use in the method. Additionally or alternatively, any one or more functional Genes (e.g., HBB, HBG1, HBG2) may be introduced into stem cells for transplantation . For example, http: / / dx.doi.org / 10.5772 / 61441; Pondarre and Badens, Ann. Biol. Clin (Paris) 72(6):639-668, 2014.

[0212] In some embodiments, the methods are used to detect the enzyme adenosine deaminase (ADA). Adenosine deaminase deficiency, a metabolic disorder whose absence causes immune deficiency May treat ADA deficiency (also called ADA-SCID (severe combined immunodeficiency)) Individuals with SCID lack virtually all immune defenses against bacteria, viruses, and fungi. They are prone to recurrent infections that can become very serious or life-threatening. These infections are not normally illnesses in people with normal immune systems. Most people with ADA deficiency have SCID is diagnosed within the first six months of life. Without treatment, these babies usually live for 2 years. In approximately 10 to 15% of cases, the onset of immunodeficiency occurs between the ages of 6 and 8 years. between 1 and 24 months (delayed onset) or into adulthood (late onset) The immunodeficiency in these later-onset cases tends to be less severe. It primarily causes recurrent upper respiratory tract and ear infections. Over time, affected individuals develop chronic lung damage. malnutrition and other health problems. , any one of the mutations in the adenosine deaminase gene that cause ADA, In addition, or alternatively, functional modifications may be made in stem cells for use in the present methods. A specific adenosine deaminase gene may be introduced into stem cells for transplantation. In another embodiment, the genetically modified stem cells are Strimvelis 登録商標 (Human ADA cDNA Autologous CD34+ cells transduced with a retroviral vector encoding the sequence (autologous CD34+ enriched cell fraction).

[0213] In some embodiments, the methods are used to detect the enzyme arylsulfatase A (ARSA). It is a lysosomal storage disease caused by a deficiency of sulfatides in the cells. Metachromatic leukodystrophy (MLD or arylsulfamethasone) is a condition characterized by the accumulation of lipids in the leukocytes. This buildup can cause nerve insulation and lead to heart problems. It particularly affects cells of the nervous system that produce myelin, a protective substance. Nerve cells covered with myelin make up the tissue called white matter. Accumulation of phatides can affect, for example, the brain and spinal cord (central nervous system) and the nerves that connect the brain and spinal cord to muscles. The nerves that transmit the signals, and the sensory cells that detect sensations such as touch, pain, heat, and sound (peripheral nervous system), which leads to the progressive destruction of white matter throughout the nervous system (leukodystrophy) In people with metachromatic leukodystrophy, damage to the white matter can affect functions such as intellectual function and the ability to walk. Motor skills progressively decline. Affected individuals also experience loss of sensation in the extremities (peripheral neuropathy), incontinence, and diarrhea. Seizures, paralysis, loss of speech, blindness, and deafness may occur. They lose awareness of their surroundings and become unresponsive. Any one of the mutations in the ARSA gene that cause MLD can be used in this method. Additionally or alternatively, a functional ARSA gene may be engineered in stem cells for the purpose of may be introduced into stem cells for transplantation.

[0214] In some embodiments, the methods are used to identify a gene that is caused by a mutation in the WASp gene. It is an X-linked recessive disorder caused by abnormal immune system function (immunodeficiency) and blood clotting. Wiskott-Aldrich syndrome (Wiskott-Aldrich syndrome) is characterized by a decreased ability to form thrombocytopenia-immunodeficiency syndrome (WAS, or dermatitis-thrombocytopenia-immunodeficiency syndrome) Wiskott-Aldrich syndrome may be treated. Patients develop microvascular disease, a decrease in the number and size of blood cell fragments involved in clotting (platelets). have microthrombocytopenia. This platelet disorder typically occurs at birth. Present from birth, easy bruising, bloody diarrhea after minor trauma In microthrombocytopenia, symptoms of thrombocytopenia may include: Small areas of bleeding may appear just under the surface of the skin, resulting in purplish spots called purpura or pinpoint spots. A rash of small red spots called bloods may develop. In some cases, the bleeding can be life-threatening. Wiskott-Aldrich syndrome can also be a life-threatening condition. Characterized by cells of the immune system (e.g., white blood cells). Changes in white blood cells are In patients with Wiskott-Aldrich syndrome, several immune and inflammatory disorders are These immune disorders vary in severity and can lead to increased risk of infections and dermatitis (redness). They are particularly susceptible to inflammatory skin diseases (inflammatory skin disorders characterized by abnormal patches of sore, irritated skin). People with Wiskott-Aldrich syndrome often suffer from rheumatoid arthritis and hemolytic anemia. These diseases occur when the immune system goes haywire, increasing the risk of developing autoimmune diseases such as rheumatoid arthritis. This occurs when the body attacks its own tissues and organs. People with the syndrome develop certain types of cancer, including cancer of immune system cells (lymphoma). Using known gene editing methods, Wiskott-Aldrich Any one or more of the mutations in the WASp gene that cause the syndrome may be used in the method. Additionally or alternatively, a functional WASp gene may be engineered in stem cells for the purpose of may be introduced into stem cells for transplantation.

[0215] In some embodiments, the methods are used to detect a deficiency in the enzyme phagocyte NADPH oxidase. It is caused by mutations in one of five different genes that result in immune deficiency. It may treat chronic granulomatous disease (CGD), which is caused by People with this condition may suffer from recurring bacterial and fungal infections. Inflammatory areas (granulomas) may develop in various tissues, causing damage to those tissues. In these patients, the lungs are the most frequently infected area; pneumonia is a common feature of this condition. People with chronic granulomatous disease are at increased risk of developing a fungal pneumonia called mulch pneumonitis. A type of fungus can develop in people who eat decaying organic matter such as mulch, hay, or dead leaves. Fever and shortness of breath occur after exposure to these organic substances and their decomposition. When exposed to large numbers of fungi, people with chronic granulomatous disease develop fungal infections in their lungs. Other common areas of infection in people with chronic granulomatous disease include the skin, liver, and lymph nodes. Furthermore, inflammation occurs in many different areas of the body in patients with CGD. Granulomas most commonly occur in the gastrointestinal and genitourinary tracts. Using editing methods, we identified mutations in the phagocyte NADPH oxidase gene that cause CGD. Any one or more of these may be modified in stem cells for use in the present method. Alternatively, a functional phagocyte NADPH oxidase gene may be inserted into the stem cells for transplantation. It may be introduced into

[0216] In certain embodiments, the methods are used to identify a gene that is caused by a mutation in the GALC gene. Globoid cell leukodystrophy (GCL) is a condition that affects the globoid cell leukodystrophy (GCL) Galactosylceramide lipidosis or Krabbe disease (K The GALC gene may be involved in the treatment of certain lipid disorders, also known as rabbe disease. It directs the production of galactosylceramidase, which breaks down proteins (e.g., galactolipids). A certain galactosylceramide, called galactosylceramide, is degraded by galactosylceramidase. Ceramide is an important component of myelin. The breakdown of galactosylceramide occurs throughout life. It is part of the normal turnover of myelin that occurs over time. Another galactolipid, called psychosine, is metabolized by galactosylceramidase. It is toxic if not broken down. Generally, GCL is involved in the growth of protective myelin sheaths on nerves. This affects the GCL, causing severe degeneration of motor skills. It is characterized by abnormal cells in the brain called globoid cells, which are large cells that grow inside the brain. Using known gene editing methods, any one of the mutations in the GALC gene that causes GCL is One or more of the following may be modified in stem cells for use in the method. Alternatively, a functional GALC gene may be introduced into stem cells for transplantation.

[0217] In some embodiments, the methods are used to detect the iduronidase alpha-L gene (IDU). It is caused by a mutation in the A gene (leading to a deficiency of the enzyme alpha-L-iduronidase) Mucopolysaccharidosis type I (also called MPS type I), a form of MPS that is commonly Treatment of the inability to metabolize complex carbohydrates known as polysaccharides into simpler molecules When IDUA enzyme activity is deficient, it accumulates in cells, especially in the lysosomes. This leads to the accumulation of glycosaminoglycans (GAGs). People with MPS1 have large heads (megalopsia). Macrocephaly), fluid accumulation in the brain (hydrocephalus), abnormalities of the heart valves, enlarged liver and spleen (hepatosplenocele) The vocal cords may be enlarged, causing a louder voice. People with MPS I have narrowed airways and upper airway obstruction. Frequent infections and short pauses in breathing during sleep (sleep apnea) People with MPS I often develop corneal clouding, which can lead to significant vision loss. Affected individuals may also have hearing loss and recurrent ear infections. People with MPS I have short stature and joint deformities (contractures) that affect mobility. Most people with severe forms of the disorder also have dysostosis multiplex, which refers to multiple skeletal abnormalities. When the canal narrows (spinal stenosis), the neck compresses and damages the spinal cord. Using known gene editing methods, mutations in the IDUA gene that cause MPS1 can be Any one or more of the mutations may be modified in stem cells for use in the present method. Additionally or alternatively, a functional IDUA gene can be introduced into stem cells for transplantation. be.

[0218] Additionally or alternatively, the compositions and methods described in this application may be used in combination with any of the methods specifically described herein. The conjugates can be used to treat malignancies or proliferative disorders, such as hematological cancers, myeloproliferative disorders, and the like. In the case of cancer treatment, the compositions and methods described herein may be used to treat: Prescribed to patients to reduce the endogenous hematopoietic stem cell population prior to hematopoietic stem cell transplantation therapy. In this case, the transplanted cells may be produced by a process of depleting endogenous cells. They may home into the provided niche and establish productive hematopoiesis. The cell population that was reduced during eradication of cancer cells, such as during chemotherapy, is now being repopulated. The compositions and methods described herein may be used to treat Exemplary hematological cancers include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, and leukemia. Myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large cell type B Cell lymphoma, non-Hodgkin's lymphoma, and neuroblastoma Other cancer symptoms, including cysts, include:

[0219] Additional conditions that may be treated using the compositions and methods described in this application include: including but not limited to adenosine deaminase deficiency and severe combined immunodeficiency, Hyperimmunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis , osteogenesis imperfecta, storage disease, thalassemia major, systemic sclerosis, systemic lupus erythematosus These include systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis. It can be obtained.

[0220] The compositions and methods described herein can be used to induce endogenous hematopoietic stem cell transplantation prior to hematopoietic stem cell transplantation therapy. Reducing the population of blood stem cells may treat autoimmune diseases. In this case, the transplanted cells reside in a niche created by the process of depleting endogenous cells. They may home to and establish productive hematopoiesis, thereby eliminating autoimmune cells. The cell population that was depleted during eradication can now be reconstituted.

[0221] Autoimmune diseases that can be treated using the compositions and methods described in this application include: , including but not limited to, psoriasis, psoriatic arthritis, type 1 diabetes (T1D), rheumatoid arthritis Human systemic lupus erythematosus (RA), human systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), Lymphocytic colitis, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis , antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune liver inflammation, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis , Baro's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, chronic fatigue, immune deficiency CFIDS, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid , Celiac sprue dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid Lupus erythematosus, autonomic neuropathy, endometriosis, essential mixed cryoglobulinemia, fibromyalgia Fibromyalgia-fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (G BS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, idiopathic Pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, leprosy Meniere's disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, Psoclonus-myoclonus syndrome (OMS), optic neuritis, Ord's thyro iditis), pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary liver cirrhosis, polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica, primary non-cancer Primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, Rheumatic Mitchell's fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-person syndrome , Takayasu's arteritis, temporal arteritis (also known as giant cell arteritis), ulcerative colitis, collagen fibrillation Colitis, uveitis, vasculitis, vitiligo, vulvar pain (vulvar vestibulitis), and Wegener's disease blastoma, among others.

[0222] C. Antibody Drug Conjugates (ADCs) 1. Antibodies As described herein, the methods of the present application involve the detection of IL-16 on hematopoietic stem cells and / or on immune cells. , specific molecules (e.g., CD2, CD5, CD7, CDwl2, CD13, CD15, CD19, CD21, CD22, CD29, CD30, CD33, CD34, CD36, CD38, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD45 , CD45RA, CD45RB, CD45RC, CD45RO, CD47, CD48, CD49b, CD49d, CD49e, CD49f, CD50, CD53, CD55, CD64a, CD68, CD71, CD72, CD73, CD81, CD82, CD85A, CD85K, CD90, CD99, CD104, CD105, CD109, CD110, CD111, CD112, CD114, CD115, CD117, CD123, CD124, CD 126, CD127, CD130, CD131, CD133, CD134, CD135, CD137, CD138, CD151, CD157, CD162 , CD164, CD168, CD172a, CD173, CD174, CD175, CD175s, CD176, CD183, CD191, CD200, CD201, CD205, CD217, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD 228, CD229, CD230, CD235a, CD235b, CD236, CD236R, CD238, CD240, CD242, CD243, CD 252, CD277, CD292, CDw293, CD295, CD298, CD309, CD318, CD324, CD325, CD338, CD34 These include the use of ADCs that target CD4, CD349, or CD350, among others. In some embodiments, the ADC targets one or more specific molecules on HSCs and / or on immune cells. The present invention also includes an antibody or antigen-binding fragment thereof that specifically binds to the Methods for generating suitable antibodies for this purpose are readily available to those skilled in the art.

[0223] a. Anti-CD117 antibody Antibodies capable of binding to CD117 (e.g., GNNK+ CD117) and their antigen-binding fragments The fragments can be used as the sole therapeutic agent or as conjugates (ADCs), e.g. (i) to treat cancers and autoimmune diseases characterized by CD117+ cells, and (ii) to treat diseases requiring transplantation therapy. To promote the engraftment of transplanted genetically modified hematopoietic stem cells in patients who require such treatment. These therapeutic activities may be achieved by targeting cells such as cancer cells, autoimmune cells, or hematopoietic stem cells. isolated anti-CD1 that binds to CD117 expressed on the surface of target cells (e.g., GNNK+ CD117). 17 antibodies, their antigen-binding fragments, binding and subsequent induction of cell death This may be due to the reduction of endogenous hematopoietic stem cells, which may lead to the transplantation of hematopoietic stem cells. It provides a niche to which cells can home, and subsequently establish productive hematopoiesis. In this way, the transplanted hematopoietic stem cells are not affected by the stem cell disorders described in this application. These cells can be successfully engrafted in patients, including human patients.

[0224] Human CD117 (also known as c-Kit, mRNA NCBI reference sequence: NM_000222.2, protein NCBI reference sequence: Antibodies and antigen-binding fragments (e.g., GNNK+CD117 (e.g., antibodies and antigen-binding fragments capable of binding to CD117) to hematopoietic stem cells To condition a patient for transplant therapy, the compositions described in this application and This method may be used in combination with other methods to detect CD117, which is present in a significant proportion of the population. Polymorphisms affecting the binding region or extracellular domain are currently not relevant for non-oncology indications. At least four isoforms of CD117 have been identified. Therefore, it is possible that additional isoforms are expressed in tumor cells. Two of the forms are localized to the intracellular domain of the protein, and two are located in the outer juxtamembrane region. Two extracellular isoforms, GNNK+ and GNNK-, share a four amino acid sequence (GNNK These isoforms differ in that they react with the ligand (SCF) either as a phosphodiesterase (GlcNAc) or as a non-phosphodiesterase (GNNK). Although it has been reported that the ligand binding to GNNK-isoforms is It has been reported that GNNK+ isoflavones enhance cellular uptake and degradation. The isoform contains GNNK+ and GNNK- proteins, and antibodies raised against this isoform It may be used as an immunogen to generate antibodies capable of binding to CD117. The amino acid sequences of human CD117 isoforms 1 and 2 are set forth in SEQ ID Nos. 145 and 146, respectively. and 146. In certain embodiments, the antibodies disclosed in the present application are anti-human CD117 (hCD1 17) The antibody can bind to both isoform 1 and isoform 2 of human CD117. Cut.

[0225] As described below, yeast libraries of human antibodies can be screened for diagnostic applications. We have identified a novel anti-CD117 antibody and its fragments for therapeutic use. 4), Antibody 55 (Ab55), Antibody 56 (Ab56), Antibody 57 (Ab57), Antibody 58 (Ab58), Antibody 61 (Ab61), Antibody 66 (Ab66), antibody 67 (Ab67), antibody 68 (Ab68), and antibody 69 (Ab69) were identified in this screen. These antibodies cross-react with human CD117 and rhesus monkey CD117. Furthermore, the antibodies disclosed in the present application bind to both isoforms of human CD117 (i.e., Isoform 1 (SEQ ID NO: 145) and isoform 2 (SEQ ID NO: 146)).

[0226] Anti-CD117 antibodies (e.g., Ab54, Ab55, Ab56, Ab57, Ab58, Ab61, Ab66, Ab67, Ab68, and The amino acid sequences of various binding regions of the CDRs of the IgG1A-specific ... The present invention provides a human anti-CD117 antibody comprising the variable region described in Table 9. Included in the display.

[0227] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 55. , variable regions). The amino acid sequence of the heavy chain variable region (VH) of antibody 55 (i.e., Ab55) is shown in SEQ ID NO: 19. (See Table 9.) The amino acid sequence of the VH CDR domain of antibody 55 is set forth in SEQ ID NO: 21 (VH CDR1). ; shown in SEQ ID NO: 22 (VH CDR2) and SEQ ID NO: 23 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of 55 is shown in SEQ ID NO: 20 (see Table 9). The amino acid sequences of the VL CDR domains of 55 are set forth in SEQ ID NO: 24 (VL CDR1); SEQ ID NO: 25 (VL CDR2); The heavy chain constant region of antibody 55 is shown in SEQ ID NO: 25 (VL CDR2) and SEQ ID NO: 26 (VL CDR3). The light chain constant region of antibody 55 is shown in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen-binding portion thereof, is The variable heavy chain CDR sets (CDR1, CDR2, and CDR3) shown in SEQ ID Nos: 21, 22, and 23 ), and the light chain variable region CDRs set forth in SEQ ID Nos: 24, 25, and 26. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of: and a heavy chain variable region set forth in SEQ ID NO: 19. nothing.

[0228] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 54. , variable regions). The amino acid sequence of the heavy chain variable region (VH) of antibody 54 (i.e., Ab54) is shown in SEQ ID NO: 29 ( (See Table 9.) The amino acid sequence of the VH CDR domain of antibody 54 is set forth in SEQ ID NO: 31 (VH CDR1). ; shown in SEQ ID NO: 32 (VH CDR2) and SEQ ID NO: 33 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of 54 is shown in SEQ ID NO: 30 (see Table 9). The amino acid sequences of the VL CDR domains of SEQ ID NO: 34 (VL CDR1); SEQ ID NO: 35 (VL CDR2); The heavy chain constant region of antibody 54 is shown in SEQ ID NO: 35 (VL CDR2) and SEQ ID NO: 36 (VL CDR3). The light chain constant region of antibody 54 is shown in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen-binding portion thereof, is The variable heavy chain CDR sets (CDR1, CDR2, and CDR3) shown in SEQ ID Nos: 31, 32, and 33 ), and the light chain variable region CDRs set forth in SEQ ID Nos: 34, 35, and 36. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of: and a heavy chain variable region set forth in SEQ ID NO: 29. nothing.

[0229] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 56. , variable regions). The amino acid sequence of the heavy chain variable region (VH) of antibody 56 (i.e., Ab56) is shown in SEQ ID NO: 39 ( (See Table 9.) The amino acid sequence of the VH CDR domain of antibody 56 is set forth in SEQ ID NO: 41 (VH CDR1). ; shown in SEQ ID NO: 42 (VH CDR2) and SEQ ID NO: 43 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of 56 is shown in SEQ ID NO: 40 (see Table 9). The amino acid sequences of the VL CDR domains of 56 are set forth in SEQ ID NO: 44 (VL CDR1); SEQ ID NO: 45 (VL CDR2); The heavy chain constant region of antibody 56 is shown in SEQ ID NO: 45 (VL CDR2) and SEQ ID NO: 46 (VL CDR3). The light chain constant region of antibody 56 is shown in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen-binding portion thereof, is The variable heavy chain CDR sets (CDR1, CDR2, and CDR3) shown in SEQ ID Nos: 41, 42, and 43 ), and the light chain variable region CDRs set forth in SEQ ID Nos: 44, 45, and 46. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of: and a heavy chain variable region set forth in SEQ ID NO: 39. nothing.

[0230] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 57. , variable regions). The amino acid sequence of the heavy chain variable region (VH) of antibody 57 (i.e., Ab57) is shown in SEQ ID NO: 49 ( (See Table 9.) The amino acid sequence of the VH CDR domain of antibody 57 is set forth in SEQ ID NO: 51 (VH CDR1). ; shown in SEQ ID NO: 52 (VH CDR2) and SEQ ID NO: 53 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of 57 is shown in SEQ ID NO: 50 (see Table 9). The amino acid sequences of the VL CDR domains of 57 are shown in SEQ ID NO: 54 (VL CDR1); SEQ ID NO: 55 (VL CDR2); The heavy chain constant region of antibody 57 is shown in SEQ ID NO: 55 (VL CDR2) and SEQ ID NO: 56 (VL CDR3). The light chain constant region of antibody 57 is shown in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen-binding portion thereof, is The variable heavy chain CDR sets (CDR1, CDR2, and CDR3) shown in SEQ ID Nos: 51, 52, and 53 ), and the light chain variable region CDRs set forth in SEQ ID Nos: 54, 55, and 56. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of: and a heavy chain variable region set forth in SEQ ID NO: 49. nothing.

[0231] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 58. , variable regions). The amino acid sequence of the heavy chain variable region (VH) of antibody 58 (i.e., Ab58) is shown in SEQ ID NO: 59 ( (See Table 9.) The amino acid sequence of the VH CDR domain of antibody 58 is set forth in SEQ ID NO: 61 (VH CDR1). shown in SEQ ID NO: 62 (VH CDR2) and SEQ ID NO: 63 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of 58 is shown in SEQ ID NO: 60 (see Table 9). The amino acid sequences of the VL CDR domains of 58 are set forth in SEQ ID NO: 64 (VL CDR1); SEQ ID NO: 65 (VL CDR2); The heavy chain constant region of antibody 58 is shown in SEQ ID NO: 65 (VL CDR2) and SEQ ID NO: 66 (VL CDR3). The light chain constant region of antibody 58 is shown in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen-binding portion thereof, is The variable heavy chain CDR sets (CDR1, CDR2, and CDR3) shown in SEQ ID Nos: 61, 62, and 63 ), and the light chain variable region CDRs set forth in SEQ ID Nos: 64, 65, and 66. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of: and a heavy chain variable region set forth in SEQ ID NO: 59. nothing.

[0232] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 61. , variable regions). The amino acid sequence of the heavy chain variable region (VH) of antibody 61 (i.e., Ab61) is shown in SEQ ID NO: 69 ( (See Table 9.) The amino acid sequence of the VH CDR domain of antibody 61 is set forth in SEQ ID NO: 71 (VH CDR1). ; shown in SEQ ID NO: 72 (VH CDR2) and SEQ ID NO: 73 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of antibody 61 is shown in SEQ ID NO: 70 (see Table 9). The amino acid sequences of the VL CDR domains of VL61 are shown in SEQ ID NO: 74 (VL CDR1); SEQ ID NO: 75 (VL CDR2); The heavy chain constant region of antibody 61 is shown in SEQ ID NO: 75 (VL CDR2) and SEQ ID NO: 76 (VL CDR3). The light chain constant region of antibody 61 is shown in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen-binding portion thereof, is The variable heavy chain CDR sets (CDR1, CDR2, and CDR3) shown in SEQ ID Nos: 71, 72, and 73 ), and the light chain variable region CDRs set forth in SEQ ID Nos: 74, 75, and 76. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of: and a heavy chain variable region set forth in SEQ ID NO: 69. nothing.

[0233] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 66. , variable regions). The amino acid sequence of the heavy chain variable region (VH) of antibody 66 (i.e., Ab66) is shown in SEQ ID NO: 79 ( (See Table 9.) The amino acid sequence of the VH CDR domain of antibody 66 is set forth in SEQ ID NO: 81 (VH CDR1). ; shown in SEQ ID NO: 82 (VH CDR2) and SEQ ID NO: 83 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of 66 is shown in SEQ ID NO: 80 (see Table 9). The amino acid sequences of the VL CDR domains of 66 are set forth in SEQ ID NO: 84 (VL CDR1); SEQ ID NO: 85 (VL CDR2); The heavy chain constant region of antibody 66 is shown in SEQ ID NO: 85 (VL CDR2) and SEQ ID NO: 86 (VL CDR3). The light chain constant region of antibody 66 is shown in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen-binding portion thereof, is The variable heavy chain CDR sets (CDR1, CDR2, and CDR3) shown in SEQ ID Nos: 81, 82, and 83 ), and the light chain variable region CDRs set forth in SEQ ID Nos: 84, 85, and 86. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of: and a heavy chain variable region set forth in SEQ ID NO: 79. nothing.

[0234] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 67. , variable regions). The amino acid sequence of the heavy chain variable region (VH) of antibody 67 is shown in SEQ ID NO: 9 (see Table 9). The amino acid sequences of the VH CDR domains of antibody 67 are SEQ ID NO: 11 (VH CDR1); SEQ ID NO: 12 ( The light chain variable region (VL) of antibody 67 is shown in SEQ ID NO: 13 (VH CDR2) and SEQ ID NO: 14 (VH CDR3). The amino acid sequence is shown in SEQ ID NO: 10 (see Table 9). The amino acid sequences are SEQ ID NO: 14 (VL CDR1); SEQ ID NO: 15 (VL CDR2) and SEQ ID NO: 16 (VL CDR3). The full-length heavy chain (HC) of antibody 67 is shown in SEQ ID NO: 110 (VL CDR3). The full-length heavy chain constant region of antibody 67 is shown in SEQ ID NO: 122. The light chain (LC) of antibody 67 is shown in SEQ ID NO: The light chain constant region of antibody 67 is shown in SEQ ID NO: 121. Thus, in certain embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO: The variable heavy chain CDR sets (CDR1, CDR2, and CDR3) shown in Q ID Nos. 11, 12, and 13, and the sequences In another embodiment, the light chain variable region CDRs are as set forth in SEQ ID Nos: 14, 15, and 16. The anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of the amino acid residues set forth in SEQ ID NO:9. and a heavy chain variable region as set forth in SEQ ID NO: 10. In embodiments, the anti-CD117 antibody has a heavy chain comprising SEQ ID NO:110 and a heavy chain comprising SEQ ID NO:111. NO):109, including light chains.

[0235] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 68. , variable regions). The amino acid sequence of the heavy chain variable region (VH) of antibody 68 (i.e., Ab68) is set forth in SEQ ID NO: 89 ( (See Table 9.) The amino acid sequence of the VH CDR domain of antibody 68 is set forth in SEQ ID NO: 91 (VH CDR1). ; shown in SEQ ID NO: 92 (VH CDR2) and SEQ ID NO: 93 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of 68 is shown in SEQ ID NO:90 (see Table 9). The amino acid sequences of the VL CDR domains of 68 are set forth in SEQ ID NO: 94 (VL CDR1); SEQ ID NO: 95 (VL CDR2); The heavy chain constant region of antibody 68 is shown in SEQ ID NO: 95 (VL CDR2) and SEQ ID NO: 96 (VL CDR3). The light chain constant region of antibody 68 is shown in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen-binding portion thereof, is The variable heavy chain CDR sets (CDR1, CDR2, and CDR3) shown in SEQ ID Nos: 91, 92, and 93 ), and the light chain variable region CDRs set forth in SEQ ID Nos: 94, 95, and 96. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of: and a heavy chain variable region set forth in SEQ ID NO: 89. nothing.

[0236] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 69. , variable regions). The amino acid sequence of the heavy chain variable region (VH) of antibody 69 (i.e., Ab69) is set forth in SEQ ID NO: 99 ( See Table 9. The amino acid sequence of the VH CDR domain of antibody 69 is set forth in SEQ ID NO: 101 (VH CDR1 ); shown in SEQ ID NO: 102 (VH CDR2) and SEQ ID NO: 103 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of antibody 69 is shown in SEQ ID NO: 100 (see Table 9). The amino acid sequences of the VL CDR domains of antibody 69 are set forth in SEQ ID NO: 104 (VL CDR1); The overexpression of antibody 69 is shown in SEQ ID NO: 105 (VL CDR2) and SEQ ID NO: 106 (VL CDR3). The light chain constant region of antibody 69 is shown in SEQ ID NO: 122. NO):121. Thus, in certain embodiments, an anti-CD117 antibody, or an antigen-binding The portion comprises the variable heavy chain CDR set (CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR19, CDR110, CDR111, CDR12, CDR13, CDR1 2, and CDR3), and the light chain variable region CDRs shown in SEQ ID Nos: 104, 105, and 106. In other embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 100, and a heavy chain set forth in SEQ ID NO: 99 variable region.

[0237] Furthermore, various results of the anti-CD117 antibodies Ab77, Ab79, Ab81, Ab85, Ab86, Ab87, Ab88, and Ab89 were obtained. The amino acid sequences of the junction regions are listed in Table 9.

[0238] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 77. , variable regions). (i.e., Ab77) heavy chain variable region (VH) amino acid sequence is shown in SEQ ID NO: 147 ( See Table 9. The amino acid sequence of the VH CDR domain of antibody 77 is SEQ ID NO: 263 (VH CD R1); shown in SEQ ID NO: 2 (VH CDR2) and SEQ ID NO: 3 (VH CDR3) The amino acid sequence of the light chain variable region (VL) of antibody 77 is shown in SEQ ID NO: 231 (Table 9). The amino acid sequence of the VL CDR domain of antibody 77 is set forth in SEQ ID NO: 264 (VL CDR1). , as shown in SEQ ID NO: 265 (VL CDR2), and SEQ ID NO: 266 (VL CDR3). The heavy chain constant region of antibody 77 is shown in SEQ ID NO: 269. The light chain constant region of antibody 77 is shown in SEQ ID NO: 269. It is set forth in SEQ ID NO: 283. Thus, in certain embodiments, anti-CD117, or The antigen-binding portion of the variable heavy chain CDR set (CDR1) shown in SEQ ID NOs: 263, 2 and 3 , CDR2 and CDR3), and the light chain variable region CDRs shown in SEQ ID NOs: 264, 265 and 266. In other embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 231, and a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 147 heavy chain variable region.

[0239] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 79. , variable regions). (i.e., Ab79) heavy chain variable region (VH) amino acid sequence is shown in SEQ ID NO: 147 ( See Table 9. The amino acid sequence of the VH CDR domain of antibody 79 is SEQ ID NO: 263 (VH CD R1); shown in SEQ ID NO: 2 (VH CDR2) and SEQ ID NO: 3 (VH CDR3) The amino acid sequence of the light chain variable region (VL) of antibody 79 is shown in SEQ ID NO: 233 (Table 9). The amino acid sequence of the VL CDR domain of antibody 79 is set forth in SEQ ID NO: 267 (VL CDR1). , as shown in SEQ ID NO: 265 (VL CDR2), and SEQ ID NO: 266 (VL CDR3). The heavy chain constant region of antibody 79 is shown in SEQ ID NO: 269. The light chain constant region of antibody 79 is shown in SEQ ID NO: 269. It is set forth in SEQ ID NO: 283. Thus, in certain embodiments, anti-CD117, or The antigen-binding portion of the variable heavy chain CDR set (CDR1) shown in SEQ ID NOs: 263, 2 and 3 , CDR2 and CDR3), and the light chain variable region CDRs shown in SEQ ID NOs: 267, 265 and 266 In other embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 233, and a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 147 heavy chain variable region.

[0240] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 81. , variable regions). (i.e., Ab81) heavy chain variable region (VH) amino acid sequence is shown in SEQ ID NO: 147 ( See Table 9. The amino acid sequence of the VH CDR domain of antibody 81 is SEQ ID NO: 263 (VH CD R1); shown in SEQ ID NO: 2 (VH CDR2) and SEQ ID NO: 3 (VH CDR3) The amino acid sequence of the light chain variable region (VL) of antibody 81 is shown in SEQ ID NO: 235 (Table 9). The amino acid sequence of the VL CDR domain of antibody 81 is set forth in SEQ ID NO: 264 (VL CDR1). , as shown in SEQ ID NO: 268 (VL CDR2), and SEQ ID NO: 266 (VL CDR3). The heavy chain constant region of antibody 81 is shown in SEQ ID NO: 269. The light chain constant region of antibody 81 is shown in SEQ ID NO: 269. It is set forth in SEQ ID NO: 283. Thus, in certain embodiments, anti-CD117, or The antigen-binding portion of the variable heavy chain CDR set (CDR1) shown in SEQ ID NOs: 263, 2 and 3 , CDR2 and CDR3), and the light chain variable region CDRs shown in SEQ ID NOs: 264, 268 and 266 In other embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 235, and a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 147 heavy chain variable region.

[0241] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 85. , variable regions). (i.e., Ab85) heavy chain variable region (VH) amino acid sequence is shown in SEQ ID NO: 243 ( See Table 9. The amino acid sequence of the VH CDR domain of antibody 85 is set forth in SEQ ID NO: 245 (VH CD R1); SEQ ID NO: 246 (VH CDR2), and SEQ ID NO: 247 (VH CDR3) The amino acid sequence of the light chain variable region (VL) of antibody 85 is shown in SEQ ID NO: 242 ( See Table 9. The amino acid sequence of the VL CDR domain of antibody 85 is set forth in SEQ ID NO: 248 (VL CDR). DR1), SEQ ID NO: 249 (VL CDR2), and SEQ ID NO: 250 (VL CDR3) The heavy chain constant region of antibody 85 is shown in SEQ ID NO: 269. The light chain constant region of antibody 85 is shown in SEQ ID NO: 270. The region is set forth in SEQ ID NO: 283. Thus, in certain embodiments, anti-CD117, or The antigen-binding portion comprises the variable heavy chain CDR sets shown in SEQ ID NOs: 245, 246 and 247. The light chain variable domains (CDR1, CDR2 and CDR3) are shown in SEQ ID NOs: 248, 249 and 250. In other embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 244, and a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 243 The heavy chain variable region is shown in

[0242] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 86. , variable regions). (i.e., Ab86) heavy chain variable region (VH) amino acid sequence is shown in SEQ ID NO: 251 ( See Table 9. The amino acid sequence of the VH CDR domain of antibody 86 is set forth in SEQ ID NO: 245 (VH CD R1); SEQ ID NO: 253 (VH CDR2), and SEQ ID NO: 3 (VH CDR3) The amino acid sequence of the light chain variable region (VL) of antibody 86 is shown in SEQ ID NO: 252 (Table 1). 9). The amino acid sequence of the VL CDR domain of antibody 86 is set forth in SEQ ID NO: 254 (VL CDR 1), SEQ ID NO: 249 (VL CDR2), and SEQ ID NO: 255 (VL CDR3). The heavy chain constant region of antibody 86 is shown in SEQ ID NO: 269. The light chain constant region of antibody 86 is shown in SEQ ID NO: 269. is shown in SEQ ID NO: 283. Thus, in certain embodiments, anti-CD117, or The antigen-binding portion comprises the variable heavy chain CDR sets (CDRs) shown in SEQ ID NOs: 245, 253 and 3. DR1, CDR2 and CDR3), and the light chain variable regions shown in SEQ ID NOs: 254, 249 and 255. In another embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: (S a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 252, and a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 251; and a heavy chain variable region.

[0243] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 87. , variable regions). (i.e., Ab87) heavy chain variable region (VH) amino acid sequence is shown in SEQ ID NO: 243 ( See Table 9. The amino acid sequence of the VH CDR domain of antibody 87 is set forth in SEQ ID NO: 245 (VH CD R1); SEQ ID NO: 246 (VH CDR2), and SEQ ID NO: 247 (VH CDR3) The amino acid sequence of the light chain variable region (VL) of antibody 87 is shown in SEQ ID NO: 256 ( See Table 9. The amino acid sequence of the VL CDR domain of antibody 87 is set forth in SEQ ID NO: 257 (VL CDR). SEQ ID NO: 5 (VL CDR1), SEQ ID NO: 5 (VL CDR2), and SEQ ID NO: 255 (VL CDR3). The heavy chain constant region of antibody 87 is shown in SEQ ID NO: 269. The light chain constant region of antibody 87 is shown in SEQ ID NO: 269. is shown in SEQ ID NO: 283. Thus, in certain embodiments, anti-CD117, or The antigen-binding portion comprises the variable heavy chain CDR sets shown in SEQ ID NOs: 245, 246 and 247. (CDR1, CDR2 and CDR3), and the light chain variable region shown in SEQ ID NOs: 257, 5 and 255 In another embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: (S a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 256, and a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 243; and a heavy chain variable region.

[0244] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 88. , variable regions). (i.e., Ab88) heavy chain variable region (VH) amino acid sequence is shown in SEQ ID NO: 258 ( See Table 9. The amino acid sequence of the VH CDR domain of antibody 88 is set forth in SEQ ID NO: 245 (VH CD R1); SEQ ID NO: 259 (VH CDR2), and SEQ ID NO: 3 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of antibody 88 is shown in SEQ ID NO: 256 (Table 1). 9). The amino acid sequence of the VL CDR domain of antibody 88 is set forth in SEQ ID NO: 257 (VL CDR 1), SEQ ID NO: 5 (VL CDR2), and SEQ ID NO: 255 (VL CDR3). The heavy chain constant region of antibody 88 is shown in SEQ ID NO: 269. The light chain constant region of antibody 88 is shown in SEQ ID NO: 269. It is set forth in SEQ ID NO: 283. Thus, in certain embodiments, anti-CD117, or The antigen-binding portion of the variable heavy chain CDR set (CDRs) shown in SEQ ID NOs: 245, 259 and 3 1, CDR2 and CDR3), and the light chain variable region CDRs shown in SEQ ID NOs: 257, 5 and 255 In other embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 256, and a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 258 heavy chain variable region.

[0245] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 89. , variable regions). (i.e., Ab89) heavy chain variable region (VH) amino acid sequence is shown in SEQ ID NO: 260 ( See Table 9). The amino acid sequence of the VH CDR domain of antibody 89 is set forth in SEQ ID NO:245 (VH CD R1); shown in SEQ ID NO: 2 (VH CDR2) and SEQ ID NO: 3 (VH CDR3) The amino acid sequence of the light chain variable region (VL) of antibody 89 is shown in SEQ ID NO: 252 (Table 9). The amino acid sequence of the VL CDR domain of antibody 89 is set forth in SEQ ID NO: 254 (VL CDR1). , as shown in SEQ ID NO: 249 (VL CDR2), and SEQ ID NO: 255 (VL CDR3). The heavy chain constant region of antibody 89 is shown in SEQ ID NO: 269. The light chain constant region of antibody 89 is shown in SEQ ID NO: 269. It is set forth in SEQ ID NO: 283. Thus, in certain embodiments, anti-CD117, or The antigen-binding portion of the variable heavy chain CDR set (CDR1) shown in SEQ ID NOs: 245, 2 and 3 , CDR2 and CDR3), and the light chain variable region CDRs shown in SEQ ID NOs: 254, 249 and 255 In other embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 252, and a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 260 heavy chain variable region.

[0246] In one embodiment, the present disclosure provides a binding region (e.g., CDRs) corresponding to the binding region of antibody 249. , variable region). The amino acid sequence of the heavy chain variable region (VH) of Ab249 (i.e., Ab249) is shown in SEQ ID NO: 238. (See Table 9.) The amino acid sequence of the VH CDR domain of antibody 249 is SEQ ID NO: 286 (VH CDR1); SEQ ID NO: 2 (VH CDR2), and SEQ ID NO: 287 (VH CDR3) The amino acid sequence of the light chain variable region (VL) of antibody 249 is shown in SEQ ID NO: 242 ( See Table 9. The amino acid sequence of the VL CDR domain of antibody 249 is set forth in SEQ ID NO: 288 (VL CDR1), SEQ ID NO: 249 (VL CDR2), and SEQ ID NO: 289 (VL CDR3) The heavy chain constant region of antibody 249 is shown in SEQ ID NO: 269. The light chain constant region of antibody 249 is shown in SEQ ID NO: 269. The region is set forth in SEQ ID NO: 283. Thus, in certain embodiments, anti-CD117, or an antigen-binding portion thereof comprising the variable heavy chain CDR sets shown in SEQ ID NOs: 286, 287 and 288; The light chain variable fragments (CDR1, CDR2 and CDR3) are shown in SEQ ID NOs: 288, 249 and 289. In other embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 242, and a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 238 The heavy chain variable region is shown in

[0247] Further, the present disclosure includes the binding regions (heavy and light chain Cs) set forth in SEQ ID NOs: 147 to 168. In one embodiment, the present invention includes an anti-CD117 antibody drug conjugate comprising a nucleotide sequence (e.g., a nucleotide sequence or a variable region) of the CD117 antibody. the anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO: 147 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 148. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 147 and the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 149 In one embodiment, the anti-CD117 antibody or its derivatives comprises a light chain variable region as shown in the sequence The original binding portion comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 147, and a heavy chain variable region shown in the sequence In one embodiment, the antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 150. The anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence set forth in SEQ ID NO:147. a heavy chain variable region as shown in Figure 1, and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 151; In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147, 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 antigen comprises a light chain variable region as shown in the sequence The binding moiety comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 147 and a heavy chain variable region shown in SEQ ID NO: 148. In one embodiment, the antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 153. The anti-CD117 antibody, or an antigen-binding portion thereof, is represented by the amino acid sequence of SEQ ID NO:147. a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 154; and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 154. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO: 0):147, and the heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:155 In one embodiment, the anti-CD117 antibody, or its antigen-binding domain, comprises a light chain variable region as shown in the sequence The fusion region is a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 147, and a heavy chain variable region shown in SEQ ID NO: 148. In one embodiment, the anti- The CD117 antibody, or an antigen-binding portion thereof, is shown in the amino acid sequence of SEQ ID NO:147. a heavy chain variable region and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 157. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of: a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 158; 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: 147, and a heavy chain variable region set forth in SEQ ID NO: In one embodiment, the anti-CD40 antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 159. The 117 antibody, or an antigen-binding portion thereof, has the amino acid sequence set forth in SEQ ID NO:147. a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 160, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 160. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence identified as SEQ ID NO: The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 147, and the amino acid sequence of SEQ ID NO: 161 In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region as shown in The heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 147, and the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 148. In one embodiment, the anti-CD11 antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 162. 7 antibody, or an antigen-binding portion thereof, has a heavy chain represented by the amino acid sequence of SEQ ID NO: 147 The antibody comprises a light chain variable region and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:163. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence set forth in SEQ ID NO: 16 The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 165 In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region as shown in The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 166 and the heavy chain variable region shown in SEQ ID NO: 167 are In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO:167. The antibody, or antigen-binding portion thereof, may comprise a heavy chain polypeptide as set forth in the amino acid sequence of SEQ ID NO:168. The light chain variable region is a light chain variable region represented by the amino acid sequence of SEQ ID NO: 169. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO: 170 and a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 171. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region. , a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 172, and In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO:173. The antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:174. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 175. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence of SEQ ID NO: 176. The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 177 In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region. The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 178, and the heavy chain variable region shown in SEQ ID NO: ):179. 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, the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 180 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 181. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, has the antigen of SEQ ID NO: 172. The heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 182. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a sequence The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 183, and SEQ ID NO: In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence of: or the antigen-binding portion thereof, the heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 185 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 186. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO:187. and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 188. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises the sequence The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 189, and the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 1 In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 90. the antigen-binding portion thereof comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 191; and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 192. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises the amino acid sequence of SEQ ID NO:193. and a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 194. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 195, and the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 196 In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence of The antigen-binding portion comprises a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 197, and and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 198. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises the amino acid sequence of SEQ ID NO: 199. The heavy chain variable region shown in the sequence and the light chain variable region shown in the amino acid sequence of SEQ ID NO: 200 In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises the sequence set forth in SEQ ID NO: a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 201, and a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 190; In one embodiment, the anti-CD117 antibody or its derivatives comprises a light chain variable region as shown in the amino acid sequence The antigen-binding portion of the heavy chain variable region of the present invention is shown in the amino acid sequence of SEQ ID NO: 202, and In one embodiment, the antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 203. In the present specification, an anti-CD117 antibody, or an antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO:204. The heavy chain variable region shown in the column and the light chain variable region shown in the amino acid sequence of SEQ ID NO: 205. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: (SE a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 206, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 207; In one embodiment, the anti-CD117 antibody or its derivatives comprises a light chain variable region as shown in the amino acid sequence The antigen-binding portion comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 208, and a In one embodiment, the antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 209. the anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO:210 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 211. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 212 and the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 213 In one embodiment, the anti-CD117 antibody or its derivatives comprises a light chain variable region as shown in the sequence The original binding portion comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 214, and a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 215. In one embodiment, the light chain variable region is set forth in the amino acid sequence of SEQ ID NO: 215. The anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence set forth in SEQ ID NO:216. a heavy chain variable region as shown in Figure 1, and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 217; In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises SEQ ID NO: a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 218, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 219; In one embodiment, the anti-CD117 antibody or its antigen comprises a light chain variable region as shown in the sequence The binding moiety comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 220 and a heavy chain variable region shown in SEQ ID NO: 230. In one embodiment, the antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 221. The anti-CD117 antibody, or an antigen-binding portion thereof, is shown in the amino acid sequence of SEQ ID NO:222. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 223. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO: 0):224, and the heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:225. In one embodiment, the anti-CD117 antibody, or its antigen-binding domain, comprises a light chain variable region as shown in the sequence The fusion region is a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 226, and a heavy chain variable region shown in SEQ ID NO: 227. In one embodiment, the anti- The CD117 antibody, or an antigen-binding portion thereof, is shown in the amino acid sequence of SEQ ID NO:147. a heavy chain variable region, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 228. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of: a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 229; 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: 147, and a heavy chain variable region set forth in SEQ ID NO: In one embodiment, the anti-CD40 antibody comprises a light chain variable region set forth in the amino acid sequence of EQ ID NO: 230. The 117 antibody, or an antigen-binding portion thereof, has the amino acid sequence set forth in SEQ ID NO:147. a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 231, In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence identified as SEQ ID NO: The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 147, and the amino acid sequence of SEQ ID NO: 232 In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region as shown in The heavy chain fragment is shown in the amino acid sequence of SEQ ID NO: 147. The light chain variable region is represented by the amino acid sequence of SEQ ID NO: 233. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO: 147 and the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 234. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region. , a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 148. In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO:235. The antibody, or an antigen-binding portion thereof, may comprise a heavy chain variable domain as set forth in the amino acid sequence of SEQ ID NO:147. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:236.

[0248] In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO: 0):147, and the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:237 In one embodiment, the anti-CD117 antibody, or its antigen-binding domain, comprises a light chain variable region as shown in the sequence The fusion region is a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 243, and a heavy chain variable region shown in SEQ ID NO: 244. In one embodiment, the anti- The CD117 antibody, or an antigen-binding portion thereof, is shown in the amino acid sequence of SEQ ID NO:251. a heavy chain variable region, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 252. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of: a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 243, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 256; 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: 258, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: In one embodiment, the anti-CD40 antibody comprises a light chain variable region set forth in the amino acid sequence of EQ ID NO: 256. The 117 antibody, or an antigen-binding portion thereof, has the amino acid sequence set forth in SEQ ID NO:260. a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 252, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 252. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence identified as SEQ ID NO: The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 238, and the amino acid sequence of SEQ ID NO: 239 In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region as shown in The heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 147, and the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO: 148. In one embodiment, the anti-CD11 antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 239. 7 antibody, or an antigen-binding portion thereof, has a heavy chain represented by the amino acid sequence of SEQ ID NO: 147 The variable region comprises a light chain variable region and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO:240. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO:23 The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 8, and the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 241 In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region as shown in The heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 238 and the heavy chain variable region shown in SEQ ID NO: 1 D NO):242.

[0249] Certain of the anti-CD117 antibodies described in this application are characterized in that the antibodies inhibit CD117 activity on CD117-expressing cells. It is a neutral antibody in that it does not substantially inhibit the activity of the antibody. The antibody can be used, for example, in an in vitro stem cell factor (SCF)-dependent cell proliferation assay (e.g., , see Example 11 described in this application). In cell proliferation assays, neutral CD117 antibodies kill CD34+ cells that divide in an SCF-dependent manner. This is unlikely because neutral antibodies inhibit the binding of SCF to CD117. This is because it blocks CD117 and does not inhibit CD117 activity.

[0250] Neutral antibodies are those that can specifically bind to human CD117. In view of this, the cytotoxins described in this application may be used for diagnostic purposes. It is also effective in killing CD117-expressing cells when conjugated to a cytotoxin. Typically, the antibody used in the conjugate has an agonist activity specific to the antibody. However, the present application describes a method for treating congenital heart disease. This is a unique approach to the development of conjugates, and in particular, the conjugates can be used to treat stem cells. This is an approach in the context of using it as a conditioning agent before cell transplantation. Antagonist antibodies alone or in combination with cytotoxins as conjugates may be used in combination with cytotoxins. On the other hand, neutral anti-CD117 antibodies may be effective if the antibody alone has the cytotoxicity. Conditioning with a conjugate containing the antibody inhibits the activity of the cytotoxin. Secondary to the effect, the antibody's internalization and affinity properties (e.g. Another alternative is to use a cytotoxin containing a hydroxyl group (hydroxyl group, dissociation rate, etc.) when the hydroxyl group is important for effective delivery of the cytotoxin. Present an alternative strategy.

[0251] Examples of neutral anti-CD117 antibodies include Ab58, Ab61, Ab66, Ab67, Ab68, and Ab69. Comparison of the amino acid sequences of the CDRs of neutral antibodies revealed that the CDRs identified by the anti-CD117 antibody CDRs A consensus sequence between two groups of neutral antibodies is revealed: Ab58 and Comparison of the heavy and light chain variable regions of Ab61 and Ab62 is described in PCT / US2018 / 057172. and is incorporated by reference in its entirety. Ab58 and Ab61 have the same light chain CDRs and HC CD They share R3 and have slight differences in HC CDR1 and HC CDR2. Consensus of HC CDR1 and CDR2 The sequences are set forth in SEQ ID Nos: 133 and 134. Ab66, Ab67, Ab68, and Ab69 are also These antibodies are neutral antibodies. The heavy and light chain variable regions of these antibodies are: No. PCT / US2018 / 057172, which is incorporated by reference in its entirety. Although Ab68 and Ab69 share the same light chain CDR and the same HC CDR3, these antibodies have There are differences within the HC CDR1 and HC CDR2 regions of these. The consensus sequences of the antibodies are set forth in SEQ ID Nos: 139 and 140, respectively.

[0252] Antagonist antibodies, such as Ab54, Ab55, Ab56, and Ab57, are also described in this application. A comparison of the amino acid sequences of the variable heavy and light chains of these antibodies is provided in PCT / US2018057172 (incorporated by reference in its entirety). 57 share the same light chain CDR and the same HC CDR3, but these antibodies have the same HC CDR1 There are differences in the HC CDR1 and HC CDR2 regions. The census sequences are set forth in SEQ ID Nos: 127 and 128, respectively.

[0253] The anti-CD117 antibodies described in this application may be full-length antibodies, bispecific antibodies, or the like. antibody), dual variable domain antibody, multi-chain or single-chain antibody, and / or specific for human CD117 These may be in the form of binding fragments that bind to the , scFv (single chain Fv), surrobodies (including surrogate light chain constructs), single domain antibodies , camelized antibodies, etc. They also include, for example, Ig A (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or Ig It may be of any isotype, such as M, or may be derived from In some embodiments, the anti-CD117 antibody is an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4). be.

[0254] Antibodies for use in conjunction with the methods described in this application include antibodies containing an Fc domain or Deleting antibody fragments, as well as humanized variants of the non-human antibodies described in this application and one or more CDRs or equivalent regions thereof of the antibodies or antibody fragments described in this application. antibody-like protein scaffolds containing the above, or all of the above (e.g., 10 Fn3 domain) Exemplary antigen-binding fragments of the above antibodies include: Examples include, inter alia, dual-variable immunoglobulin domains (DVIGs) lin domain), single-chain Fv molecules (scFv), diabodies, triabodies, nanobodies, antibody-like Protein scaffolds, Fv fragments, Fab fragments, F(ab')2 molecules, and proteins dem di-scFv.

[0255] In one embodiment, an anti-CD117 antibody is provided that comprises one or more radiolabeled amino acids. Radiolabeled anti-CD117 antibodies can be used for both diagnostic and therapeutic purposes (radiolabeled Conjugation to radiolabeled molecules is another possible feature. Non-limiting examples of peptide labeling include: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc and 12 5 I, 131 I and 186 Radiolabeled amino acids and related Methods for preparing derivatives of related peptides are known in the art (see, for example, Junghans et al., Cancer Chemotherapy and Biotherapy 655-686, 2d ed., Chafner and Longo (eds.), Lippin Cott Raven (1996) and U.S. Patent No. 4,681,581, U.S. Patent No. 4,735,210, U.S. Patent No. 5,10 No. 1,827, U.S. Patent No. 5,102,990 [US RE35,500], U.S. Patent No. 5,648,471, U.S. Patent (See US Pat. No. 5,697,902.) For example, radioisotopes can be bound by the chloramine T method. There are cases where they are combined.

[0256] The anti-CD117 antibodies or binding fragments described in this application may also be used as antibodies and / or fragments of Modifications and / or mutations (e.g., as known to those skilled in the art) that change the properties of the fragment (including those that increase half-life, increase or decrease ADCC, etc.) This sometimes happens.

[0257] In one embodiment, the anti-CD117 antibody, or binding fragment thereof, is a variant ( or modified) Fc region, wherein the variant Fc region has at least one Fc region having ... at least one amino acid modification such that the molecule has altered affinity for FcγR Specific amino acid positions within the Fc region have been identified by crystallographic studies in 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). In some embodiments, The anti-CD117 antibodies described in the application are directly linked to FcγRs based on structural and crystallographic analysis. The Fc region may comprise a variant Fc region in which at least one residue in the Fc region that contacts the Fc region is altered. In embodiments, the Fc region of the anti-CD117 antibody (or fragment thereof) is selected from the group consisting of the Fc region of the anti-CD117 antibody (or fragment thereof) and the Fc region of the anti-CD117 antibody (or fragment thereof) described in Kabat et al., Se Quences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH 1, MD (1991) (expressly incorporated herein by reference) The "EU index as in Kabat" is refers to the numbering of human IgG1 EU antibodies. The EU numbering system refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, which is incorporated herein by reference in its entirety. In one embodiment, the Fc region comprises a D265A mutation. In another embodiment, the Fc region comprises a D265C mutation. In some embodiments, the Fc region of the anti-CD117 antibody (or fragment thereof) is In one embodiment, the amino acid substitution at amino acid 234 according to the EU index is: The Fc region comprises a L234A mutation. In some embodiments, the anti-CD117 antibody (or its The Fc region of the IgG1A-specific Fc-like peptide fragment is located at amino acid 235 according to the EU index as in Kabat. In one embodiment, the Fc region comprises a L235A mutation. In one embodiment, the Fc region comprises the L234A and L235A mutations. The Fc region of the antibody for the ADC described contains D265C, L234A, and L235A mutations.

[0258] In certain embodiments, the variant IgG Fc domain comprises one or more amino acid substitutions. Decreased binding affinity to FcγR and / or C1q compared to wild-type Fc domains containing The Fc binding interaction may be achieved by one or more amino acid substitutions that result in the Fc binding interaction being either absent or absent. Various effector functions and downstream signaling events (e.g., antibody-dependent cell-mediated cytotoxicity) Antibody dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell damage These include, but are not limited to, complement dependent cytotoxicity (CDC). Therefore, in certain embodiments, antibodies (e.g., antibodies that contain an altered Fc region) For example, mutations L234A, L235A, and D265C result in a substantial reduction in effector function. Or disappear.

[0259] Affinity for the Fc region can be determined using various techniques known in the art (e.g., limited However, equilibrium methods (e.g., enzyme-linked immunosorbent assay) bent assay (ELISA));KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 3 73:52-60, 2008; radioimmunoassay (RIA) or surface plasmon kinetics-based assays (e.g., BIACORE®) TM analysis or Octet TM analysis (forteBIO)), as well as other methods [indirect binding assay, competitive binding assay, B. Fluorescence resonance energy transfer (FRET), gel electrolysis can be measured using electrophoresis and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more components of interest, and / or various detection methods (e.g., but not limited to, chromogenic labels, fluorescent labels, Fluorescent or isotopic labels may be used to determine binding affinity and kinetics. For a detailed description of this topic, see Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott t-Raven, Philadelphia (1999) (which focuses on antibody-immunogen interactions) An example of a competitive binding assay is a radioimmunoassay. Immunoassays involve the combination of a labeled antigen and the antibody of interest with increasing amounts of unlabeled antigen. and detecting the antibody bound to the labeled antigen. From the data, Scatchard plot analysis was performed to identify specific antigens. The affinity and binding off-rate of the antibody of interest for the second Competition with antibodies may also be measured using radioimmunoassays. The antigen is then mixed with an antibody of interest conjugated to a labeled compound and increasing amounts of an unlabeled second antibody. The mixture is incubated in the presence of the

[0260] The antibodies of the present disclosure can be further modified to produce 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: 175 9-69), (Datta-Mannan et al. (2007) Drug Metab Dispos 35: 86-94), (Vaccaro et al. (2 005) Nat Biotechnol 23: 1283-8), (Yeung et al. (2010) Cancer Res 70: 3269-77) and ( Further Fc mutations, such as those described in Kim et al. (1999) Eur J Immunol 29: 2819-25, may be introduced. By incorporating the modified Fc region, the half-life of the antibody can be increased (e.g., compared to an antibody with an unmodified Fc region). (Compared to the previous example) may be further adjusted, as well as positions 250, 252, 253, 254, 256, 257, 307, 376 , 380, 428, 434, and 435. Exemplary compounds that may be produced alone or in combination include: The most common mutations were T250Q, M252Y, 1253A, S254T, T256E, P2571, T307A, D376V, E380A, M428L, These mutations are H433K, N434S, N434A, N434H, N434F, H435A, and H435R.

[0261] Thus, in one embodiment, the Fc region comprises a mutation that results in a decreased half-life. Antibodies with short half-lives are useful in certain situations where the antibody is expected to function as a short-lived therapeutic agent. Examples of such antibodies include those described in the present application, in which the antibodies are administered followed by HSC administration. Ideally, unlike endogenous stem cells, Before administering HSCs, which also generally express CD117 but are not the target of anti-CD117 antibodies In one embodiment, the antibody should be substantially cleared. The region contains a mutation at position 435 (EU index as in Kabat). , the mutation is H435A mutation.

[0262] In one embodiment, the anti-CD117 antibodies described in the present application have a half-life of about 24 hours or less, a half-life of about 2 Half-life of less than 2 hours, half-life of less than about 21 hours, half-life of less than about 20 hours, half-life of less than about 19 hours Decay life, Half-life of approximately 18 hours or less, Half-life of approximately 17 hours or less, Half-life of approximately 16 hours or less, Approximately 15 hours a half-life of about 14 hours or less, a half-life of about 13 hours or less, a half-life of about 12 hours or less, In one embodiment, the compound has a half-life of about 11 hours or less, or a half-life of about 10 hours or less. The half-life of the antibody is about 11 hours to about 24 hours; about 12 hours to about 22 hours; about 10 hours to about 20 hours. about 8 hours to about 18 hours; or about 14 hours to about 24 hours. The anti-CD117 antibodies described herein may be used in a range of treatments, including treatments for about 1 to 5 hours, about 5 to 10 hours, about 10 to 15 hours, It has a half-life (eg, in humans) of about 15 to 20 hours, or about 20 to 25 hours.

[0263] In some embodiments, the Fc region confers a reduced half-life and enhances the efficacy of the antibody. Contains two or more mutations that significantly reduce or completely eliminate the function of the receptor. In this embodiment, the Fc region contains mutations that result in decreased half-life, and (e.g., structural and at least one residue that can make direct contact with FcγR (based on crystallographic analysis). In one embodiment, the Fc region comprises a H435A mutation, a L234A mutation, and a L235A mutation. In one embodiment, the Fc region comprises the mutations H435A and D265C. In embodiments, the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, and a D265C mutation. .

[0264] In some embodiments, the anti-CD117 antibody or antigen-binding fragment thereof is via cysteine ​​residues in the Fc domain of the antibody or antigen-binding fragment thereof. In some embodiments, the antibody is conjugated to a toxin (e.g., an amatoxin). The cysteine ​​residue is mutated in the Fc domain of the antibody or antigen-binding fragment thereof. For example, the cysteine ​​residues are introduced by Cys118, Cys239, and Cys265. In one embodiment, the anti-CD117 antibody (or a flag thereof) may be selected from the group consisting of: The Fc region of the IgG1 gene is the amino acid sequence at amino acid 265 according to the EU index as per Kabat. In one embodiment, the Fc region comprises a D265C mutation. The Fc region comprises a D265C and a H435A mutation. In one embodiment, the Fc region comprises the mutations D265C, L234A, L235A, and L234B. and H435A mutation. In one embodiment, the anti-CD117 antibody, or its antigen-binding fragment, The Fc region of this fragment is at amino acid 239 according to the EU index as per Kabat. In one embodiment, the Fc region comprises a S239C mutation. In another embodiment, the Fc region comprises a L234A mutation, a L235A mutation, a S239C mutation, and a D265A mutation. In one embodiment, the Fc region comprises the S239C and H435A mutations. In yet another embodiment, the Fc region comprises a L234A mutation, a L235A mutation, and a S239C mutation. In yet another embodiment, the F The c region contains the H435A mutation, the L234A mutation, the L235A mutation, the S239C mutation, and the D265A mutation.

[0265] In particular, Fc amino acid positions refer to the EU numbering index unless otherwise indicated. do.

[0266] In some embodiments of these aspects, the cysteine ​​residue is is naturally present in the Fc domain of the antigen-binding fragment. For example, the Fc domain may be an IgG Fc domain, such as a human IgG1 Fc domain, and the cysteine ​​residue is C It may be selected from the group consisting of Cys261, Csy321, Cys367, and Cys425.

[0267] For example, in one embodiment, the Fc region of antibody 67 is modified to include a D265C mutation. (e.g., SEQ ID NO: 111). In another embodiment, the Fc region of antibody 67 comprises D26 5C, L234A, and L235A mutations (e.g., SEQ ID NO: 112). In yet another embodiment, the Fc region of antibody 67 is modified to include D265C and H435A mutations. (e.g., SEQ ID NO: 113). In a further embodiment, the Fc region of antibody 67 is modified to include D265C, L234A, L235A, and H435A mutations (e.g., SEQ ID NO: (SE Q ID NO):114).

[0268] With respect to antibody 55, in one embodiment, the Fc region of antibody 55 is modified to include a D265C mutation. (e.g., SEQ ID NO: 117). In another embodiment, the Fc region of antibody 55 is , D265C, L234A, and L235A mutations (e.g., SEQ ID NO: 118). In yet another embodiment, the Fc region of antibody 55 is configured to contain D265C and H435A mutations. In a further embodiment, the Fc of antibody 55 is modified (e.g., SEQ ID NO: 119). The region is modified to include D265C, L234A, L235A, and H435A mutations (e.g., SEQ ID NO: No. (SEQ ID NO: 120).

[0269] Antibody 54, antibody 55, antibody 56, antibody 57, antibody 58, antibody 61, antibody 66, antibody 67, antibody 68, or antibody The Fc region of any one of antibodies 69 contains a D265C mutation (e.g., SEQ ID NO: 123). D265C, L234A, and L235A mutations (e.g., in the case of SEQ ID NO: 124); D265C and H435A mutation (e.g., in the case of SEQ ID NO: 125); or D265C, L234A, L235 A, and H435A mutation (e.g., in the case of SEQ ID NO: 126). obtain.

[0270] The variant Fc domains described in this application are characterized according to the amino acid modifications that make them up: All amino acid substitutions discussed in this application with respect to the Fc region are numbered as follows: The sequence always follows the EU index. Thus, for example, D265C is an E An Fc variant in which the aspartic acid (D) at U position 265 is replaced with a cysteine ​​(C). For example, D265C / L234A / L235A replaces EU positions 265 (D to C), 234 ( 235 (L to A), and 235 (L to A). Also, the variant EU amino acid position is designated according to its final amino acid composition. For example, the L234A / L235A mutation is sometimes called LALA. The order of substitutions is arbitrary. Please note that

[0271] In one embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, is Identifies at least about 90%, about 95%, about 96%, about 97%, about 98%, or more identical to the disclosed SEQ ID Nos. or the anti-CD117 antibody, or antigen-binding fragments thereof, having at least the same sequence as the SEQ ID Nos. disclosed in the present application. and having an amino acid sequence that is about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of the present invention. The variable region framework regions described in the application are shown in SEQ ID NO: s).

[0272] In certain embodiments, the anti-CD117 antibody, or antigen-binding fragment thereof, is a conjugate. It has a specific dissociation rate that is particularly useful when used as part of a gate. For example, In certain embodiments, the anti-CD117 antibody is directed against human CD117 and / or rhesus CD117. and 1×10 as measured by biolayer interferometry (BLI). -2 From 1×10 -3 , 1×10 -3 From 1×10 -4 , 1×10 -5 From 1×10 -6 , 1×10 -6 From 1×10 -7 or 1 x 10 -7 From 1×10 -8 Dissociation of In some embodiments, the antibody or antigen-binding fragment thereof has a rate constant (Koff). Concentrations are measured by Bio-Layer Interferometry (BLI) assays. 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM and CD117 (e.g., human CD117 and / or binds to rhesus monkey CD117).

[0273] The antibodies, and binding fragments thereof, disclosed in this application are described in more detail below. As indicated, it may be used as a conjugate.

[0274] 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 in the present application may be produced using Such nucleic acids are provided which encode the amino acid sequence comprising the VL of the antibody. amino acid sequence and / or amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody) In a further embodiment, one or more vectors comprising such nucleic acids may encode In a further embodiment, such a nucleic acid is provided. In one such embodiment, the host cell comprises: (e.g., transformed with): (1) an amino acid sequence containing the VL of the present antibody and the present antibody (2) a vector containing a nucleic acid encoding an amino acid sequence containing the VH of the antibody; a first vector containing a nucleic acid encoding an amino acid sequence comprising the VH of the antibody; a second vector comprising a nucleic acid encoding the sequence. In one embodiment, the host cell is a eukaryotic Organisms, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0 In one embodiment, a method for producing an anti-CLL-1 antibody is provided, wherein The method comprises inoculating a host cell containing nucleic acid encoding an antibody as provided above into the Culturing, and optionally culturing, the host cells (or and recovering the antibody from the host cell culture medium.

[0275] To recombinantly produce an anti-CD117 antibody, a nucleic acid encoding the antibody (e.g., as described above) is prepared. Such nucleic acids are isolated and further cloned and / or expressed in host cells. Such nucleic acids can be inserted into one or more vectors using conventional techniques (e.g., For example, oligonucleotides capable of specifically binding to the genes encoding the heavy and light chains of the antibody. Can be easily isolated and sequenced (by using probes) It is possible.

[0276] Suitable host cells for cloning or expressing antibody-encoding vectors include For example, antibodies may be prepared from a variety of cells, including prokaryotic or eukaryotic cells, as described herein. It may also be produced in bacteria when ligation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g. See Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), See also pp. 245-254, which describes the expression of antibody fragments in E. coli. After expression, the antibody is extracted from the bacterial cell paste in a soluble fraction. It may be isolated and further purified.

[0277] Vertebrate cells can also be used as hosts, e.g., grown in suspension. Other examples of useful mammalian host cell lines include: SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney cell line (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., Mather, Biol. Reprod. 23:243-2 TM4 cells as described in

[51] (1980); monkey kidney cells (CV1); African green monkey kidney cells cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat Human liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse breast cancer (MMT 060 562); TRI cells (described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); Other useful mammalian host cell lines include: Chinese hamster ovary (CHO) cells (DHFR-CHO cells (Urlaub et al., Proc. Natl. Aca d. Sci. USA 77:4216 (1980)); and myeloma cell lines (e.g., Y0, NS0, and Sp2 / 0 A review of certain mammalian host cell lines suitable for antibody production is available at For more information, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BK C Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0278] In one embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, is at least about 90%, about 95%, about 96%, about 97%, about 98% identical to the SEQ ID Nos. or a variable region having an amino acid sequence that is about 99% identical to said anti-CD117 antibody. or antigen-binding fragments thereof, may have at least one sequence identified as a SEQ ID Nos. disclosed herein. have an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical , SEQ ID NO: (S Contains CDRs containing E...

Claims

1. A method for administering genetically modified stem cells to a human subject in need thereof, comprising administering the genetically modified stem cells to a human subject in need thereof. The method includes: a) Binding to cell surface molecules expressed on hematopoietic stem cells (HSCs) and / or immune cells administering to the human subject an antibody-drug conjugate (ADC) comprising the antibody-drug conjugate (ADC) Depleting HSCs and / or immune cells from a human subject; and b) administering to said human subject a graft comprising a population of genetically modified stem cells.

2. A method of treating a human subject using genetically modified cells, said method comprising: administering a transplant comprising the genetically modified stem cell population to a human subject in need thereof; wherein the human subject is and antibody-drug conjugates (ADCs) that bind to cell surface molecules that are I have received gambling treatment.

3. 3. The method of claim 1 or 2, wherein the genetically modified stem cells are autologous stem cells. is.

4. 3. The method of claim 1 or 2, wherein the genetically modified stem cells are allogeneic stem cells. It is a cell.

5. 3. The method of claim 1 or 2, wherein the genetically modified stem cells are HSCs.

6. 6. The method of claim 1, wherein the subject is diagnosed with cancer, abnormal hemoglobin, or suffer from a vascular disease, myelodysplastic disorder, immunodeficiency disorder, or metabolic disorder.

7. 7. The method of claim 6, wherein the hemoglobinopathy disorder is sickle cell anemia, Racemia, Fanconi anemia, aplastic anemia, or Wiskott-Aldrich syndrome, Any one or more of the following is selected.

8. 7. The method of claim 6, wherein the immunodeficiency disorder is a congenital immunodeficiency disorder or an acquired immunodeficiency disorder. It's called epidemic deficiency.

9. 7. The method of claim 6, wherein the acquired immunodeficiency disease is caused by human immunodeficiency virus or Acquired immunodeficiency syndrome (AIDS).

10. 7. The method of claim 6, wherein the metabolic disorder is glycogen storage disease, mucopolysaccharidosis, Gaucher disease, Hurler disease, sphingolipidosis, globoid cell leukodystrophy one or more of the following: id cell leukodystrophy, or metachromatic leukodystrophy are selected.

11. 7. The method of claim 6, wherein the cancer is leukemia, lymphoma, multiple myeloma, or Neuroblastoma.

12. 7. The method of claim 6, wherein the cancer is a hematological cancer.

13. 13. The method of claim 12, wherein the hematological cancer is acute myeloid leukemia, acute lymphoblastic leukemia, or leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, or multiple myeloma.

14. 6. The method of any one of claims 1 to 5, wherein the subject is adenosine deaminase (ADDM)-dependent neurotransmitter. enzyme deficiency, severe combined immunodeficiency, hyperimmunoglobulin M syndrome, Chediak-Higashi disease , hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major , systemic sclerosis, systemic lupus erythematosus, multiple sclerosis and juvenile rheumatoid arthritis. There are.

15. 6. The method of any one of claims 1 to 5, wherein the subject is suffering from an autoimmune disorder. is doing.

16. 16. The method of claim 15, wherein the autoimmune disorder is multiple sclerosis, human systemic encephalopathy, Treatment of rheumatoid arthritis, inflammatory bowel disease, psoriasis, type 1 diabetes (type 1 diabetes), acute Disseminated encephalomyelitis, Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid syndrome, Aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune Epidemic lymphoproliferative syndrome, autoimmune oophoritis, Barrow's disease, Behçet's disease, bullous pemphigoid , cardiomyopathy, Chagas disease, chronic fatigue immune deficiency syndrome, chronic inflammatory demyelinating polyneuropathy -, Crohn's disease, cicatricial pemphigoid, celiac sprue dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythematosus, autonomic neuropathy, endometriosis, essential Mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome, Gray's syndrome Busu disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute hematoma Thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis , Kawasaki disease, Lichen planus, Lyme disease, Meniere's disease, Mixed connective tissue disease, Myasthenia gravis, Neuromuscular atrophy - Myotonia, opsoclonus-myoclonus syndrome, optic neuritis, ordothyroiditis (Ord's thyroiditis), pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyopathy inflammation, primary biliary cirrhosis, polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica , primary agammaglobulinemia, Raynaud's phenomenon, Raynaud's syndrome Sjogren's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff neck Parson's syndrome, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, Select one or more of the following: vulvodynia, chronic granulomatous disease, or Wegener's granulomatosis It is selected.

17. 17. The method of any one of claims 1 to 16, wherein the population of stem cells is a target It has been genetically modified to alter the target gene.

18. 18. The method of claim 17, wherein the target gene is beta-globin, cancer Maglobin, adenosine deaminase, arylsulfatase A, WASp gene, phagocytosis Cellular NADPH oxidase, galactosyl ceramidase, beta- Galactosidase, beta-hexosaminidase, alpha-L iduronidase, ATM serine / Threonine kinase, ribosome maturation protein SBDS, or CCR5 Selected from above.

19. 19. The method of any one of claims 1 to 18, wherein the population of genetically modified stem cells The grafts containing the gene were modified using a gene editing system.

20. 20. The method of claim 19, wherein the gene editing system is a CRISPR / Cas system. be.

21. 21. The method of any one of claims 1 to 20, wherein the ADC is selected from the group consisting of CD2, CD5, CD7, CD wl2, CD13, CD15, CD19, CD21, CD22, CD29, CD30, CD33, CD34, CD36, CD38, CD40, CD4 1, CD42a, CD42b, CD42c, CD42d, CD43, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD48, CD49b, CD49d, CD49e, CD49f, CD50, CD53, CD55, CD64a, CD68, CD71, CD72, CD73, CD 81, CD82, CD85A, CD85K, CD90, CD99, CD104, CD105, CD109, CD110, CD111, CD112, CD 114, CD115, CD117, CD123, CD124, CD126, CD127, CD130, CD131, CD133, CD135, CD137 , CD138, CD151, CD157, CD162, CD164, CD168, CD172a, CD173, CD174, CD175, CD175s, CD176, CD183, CD191, CD200, CD201, CD205, CD217, CD220, CD221, CD222, CD223, CD 224, CD225, CD226, CD227, CD228, CD229, CD230, CD235a, CD235b, CD236, CD236R, CD 238, CD240, CD242, CD243, CD277, CD292, CDw293, CD295, CD298, CD309, CD318, CD32 4, one or more cell surface molecules selected from CD325, CD338, CD344, CD349, or CD350. and antibodies or antigen-binding fragments thereof that bind to the

22. 21. The method of any one of claims 1 to 20, wherein the ADC binds to CD117. An antibody or antigen-binding fragment thereof.

23. 21. The method of any one of claims 1 to 20, wherein the ADC is administered to the subject by CD1 The antibody is administered in an amount sufficient to reduce the population of 17+ cells.

24. 23. The method of claim 22, wherein the CD117 is GNNK+ CD117.

25. 23. The method of claim 22, wherein the anti-CD117 antibody or antigen-binding fragment thereof includes: (a) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 32, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 33; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 34, an amino acid sequence set forth in SEQ ID NO: 35, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 36; a light chain variable region comprising a CDR3 domain containing (b) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 21, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 23; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 24, an amino acid sequence set forth in SEQ ID NO: 25, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 26; a light chain variable region comprising a CDR3 domain containing (c) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 41, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 42, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 43; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 44, an amino acid sequence set forth in SEQ ID NO: 45, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 46; a light chain variable region comprising a CDR3 domain containing (d) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 51, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 52, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 53; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 54, an amino acid sequence set forth in SEQ ID NO: 55, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 56; a light chain variable region comprising a CDR3 domain containing (e) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 61, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 62, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 63; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 64, an amino acid sequence set forth in SEQ ID NO: 65, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 66; a light chain variable region comprising a CDR3 domain containing (f) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 71, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 72, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 73; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 74, an amino acid sequence set forth in SEQ ID NO: 75, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 76; a light chain variable region comprising a CDR3 domain containing (g) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 81, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 82, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 83; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 84, an amino acid sequence set forth in SEQ ID NO: 85, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 86; a light chain variable region comprising a CDR3 domain containing (h) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 14, an amino acid sequence set forth in SEQ ID NO: 15, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 16; a light chain variable region comprising a CDR3 domain containing (i) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 91, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 92, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 93; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 94, an amino acid sequence set forth in SEQ ID NO: 95, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 96; a light chain variable region comprising a CDR3 domain containing (j) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 101, SEQ ID NO: SEQ ID NO: 102, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 10 a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:3; a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 104, a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 105, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:

106. a light chain variable region comprising a CDR3 domain comprising the sequence (k) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 245, SEQ ID NO: SEQ ID NO: 246, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 24 a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 7; a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 248, a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 249, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:

250. a light chain variable region comprising a CDR3 domain comprising the sequence

26. 23. The method of claim 22, wherein the anti-CD117 antibody or antigen-binding fragment thereof teeth, CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 127, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 128, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 129 a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 130, SEQ ID NO: 131; and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:

132. and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence.

27. 23. The method of claim 22, wherein the anti-CD117 antibody or antigen-binding fragment thereof includes: (a) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 133, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 134, and SEQ ID NO: 135; a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 136, a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 137, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:

138. a light chain variable region comprising a CDR3 domain comprising a sequence; or (b) a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 139, SEQ ID NO: a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 140, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 141 a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 142, a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 143, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:

144. The light chain variable region includes a CDR3 domain, which includes a sequence.

28. 23. The method of claim 22, wherein the anti-CD117 antibody or antigen-binding fragment thereof includes: (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 30; (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 20; (c) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 39, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40; (d) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 49, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 50; (e) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 59, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 60; (f) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 69, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70; (g) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 79, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 80; (h) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence set forth in (SEQ ID NO): 10; (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 90; (j) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 99, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 100; or (k) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 243, and A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

244.

29. 29. The method of any one of claims 22 to 28, wherein the anti-CD117 antibody or its antibody The original binding fragments were identified by bio-layer interferometry (BLI). When measured, it is 1×10 -2 From 1×10 -3 , 1×10 -3 From 1×10 -4 , 1×10 -5 From 1×10 -6 , 1×1 0 -6 From 1×10 -7 , or 1 × 10 -7 From 1×10 -8 , the dissociation rate (K OFF )

30. 29. The method of any one of claims 22 to 28, wherein the antibody or its antigen-binding Fragments are measured by bio-layer interferometry (BLI) 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less Lower, about 4 nM or less, about 2 nM or less, about 1 nM or less, K D and binds to CD117.

31. 29. The method of any one of claims 21 to 28, wherein the antibody or its antigen-binding The fragment is human.

32. 29. The method of any one of claims 21 to 28, wherein the antibody or its antigen-binding The fragment is an intact antibody.

33. 29. The method of any one of claims 21 to 28, wherein the antibody or its antigen-binding The fragment is an IgG.

34. 29. The method of any one of claims 21 to 28, wherein the antibody or its antigen-binding The fragment is an IgG1 or IgG4.

35. 29. The method of any one of claims 21 to 28, wherein the antibody or its antigen-binding The fragment is a monoclonal antibody.

36. 29. The method of any one of claims 21 to 28, wherein the antibody or its antigen-binding The fragment comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO:

122. and / or a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO:

121. nothing.

37. 29. The method of any one of claims 21 to 28, wherein the antibody or its antigen-binding The fragment consists of D265C, H435A, L234A, and L235A (numbered according to the EU index). an Fc region comprising at least one amino acid substitution selected from the group consisting of:

38. 38. The method of claim 37, wherein the Fc region comprises the amino acid substitutions D265C, L234A, and L2 35A (numbering according to the EU index), including

39. 23. The method of claim 22, wherein the anti-CD117 antibody or antigen-binding fragment thereof a light chain comprising the amino acid sequence set forth in SEQ ID NO: 109, and a SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 113, and SEQ ID NO:

114. , including.

40. 23. The method of claim 22, wherein the anti-CD117 antibody or antigen-binding fragment thereof is a light chain comprising the amino acid sequence set forth in SEQ ID NO: 115, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 11 a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, and SEQ ID NO: 120; Includes.

41. 23. The method of claim 22, wherein the anti-CD117 antibody or antigen-binding fragment thereof is a light chain comprising the amino acid sequence set forth in SEQ ID NO: 284, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

285. SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, and SEQ ID NO: ):278, and a heavy chain comprising an amino acid sequence selected from the group consisting of:

42. 23. The method of claim 22, wherein the anti-CD117 antibody or antigen-binding fragment thereof is , including: HC-CDR1, HC-CDR2, and HC-CDR3 or Ab55, Ab54, Ab56, Ab57, Ab58, Ab61, Ab66, Ab67, Ab68, Ab69, Ab85, Ab86, Ab87, Ab88, Ab89, Ab77, Ab79, Ab81, Ab85, young or Ab249, and a heavy chain comprising a variable region sequence derived from the heavy chain variable region of LC-CDR1, LC-CDR2, and LC-CDR3 or Ab55, Ab54, Ab56, Ab57, Ab58, Ab61, Ab66, Ab67, Ab68, Ab69, Ab85, Ab86, Ab87, Ab88, Ab89, Ab77, Ab79, Ab81, Ab85, or Ab249 a light chain comprising a variable region sequence derived from the light chain variable region of HC-CDR1, HC-CDR2, and HC-CDR3 or SEQ ID NOs: 147, 164, 166, 168, 170, 172, 174, 176, 178, 180, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 238, if a heavy chain comprising a variable region derived from the heavy chain variable region amino acid sequence of 243, and DR1, LC-CDR2, and LC-CDR3 or SEQ ID NOs: 148, 149, 150, 151, 152 , 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 165, 167, 169, 171, 173 , 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 203, 205 , 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 228, 229, 230, 231, 232 , 233, 234, 235, 236, 237, 239, 240, 241, 242, or 244. a light chain comprising a variable region derived from the amino acid sequence of

43. 43. The method of any one of claims 1 to 42, wherein the ADC has the formula Ab-(ZL-Cy) n to is represented by, where: Ab is the antibody or antigen-binding fragment thereof; L is a linker; Z is a combination of a reactive substituent on L and a reactive substituent on the antibody or antigen-binding fragment thereof. A chemical substructure formed by a coupling reaction between a substituent and a Cy includes amatoxin, Pseudomonas exotoxin A, debouganin, diphtheria toxin, and sa Porin, maytansine, maytansinoid, pyrrolobenzodiazepine, pyrrolobenzodiazepine Zepine dimer, indolinobenzodiazepine, indolinobenzodiazepine dimer, potassium a cell selected from the group consisting of caremicins, auristatins, and anthracyclines. is a cytotoxin; and n is an integer from about 1 to about 20 (which represents the average number of cytotoxins per antibody). ）。

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