Methods for allogeneic hematopoietic stem cell transplantation

Abstract

To provide compositions and methods useful for the depletion of CD117+ or CD45+ cells and for the treatment of various hematopoietic diseases, metabolic disorders, cancers, and autoimmune diseases, among others.SOLUTION: The compositions and methods of the present invention can be used to treat a disorder, for instance, by depleting a population of CD117+ or CD45+ cancer cells or autoimmune cells. The compositions and methods of the present invention can also be used to prepare a patient for allogeneic hematopoietic stem cell transplant therapy and to improve engraftment of allogeneic hematopoietic stem cell transplants by selectively depleting endogenous hematopoietic stem cells prior to the transplant procedure.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application is a continuation of U.S. Provisional Application No. 62 / 752,828, filed October 30, 2018, and U.S. Provisional Application No. 62 / 752,828, filed November 30, 2018. U.S. Provisional Application No. 62 / 773,873 filed on August 2, 2019, and U.S. Provisional Application No. 62 / 8 No. 82,362, the entire contents of each of the foregoing priority applications are incorporated herein by reference. INCORPORATED INTO THE SPECIFICATION.

[0002] [Technical field] The present disclosure provides a method for detecting hematopoietic stem cells that can bind to CD117 or CD45 expressed by hematopoietic cells such as hematopoietic stem cells. and the administration of antibody drug conjugates containing the same to various disease states (e.g., blood disorders, in particular). The present invention relates to the treatment of patients suffering from various conditions, including inflammatory bowel diseases, metabolic disorders, cancer, and autoimmune diseases. [Background technology]

[0003] Allogeneic cell therapy involves transplanting cells into a patient, where the transplanted cells The cells are derived from a donor other than the patient. Common types of related donors include HLA-matched siblings and matched unrelated donors. donor, partially matched family member donor, related cord blood donor, and The ultimate goal of cell therapy is to is a promising allogeneic cell therapy that can form the basis of "off the shelf" The aim is to identify the method (Brandenberger, et al. (2011). BioProcess International. 9 (suppl. I): 30-37), thereby expanding the use of allogeneic cell therapy.

[0004] Despite their promise, the therapeutic use of allogeneic cells currently remains limited. However, there are complications that make this treatment difficult. In an immunocompetent host, the transplanted allogeneic These cells are rapidly rejected, resulting in host versus graft rejection (HvG). The HvG substantially reduces the effectiveness of the transferred cells. This may result in adverse events in the recipient and may lead to allogeneic This would limit the use of allogeneic cells. Currently, allogeneic hematopoietic stem cell transplants are not available. promotes the engraftment of transplanted cells, thereby preserving the pluripotency and hematopoietic function of these cells after transplantation. Such compositions and methods are needed. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides antibodies or ADCs useful in conditioning procedures, wherein a patient , ready to receive a graft containing allogeneic hematopoietic stem cells. According to the study, patients are diagnosed with hematopoietic cell (e.g., hematopoietic stem cell)-expressed antigens (e.g., CD117 [e.g., For example, an ADC, antibody, or antigen-binding fragment thereof that can bind to GNNK+ CD117 or CD45. The fragment is administered to the patient in combination with an immunosuppressant to inhibit allogeneic leukemia. The patient may be conditioned for blood stem cell transplantation therapy. As described above, the antibody may be coupled to a cytotoxin to form an antibody drug conjugate (ADC). May be covalently conjugated. [Means for solving the problem]

[0006] In one aspect, the present invention provides a population of CD117+ cells in a human patient in need of hematopoietic stem cell transplantation. 10. A method for reducing the risk of HIV infection in a patient before receiving a transplant comprising allogeneic hematopoietic stem cells. administering to said patient an effective amount of an anti-CD117 antibody drug conjugate and an immunosuppressant. The present invention provides a method comprising the steps of:

[0007] In another aspect, the present invention provides a method comprising: administering to a human patient a population of CD117+ cells in said patient. administering an anti-CD117 antibody drug conjugate and an immunosuppressant in an amount sufficient to reduce the number of and thereafter administering to said patient a graft comprising allogeneic hematopoietic stem cells. To provide.

[0008] In another aspect, the present invention provides a method for the treatment of hematopoietic stem cells by administering to a human patient a graft comprising allogeneic hematopoietic stem cells. wherein said patient is administered a therapeutic agent sufficient to reduce the population of hematopoietic stem cells in said patient. and a patient previously administered an anti-CD117 antibody drug conjugate and an immunosuppressant in an adequate amount. To provide.

[0009] In some embodiments, the CD117 is GNNK+ CD117.

[0010] In another aspect, the present invention provides a population of CD45+ cells in a human patient in need of hematopoietic stem cell transplantation. 10. A method for reducing the risk of HIV infection in a patient before receiving a transplant comprising allogeneic hematopoietic stem cells. administering to said patient an effective amount of an anti-CD45 antibody drug conjugate and an immunosuppressant. The present invention provides a method comprising the steps of:

[0011] In another aspect, the present invention provides a method comprising: administering to a human patient a CD45+ cell population in said patient. administering an anti-CD45 antibody drug conjugate and an immunosuppressant in an amount sufficient to reduce the number of and thereafter administering to said patient a graft comprising allogeneic hematopoietic stem cells. To provide.

[0012] In another aspect, the present invention provides a method for the treatment of hematopoietic stem cells by administering to a human patient a graft comprising allogeneic hematopoietic stem cells. wherein said patient is administered a therapeutic agent sufficient to reduce the population of hematopoietic stem cells in said patient. and a patient previously administered an anti-CD45 antibody drug conjugate and an immunosuppressant in an adequate amount. To provide.

[0013] In another aspect, the present invention provides a method for the treatment of hematopoietic stem cells by administering to a human patient a graft comprising allogeneic hematopoietic stem cells. wherein said patient is administered a therapeutic agent sufficient to reduce the population of hematopoietic stem cells in said patient. The present invention provides a method for treating a patient who has been previously administered an anti-CD45 antibody drug conjugate in an amount sufficient to treat the patient.

[0014] In some embodiments, the method comprises administering to the patient after the patient has received the transplant. The method further comprises administering the immunosuppressant to

[0015] In another aspect, the present invention provides a population of CD117+ cells in a human patient in need of hematopoietic stem cell transplantation. 1. A method for reducing CD117+ in said human patient, comprising: (a) administering to said human patient a method for reducing CD117+ in said patient; administering an anti-CD117 antibody drug conjugate in an amount sufficient to reduce the population of cells; (b) administering to said human patient a graft comprising allogeneic hematopoietic stem cells; and (c ) then administering an immunosuppressant to said patient.

[0016] In a further aspect, the present invention provides a method for the collection of CD45+ cells in a human patient in need of hematopoietic stem cell transplantation. 1. A method for reducing a CD45 population in a human patient, the method comprising: (a) administering to said human patient an injection of CD45 and administering an anti-CD45 antibody drug conjugate in an amount sufficient to reduce the population of CD45+ cells. (b) administering to said human patient a graft comprising allogeneic hematopoietic stem cells; and (c ) then administering an immunosuppressant to said patient.

[0017] In some embodiments, the graft is an MHC-matched (e.g., HLA-matched) allogeneic transplant. Thus, in some embodiments, the graft comprises hematopoietic stem cells of all HLA antigens. comprises allogeneic hematopoietic stem cells that are compatible with the HLA antigens of said human patient.

[0018] In certain embodiments, the graft is resistant to at least one HLA antigen of the patient. In certain embodiments, the allogeneic hematopoietic stem cells comprise HLA-mismatched The allogeneic hematopoietic stem cells have at least two HLA-mismatches to the patient's HLA antigens. In certain embodiments, the allogeneic hematopoietic stem cells are directed against the patient's HLA antigens. In certain embodiments, the allogeneic antibody comprises at least three HLA-mismatches. The hematopoietic stem cells of the present invention contain at least four HLA-mismatches to the HLA antigens of said patient. In certain embodiments, the allogeneic hematopoietic stem cells are selected from the group consisting of: In certain embodiments, the allogeneic hematopoietic stem cell line comprises at least five HLA-mismatches. The stem cells contain at least six HLA-mismatches to the patient's HLA antigens. In certain embodiments, the allogeneic hematopoietic stem cells are at least partially homologous to the patient's HLA antigens. In certain embodiments, the allogeneic hematopoietic stem cells contain seven HLA-mismatches. The antibody contains at least eight HLA-mismatches to the patient's HLA antigens. In some embodiments, the allogeneic hematopoietic stem cells are at least 9 homologous to the patient's HLA antigens. In certain embodiments, the allogeneic hematopoietic stem cells comprise an HLA-mismatch of In certain embodiments, the antibody comprises a complete HLA-mismatch against the patient's HLA antigens. The allogeneic hematopoietic stem cells are HLA-mismatched to the patient's HLA antigens by between one and four mismatches. , between 1 and 3 HLA-mismatches, between 1 and 2 HLA-mismatches, between 2 and 4 HLA-mismatches of 1, 2, or 3, or 3, or 4 Includes:

[0019] In some embodiments, the graft is adapted to the patient's minor histocompatibility antigens (miHA). In contrast, allogeneic hematopoietic stem cells containing at least one minor histocompatibility antigen-mismatch Contains cells.

[0020] In some embodiments, the graft comprises HLA-mismatched allogeneic hematopoietic stem cells. nothing.

[0021] In some embodiments, the method comprises obtaining at least 80% donor chimerism. In some embodiments, the method is effective to establish a chromatic aberration. It is also effective in establishing 85% donor chimerism. In embodiments, the method ensures at least 90% donor chimerism. In some embodiments, the method is effective in establishing at least 95% of the donor population. In some embodiments, the method is effective in establishing donor chimerism. The donor chimerism was confirmed at least 6, 7, 8, and 9 weeks after transplantation. In some embodiments, donor chimerism is assessed at week 1, week 2, or week 30. Peripheral myeloid chimerism is a type of peripheral myeloid chimerism. In embodiments, the donor chimerism is T-cell chimerism. chimerism).

[0022] In some embodiments, the immunosuppressant is cyclophosphamide. In embodiments, the immunosuppressant is 30F11. Inhibitors include cyclophosphamide (Cytoxan), e.g., low-dose Cytoxan In some embodiments, the immunosuppressant is 30F11 and cyclophosphamide. In some embodiments, the immunosuppressant is total body irradiation. In some embodiments, the immunosuppressant ( For example, Cytoxan is administered after transplantation. An immunosuppressant (e.g., 30F11) is administered prior to transplant. The inhibitor is administered substantially simultaneously with the patient receiving the transplant.

[0023] In some embodiments, the conjugate, after administration to the patient, It is taken up into the cell by autoimmune cells or hematopoietic stem cells.

[0024] In some embodiments, the concentration of the conjugate is substantially cleared from the blood of the patient. After clearance, a graft containing hematopoietic stem cells is administered to the patient.

[0025] In some embodiments, the method further comprises administering the hematopoietic stem cells to the patient more than two days after transplantation of the hematopoietic stem cells into the patient. The hematopoietic stem cells or their progeny maintain the functional potential of hematopoietic stem cells.

[0026] In some embodiments, after transplantation of said hematopoietic stem cells into said patient, The cells or their progeny can localize to hematopoietic tissue and / or re-establish hematopoiesis. can be done.

[0027] In some embodiments, when transplanted into the patient, the hematopoietic stem cells produce megakaryocytes, hematopoietic stem cells, and / or hematopoietic stem cells. thrombocytes, platelets, red blood cells, mast cells, myeloblasts, basophils, Neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages a cell selected from the group consisting of a dendritic cell, a natural killer cell, a T-lymphocyte, and a B-lymphocyte; The population of cells to be selected is recovered.

[0028] In some embodiments, the patient is suffering from a stem cell disorder.

[0029] In some embodiments, the patient has a hemoglobinopathy disorder, an autoimmune disorder, a bone marrow disorder, Suffering from a dysplastic, immunodeficiency, or metabolic disorder.

[0030] In some embodiments, the patient is suffering from cancer.

[0031] In some embodiments, the ADC comprises a heavy chain / light chain (HC / LC) CDR set (CDR1, CDR2, or CDR3), or an HC / LC variable region set as set forth in Table 3.

[0032] In some embodiments, the antibody of the conjugate is a bio-layer interferometer. 1 x 10 as measured by Rayleigh interferometry -2 From 1 x 10 -3 , 1 x 10 -3 from 1 x 10 -4 , 1 x 10 -5 From 1 x 10 -6 , 1 x 10 -6 From 1 x 10 -7 , or 1 x 10 -7 From 1 x 10 -8 Dissociation rate (K OFF )

[0033] In some embodiments, the antibody of the conjugate is a bio-layer interferometer. When measured by Rayler interferometry, the values ​​were approximately 100 nM or less, approximately 90 nM or less, and approximately 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, K of about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nM or less, about 2 nM or less, about 1 nM or less D So, C Binds to D117.

[0034] In some embodiments, the antibody of the conjugate is a human antibody.

[0035] In some embodiments, the antibody of the conjugate is an intact antibody.

[0036] In some embodiments, the antibody of the conjugate is an IgG. In some embodiments, the IgG is an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, or an IgG1 isotype. It is the gG4 isotype.

[0037] In some embodiments, the antibody is conjugated to a cytotoxin via a linker. In some embodiments, the cytotoxin is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is an amatoxin. In some embodiments, the RNA polymerase inhibitor is amanitin. In this embodiment, the amanitin may be α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, From chin, amanine, amaninamide, amanulin, amanuric acid, and proamanuline In some embodiments, the cytotoxin is selected from the group consisting of Pseudomonas exotoxin Substance A, debouganin, diphtheria toxin, saporin, maytansin, maytansinoid , auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, Tuocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indoli Indolinobenzodiazepines, indolinobenzodiazepine dimers, and indolinobenzodiazepines In some embodiments, the auristatin is selected from the group consisting of: a pin pseudodimer; is MMAE or MMAF.

[0038] In some embodiments, the antibody is modified by a cysteine ​​residue in the Fc domain of the antibody. In some embodiments, the cysteine ​​residue is conjugated to the toxin. The group is introduced by amino acid substitution in the Fc domain of the antibody. In some embodiments, the amino acid substitution is D265C. [Brief explanation of the drawings]

[0039] [Figure 1] Figures 1A–1J illustrate the design and results of an in vivo study in which ADC conditioning using anti-CD45 ADC (104-saporin; "CD45-SAP") or anti-CD117 ADC (2B8-saporin; "CD117-SAP") was performed prior to transplantation of murine minor-mismatched grafts of Balb / c donor cells into DBA / 2 recipients, in combination with 30F11 and posttransplantation cytoxan. Figures 1A and 1B show a schematic diagram of the in vivo mouse model (Figure 1A) and the dosing schedules for the various experimental groups (Figure 1B). Transplant recipients were administered CD45-SAP, CD117-SAP, or control treatment (e.g., 2 Gy TBI or no TBI) in combination with pretransplant immunosuppressant (30F11) and posttransplantation cytoxan. Figure 1C illustrates the extent of bone marrow depletion (as measured by the number of long-term HSCs (LT-HSCs) per femur (y-axis)) 7 days after treatment in C57BL / 6 mice, with treatment condition on the horizontal axis. Figure 1D illustrates the percentage of overall blood donor chimerism (CD45.1+) in peripheral blood 12 weeks after transplantation. Figure 1E graphically illustrates the percentage of bone marrow chimerism, B cell chimerism, and T cell chimerism 12 weeks after transplantation. [Figure 2]Figures 2A–2C illustrate the results of an in vivo study in which DBA / 2 recipients were pre-transplanted with a murine minor-mismatched graft of Balb / cByJ donor cells using anti-CD45 ADC (104-saporin; "CD45-SAP") conditioning, combined with post-transplant cytoxin, for Balb / cByJ donor cells. Figures 2A–2C also illustrate the treatment mode in DBA / 2 mice transplanted with CD45.1+ cells, as well as the percentage of total donor chimerism (y-axis) in the transplant recipients 8 weeks post-transplant. The percentage of peripheral donor bone marrow chimerism (Figure 2A) and the percentage of donor T-cell chimerism (Figure 2B) are also shown. FIG. 2D graphically depicts the number of donor-derived long-term-HSCs (LT-HSCs) per femur (y-axis) in transplant recipients 12 weeks after transplantation. [Figure 3] Figures 3A-3B graphically show the results of an in vivo depletion assay demonstrating that CD45-ADC effectively depletes mouse HSCs and lymphocytes in C57 mice. Figure 3A is a summary of an in vivo study evaluating the depletion of mouse HSCs by anti-CD45-ADC (CD45-saporin or "CD45-SAP"). Figure 3B shows the flow cytometry gating strategy and results demonstrating that CD45-SAP depleted long-term HSCs in bone marrow harvested on day 7. Figure 3C graphically shows the levels of long-term HSCs in bone marrow 7 days after administration of PBS, isotype-SAP, or CD45-SAP. Figure 3D graphically shows the levels of peripheral lymphocytes 7 days after administration of PBS, isotype-SAP, or CD45-SAP. An asterisk (*) indicates p<0.05 compared to the given control group. [Figure 4]Figures 4A–4C graphically show the results of in vivo studies of a mouse model of fully mismatched bone marrow transplantation. C57Bl / 6 (H-2b, CD45.2+) mice were transplanted with Balb / c (H-2d, CD45.1+) bone marrow conditioned with anti-CD45-ADC (anti-CD45-PDB or "CD45-PBD") alone or in combination with anti-CD4 and anti-CD8 antibodies. Figure 4A graphically shows the percentage of donor chimerism in the blood of transplant recipients 3 and 8 weeks after transplantation, as detected using the CD45.1+ antigen. Figure 4B graphically shows the percentage of peripheral donor bone marrow chimerism, B cell chimerism, and T cell chimerism at 8 weeks after transplantation, with the horizontal axis representing the treatment mode in the transplant recipient. Figures 4C and 4D graphically show the total cell numbers (CD45+) in the peripheral blood (Figure 4C) and spleen (Figure 4D) two days after ADC administration. [Figure 5]Figures 5A–5G graphically show the results of an in vivo study of a mouse model of fully mismatched bone marrow transplantation. C57Bl / 6 (H-2b, CD45.2+) mice were conditioned with anti-CD45-ADC (104-PBD) alone or in combination with low-dose TBI and transplanted with Balb / c (H-2d, CD45.1+) bone marrow. Figure 5A graphs the number of long-term HSCs (LT-HSCs) per femur (y-axis) at 2 days after ADC administration in transplant recipients, with the horizontal axis representing treatment conditions for varying levels of irradiation. Figures 5B–5E graphically show the extent of bone marrow depletion (cells per femur (y-axis)) of total CD45+ cells (Figure 5B), myeloid cells (Figure 5C), B cells (Figure 5D), or T cells (Figure 5E) in transplant recipients at 2 days after ADC administration, with the horizontal axis representing treatment conditions for varying levels of irradiation. Figure 5F graphically shows the percentage of donor chimerism in the peripheral blood of transplant recipients at 4 weeks post-transplant. Figure 5G graphically shows the percentage of bone marrow chimerism, B cell chimerism, and T cell chimerism in transplant recipients at 4 weeks post-transplant. DETAILED DESCRIPTION OF THE INVENTION

[0040] [Detailed explanation] The present invention provides antibodies or ADCs useful in conditioning procedures, wherein a patient The patient is prepared to receive a graft containing allogeneic hematopoietic stem cells. According to the methods described herein, the engraftment of allogeneic hematopoietic stem cells is promoted. The subject is interested in identifying antigens expressed by hematopoietic cells (e.g., hematopoietic stem cells) (e.g., CD117, e.g., GNNK+ CD1 The ADC, antibody, or antigen-binding fragment thereof capable of binding to CD45 (CD17 or CD45) is used as an immunosuppressant. In an allogeneic hematopoietic stem cell transplantation therapy, the compound is administered to the patient in combination with an immunosuppressant. As described herein, the anti- The antibody may be covalently conjugated to a cytotoxin to form an antibody drug conjugate (ADC). ADCs, antibodies, and the like that can bind one or more of the above antigens may be used. or a drug-antibody conjugate, When administered in combination with immunosuppressants to patients with rheumatoid arthritis, for example, endogenous hematopoietic stem cells are selectively activated. gradually decreases, thereby creating a void that can be filled by the transplant of exogenous hematopoietic stem cells. This may enhance the survival of allogeneic hematopoietic stem cell grafts.

[0041] definition As used herein, the term "about" refers to a value 5% above or below the stated value. say.

[0042] As used herein, the term "allogeneic" when used in the context of transplantation Transplantation from a genetically dissimilar donor to a recipient of the same species Used to define a cell (or tissue or organ).

[0043] As used herein, the term "autologous" refers to both the donor and the recipient. The patient refers to a cell or graft of the same subject.

[0044] As used herein, the term "xenogeneic" refers to a combination of donor and recipient. This refers to cells that are of a different species (pient).

[0045] As used herein, the term "immune cells" includes, but is not limited to, hematopoietic cells. It is intended to include cells that are of origin and that play a role in the immune response. Examples include, but are not limited to, T cells and natural killer (NK) cells. Natural killer cells are well known in the art. Examples of NK cell lines include NK-92 cells. Examples of immune cells include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells. May be allogeneic or autologous.

[0046] As used herein, the term "antibody" refers to an antibody that specifically binds to or binds to a particular antigen. refers to an immunoglobulin molecule that is immunologically reactive with a specific antigen. Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, and the like. specific antibodies (e.g., bispecific antibodies), genetically engineered antibodies, and Other modified forms of antibodies (e.g., but not limited to, chimeric antibodies, humanized Antibodies, heteroconjugate antibodies [e.g., bi-, tri-, and quad-specific antibodies, diabodies, triabodies, and tetrabodies], and antibody fragments [i.e. antigen-binding fragments of antibodies [e.g., Fab', F(ab')2, Fab, Fv, rlgG, and scFv] fragments, etc.], as long as they exhibit the desired antigen-binding activity). .

[0047] The antibodies of the present invention are generally isolated or recombinant. As used herein, a gene is a protein that is identified and isolated from a cell or cell culture that expresses it, and The term "isolated antibody" refers to a polypeptide (e.g., antibody) that has been isolated and / or recovered. Typically, an isolated antibody is a small amount of An "isolated antibody" is prepared by at least one purification step. It refers to an antibody that is substantially free of other antibodies with the same antigen specificity. For example, The isolated antibody that binds to CD117 is substantially free of antibodies that specifically bind to antigens other than CD117. Similarly, an isolated antibody that specifically binds CD45 may not specifically bind to an antigen other than CD45. It is substantially free of antibodies that bind to it.

[0048] As used herein, the term "monoclonal antibody" refers to any eukaryotic, prokaryotic, or from a single clone, including a phage clone, available or known in the art It refers to antibodies obtained by any means, as well as those produced by hybridoma technology. Monoclonal antibodies useful in the present disclosure are not limited to antibodies derived from hybridomas, recombinant and phage display technology, or a combination thereof. Unless otherwise specified, the term "mono- Monoclonal antibodies (mAbs) are capable of binding specifically to target proteins. , including intact molecules and antibody fragments (e.g., Fab and F(ab')2 fragments, etc. As used herein, Fab and F(ab')2 fragments are meant to include both Fab and F(ab')2 fragments. An Fc fragment refers to an antibody fragment that lacks the Fc fragment of an intact antibody. In some embodiments, the antibody fragment comprises an Fc region.

[0049] Generally, antibodies comprise a heavy chain and a light chain that contain an antigen-binding region. Each heavy chain comprises a heavy chain variable region (herein, abbreviated as HCVR or VH) and a heavy chain constant region. Each light chain consists of three domains (CH1, CH2, and CH3). The light chain constant region is composed of a light chain constant region (abbreviated as LCVR or VL in the specification) and a light chain constant region. The VH and VL regions are composed of two domains (CL). The VH and VL regions are composed of two hypervariable regions (complementarity determining regions). These regions can be further subdivided into more conserved regions (called framework regions (FRs)) and more conserved regions (called central and secondary Each VH and VL consists of three CDRs and four FRs, and the amino-terminal From the carboxyl end to the carboxyl end, they are arranged in the following order: FR1, CDR1, FR2, CDR2, FR 3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody is a region that allows the immunoglobulin to bind to host tissues or factors (e.g., immune system). various cells of the system (e.g., effector cells) and the first component of the classical complement system (C1q, etc.) It may act as an intermediary between two substances.

[0050] As used herein, the term "antigen-binding fragment" refers to a fragment that is specific to a target antigen. The term "antigen-binding portion" refers to one or more portions of an antibody that retain the antigen-binding function of an antibody. This can be achieved by fragments of full-length antibodies. Such antibody fragments can be, for example, , Fab, F(ab')2, scFv, diabody, triabody, affibody, nanobody, The term "antigen-binding fragment" of an antibody may be a tammer or domain antibody. Examples of combined fragments encompassed by the term include, but are not limited to: (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; ii) F(ab')2 fragment, two Fab fragments linked by a disulfide bridge at the hinge region (iii) a bivalent fragment comprising the VH and CH1 domains; and (iv) an Fd fragment comprising the VH and CH1 domains. (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a VH and VL domain (vi) a dAb fragment consisting of a VH domain (e.g., Ward et al., Nature (vii) dAbs consisting of VH or VL domains; (viii) isolated and (ix) optionally linked by a synthetic linker. A combination of two or more (e.g., two, three, four, five, or six) isolated CDRs. In addition, the two domains of the Fv fragment (VL and VH) are encoded by separate genes. However, by using recombinant methods, they can be linked by a linker to form the resulting As a result, it is possible to obtain a single protein chain (the VL and VH domains pair to form a monovalent molecule). (known as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988). 8 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 screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be synthesized using recombinant DNA techniques, intact immune cells, or other methods. by enzymatic or chemical cleavage of globulins, or in some cases by methods known in the art. It can be produced by known chemical peptide synthesis procedures.

[0051] As used herein, the term "anti-CD117 antibody" or "antibody that binds to CD117" refers to The antibodies are believed to be useful as diagnostic and / or therapeutic agents targeting CD117. refers to an antibody that is capable of binding to CD117 with sufficient affinity.

[0052] As used herein, the term "anti-CD45 antibody" or "antibody that binds to CD45" refers to The antibodies are useful as diagnostic and / or therapeutic agents targeting CD45. Refers to an antibody that is capable of binding to CD45 with sufficient affinity.

[0053] As used herein, the term "diabody" refers to a molecule comprising two polypeptide chains. refers to a bivalent antibody, in which each polypeptide chain is connected to a V on the same peptide chain. H and V L Domain A linker (e.g., consisting of five amino acids) that is so short that intramolecular association of the V linked by a linker H and V L This configuration includes the domain. The domains pair with complementary domains on another polypeptide chain to form homodimeric structures. Thus, the term "triabody" refers to a trivalent antibody containing three peptide chains, Each of them is a V within the same peptide chain. H and V L To enable intramolecular association of domains, linked by a linker that is too short (e.g., a linker consisting of 1 to 2 amino acids) One V HDomain and one V L The peptide thus constructed contains the In order for the peptide to fold into its native structure, the V H Domain and V L They trimerize so that the domains are spatially close to each other (e.g., Holliger et al., Pr (See J. Natl. Acad. Sci. USA 90:6444-48, 1993).

[0054] As used herein, the term "bispecific antibody" can bind to, for example, at least two different antigens or two different epitopes. The term "antibody" refers to a monoclonal antibody, e.g., a human antibody or a humanized antibody, capable of binding to a target molecule, e.g., a human antibody or a humanized antibody. One of them is directed against a hematopoietic stem cell surface antigen, for example, CD117 (e.g., GNNK+ CD117) or CD45. and the other, receptors involved in signal transduction pathways that enhance cell proliferation. Various hematopoietic stem cell surface antigens or other cell surfaces, such as receptor subunits In some embodiments, the binding specificity of the may be directed to unique, non-overlapping epitopes on the same target antigen. (i.e., biparatopic antibody). As used herein An "intact" or "full-length" antibody is a fragment of a protein that is interconnected by disulfide bonds. Refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides. Each heavy chain is The antibody is composed of a heavy chain variable region (abbreviated as HCVR or VH herein) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). The light chain is composed of a variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The constant region is composed of one domain (CL). The VH and VL regions are hypervariable regions. The more conserved regions can be further subdivided into complementary regions (called complementarity-determining regions (CDRs)). Each VH and VL consists of three CDRs and four FRs. The sequences are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR 1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains are responsible for interacting with an antigen. The constant region of the antibody contains a binding domain that allows the immunoglobulin to bind to host tissues or factors ( For example, various cells of the immune system (e.g., effector cells) and the first members of the classical complement system It may mediate the binding of C1q to other components of the IL-1 receptor.

[0055] As used herein, the term "complementarity determining region" (CDR) refers to the light and heavy chains of an antibody. The term refers to the hypervariable regions found in both the ribosomal and ribosomal variable domains. The remaining part is called framework region (FR). The positions of amino acids representing the hypervariable region of an antibody are: It may vary depending on the context and various definitions known in the art. Some positions within the gene may be considered hybrid hypervariable positions, and these positions A position may be considered within a hypervariable region under one set of criteria, while under another set of criteria, One or more of these positions may be considered to be outside the hypervariable region under the criteria of They may also be found in extended hypervariable regions. These hybrid hypervariable positions may contain modifications. Each of the main domains has a predominantly β-sheet structure and is connected by three CDRs (the CDRs are -Connecting the sheet structure and possibly forming part of it, forming loops), four The CDRs in each chain are arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the framework region is The CDRs are closely packed together by the CDR domains and, together with the CDRs from the other antibody chains, form the target of the antibody. contributes to the formation of the target binding site (Kabat et al., Sequences of Proteins of Immunology (See "Medical Interest," National Institute of Health, Bethesda, MD., 1987). In certain embodiments, the numbering of the amino acid residues in the immunoglobulin is The immunoglobulin amino acid residue numbering system of Kabat et al. Any antibody numbering scheme may be used, including but not limited to IMGT and Chothia. may be used).

[0056] The term "specifically binds" as used herein means that an antibody (or ADC) specifically binds to a target molecule. The ability to recognize and bind to specific protein structures (epitopes) rather than proteins in general If the antibody is specific for epitope "A", it includes the labeled "A" and the antibody. In the reaction, the presence of a molecule containing epitope A (or free, unlabeled A) activates the antibody. The amount of labeled A that binds to the antibody decreases. For example, an antibody (if labeled) If the antibody can be competed away from its target by an unlabeled antibody In some embodiments, an antibody "specifically binds" to a target (e.g., For example, antigens expressed by hematopoietic stem cells [e.g., CD117 (e.g., GNNK+ CD117), or CD45 ], or antigens expressed by mature immune cells [e.g., T-cells] [e.g., CD4, or CD8]) and wherein the antibody is at least about 10 -4 M, about 10 -5 M, about 10 -6 M, about 10 -7 M, about 10 -8 M, about 10 - 9 M, about 10 -10 M, about 10 -11 M, about 10 -12 M or less (less than about 10 -12 A number less than means, for example, 10 -13 ) for the target D specifically binds when In one embodiment, the term "specifically binds" means to bind to a molecule that specifically binds to at least about 1 x 10 -6 M, approx. 1x10 -7 M , approx. 1x10 -8 M, approx. 1x10 -9 M, approx. 1x10 -10 M, approx. 1 x 10 -11 M, approx. 1x10 -12 M or higher binds to antigens with a Kd greater than its affinity for nonspecific antigens K refers to the ability of an antibody to bind to an antigen with twice the affinity as K. D is measured according to standard Bio-Layer Interferometry (BLI) However, the antibody may be capable of specifically binding to two or more antigens that are related in sequence. It should be understood that, for example, in certain embodiments, the antibody may be directed against an antigen (e.g., CD117[GNNK+ CD117], or CD45) human and non-human (e.g., mouse or non-human primate) orthologs It may specifically bind to both LOGs.

[0057] As used herein, the term "chimeric" antibody refers to a non-human IgG antibody, such as a rat or mouse antibody. -variable sequences derived from human immunoglobulins, and typically human immunoglobulin templates A chimeric antibody refers to an antibody having a human immunoglobulin constant region selected from the group consisting of: Methods for production are known in the art, see, e.g., Morrison, 1985, Science 22 9(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patents 5,807,715; 4,816,567; and 4,816,397 The terms "Fc," "Fc region," "Fc domain," and "IgG Fc domain" are used herein. As used herein, a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region refers to the portion of an immunoglobulin (e.g., an IgG molecule) that is responsible for the division of the immune system. It contains the C-terminal halves of the two heavy chains of an IgG molecule, linked by sulfide bonds. Although it does not have antigen binding activity, it has carbohydrate moieties and Fc receptors, including complement and FcRn receptors. For example, the Fc region comprises a second constant domain, CH2 ( for example, residues at EU positions 231-340 of human IgG1) and the third constant domain CH3 (for example, residues at EU positions 231-340 of human IgG1 As used herein, the Fc region includes the "lower" and "lower" regions of the Fc antigen (residues 341 to 447 of the Fc antigen). - lower hinge region (e.g., residues 233-239 of EU position of human IgG1) nothing.

[0058] 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 man).

[0059] As used herein, the term "altered Fc region" or "variant Fc region" refers to a Contains one or more amino acid substitutions, deletions, insertions or modifications introduced anywhere within the Fc region. In certain embodiments, a variant IgG Fc domain comprises one or more The Fc domain has a higher affinity for Fc gamma R and / or C1q compared to a wild-type Fc domain that does not contain the amino acid substitutions. Contains one or more amino acid substitutions that result in reduced or abolished binding affinity to the target polypeptide. Furthermore, Fc binding interactions mediate various effector functions and downstream signaling events ( antibody dependent cell-mediated cytotoxicity (ADCC) ) and complement dependent cytotoxicity (CDC), etc. Thus, in certain embodiments, the variant Fc The antibody containing domain (e.g., antibody, fusion protein, or conjugate) may comprise one or more does not contain amino acid substitutions, deletions, insertions, or modifications of an unmodified Fc region containing naturally occurring amino acid residues) but with the same amino acids at least one or more Fc ligands (e.g., Fc gamma R).

[0060] The variant Fc domains described herein are characterized according to the amino acid modifications that make them up. For all amino acid substitutions discussed herein with respect to the Fc region, the number The coding always follows the EU index as in Kabat. So, for example, D265C is the parent For the Fc domain, an Fc bacilloproteinase (BAB) in which the aspartic acid (D) at EU position 265 is replaced with a cysteine ​​(C) is used. Similarly, for example, D265C / L234A / L235A is an alternative to the EU position relative to the parent Fc domain. Fc variants with substitutions at positions 265 (D to C), 234 (L to A), and 235 (L to A) are defined. A variant may also be defined as a variant of the final amino acid composition at the mutated EU amino acid position. For example, the L234A / L235A mutation is sometimes called "LALA." As a further example, the E233P.L234V.L235A.delG236 (deletion of 236) mutation is referred to as "EPLVLAdelG." As yet another example, the I253A.H310A.H435A mutation is sometimes referred to as "IHH." Note that the order in which the substitutions are shown is arbitrary.

[0061] The term "Fc gamma receptor" or "Fc gamma R" as used herein refers to an Fc gamma receptor. Binds to the Fc region of antibodies and is a protein encoded by the Fc gamma R gene In humans, this family is not limited to Although there is no evidence for Fc gamma RI (CD64) (e.g., the isoforms Fc gamma RIa, Fc gamma RIb, and and Fc gamma Rlc; Fc gamma RII (CD32) (e.g., the isoform Fc gamma Rlla [e.g., allotypes H131 and R131], Fc gamma RIIb [e.g., Fc gamma RIIb-1 and Fc gamma RIIb-2], Fc gamma RIIb-2], and Fc gamma RIIc, etc.; and Fc gamma RIII (CD16) (e.g., isoforms Forms Fc gamma RIIIa [e.g., allotypes V158 and F158] and Fc gamma RIIIb [e.g., For example, allotypes Fc gamma RIIIb-NA1 and Fc gamma RIIIb-NA2], and any undiscovered human Fc gamma R or an isoform or allotype of Fc gamma R. Fc gamma R includes Any living organism (e.g., but not limited to, humans, mice, rats, rabbits, and Mouse Fc gamma R may be derived from, but is not limited to, Fc gamma RI (CD64), Fc gamma RII (CD32), Fc gamma RIII (CD16), and Fc gamma RIII-2 (CD 16-2), and any undiscovered mouse Fc gamma R or Fc gamma R isoform or Allotypes, including:

[0062] The term "effector function" as used herein refers to the interaction of an Fc domain with an Fc receptor. Effector functions are limited to the following: Antibody-specific antibodies include, but are not limited to, ADCC, ADCP, and CDC. "Effector cells" are cells that express one or more Fc receptors and have one or more effector functions. Effector cells are cells of the immune system that mediate the immune response. However, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, and platelets (pla telets), B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and gamma delta T cells, as well as any living organism (e.g., but not limited to) The virus is derived from a variety of animals (humans, mice, rats, rabbits, and monkeys, but not from humans).

[0063] As used herein, the terms "silent," "silenced," " " or "silencing" refers to the FcγR silencing of the same antibody containing an unmodified Fc region. The antibodies described herein exhibit reduced binding to Fc gamma receptors (FcγRs) compared to binding to FcγRs. The term "antibody" refers to an antibody having an altered Fc region (e.g., an antibody having an altered Fc region, e ... The reduction in binding to FcγRs is less than that of the same antibody comprising an Fc region that does not contain the FcγR. at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least In some embodiments, the Fc silenced The antibody does not exhibit detectable binding to FcγR. Binding can be performed using a variety of techniques known in the art, including, but not limited to, equilibration methods. [e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytica Biochemistry, Vol. 373:52-60, 2008; radioimmunoassay (RIA)], or surface plate Electron resonance assays or other mechanism-based kinetic assays [e.g., BIACORE ( analysis or Octet® analysis (forteBIO)], as well as other methods [e.g., Indirect binding assay, competitive binding assay, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration, etc.). These and other methods may utilize labels present on one or more of the components being evaluated. and / or various detection methods (e.g., but not limited to, chromogenic labels, Fluorescent, luminescent, or isotopic labels may be used. Regarding dynamics, see Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Ra ven, Philadelphia (1999), which focuses on antibody-immunogen interactions. An example of a competitive binding assay involves combining a labeled antigen with an antibody of interest in a non-labeled manner. Incubating in the presence of increasing amounts of a target antigen; The data is a radioimmunoassay that involves detecting antibodies bound to specific antigens. From this, the affinity of the antibody of interest for a specific antigen can be determined by Scatchard plot analysis. The binding off-rate may be determined. Competition with a second antibody may be determined using radioimmunoassays. It may also be measured using an immunoassay, in which the antigen is coupled to a labeled compound. in the presence of a conjugated antibody of interest and increasing amounts of an unlabeled second antibody. , and incubate.

[0064] As used herein, the term "the same antibody comprising an unmodified Fc region" refers to the same antibody as listed above. lacking the amino acid substitutions (e.g., D265C, L234A, L235A, and / or H435A) but otherwise refers to an antibody that has the same amino acid sequence as the Fc-modified antibody to which it is being compared.

[0065] 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., Nature 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 Fc-fusion proteins and Fc-conjugated proteins are used to induce cell-mediated cell death. It is contemplated that this will result in cell damage.

[0066] 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 herein as ADCC activity. The ability of a target cell to mediate lysis can be assayed. To achieve this, a polypeptide of interest (e.g., an antibody) is transfected into a host cell, along with immune effector cells. The target cells are then added to the target cells, resulting in cytolysis of the target cells. Generally, lysed cells are subjected to labelling (e.g., radioactive substrates, fluorescent dyes or natural intracellular The effect of the antibody is detected by the release of a specific protein. The target cells include peripheral blood mononuclear cells (PBMCs) 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. (1987); Wilkinson et al., J. Immunol. Methods 258:183 (2001); Patel et al., J. I Munol. Methods 184:29 (1995). Alternatively, or in addition, the A of the antibody of interest may be DCC activity can be measured in vivo (e.g., Clynes et al., Proc. Natl. Acad. Sci. USA 95:652 ( The efficacy of the present invention may be evaluated in animal models such as those disclosed in the literature (e.g., in the US Pat. No. 6,1998).

[0067] As used herein, the terms "conditioning" and "conditioning "Priming" refers to the process by which a patient undergoes a transplant (e.g., a transplant containing hematopoietic stem cells) to receive the transplant. This refers to the process of preparing the hematopoietic stem cell graft for engraftment. is enhanced (e.g., after a conditioning procedure and subsequent hematopoietic stem cell transplantation). , the amount of viable hematopoietic stem cells in blood samples isolated from patients is continuously increased According to the method described herein, antigens expressed by hematopoietic stem cells ( For example, an ADC, antibody, or its derivatives that can bind to CD117 (e.g., GNNK+ CD117) or CD45. The antigen-binding fragments can be administered to patients to prepare them for hematopoietic stem cell transplantation therapy. The patient may be conditioned. As described herein, the antibody may be administered to a patient in need thereof. It may be covalently conjugated to a cytotoxin to form an ADC. an ADC, antibody, or antigen-binding fragment thereof capable of binding to one or more of the antigens of Administration to patients in need of transplantation therapy can, for example, selectively reduce endogenous hematopoietic stem cells. thereby creating a void that can be filled by the transplant of exogenous hematopoietic stem cells. This may promote the engraftment of hematopoietic stem cell grafts.

[0068] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to achieve a desired result. or to have an effect against autoimmune disease or cancer. Point.

[0069] As used herein, the term "half-life" refers to the time it takes for the plasma concentration of an antibody drug in the body to decrease by half. This 50% decrease in serum concentration is the time it takes for the amount of drug in circulation to decrease by 50%. Reflects.

[0070] As used herein, the term "human antibody" refers to an antibody that is a human antibody derived from a human germline immunoglobulin sequence. A human antibody is intended to include antibodies having variable and constant regions derived from a human. Amino acid residues not encoded by the germline immunoglobulin sequence (e.g., in vivo Mutations introduced by random or site-directed mutagenesis in vitro or in vivo However, mutations introduced by gene rearrangement or somatic mutation in the host may be present. However, as used herein, the term "human antibody" includes antibodies produced by the human genome of another mammalian species, such as a mouse. It is intended to include antibodies in which CDR sequences derived from a human germline have been grafted onto human framework sequences. Human antibodies have not been produced in human cells (e.g., by recombinant expression) or functionally A non-human immunoglobulin capable of expressing rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Human antibodies can be produced by mammalian cells or by prokaryotic or eukaryotic cells. If so, it may contain a linker peptide not found in natural human antibodies. For example, Fvs may contain from two to about eight linker peptides, such as glycine or other amino acid residues. The linker peptide may comprise a linker peptide between the heavy chain variable region and the light chain variable region. Such linker peptides are considered to be of human origin. The present study was carried out using a phage display antibody library derived from human immunoglobulin sequences. Human antibodies may be produced by a variety of methods known in the art, including the Rey method. also cannot express functional endogenous immunoglobulins, but can express human immunoglobulins. To produce the phospholipase A (PGA) gene using transgenic mice capable of expressing the PGA gene. (e.g., PCT Publication Nos. WO 1998 / 24893; WO 1992 / 01047; WO 1996 / 34096; WO 1996 / 3 3735; 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. (This refers to lighting.)

[0071] "Humanized" forms of non-human (e.g., murine) antibodies are derived from non-human immunoglobulins. Generally, humanized antibodies are chimeric immunoglobulins that contain at least the sequence It comprises substantially all of one, and typically two, variable domains, including all or part of the CDR regions. Substantially all of the FR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region corresponds to that of a non-human immunoglobulin. are of human immunoglobulin sequences. The humanized antibody also contains immunoglobulin constant regions. At least a portion of the Fc region, typically at least one of the human immunoglobulin consensus sequence Methods for humanizing antibodies are known in the art. For example, Riechmann et al., 1988, Nature 332:323-7; U.S. Patents 5,530,101; 5,585,089; 5,693,7 61; 5,693,762; and 6,180,370 to Queen et al.; EP239400; PCT Publication WO 91 / 09967; U.S. National Patent 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; S tudnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. A See Cad. Sci. 91:969-973; and U.S. Patent No. 5,565,332.

[0072] As used herein, the term "engraftment potential" refers to the ability of a hematopoietic stem cell to engraft. It refers to the ability of hematopoietic cells and hematopoietic progenitor cells to repopulate tissues, and such cells The term refers to cells, whether naturally circulating or provided by transplantation. The mechanisms surrounding engraftment, such as tissue homing and colonization of cells into tissues of interest, The engraftment efficiency or engraftment rate is a term commonly known to those skilled in the art. The activity can be assessed or quantified using any known clinically accepted parameter, e.g. , evaluating competitive repopulating units (CRUs); stem cells Markers are removed in tissues where the cells have homed, colonized, or engrafted. or disease progression, hematopoietic stem and progenitor cells assessing the progression of the subject by survival of the recipient or survival of the recipient. Engraftment can also be assessed by measuring the number of white blood cells in the peripheral blood after transplantation. Engraftment is the recovery rate of bone marrow cells from donor cells in a bone marrow aspirate sample. It can also be evaluated by measuring.

[0073] As used herein, the term "hematopoietic stem cell" ("HSC") refers to a cell that has the ability to self-renew. , and granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, blood cells, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes) , platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia , osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Immature blood cells have the ability to differentiate into mature blood cells, including various lineages. Such cells express CD34 + May contain CD34 cells. + The cells express the CD34 cell surface marker. In humans, CD34+ cells possess the properties of stem cells as defined above. In mice, HSCs are thought to comprise a subpopulation of cells that are CD34-. HSC also refers to long-term refurbished HSC (LT-HSC) and short-term refurbished HSC (ST-HSC). HSCs are differentiated based on their functional potential and expression of cell surface markers. Human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (CD2, CD3, CD4, CD7, CD8 Negative for mature lineage markers including CD10, CD11B, CD19, CD20, CD56, CD235A, etc. In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamfl / CD150 +, CD48-, and lin- (mature lineages including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra, etc.) ST-HSCs are CD34+, SCA-1+, C-kit+, CD 135-, Slamfl / CD150+, and lin- (Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra, etc.) In addition, ST-HSCs are negative for mature lineage markers, including Under certain conditions, LT-HSCs are less quiescent and more proliferative than LT-HSCs. However, LT-HSCs have the ability to self-renew. Higher (i.e., LT-HSCs survive throughout adulthood and are continuously passed through recipients can be transplanted), whereas ST-HSCs have limited self-renewal capacity (i.e., ST-HSCs (They survive for a limited period of time and cannot be transplanted serially). ST-HSCs can be used in the methods described herein because they are highly proliferative. These are particularly useful because they can generate differentiated progeny more quickly.

[0074] As used herein, the term "anti-hematopoietic cell antibody" refers to a dy)" or "anti-HC antibody" is an antibody that binds to an antigen expressed by hematopoietic stem cells (e.g., CD117 [e.g., GNNK+ CD117], or CD45), or antigens expressed by mature immune cells (e.g., T-cells) (e.g., It refers to an antibody that specifically binds to a target antigen (e.g., CD45).

[0075] As used herein, the term "functional potential of hematopoietic stem cells" refers to: 1) pluripotency (multipotency) ti-potency) (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., (This refers to the ability to differentiate into multiple different blood lineages, including, for example, NK cells, T cells, and B cells.) 2) self-renewal (which is the process by which hematopoietic stem cells give rise to daughter cells with the same potential as the parent cell) Furthermore, this ability does not wear out and occurs repeatedly throughout an individual's lifespan. and 3) hematopoietic stem cells or their progeny, when reintroduced into the transplant recipient, can induce hematopoiesis. hematopoiesis, including the ability to home to stem cell niches and re-establish productive and sustained hematopoiesis. Refers to the functional properties of stem cells.

[0076] As used herein, the term "donor chimerism" refers to the The lymphohematopoietic system of the recipient (i.e., host) of allogeneic hematopoietic stem cell transplantation For example, 85% donor chimerism Hematopoietic stem cell transplantation (HSCT) is a type of lymphoid tumor regeneration that contains 85% donor cells after allogeneic hematopoietic stem cell transplantation. In some embodiments, the methods herein involve in vivo At least 80% donor chimerism, at least 85% donor chimerism It is effective in establishing chimerism (no chimerism) or at least 90% chimerism. The degree of chimerism (e.g., the proportion of donor stem cells in the host) can be assessed using a number of standard methods. In the host, the presence of a sex chromosome specific marker or the like may be detected by standard methods. The presence of donor markers can be confirmed, for example, by standard cytogenetic analysis, appropriate Polymerase chain reaction (PCR) using primers, variable number tandem repeat-PCR (VNTR -PCR), microsatellite markers or other fingerprinting techniques, or may be determined using fluorescence in situ hybridization (FISH). Donor chimerism can also be achieved by, for example, standard complement-dependent microcytotoxicity. by measuring the proportion of cells of the donor type in the host's blood using a toxicity test. However, judgment may be made even if

[0077] As used herein, the term "mismatched" (e.g., "MHC-mismatched," "H "miHA-mismatch" or "miHA-mismatch") is a mismatch in the context of hematopoietic stem cell transplantation. Recipient expressed on allogeneic cells (or tissues or organs) (e.g., donor cells) and at least one dissimilar (e.g., non-identical) cell surface antigen variant. In some embodiments, allogeneic transplants refer to the presence of antigens in the transplant recipient. This may contain "minor mismatches" with respect to the original entry. "Mismatches" include individual differences in cell surface antigens other than MHC antigens or HLA antigens. Minor mismatches include differences in minor histocompatibility antigens. In some embodiments, the allogeneic transplant is a "meta-transplant" to the transplant recipient. Such a "major mismatch" may include a "major mismatch" In the MHC haplotype or in the HLA haplotype between the graft and the recipient In an exemplary embodiment, an allogeneic transplant is a transplant recipient share the same MHC or HLA haplotype as the target, but with one or more minor differences (referred to herein as "minor-mismatched allografts") In another exemplary embodiment, the allogeneic graft comprises one or more major myocardiums. A "complete" mismatch may contain a mismatch, either alone or in addition to one or more minor mismatches. A "full mismatch" allograft is one or more major mismatches and Refers to allogeneic grafts containing one or more minor mismatches. The presence of minor mismatches can be confirmed by conventional assays used in the art (e.g. may be determined by serological, genomic, or molecular analysis In some embodiments, at least one major histocompatibility complex antigen is mismatched to the allele expressed by the recipient, or Additionally, at least one minor histocompatibility antigen is expressed by the recipient. It is a mismatch to the corresponding allele.

[0078] As used herein, the terms "subject" and "patient" refer to a subject, as described herein. refers to a living organism, such as a human, that is being treated for a specific disease or condition. Patients such as patients undergo hematopoietic stem cell transplantation therapy to promote engraftment of exogenous hematopoietic stem cells. Treatment may be given before the law.

[0079] As used herein, the term "donor" refers to a person who provides cells or other organs to a recipient. refers to a human or animal from which one or more cells have been isolated prior to administering to the human or animal progeny thereof. The cells may be, for example, a population of hematopoietic stem cells.

[0080] As used herein, the term "recipient" refers to a population of hematopoietic stem cells. The term "transplant" refers to a patient receiving a transplant, such as a transplant containing cells. For example, they can be autologous, syngeneic, or allogeneic cells.

[0081] As used herein, the term "endogenous" refers to a gene that is naturally occurring in a particular organism, such as a human patient. naturally occurring molecules, cells, tissues, or organs (e.g., hematopoietic stem cells, megakaryocytes, platelets ( thrombocyte, platelet, red blood cell, mast cell, myeloblast, basophil, neutrophil, Eosinophils, microglial cells, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, Cells of the hematopoietic lineage, such as dendritic cells, natural killer cells, T-lymphocytes, or B-lymphocytes This describes substances such as vesicles.

[0082] As used herein, the term "sample" refers to a specimen (e.g., a sample) taken from a subject. , blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or dermis), pancreatic juice, chorionic villus samples, and cells).

[0083] As used herein, the term "scFv" refers to a fragment comprising the variable domain of a heavy chain and a light chain from an antibody. The scFv fragment refers to a single-chain Fv antibody in which the variable domains of the Fv fragment and the Fv fragment are linked to form a single chain. The variable region of the antibody light chain (V 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 CDR-H 3) The V of the scFv fragment L Area and V H Connecting regions The linker is a peptide linker made up of amino acids that make up proteins. Alternative linkers have been proposed 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 achieve this, a polyethylene glycol-containing linker or repeating glycine and hydrophilic linkers such as polypeptides containing serine residues), biophysical properties of the molecule To improve stability (e.g., systems that form intramolecular or intermolecular disulfide bonds), linkers containing methylamino acid residues), or to reduce the immunogenicity of the scFv fragment (e.g. For example, a linker containing a glycosylation site may be used. The variable regions of the scFv molecules to be 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 (corresponding to In order to preserve or enhance the ability of the antibody to bind to the antigen recognized by the antibody, Nucleotide or amino acid substitutions resulting in conservative substitutions or changes in the CDRs and / or may be performed on backbone residues).

[0084] As used herein, the phrase "substantially cleared from the blood" refers to a therapeutic administering an agent (e.g., an anti-CD117 antibody, an anti-CD45 antibody, or an antigen-binding fragment thereof) to a patient; The concentration of the therapeutic agent in a blood sample isolated from the patient at a time point after administration. If the intensity is such that the therapeutic agent cannot be detected by conventional methods (e.g., The therapeutic agent is a nucleotide of the device or assay used to detect the therapeutic agent. This refers to cases where the detection threshold is exceeded and the detection is impossible. Various techniques (e.g., ELISA, known in the art or described herein) can be used. antibody, antibody fragment, and protein ligation assays (e.g., antibody-based detection assays) May be used to detect antibodies or antibody fragments Further assays include immunoprecipitation and immunoblotting, among others, which are well known in the art. Assays are included.

[0085] As used herein, the term "transfection" refers to electroporation. fusion, lipofection, calcium phosphate precipitation, DEAE-dextran transfection Commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as recombinant DNA technology. The term "technology" refers to any of a wide variety of technologies that are used commercially.

[0086] As used herein, the terms "to treat" or "treatment" refer to "Treatment" means reducing the severity and / or frequency of disease symptoms, alleviating disease symptoms and / or eliminating the underlying cause of said symptoms, the frequency of disease symptoms and / or their underlying causes or reducing the likelihood of death, as well as the damage caused directly or indirectly by the disease. improving or repairing the wound, any improvement in any outcome of the disease (e.g., increased survival time) the side effects of reduced morbidity and / or alternative treatment modalities As is readily understood in the art, Complete eradication of the disease is preferred, but is not a requirement for therapeutic intervention. Desired clinical outcomes include, but are not limited to, those described herein. Exogenous factors were detected in patients after antibody conditioning therapy and subsequent hematopoietic stem cell transplantation. Further beneficial outcomes include enhanced hematopoietic cell engraftment. In patients requiring transplantation, a conditioning regimen followed by exogenous hematopoietic stem cell transplantation is used. After administering the cell graft to the patient, an increase in the number of hematopoietic stem cells or a relative concentration Beneficial results of the therapies described herein also include increased After conditioning therapy and subsequent hematopoietic stem cell transplantation, one or more cells of the hematopoietic lineage are Cells (megakaryocytes, thrombocytes, platelets, red blood cells, mast cells, myeloblasts) , basophils, neutrophils, eosinophils, microglial cells, granulocytes, monocytes, osteoclasts, antigen-presenting cells alveoli, macrophages, dendritic cells, natural killer cells, T lymphocytes, or B lymphocytes, etc. This may include an increase in the number of cells or an increase in the relative concentration of Additional beneficial results include the proliferation of cancer cells (e.g., CD117+ leukemia cells) or autoimmune cells (e.g., CD117+ autoimmune diseases, such as CD117+ T-cells that express T-cell receptors that cross-react with self-antigens These may include a reduction in the abundance of disease-causing cell populations, such as the lymphocyte (proliferative lymphocyte) population. To the extent that the methods of the present disclosure are directed to preventing a disorder, the term "prevent" does not include the term "preventing" or "preventing" as used herein. It is understood that this does not require that the condition be completely prevented. When used, the term prevent refers to identifying a population susceptible to a disorder and preventing the onset of the disease. The term refers to a disease that can be treated by a person skilled in the art, such as administering a compound of the present disclosure to a patient before the onset of the disease. This does not mean that the above conditions are completely avoided.

[0087] As used herein, a patient "in need" of a hematopoietic stem cell transplant is one or more hematopoietic stem cell transplant recipients. Patients who exhibit defects or deficiencies in the types of fluid cells, as well as those with stem cell disorders, autoimmune diseases, cancer, or Hematopoietic stem cells are generally classified into two types: 1) multipotent (pluripotent) and 2) pluripotent (pluripotent) stem cells. -potency), thus including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, erythrocytes), platelets (e.g., thrombocytes) megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes , macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells 2) can differentiate into multiple different blood lineages, including autologous, B-cells, and T-cells; capable of replicating, thus giving rise to daughter cells with potential equivalent to that of the mother cell; and 3) When reintroduced into the transplant recipient, they home to the hematopoietic stem cell niche and become productive and persistent. Thus, the ability to re-establish normal hematopoiesis in one or more cell types of the hematopoietic lineage. Reconstitute defective or missing cell populations in vivo in patients with defective or missing cells. For example, the patient may be suffering from cancer and the hematopoietic stem cells may be administered to treat the cancer. The defect may be a chemotherapeutic agent that selectively or non-specifically reduces the cancerous cell population. It may also be caused by administering steroid or other drugs. Alternatively, the patient may have sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and hemoglobinopathies such as Wiskott-Aldrich syndrome (e.g., non-malignant hemoglobinopathies). The subject may be suffering from adenosine deaminase (ADD) severe steroid syndrome (SASS). Combined immunodeficiency (ADA SCID), HIV / AIDS, metachromatic leukodystrophy, Diamond-Brown Subjects suffering from Schwachmann anemia and Shwachmann-Diamond syndrome The subject may have an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune disease. Additionally or alternatively, the subject may have or be affected by have or are affected by a malignant tumor such as neuroblastoma or blood cancer For example, the subject may have leukemia, lymphoma, or myeloma. In some embodiments, the subject has acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, Leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma or non-Hodgkin lymphoma In some embodiments, the patient has non-Hodgkin's lymphoma. The subject has myelodysplastic syndrome. In some embodiments, the subject has scleroderma, multifocal sclerosis, or fibromyalgia. autoimmune diseases such as idiopathic sclerosis, ulcerative colitis, Crohn's disease, type I diabetes, or any of the diseases described herein In some embodiments, the subject has another autoimmune condition described above. In some embodiments, the subject is in need of metabolic T-cell receptor (CART) therapy. The subject may have or be affected by a glycogen storage disorder. Mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidosis, metachromatic leukodystrophy, or a metabolic disorder selected from the group consisting of: any other disease or disorder that may be acquired (including but not limited to severe combined immunodeficiency syndrome, General illness, Wiskott-Aldrich syndrome, Hyperimmunoglobulin M syndrome, Chediac Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia Sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile onset Rheumatoid Arthritis), and "Bone and Marrow Transplantation for Non-Malignant Disease", ASH Education Book, 1:319-338 (2000) (This disclosure is intended to be used to prescribe hematopoietic stem cell transplantation therapy. and relates to conditions that may be treated by the methods disclosed herein, which are incorporated by reference in their entirety. suffer from or are affected by a disease or disorder listed in Additionally or alternatively, a patient "in need" of a hematopoietic stem cell transplant may be Patients with or without one of the pathologies (but who nevertheless have megakaryocytes) , thrombocyte, platelet, red blood cell, mast cell, myeloblast, basophil , neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages Within the hematopoietic lineage, including lymphocytes, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes a reduction in the levels of one or more endogenous cell types [e.g., levels in an otherwise healthy subject] Some of the skilled artisans may, for example, Flow cytometry and fluorescence-activated cell sorting (FAC), among other procedures known in the surgical field, are used. S) The method provides a method for determining the level of one or more of the above cell types or other blood cell types. and whether the level is decreased relative to otherwise healthy subjects can be easily determined. do.

[0088] As used herein, the term "immunosuppressive agent" or "Immunosuppressant" is a drug that suppresses the immune system of hematopoietic transplant recipients. This refers to substances that act to suppress or mask the system, including cytokine production. suppressing the expression of autoantigens, downregulating or suppressing the expression of autoantigens, or inhibiting the expression of MHC These would include substances that mask antigens. Examples of such agents include calcineurin, Phospho / MTOR inhibitors (e.g., tacrolimus, sirolimus, rapamycin, cyclosporine, everolimus), molecules that block costimulation (e.g., CTLA4-Ig, anti-CD40L), and reduce NK Drugs, anti-thymocyte globulin (ATG), alkylating agents (e.g. , nitrogen mustards, e.g., cyclophosphamide; nitrosoureas (e.g., , carmustine); platinum compounds), methotrexate, anti-TCR agents (e.g., muromonab-CD3) , anti-CD20 antibodies (e.g., rituximab, ocrelizumab, ofatumumab, and veltuzumab) fludarabine, Campath (alemtuzumab), 2-amino-6-aryl-5-substituted Pyrimidines (see U.S. Pat. No. 4,665,077, the disclosure of which is incorporated herein by reference) ), azathioprine (or, if adverse reactions to azathioprine occur, cyclophosphamide, bromocriptine; glutaraldehyde (which is described in U.S. Patent 4,120,649) masking MHC antigens, as described above); anti-idiotypic antibodies against MHC antigens; cyclosporin A; one or more steroids (e.g., corticosteroids, e.g., prednisolone); anti-interferon; anti-inflammatory drugs ... Anti-tumor necrosis factor-γ antibody; anti-tumor necrosis factor-α antibody; anti-tumor necrosis factor-β antibody; anti-interleukin anti-IL-2 receptor antibodies; xenogeneic anti-lymphocyte antibodies pan-T antibodies, e.g., OKT-3 monoclonal antibody; antibodies against CD4; antibodies against CD8 antibodies against CD45 (e.g., 30-F11, YTH24.5 and / or YTH54.12 (e.g., YTH a combination of YTH54.5 and YTH54.12); streptokinase; streptodornase; or host-derived Further immunosuppressants include, but are not limited to, RNA or DNA. However, total body irradiation (TBI), low-dose TBI, and / or cytoxin (Cytoxan) is an example.

[0089] As used herein, the terms "variant" and "derivative" ) are used interchangeably and refer to the compounds, peptides, proteins described herein. The term "antibody" refers to naturally occurring, synthetic, and semi-synthetic analogs of , or other substrates. Variants or derivatives of compounds, peptides, proteins, or other substances described in The biological activity of the substance may be retained or improved.

[0090] As used herein, the phrase "stem cell disorder" refers to a condition that conditions a target tissue of a subject. and / or by partitioning the target tissue to remove endogenous stem cell populations. by removing (e.g., removing an endogenous hematopoietic stem cell population or hematopoietic stem cells from the subject's bone marrow tissue) by removing progenitor cell populations) and / or by introducing stem cells into the target tissue of interest. Any disease, disorder, or condition that can be treated or cured by engraftment or transplantation For example, type 1 diabetes has been shown to be cured by hematopoietic stem cell transplantation. and by conditioning in accordance with the compositions and methods described herein. can benefit from the use of the compositions and methods described herein. Additional disorders include, but are not limited to, sickle cell anemia, thalassemia, Fanconi anemia, Anemia, aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV / AIDS, heterochromatin Leukodystrophy, Diamond-Blackfan anemia, and Shwachman-Dia Mondo syndrome. Further diseases that can be treated using stem cell transplantation include inherited blood disorders (e.g. (e.g., sickle cell anemia) and autoimmune diseases such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease. Autoimmune diseases are also included. Further diseases that can be treated using the method include malignant tumors (e.g., neuroblastoma), or hematological cancers (e.g., For example, the cancer may be acute myeloid leukemia, lymphoma, or myeloma. Leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, Diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma ) using the conditioning and / or transplantation methods described herein. Further diseases that can be treated include myelodysplastic syndromes. The subject has or is affected by a metabolic storage disorder. For example, the subject , glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidosis, heterochromatin leukodystrophy, or a metabolic disorder disclosed herein. Any other disease or disorder that can benefit from treatment and therapy, including but not limited to: No, but 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 is for the purpose of providing information on the possibility of treating hematopoietic stem cell transplantation therapy. and which are incorporated herein by reference in their entirety. may be suffering from or affected by a disease or disorder that is

[0091] As used herein, the term "vector" includes plasmids, DNA vectors, and nucleic acid vectors, such as nucleotides, RNA vectors, viruses, or other suitable replicons. The expression vectors described herein can be used to express polynucleotide sequences as well as, for example, and / or by integrating these polynucleotide sequences into the genome of mammalian cells. The antibody of the present invention may contain additional sequence elements that are used to incorporate the sequence. Certain vectors that can be used to express antibody fragments include: Plasmids containing regulatory sequences, such as promoter regions and enhancers, that direct gene transcription Other useful vectors for expressing antibodies and antibody fragments include enhance the rate of translation of these genes or improve the stability or nucleation of the mRNA resulting from gene transcription. These sequence elements include polynucleotide sequences that improve the expression of the 5' and 3' untranslated regions and polynucleotides that allow efficient transcription of genes carried on the vector The expression vectors described herein may also contain a denylation signal site. A polynucleotide encoding a marker for selecting cells containing a vector such as Examples of suitable markers include ampicillin, chloramphenicol, Resistance to antibiotics such as kanamycin and nourseothricin Examples of genes that regulate the expression of ATP include:

[0092] As used herein, the term "conjugate" or "antibody drug conjugate" " or "ADC" refers to an antibody conjugated to a cytotoxin. An ADC is an antibody, or its antigen-binding fragment. The reactive functional groups of one molecule, such as a cytotoxin, and the reactive functional groups of another molecule, such as a cytotoxin, are described herein. Conjugates are formed by chemical bonding with suitable reactive functional groups on the molecule. A linker may be included between the two molecules that are linked together (e.g., between an antibody and a cytotoxin). Examples of linkers that can be used to form conjugates include peptide-containing linkers. - (e.g., containing naturally occurring or non-naturally occurring amino acids, e.g., D-amino acids) Linkers include those described herein and known in the art. It is known that various strategies can be used to prepare Depending on the reactive moiety, the linker can be modified by, for example, enzymatic hydrolysis, photolysis, or cleavage under acidic conditions. Hydrolysis, basic hydrolysis, oxidation, disulfide reduction, nucleophilic cleavage, or organogold Genetic cleavage can result in cleavage (e.g., Leriche et al., Bioorg. Med. Chem., 2 0:571-582, 2012).

[0093] As used herein, the term "microtubule-binding agent" refers to an agent that induces mitosis and Compounds that act by disrupting the microtubule network essential for cell function during and after interphase Examples of microtubule-binding agents include maytansin, maytansinoids, and their derivatives. cyclins (such as those described herein or known in the art), vinblastine, vinblastine, Vinblastine sulfate, vincristine, vincristine sulfate, vindesine, and vinorelbine Vinca alkaloids such as benzodiazepines, taxanes such as docetaxel and paclitaxel, disco Macrolides such as dermolide, colchicine, and epothilones, and their Derivatives thereof (such as epothilone B or its derivatives) are included, but are not limited to these.

[0094] As used herein, the term "amatoxin" refers to the compound found in Amanita phalloides mushrooms. A member of the amatoxin family of peptides produced by, or a variant thereof or derivatives thereof (e.g., variants or derivatives thereof capable of inhibiting RNA polymerase II activity) Amatoxyl derivatives useful in conjunction with the compositions and methods described herein Examples of compounds include, but are not limited to, compounds represented by formula (III), (IIIA), (IIIB) and (IIIC): ) compounds (each as described herein below) (e.g., α-amanitin, β- Amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, Examples of compounds include amanuric acid, amanuric acid, and proamanurin. In this way, the amatoxin can be linked to the antibody, or its antibody, for example, via a linker moiety (L). The antibody may be conjugated to a protease-binding fragment (thereby forming an ADC). Exemplary amatoxin conjugation methods and methods useful in such processes are The antibodies or antigen-binding fragments according to the compositions and methods are described below. Exemplary linker-containing amatoxins useful for conjugation to the α-amyloid β ... Posted.

[0095] As used herein, the term "acyl" refers to -C(=O)R, where R is an acyl group of the present invention. As defined herein, hydrogen ("aldehyde"), alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl. Non-limiting examples Examples include formyl, acetyl, propanoyl, benzoyl, and acryloyl. can be.

[0096] As used herein, the term "alkyl" refers to an alkyl group having, for example, 1 to 20 carbon atoms in the chain. Examples of alkyl groups include methyl, ethyl, and methyl groups. , n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl Examples include ethyl, isopentyl, tert-pentyl, hexyl, and isohexyl.

[0097] As used herein, the term "alkylene" refers to a straight or branched chain divalent alkyl The divalent positions may be on the same atom or on different atoms within the alkyl chain. Examples of alkylene include methylene, ethylene, propylene, and isopropylene. Examples include:

[0098] As used herein, the term "heteroalkyl" refers to a heteroalkyl group having, for example, 1 to 20 alkyl groups in the chain. carbon atoms and one or more heteroatoms in the chain (e.g., oxygen, nitrogen, or refers to a straight or branched chain alkyl group containing sulfur.

[0099] As used herein, the term "heteroalkylene" refers to a straight or branched chain divalent heteroalkylene. The divalent positions may be on the same atom within the heteroalkyl chain, but may be different. The divalent positions may be on one or more heteroatoms.

[0100] As used herein, the term "alkenyl" refers to an alkyl group having, for example, 2 to 20 carbon atoms in the chain. It refers to a straight or branched chain alkenyl group having 2 or more atoms. Examples of alkenyl groups include vinyl propenyl, isopropenyl, butenyl, tert-butylenyl, hexenyl, and the like. do.

[0101] As used herein, the term "alkenylene" refers to a straight or branched chain divalent alkenylene. The divalent positions may be on the same atom within the alkenyl chain or on different atoms. Examples of alkenylene include ethenylene, propenylene, and isopropenylene. Examples of suitable alkylenes include olefins, butenylene, and the like.

[0102] As used herein, the term "heteroalkenyl" refers to a heteroaryl group having, for example, 2 to 20 alkyl groups in the chain. carbon atoms in the chain, and further containing one or more heteroatoms (such as oxygen, nitrogen, or refers to straight or branched chain alkenyl groups containing sulfur.

[0103] As used herein, the term "heteroalkenylene" refers to a straight or branched chain bivalent The divalent positions are on the same atom within the heteroalkenyl chain. The divalent positions may be on one or more heteroatoms. do.

[0104] As used herein, the term "alkynyl" refers to an alkynyl group having, for example, 2 to 20 carbon atoms in the chain. It refers to a straight or branched chain alkynyl group having 1 to 3 carbon atoms. Examples of alkynyl groups include propane, methyl ... Examples include arginyl, butynyl, pentynyl, hexynyl, and the like.

[0105] As used herein, the term "alkynylene" refers to a straight or branched chain divalent alkyl group. The divalent positions may be on the same atom within the alkynyl chain or on different atoms. It's okay to have it.

[0106] As used herein, the term "heteroalkynyl" refers to a heteroalkynyl group having, for example, 2 to 20 alkyl groups in the chain. carbon atoms in the chain, and further containing one or more heteroatoms (such as oxygen, nitrogen, or refers to straight or branched chain alkynyl groups containing sulfur.

[0107] As used herein, the term "heteroalkynylene" refers to a straight or branched chain bivalent The bivalent positions are on the same atom within the heteroalkynyl chain. The divalent positions may be on one or more heteroatoms. do.

[0108] As used herein, the term "cycloalkyl" refers to a saturated and For example, monocyclic or fused, bridged, or spiropolycyclic ring structures having 3 to 12 carbon ring atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclopentyl. cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[3.1.0]hexane, etc. Examples include:

[0109] As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl group. The bivalent positions may be on the same atom or on different atoms within the ring structure. Examples of cycloalkylene include cyclopropylene, cyclobutylene, and cyclopentylene. Examples of suitable alkyl ethers include cyclohexylene and cyclohexylene.

[0110] As used herein, the term "heterocyloalkyl" refers to a saturated and consisting of, for example, carbon atoms and, for example, nitrogen, oxygen, and and heteroatoms selected from sulfur and having 3 to 12 ring atoms per ring structure. The term "monocyclic, or fused, bridged, or spiropolycyclic ring structures" refers to ring structures such as It may contain one or more oxo groups on the carbon, nitrogen, or sulfur ring members. Examples of cycloalkyl include, but are not limited to, dihydropyridyl, tetrahydropyridyl, Hydropyridyl (piperidyl), tetrahydrothiophenyl, piperidinyl, 4-piperidonyl pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydropyranyl, biphenyl tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydropyranyl, Trihydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, pipet Examples include rhazinyl, quinuclidinyl, and morpholinyl.

[0111] As used herein, the term "heterocycloalkylene" refers to a divalent heterocycloalkylene. The divalent positions may be on the same atom within the ring structure or on different atoms. It's okay to have it.

[0112] As used herein, the term "aryl" refers to an aryl group containing, for example, 6 to 19 carbon atoms. Aryl groups include phenyl, fluorenyl, naphthyl, etc. The divalent position may be one or more heteroatoms. It may be a child.

[0113] As used herein, the term "arylene" refers to a divalent aryl group. The positions may be on the same atom or on different atoms.

[0114] As used herein, "heteroaralkyl" refers to a heteroaryl group having a carbon atom (typically a terminal or sp 3 charcoal A non-cyclic heteroaryl group in which one of the hydrogen atoms bonded to the heteroaryl group is replaced by a heteroaryl radical. Typical heteroarylalkyl groups include, but are not limited to: Examples of the methyl group include, but are not limited to, 2-benzimidazolylmethyl and 2-furylethyl. Heteroarylalkyl groups contain 6 to 20 carbon atoms, e.g., heteroarylalkyl The alkyl portion of the group (including alkanyl, alkenyl, or alkynyl groups) may have 1 to 6 carbon atoms, and the heteroaryl moiety is 5 to 14 carbon atoms, and N, O, P, and S. The heteroarylalkyl group has 1 to 3 heteroatoms selected from the group consisting of aryl, aryl, aryl and aryl. The aryl moiety may be a single ring having 3 to 7 ring members (2 to 6 carbon atoms); has 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 hetero atoms selected from N, O, P, and S) It can be a bicyclic ring with 4,5, 5, 5, 6, or 6, 6 atoms (for example: a bicyclic ring [4,5], [5,5], [5,6], or [6,6] ring). stem).

[0115] As used herein, the term "heterocycloalkyl" refers to a heterocycloalkyl group that is saturated and selected from, for example, carbon atoms and, for example, nitrogen, oxygen, and sulfur, among others. monocyclic or refers to a fused, bridged, or spiro polycyclic ring structure. The ring structure may be, for example, a ring structure containing carbon, nitrogen, or may contain one or more oxo groups on the sulfur ring member. Examples of cyclohexyl methyl ether include, but are not limited to, dihydropyridyl, tetrahydropyridyl ( piperidyl), tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydro propylanyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydroiso Quinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinunyl Examples include clidinyl and morpholinyl.

[0116] As used herein, the term "heterocycloalkylene" refers to a divalent heterocycloalkylene. The divalent positions may be on the same atom or on different atoms within the ring structure. It's okay to have it.

[0117] As used herein, the term "aryl" refers to an aryl group containing, for example, 6 to 19 carbon atoms. The aryl group refers to a monocyclic or polycyclic aromatic ring system including phenyl, fluorenyl, The divalent position may contain one or more heteroatoms, including, but not limited to, naphthyl, etc. This may be the case.

[0118] As used herein, the term "arylene" refers to a divalent aryl group. The positions may be on the same atom or on different atoms.

[0119] As used herein, the term "heteroaryl" refers to a monocyclic heteroaromatic or bicyclic heteroaromatic ring. A cyclic or tricyclic fused-ring heteroaromatic group, wherein one or more ring atoms is a heteroatom. Heteroaryl groups include pyridyl, pyrrolyl, and the like. , furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, i Sothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxazolyl Azolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl , 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl Isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, iodobenzofuryl, Indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyri Zyl, benzothiazolyl, benzoxazolyl, quinolizinyl, quinazolinyl, phthalazide quinoxalinyl, cinnolinyl, napthyridinyl, pyrido[3,4-b]pyridyl, pyridyl do[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-Tetrahydroquinolyl, 5,6,7,8-Tetrahydroisoquinolyl, Purinyl, Pteryl Examples thereof include diphenyl, carbazolyl, xanthenyl, and benzoquinolyl.

[0120] As used herein, the term "heteroarylene" refers to a divalent heteroaryl group. The divalent positions may be on the same atom or on different atoms. may be one or more heteroatoms.

[0121] 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.

[0122] 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, 5-pyridyl, 6- Pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyridazinyl Pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl thiazolyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl Examples include:

[0123] 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. Rings include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, and 1-imidazolyl. , 1-pyrazolyl, and 1-piperidinyl.

[0124] Unless otherwise constrained by the definition of the individual substituents, the above chemical substructures, e.g. For example, "alkyl," "alkylene," "heteroalkyl," "heteroalkylene," alkenyl," "alkenylene," "heteroalkenyl," "heteroalkenylene," alkynyl, alkynylene, heteroalkynyl, heteroalkynylene, "Cycloalkyl", "cycloalkylene", "heterocycloalkyl", "heterocyclo "aryl," "arylene," "heteroaryl," and "heteroaromatic" The "arylene" group and the like are optionally substituted with, for example, alkyl, alkenyl, alkynyl, cyclohexyl, alkyl, heterocycloalkyl, alkyl-aryl, alkyl-heteroaryl, Alkyl cycloalkyl, alkyl heterocycloalkyl, amino, ammonium, Acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea Iodide, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy Ci, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, methyl It may be substituted with 1 to 5 substituents selected from the group consisting of capryl, nitro, etc. Typical substituents include, but are not limited to, -X, -R, -OH, -OR, -SH, -SR, NH, -NH R, -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(=N where each X is selected from F, Cl, Br, and I, in each case and each R is alkyl, aryl, heterocycloalkyl, or heteroaryl. a heteroaryl, a protecting group, and a prodrug moiety, each independently selected in each case from Whenever a group is described as being "optionally substituted," the group is, in each occurrence independently, optionally substituted with one or more of the above substituents. The substitution may be ring-closing with adjacent substituents (e.g., ring-closing of adjacent functional substituents), e.g., Lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals, amines nals, and hemiaminals (which can be formed by ring closure, e.g., to provide a protecting group) This may include circumstances that form a

[0125] 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".

[0126] As used herein, the term "coupling reaction" refers to the process of combining suitable compounds to react with each other. Two or more substituents react to form molecular fragments attached to each substituent (e.g., Coupling Reactions: A chemical reaction that forms covalently (or covalently) bonded chemical substructures. The inhibitor may include a cytotoxin (e.g., a cytotoxin known in the art or described herein). 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 (such as GNNK+ CD117) specific antibodies known in the art or described herein. Antibody, antigen-binding fragment thereof) with appropriate reactivity bound to the fragment Examples of suitable reactive substituents include nucleophilic / electrophilic pairs ( For example, in particular, thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α, β-unsaturated carbonyl pairs), diene / dienophile pairs (e.g., azide / alkoxy, among others). Coupling reactions include, but are not limited to, thiol- Alkylation, hydroxyl alkylation, amine alkylation, amine condensation, amidation, Esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cyclization, among others) addition, [3+2] Huisgen cycloaddition), aromatic nucleophilic substitution, aromatic electrophilic substitution, and related art Other response modalities known in the art or described herein are included.

[0127] As used herein, "CRU (competitive repopulation unit)" refers to a g unit)) is a unit of measurement for long-term engrafting stem cells that can be detected after in vivo transplantation. This refers to the following.

[0128] As used herein, "drug-to-antibody ratio" or "DAR" " refers to the number of cytotoxins (e.g., amatoxins) attached to the antibodies of the ADC. Depending on the number of sites, higher loadings are possible, but the DAR of the ADC should be in the range of 1 to 8. In certain embodiments, the ADCs described herein include 1, 2, 3, 4, 5, 6, 7, or has a DAR of 8.

[0129] The substituent is shown as a di-radical (i.e., the positions of both bonds to the rest of the molecule are Wherever a group has a substituent, said substituents may be attached in any orientation unless otherwise specified. It should be understood that this is possible.

[0130] Treatment method Disclosed herein are methods for treating allogeneic transplants, such as allogeneic hematopoietic stem cell (HSC) transplants. Methods for reducing the population of CD117+ cells and / or the population of CD45+ cells in a patient in need thereof Increased levels of allogeneic cell engraftment in recipient subjects Also provided herein are methods for performing allogeneic transplantation. Various transplant-related disorders (especially, for example, cell-type diseases in the hematopoietic system, cancer, autoimmune to treat diseases, metabolic disorders, graft-versus-host disease, host-versus-graft rejection, and stem cell disorders The compositions and methods described herein may be used to treat (i) conditions that cause pathology. Populations of cells (e.g., cancer cells [e.g., leukemia cells] and autoimmune cells [e.g., autoimmune (ii) may directly deplete the transplanted immune system (e.g., populations of immune-reactive T-cells) and / or Promotes engraftment of transplanted hematopoietic stem cells by providing a niche for cell homing These may reduce the endogenous hematopoietic stem cell population, as seen in transplants (e.g., HSC Depleting endogenous hematopoietic cells in a subject in need of transplantation (transplantation) involves the depletion of endogenous hematopoietic stem cells. ADCs, antibodies, or antigen-binding fragments thereof, capable of binding to antigens expressed by This can be achieved by administering the drug to patients to prepare them for transplantation therapy. When administered, this treatment selectively reduces the endogenous hematopoietic stem cell population. This creates space in the bone marrow and other hematopoietic tissues, which are then transplanted. The vacancy may be filled by exogenous hematopoietic stem cells (e.g., CD1 17+ [e.g., GNNK+ CD117] or CD45+ cells), or antigens expressed by immune cells (e.g., It binds to antigens (e.g., CD45) expressed by mature immune cells (e.g., T-cells). ADCs, antibodies, or antigen-binding fragments thereof that can be administered to patients to reduce the number of cells. Therefore, antigens expressed by hematopoietic stem cells (e.g., CD117) can have a beneficial effect. [e.g., GNNK+ CD117] or CD45), or immune cells (e.g., mature immune cells) (e.g., T- ADCs, antibodies, or their antigen-binding molecules that bind to antigens (e.g., CD45) expressed by cells (e.g., cells) The recombinant fragment is administered to a patient suffering from cancer or an autoimmune disease to inhibit the growth of cancer cells or It may directly reduce the population of autoimmune cells, as well as reduce the proliferation of transplanted cells (e.g., In order to promote the survival and engraftment potential of hematopoietic stem cells, It may be administered to patients in need of treatment.

[0131] Transplant patients may receive autologous grafts (the grafts contain the subject's own cells). In another embodiment, the transplant patient receives an allogeneic graft (the graft is obtained from another individual). In the case of allogeneic transplants, a patient may receive a transplant (containing cells derived from another individual or cells from another individual). The engraftment of transplanted cells may be mediated by the host's immune cells, potentially resulting in an immune response to the graft (host cell death). Graft-versus-graft disease), or an immune response against host cells mediated by immune cells present in the graft The transplant process is complicated by the possibility of graft versus host disease. With respect to the recipient patient, the degree of difference in the antigenic composition of the graft varies. The likelihood of the above complications increases. Therefore, allogeneic transplantation is usually performed with a focus on HLA antigens and minor It is performed between patients with the highest similarity of histocompatibility antigens. A very high degree of antigenic similarity is required between the recipient and the target, so a suitable match is required. People who cannot receive this treatment because a suitable donor is not available and who need a transplant There are patients who do this.

[0132] The methods provided herein include: (i) an ADC capable of binding to CD117 or CD45; and (ii) ) immunosuppressive drugs, to condition patients requiring allogeneic transplants. This results in a high degree of antigen mismatch between the allogeneic cells and the transplant recipient. The discovery that allogeneic donor cell engraftment is significantly increased, including in situations involving Without wishing to be bound by theory, it is believed that the immunosuppressive Antibody inhibitors inhibit the activity of residual immune cells (e.g., residual T cells) present in patients after ADC administration. It is thought that this may inhibit the growth of autologous cells, which may limit the engraftment of autologous cells. Administration of ADC in combination with immunosuppressants increases autologous donor cell engraftment and This leads to increased donor chimerism. In some embodiments, the method increases the engraftment of autologous hematopoietic stem cells and reduces the number of stem cells in the bone marrow and peripheral blood. Donor chimerism (e.g., myeloid chimerism, B-cell chimerism, and It may be used to increase immune responses (e.g., immune responses and T cell chimerism).

[0133] As described herein, hematopoietic stem cell transplantation therapy involves engrafting one or more blood cell types. It may be prescribed to a subject in need of treatment to induce or regenerate hematopoietic stem cells. Cells generally exhibit multi-potency and therefore include, but are not limited to: Granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, microcytes) platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts They differentiate into several different blood lineages, including cells, and lymphocytes (e.g., NK cells, B cells, and T cells). Furthermore, hematopoietic stem cells have the ability to self-renew and therefore can reproduce at the same rate as their parent cells. These give rise to daughter cells that have the potential to be reintroduced into the transplant recipient. Once activated, they retain the ability to home to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis. It can be a feature.

[0134] Therefore, to reconstitute defective or missing cell populations in vivo, hematopoietic lineage administering hematopoietic stem cells to patients with defects or deficiencies in one or more cell types of thereby treating conditions associated with defective or reduced populations of endogenous blood cells. Therefore, the compositions and methods described herein can be used to treat non-malignant hemoglobinopathies ( For example, sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiscott's anemia. To treat hemoglobinopathies selected from the group consisting of Tow-Aldrich syndrome Additionally or alternatively, the compositions and methods described herein can be used to treat congenital immune Additionally or alternatively, the compounds described herein may be used to treat immune deficiencies, such as immunodeficiency syndrome. The compositions and methods described herein can be used to treat acquired immune deficiency diseases (e.g., from the group consisting of HIV and AIDS). The compositions and methods described herein may be used to treat selected acquired immune deficiencies. and methods can be used to treat metabolic disorders (e.g., glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, a disease selected from the group consisting of Hurler's disease, sphingolipidosis, and metachromatic leukodystrophy; It may be used to treat metabolic disorders.

[0135] Additionally, or alternatively, the compositions and methods described herein can be used to treat malignant tumors or May treat proliferative disorders (e.g., blood cancers, myeloproliferative disorders). In some cases, the compositions and methods described herein may be used to stimulate endogenous hematopoiesis prior to hematopoietic stem cell transplantation therapy. The patient may be given a therapy to reduce the stem cell population, in which case the transplanted Cells home to the niche created by the endogenous cell depletion step. This may establish productive hematopoiesis, which in turn may eradicate cancer cells ( Depleted cell populations can be reconstituted during certain treatments (e.g., during systemic chemotherapy). Exemplary hematological cancers that can be treated using the compositions and methods described herein include: These include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, and Leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non- Non-Hodgkin's lymphoma and other cancers, including neuroblastoma Sexual symptoms include:

[0136] Additional conditions that may be treated using the compositions and methods described herein 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. can be done.

[0137] The antibodies, or antigen-binding fragments thereof, and conjugates described herein can be used to For example, the methods described herein may be used to induce solid organ transplant tolerance. The compositions and methods can be used to reduce or deplete a population of cells from a target tissue. This may result in a decrease in hematopoietic stem cells from the bone marrow stem cell niche. After this depletion of cells from the tissue, a population of stem or progenitor cells (e.g., For example, hematopoietic stem cells from an organ donor may be administered to the transplant recipient; and After engraftment of such stem or progenitor cells, transient or stable mixed chimerism occurs. This allows for long-term transplant tolerance without the need for additional immunosuppressive drugs. For example, the compositions and methods described herein can be used to prepare solid organ transplants. May induce transplant tolerance in patients (e.g., kidney transplants, lung transplants, among others). The compositions and methods described herein can be used in, for example, temporary transplants. Low rates of successful or stable donor engraftment are sufficient to induce long-term tolerance of the transplanted organ. and therefore are sufficiently suitable for use in connection with inducing solid organ transplant tolerance. Suitable.

[0138] Additionally, the compositions and methods described herein can be used to identify CD117+ (e.g., GNNK+ CD117) Or they may directly treat cancers, such as cancers characterized by cells that are CD45+. For example, Using the compositions and methods described herein, for example, CD117+ leukemia cells In patients with leukemia, CD117+ cancerous cells (e.g., leukemic cells) By reducing Cancers may be treated directly. Exemplary cancers that may be treated in this manner include: Acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloid leukemia Myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma These include blood cancers such as lymphoma.

[0139] Additionally, the compositions and methods described herein can be used to treat autoimmune disorders. For example, antibodies, or antigen-binding fragments thereof, can be used to kill CD45+ immune cells. For example, the compound may be administered to a subject, such as a human patient, suffering from an autoimmune disorder to For example, CD45+ immune cells specifically bind to self-antigens and initiate immune responses against self-antigens. These may be autoreactive lymphocytes such as T-cells that express T-cell receptors. The compositions and methods described herein reduce autoreactive CD45+ cells may be used to treat autoimmune conditions such as those described below. Alternatively, the compositions and methods described herein may be used to treat hematopoietic stem cell transplantation therapy. and to treat autoimmune diseases by reducing the endogenous hematopoietic stem cell population. In some cases, the transplanted cells are produced by a process that reduces the endogenous cells. These cells may home to the designated niche and establish productive hematopoiesis. The cell population that was depleted during eradication of the cells can now be reconstituted.

[0140] The antibody or antibody-drug conjugate may be administered as needed prior to transplantation of cells or solid organs into a patient. In one embodiment, an anti-CD45 ADC or an anti-CD117 ADC may be administered to a human patient. C is administered prior to transplantation of cells or solid organs (e.g., about 3 days, about 2 days, about 12 hours, or about 12 hours before). (before 12 hours to 3 days before, about 1 day before ... In one embodiment, the graft is administered to a human patient receiving the ADC from the patient's blood. The compound is administered to the patient after it has been cleared or substantially cleared from the body.

[0141] By administering immunosuppressants, the methods described herein also provide a host versus graft response. (HvG) reactions. Failure after hematopoietic stem cell transplantation (e.g., failure after hematopoietic stem cell transplantation) is generally due to the initial failure of donor cells to engraft. This manifests as either a lack of donor cells or a loss of donor cells after initial engraftment. (For a review, see Mattsson et al. (2008) Biol Blood Marrow Transp lant. 14(Suppl 1): 165-170).

[0142] Preventing host-versus-graft (HvG) reactions and thereby the risk of allograft failure To prevent or reduce the immune response, various immunosuppressants are administered in combination with anti-CD117 antibodies, anti-CD45 antibodies, or their respective May be used in combination with antibody-drug conjugates. Patients at risk for HvG reactions In patients with MHC-mismatch (e.g., HLA-mismatch), the use of immunosuppressants may lead to a higher degree of MHC-mismatch. of donor cells with minor histocompatibility antigen (miHA)-mismatches Engraftment becomes possible.

[0143] In some embodiments, an anti-CD117 antibody, an anti-CD45 antibody, or an antibody-drug conjugate thereof Infusion with one or more immunosuppressants (e.g., one, two, or three immunosuppressants) In some embodiments, an anti-CD117 antibody, an anti-CD45 antibody, or an antibody-drug thereof is administered. The monoconjugate can be administered in combination with two or more immunosuppressants (e.g., those described herein). Administer in combination with

[0144] In one embodiment, an anti-CD117 antibody, an anti-CD45 antibody, or an antibody-drug conjugate thereof The compound is administered in combination with an immunoreducing drug that allows for the depletion of B-cells and / or T-cells.

[0145] In some embodiments, the immune-reducing agent is an anti-CD4 antibody, an anti-CD8 antibody, or an anti-CD4 Examples of anti-CD4 antibodies include those known in the art, such as anti-CD8 antibodies and anti-CD4 antibodies. For example, ibalizumab (also known as Trogarzo, TMB-355, TNX-355, or Hu5A8) see, for example, US9790276 and US9587022B2, which are incorporated herein by reference. zanolimumab (also known as HuMax-CD4 or 6G5.2; see, e.g., WO199 7013852, which is incorporated herein by reference), tregalizumab (BT-061 Also known as cyclohexyl benzoate; see, e.g., US 7,452,981, which is incorporated herein by reference. priliximab (also known as Centara, cM-T412, CEN 000029, MT 412) MTRX1011A (see, e.g., WO2008134046, which is incorporated herein by reference). (also known as OKT-4A), clenoliximab (IDEC-151), , also known as BB-217969), keliximab (IDEC CE9.1, also known as SB210396), ), M-T413, and TRX1 (see, e.g., WO2002102853, which is incorporated herein by reference). Examples of anti-CD8 antibodies include those described in the art. For example, WO2019033043, WO2017134306, WO2019032661, WO20190231 48, WO2014025828, US10414820, and US10377826 ( This is incorporated herein by reference. be absorbed ) In certain embodiments, the immunogens include the anti-CD8 antibodies described in The inhibitor is a lymphocyte depleting antibody. For example, the lymphocyte depleting antibody is an anti-CD45 antibody (e.g., For example, clone 30-F11, which allows for a potent depletion of peripheral B- and T-cells. These antibodies may be intact antibodies that mimic ATG by relying on effector functions. do.

[0146] In other embodiments, an anti-CD117 antibody, an anti-CD45 antibody, or an antibody-drug conjugate thereof. The drug is administered in combination with cyclophosphamide (Cytoxan, e.g., low-dose Cytoxan).

[0147] In still further embodiments, an anti-CD117 antibody, an anti-CD45 antibody, or an antibody-drug conjugate thereof administered in combination with total body irradiation (TBI, e.g., low-dose TBI) Conventional conditioning protocols involve administering high doses of erythromycin prior to receiving an allogeneic transplant. In some embodiments of the methods provided herein, the TBI may be When used in combination with an anti-CD117 antibody, an anti-CD45 antibody, or an antibody-drug conjugate thereof, Reduced dose TBI can be used to effectively condition patients in preparation for allogeneic transplant therapy. Thus, in some embodiments, the present invention provides a method for treating allogeneic transplants. Methods to reduce the level of TBI used to condition patients for treatment The method comprises administering an anti-CD117 ADC and / or an anti-CD45 ADC described herein to a patient receiving a low-dose TBI. In one embodiment, the method comprises administering to a patient a combination of the above-mentioned compounds, the combination comprising administering to a patient a dose of 100 mg / kg of TBI to treat a TBI-related condition. 5Gy or less, 4.5Gy or less, 4Gy or less, 3.5Gy or less, 3Gy or less, 2.5Gy or less, 2Gy or less, 1.5Gy or less In some embodiments, the level of TBI is about 5 Gy, about 4 Gy, or less. 5 Gy, about 4 Gy, about 3.5 Gy, about 3 Gy, about 2.5 Gy, about 2 Gy, about 1.5 Gy, about 1 Gy, or about 0.5 Gy.

[0148] In other embodiments, an anti-CD117 antibody, an anti-CD45 antibody, or an antibody-drug conjugate thereof. The effector function (i.e., complement-dependent cytotoxicity) ity (CDC)) or antibody-dependent cellular cytotoxicity (ADC) C))) and unconjugated anti-CD45 antibodies that can deplete CD45+ cells via Administer in combination.

[0149] In other embodiments, anti-CD117 ADCs and / or anti-CD45 ADCs are administered in the manner provided herein. May be used in combination with one or more of the following immunosuppressants, as directed by law: calcineurin / MTOR inhibitors (e.g., tacrolimus, sirolimus, rapamycin, cyclosporine, everolimus) Mus), molecules that block costimulation (e.g., CTLA4-Ig, anti-CD40L), drugs that reduce NK, anti- anti-thymocyte globulin (ATG), alkylating agents (e.g., nitro Gen mustards, e.g., cyclophosphamide; nitrosoureas (e.g., Calmus) platinum compounds), methotrexate, anti-TCR agents (e.g., muromonab-CD3), anti-CD20 antibodies (e.g., rituximab, ocrelizumab, ofatumumab, and veltuzumab), Darabine, Campath (alemtuzumab), 2-amino-6-aryl-5-substituted pyrimidine (See U.S. Patent 4,665,077, the disclosure of which is incorporated herein by reference), azathioprine (or cyclophosphamide if you have an adverse reaction to azathioprine) ; bromocriptine; glutaraldehyde (which is described in U.S. Patent 4,120,649) MHC antigens are used in mice (as described above); anti-idiotype antibodies against MHC antigens; cyclosporin one or more steroids (e.g., corticosteroids, e.g., prednisone, methicone, tilprednisolone, hydrocortisone, and dexamethasone); anti-interferon- gamma antibodies; anti-tumor necrosis factor-alpha antibodies; anti-tumor necrosis factor-beta antibodies; anti-interleukin-2 antibodies; Anti-cytokine receptor antibodies, such as anti-IL-2 receptor antibodies; heterologous anti-lymphocyte globulins pan-T antibodies, such as OKT-3 monoclonal antibody; antibodies against CD4; antibodies against CD8; Antibodies against CD45 (e.g., 30-F11, YTH24.5 and / or YTH54.12 (e.g., YTH24.5 and YT H54.12 combination); streptokinase; streptodornase; or RNA or DNA.

[0150] In one exemplary embodiment, the patient is treated with TBI, cytoxan, anti-CD4 antibody, anti-CD8 antibody, Conditioning with anti-CD117-PBD ADC in combination with a CD117-PBD ADC, or a combination thereof do.

[0151] In another exemplary embodiment, the patient is treated with TBI, cytoxan, anti-CD4 antibody, anti-CD8 antibody, Conditioning with anti-CD45-PBD ADC in combination with a CD45 antibody or a combination thereof do.

[0152] The aforementioned immunosuppressants (for example, but not limited to, anti-CD4 antibodies, anti-CD8 antibodies, Transplantation of allogeneic cells (e.g., allogeneic HSCs) In some embodiments, the immunosuppressant may be administered to the patient prior to receiving the immunosuppressant. In some embodiments, the immunosuppressant is administered pre-transplant and post-transplant. Both are administered to the subject.

[0153] In certain embodiments, the antibodies or ADCs described herein can be used to detect mismatches. In some embodiments, the donor is a subject receiving an allogeneic transplant. ) is the mismatched donor. (e.g., serological, genomic, or molecular analysis of a defined number of MHC or miHA antigens) In a typical assay, mismatched donor cells, organs, or tissues The tissue has at least one variant that is different from the variant expressed by the recipient. Dissimilar (e.g., non-identical) major histocompatibility complex (MHC) antigens (i.e., in humans Human leukocyte antigens (HLA) (e.g., class I, class II, or class III MHC antigens or minor In an exemplary embodiment, the allograft comprises a graft-specific histocompatibility antigen (miHA). Shares the same MHC or HLA haplotype as the recipient, but has one or more minor This may include minor mismatches (e.g., minor mismatched allografts). In an exemplary embodiment, the allograft comprises, alone or in combination with one or more minor In another exemplary embodiment, the mismatches include one or more major mismatches. the allograft has one or more major mismatches and one or more minor mismatches; It is a "full mismatch" allograft, including a match.

[0154] MHC proteins are the signaling molecules between lymphocytes and antigen-presenting cells or disease cells in an immune response. The MHC protein binds peptides and mediates T cell receptor signaling. The proteins encoded by the MHC genes are: They are expressed on the surface of cells and contain self-antigens (peptide fragments derived from the cell itself) and non-self-antigens. The antigen presents both the antigen (e.g., fragments of an invading microorganism) and the antigen to T cells.

[0155] The MHC region is divided into three subgroups: class I, class II, and class III. MHC class I proteins contain α-chains and β2-microglobulin (i.e., B2M), and are cytotoxic. They present antigen fragments to T cells. Many immune system cells have antigens on their surface, particularly antigens. On the surface of presenting cells, MHC class II proteins contain α- and β-chains and bind antigen fragments. The MHC class III region presents antigens to T-helper cells. MHC encodes certain components of the body, including cytokines. are polygenic (there are several MHC class I and II genes) and polymorphic (there are Both (each gene has multiple alleles) and

[0156] In humans, the major histocompatibility complex is called the human leukocyte antigen (HLA) complex. Each class of HC is represented by several loci in humans: e.g., in class I Regarding HLA-A (human leukocyte antigen (HLA)-A), HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA -H, HLA-J, HLA-K, HLA-L, HLA-P and HLA-V, and for class II, HLA-DRA, HLA-DRB1-9, HLA-, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DMA, HLA-DMB, HLA -DOA, and HLA-DOB. MHC is highly polymorphic: within the human population, there are many different loci for each. There are numerous haplotypes containing different alleles in each class. Class I and Class II Both different polymorphic MHC alleles have different peptide specificities: Alleles encode proteins that bind to peptides that display specific sequence patterns. Testing for HLA genomic loci and HLA alleles or proteins in humans Methods are known in the art (e.g., Choo et al. (2007). Yonsei medical jo urnal. 48.1: 11-23; Shiina et al. (2009). Journal of human genetics. 54.1: 15; Petersdorf. (2013). Blood. 122.11: 1863-1872; and Bertaina and Andreani. (2018 ). International journal of molecular sciences. 19.2: 621, see (Incorporated herein by reference).

[0157] In some embodiments, at least one major histocompatibility complex antigen (e.g., HLA antigens) are expressed in the donor (d) in the subject receiving the transplant according to the methods provided herein. In certain embodiments, the MHC antigen is a mismatch to an MHC class I antigen. In a specific embodiment, the MHC antigen is a B2M, HLA-A, or HLA class II molecule. -B, HLA- C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DPA1, HLA- DPA2 , HLA-DQA1, and / or HLA-DQB1, or any combination thereof. In embodiments, the graft has at least one HLA-mismatch compared to the HLA antigen in the human patient. For example, in certain cases, the allogeneic hematopoietic stem cells The hematopoietic stem cells of the system have at least one, at least two, or more HLA antigens compared to the HLA antigens in the human patient. one, at least three, at least four, at least five, at least six, at least seven, Contains at least 8, at least 9, or more than 9 HLA-mismatches. In an embodiment, the allogeneic hematopoietic stem cells are fully HLA-mismatched compared to HLA antigens in a human patient. Includes the switch.

[0158] Alternatively or additionally, the at least one minor histocompatibility antigen may be any of the antigens provided herein. According to the method, in a subject receiving a transplant, a mismatch with a donor is In some embodiments, the graft has at least one miHA antigen that is at least 100% identical to the miHA antigen in the human patient. In certain instances, the miHA-mismatched allogeneic hematopoietic stem cells may be derived from a single miHA-mismatched stem cell. In this study, the allogeneic hematopoietic stem cells were compared to the miHA antigen in a human patient and showed at least one At least two, at least three, at least four, at least five, at least six, The miHA fragment contains at least 7, at least 8, at least 9, or more than 9 miHA-mismatches. In certain embodiments, the minor histocompatibility antigens are HA-1, HA-2, HA-8, HA-3, HBV, -1, HY-Al, HY-A2, HY-B7, HY-B8, HY-B60, or HY-DQ5 protein. Examples of histocompatibility antigens are known in the art (e.g., Perreault et al. (1990)). Blood. 76.7: 1269-1280; Martin et al. (2017). Blood. 129.6: 791-798; and the United States Patent US10414813B2, which are incorporated herein by reference).

[0159] The methods described herein can be used to produce anti-CD117 ADCs, anti-CD45 ADCs, or immunosuppressants. Allogeneic transplant recipients compared with patients receiving either alone The level of donor cell chimerism in the system may be increased. The method further comprises obtaining at least 80% donor-specific markers in the transplant recipient. Donor chimerism (e.g., at least 80%, at least 85%, at least 90%, Establish donor chimerism of at least 95% or at least 99% The level of donor chimerism after allogeneic HSC transplantation For example, total chimerism, bone marrow chimerism, peripheral myeloid chimerism chimerism, B-cell chimerism, or T-cell chimerism be.

[0160] Route and method of administration The antibodies, antigen-binding fragments thereof, or ADCs described herein can be administered in a variety of dosage forms. Patients (e.g., those suffering from cancer, autoimmune diseases, or those requiring hematopoietic stem cell transplantation therapy) For example, the antibodies described herein, their antigen-binding fragments, or ADCs to patients suffering from cancer, autoimmune diseases, or hematopoietic stem cell transplantation A patient in need of therapy is administered an aqueous solution (e.g., containing one or more pharmaceutically acceptable excipients). The compositions and methods described herein may be administered in the form of a pharmaceutical composition, such as an aqueous solution. Examples of pharmaceutically acceptable excipients for the preparation of the aqueous solution include viscosity modifiers. , may be sterilized using techniques known in the art.

[0161] Anti-HC antibody (e.g., anti-CD117 antibody or anti-CD45 antibody), or a conjugate thereof (e.g., an ADC described herein) can be used in pharmaceutical formulations containing such antibodies or ADCs in combination with one or more By mixing with the above optional pharmaceutically acceptable carriers (Remington's Pharmac (1980)), in the form of a lyophilized preparation or an aqueous solution. Pharmaceutically acceptable carriers are generally used in the formulation at the dosages and concentrations used. Non-toxic to insects, including but not limited to: Buffers such as phosphate, citric acid, and other organic acids; ascorbic acid and methionine Contains antioxidants; preservatives (octadecyldimethylbenzyl ammonium chloride; hexadecyl chloride) Samethonium; Benzalkonium chloride; Benzethonium chloride; Phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; Catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, or immunoglobulins Proteins such as globulin; hydrophilic polymers such as polyvinylpyrrolidone; glycine, amino acids such as glutamine, asparagine, histidine, arginine, or lysine; Monosaccharides, disaccharides, and other sugars, such as glucose, mannose, or dextrin; Chelating agents such as EDTA; sucrose, mannitol, trehalose, or sorbitol any sugars; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes) ); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0162] The antibodies, antigen-binding fragments, or ADCs described herein can be administered orally, transdermally, subcutaneously, intranasally, or intravenously. Administration may be by a variety of routes, including intravenous, intramuscular, intraocular, or parenteral. In any given case, the most suitable route of administration will depend on the specific antibody or antigen binding agent being administered. a compound fragment, a patient, a pharmaceutical formulation method, an administration method (e.g., administration time and administration route), a patient The dosage depends on the age, weight, sex, severity of the disease being treated, the patient's diet, and the patient's rate of excretion. do.

[0163] An effective dose of the antibodies, or antigen-binding fragments thereof, described herein may be administered, for example, in a single dose ( For example, about 0.001 to about 0.001 mg / kg body weight per bolus, multiple dose, or continuous infusion. 100 mg / kg, so that the antibody, or antigen-binding fragment thereof, The blood concentration may be about 0.0001 to about 5000 μg / mL. , cancer, autoimmune disease, or in preparation for hematopoietic stem cell transplantation. A subject (e.g., a human) undergoing conditioning therapy is given a daily, weekly, or monthly It may be administered one or more times (for example, 2 to 10 times).

[0164] In one embodiment, the anti-HC ADC (e.g., linked to a cell via a linker) administered to the human patient The dose of the toxin-conjugated anti-CD117 antibody or anti-CD45 antibody is about 0.1 mg / kg to about 0.3 mg / kg. mg / kg.

[0165] In one embodiment, the anti-HC ADC (e.g., linked to a cell via a linker) administered to the human patient The dose of the toxin-conjugated anti-CD117 antibody or anti-CD45 antibody is about 0.15 mg / kg to about 0. 3 mg / kg.

[0166] In one embodiment, the anti-HC ADC (e.g., linked to a cell via a linker) administered to the human patient The dose of the toxin-conjugated anti-CD117 antibody or anti-CD45 antibody is about 0.15 mg / kg to about 0. 25 mg / kg.

[0167] In one embodiment, the anti-HC ADC (e.g., linked to a cell via a linker) administered to the human patient The dose of the toxin-conjugated anti-CD117 antibody or anti-CD45 antibody is about 0.2 mg / kg to about 0.3 mg / kg. mg / kg.

[0168] In one embodiment, the anti-HC ADC (e.g., linked to a cell via a linker) administered to the human patient The dose of the toxin-conjugated anti-CD117 antibody or anti-CD45 antibody is about 0.25 mg / kg to about 0. 3 mg / kg.

[0169] In one embodiment, the anti-HC ADC (e.g., linked to a cell via a linker) administered to the human patient The dose of anti-CD117 antibody or anti-CD45 antibody conjugated to a toxin is about 0.1 mg / kg. .

[0170] In one embodiment, the anti-HC ADC (e.g., linked to a cell via a linker) administered to the human patient The dose of anti-CD117 antibody or anti-CD45 antibody conjugated to a toxin is about 0.2 mg / kg. .

[0171] In one embodiment, the anti-HC ADC (e.g., linked to a cell via a linker) administered to the human patient The dose of anti-CD117 antibody or anti-CD45 antibody conjugated to a toxin is about 0.3 mg / kg. .

[0172] In one embodiment, the dose of an anti-HC ADC described herein administered to the human patient is about 0.001 mg / kg~10 mg / kg, approx. 0.01 mg / kg~9.5 mg / kg, approx. 0.1 mg / kg~9 mg / kg, approx. 0.1 m g / kg~8.5 mg / kg, approx. 0.1 mg / kg~8 mg / kg, approx. 0.1 mg / kg~7.5 mg / kg, approx. 0.1 mg / kg~7 mg / kg, about 0.1 mg / kg~6.5 mg / kg, about 0.1 mg / kg~6 mg / kg, about 0.1 mg / kg~5.5 mg / kg, Approx. 0.1 mg / kg~5 mg / kg, Approx. 0.1 mg / kg~4.5 mg / kg, Approx. 0.1 mg / kg~4 mg / kg, Approx. 0.5 m g / kg~3.5 mg / kg, approx. 0.5 mg / kg~3 mg / kg, approx. 1 mg / kg~10 mg / kg, approx. 1 mg / kg~9 mg / k g, approx. 1 mg / kg~8 mg / kg, approx. 1 mg / kg~7 mg / kg, approx. 1 mg / kg~6 mg / kg, approx. 1 mg / kg~5 mg / kg, about 1 mg / kg to 4 mg / kg, or about 1 mg / kg to 3 mg / kg.

[0173] In some embodiments, the compound is administered to a human patient for treatment or conditioning. The anti-HC ADCs described herein may be administered for up to 24 hours, up to 22 hours, up to 20 hours, up to 18 hours, or Less than 16 hours, Less than 14 hours, Less than 13 hours, Less than 12 hours, Less than 11 hours, 10 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, or 5 hours or less In one embodiment, the anti-HC ADC has a half-life of 5 hours to 7 hours; ~9 hours;15 hours~11 hours;5 hours~13 hours;5 hours~15 hours;5 hours~20 hours;5 7 hours to 24 hours; 9 hours to 24 hours; 11 hours to 24 hours; 12 hours to 22 hours hours; 10 hours to 20 hours; 8 hours to 18 hours; or 14 hours to 24 hours.

[0174] In some embodiments, the methods disclosed herein may be used to condition Hepatotoxicity is minimized in patients receiving ADCs. For example, certain In embodiments, the methods disclosed herein are performed for 24 hours, 48 ​​hours, 72 hours, or 96 hours. Over time, liver marker levels in these patients remained below known toxic levels. In other embodiments, the methods disclosed herein can be used to and (iii) for more than 72 hours, or 96 hours, the liver marker levels in the patient are within the reference range. In certain embodiments, the methods disclosed herein If the liver marker levels increased beyond 24, 48, 72, or 96 hours, More than 1.5 times above the reference range, more than 3 times above the reference range, and more than 5 times above the reference range The difference should not be more than 10 times the reference range or more than 10 times the reference range. Examples of liver markers that may be present include alanine aminotransaminase (ALT), Examples include lactate dehydrogenase (LDH) and aspartate aminotransaminase (AST). In certain embodiments, the administration of the compound as described herein (i.e., two doses instead of a single dose) may be performed. By administering an ADC (administered at a dose of 0.1 mg / kg / day), liver markers (e.g., AST, LDH, and In some cases, liver markers of toxicity are elevated. However, if the temperature is too high for a certain period (e.g., about 12 hours, about 18 hours, about 24 hours, about 36 hours, approximately 48 hours, approximately 72 hours, approximately 3 days, approximately 3.5 days, approximately 4 days, approximately 4.5 days, approximately 5 days, approximately 5.5 days, approximately 6 the liver marker is within a range of 1 day, about 6.5 days, about 7 days, about 7.5 days, or less than 1 week; The levels return to normal levels that are not associated with liver toxicity. For example, in humans (average adult males), In this study, normal non-toxic levels of ALT were 7 to 55 units per liter (U / L); normal levels of AST were 7 to 55 units per liter (U / L). A non-toxic level is 8-48 U / L. In certain embodiments, the patient's blood AST, ALT, or L At least one of the DH levels is measured by administering a first dose of an ADC to the patient and For example, the patient receives the first dose and no toxic levels are reached within 14 days of receiving the first dose. and administering a second dose within, for example, 5, 10, or 14 days after administering the first dose. A second dose, a third dose, a fourth dose, or a greater number of doses may be administered. at least one of AST, ALT, or LDH levels in the patient's blood is increased after administering the first dose to the patient; and 14 days after the first dose, no toxic levels are reached. .

[0175] In certain embodiments, at least one of the patient's blood AST, ALT, or LDH levels is , not elevated above normal levels, not elevated above normal levels by more than 1.5 times, normal levels not more than 3-fold increase above normal levels, not more than 5-fold increase above normal levels, or not more than 10-fold increase above normal levels does not rise above

[0176] In the case of a conditioning procedure before hematopoietic stem cell transplantation, the antibody or its anti- The original binding fragment is then injected at a time that optimally promotes engraftment of exogenous hematopoietic stem cells, e.g. For example, about 1 hour to about 1 week (e.g., about 1 hour, about 2 weeks) after administration of the exogenous hematopoietic stem cell graft. Hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, Approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, Approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 2 days, approximately 3 days, approximately 4 days The patient may be administered the drug about 5 days, about 6 days, about 7 days) or more prior to the administration of the drug. Ranges inclusive of the recited numbers are also encompassed in the intended manner.

[0177] Combining the above dose ranges with anti-HC ADCs having half-lives listed herein There is.

[0178] Using the methods disclosed herein, a physician of ordinary skill in the art can administer hematopoietic stem cell transplant therapy to a patient in need of hematopoietic stem cell transplant therapy. In a human patient, hematopoietic stem cell-expressed antigens (e.g., CD117 [e.g., GNNK+ CD117]) are expressed. , or CD45) or antigens expressed by mature immune cells (e.g., T-cells, etc.) (e.g., CD 45), or an ADC, antibody, or antigen-binding fragment thereof that binds to this antibody may be administered. In one embodiment, prior to administering the exogenous hematopoietic stem cell graft, a method for promoting engraftment of the hematopoietic stem cell graft is performed. The antibody may deplete the endogenous hematopoietic stem cell population so that the hematopoietic stem cells are promoted. Covalently attached to a toxin, such as a cytotoxic molecule described herein or known in the art. For example, an anti-CD117 antibody or its antigen-binding fragment may be conjugated to the antibody. fragment (e.g., an anti-HC antibody [e.g., an anti-CD117 antibody or an anti-CD45 antibody] or its antigen-binding fragments) with cytotoxins (e.g., Pseudomonas exotoxin A, de bouganin, Diphtheria toxin, amatoxins, e.g., gamma-amanitin, alpha-amanitin, saporin, may Tansin, maytansinoids, auristatins, anthracyclines, calicheamicin , irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzo Diazepine dimers, indolinobenzodiazepines, and indolinobenzodiazepine dimers The conjugate may be covalently conjugated to a hydroxybenzoate (or a variant thereof). The adjugate may be prepared by covalent bond formation techniques as described herein or known in the art. The antibody, antigen-binding fragment thereof, or drug may then be used to - the antibody conjugate is administered to exogenous hematopoietic stem cells (e.g., autologous, syngeneic, or allogeneic hematopoietic stem cells, etc.) to the patient, for example, by intravenous administration, before transplantation into the patient. Sometimes I do.

[0179] The anti-HC antibody (e.g., anti-CD117 antibody or anti-CD45 antibody), its antigen-binding fragment The drug or drug-antibody conjugate is administered to stimulate endogenous hematopoietic stem cells prior to hematopoietic stem cell transplantation. The amount of cells can be reduced to, for example, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% It may be administered in an amount sufficient to reduce the condition by about 95% or more. Characteristic hematopoiesis was observed in blood samples taken from the patient at various intervals during the course of the immunization therapy. Cells expressing stem cell surface antigens can be identified by conventional techniques known in the art, such as FACS analysis. For example, a physician skilled in the art may monitor the decline in hematopoietic stem cell numbers. At various times during the conditioning regimen, blood samples are taken from the patient and hematopoietic stem cells are analyzed. FACS analysis is performed using an antibody that binds to a marker antigen to detect hematopoiesis in the sample. By elucidating the relative concentrations of stem cells, we can measure the extent to which endogenous hematopoietic stem cells have been depleted. According to some embodiments, an anti-HC antibody (e.g., an anti-CD117 antibody, or anti-CD45 antibodies), their antigen-binding fragments, or drug-antibody conjugates. When the concentration of hematopoietic stem cells reaches a nadir in response to the conditioning therapy, The nurse may terminate the conditioning regimen and prepare for hematopoietic stem cell transplantation. , preparations may be initiated for the patient.

[0180] Anti-HC antibodies (e.g., anti-CD117 antibodies or anti-CD45 antibodies), antigen-binding fragments thereof or a drug-antibody conjugate, in one or more pharmaceutically acceptable excipients (e.g., viscosity The aqueous solution may be administered to the patient as an aqueous solution containing the drug (e.g., a drug adjusting agent). The container may be sterilized using techniques described herein or known in the art. For example, the antibody, antigen-binding fragment thereof, may be administered to the patient prior to administering the hematopoietic stem cell graft. or a drug-antibody conjugate is administered to the patient at a dose of, for example, about 0.001 mg / kg to about 100 mg / kg. g, about 0.001 mg / kg~about 10 mg / kg, about 0.01 mg / kg~9.5 mg / kg, about 0.1 mg / kg~9 mg / kg, Approx. 0.1 mg / kg~8.5 mg / kg, Approx. 0.1 mg / kg~8 mg / kg, Approx. 0.1 mg / kg~7.5 mg / kg, Approx. 0.1 mg / kg~7 mg / kg, approx. 0.1 mg / kg~6.5 mg / kg, approx. 0.1 mg / kg~6 mg / kg, approx. 0.1 mg / kg~5 .5 mg / kg, about 0.1 mg / kg~5 mg / kg, about 0.1 mg / kg~4.5 mg / kg, about 0.1 mg / kg~4 mg / kg, Approx. 0.5 mg / kg~3.5 mg / kg, Approx. 0.5 mg / kg~3 mg / kg, Approx. 1 mg / kg~10 mg / kg, Approx. 1 mg / k g~9 mg / kg, about 1 mg / kg~8 mg / kg, about 1 mg / kg~7 mg / kg, about 1 mg / kg~6 mg / kg, about 1 The dose should be approximately 1 mg / kg to 5 mg / kg, approximately 1 mg / kg to 4 mg / kg, or approximately 1 mg / kg to 3 mg / kg. At a time when exogenous hematopoietic stem cell engraftment is optimally promoted, e.g. About 1 hour to about 1 week (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, Approx. 12 hours, Approx. 13 hours, Approx. 14 hours, Approx. 15 hours, Approx. 16 hours, Approx. 17 hours, Approx. 18 hours, Approx. 19 hours, Approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, about 6 days, or about 7 days), or more before the antibody, antigen-binding fragment thereof, Alternatively, a drug-antibody conjugate may be administered to the patient.

[0181] Immunosuppressive therapy typically involves administering an effective amount of an immunosuppressant. Pharmaceutical compositions are formulated and administered in a fashion consistent with good medical practice. Factors to consider in this context include the individual patient's clinical presentation, the reason for transplant, and the drug These include the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to the practitioner. The effective amount in this context is determined by taking such considerations into account and by determining whether the host is capable of This is the minimum amount necessary to prevent an immune response that may result in graft rejection. Such an amount is preferably an amount that is toxic to said host or that renders said host susceptible to infection. The amount of immunosuppressant required for the purposes of this specification is less than the amount that renders the patient significantly more sensitive. may be less than what is normally required for unprepared transplanted grafts. and depends on the individual circumstances surrounding the graft and the type of immunosuppressant used. .

[0182] However, as noted above, these suggested immunosuppressant doses are subject to a great deal of therapeutic discretion. An important factor in selecting the appropriate dose and scheduling is the The outcome is that the graft survives. For example, hyperacute graft rejection (which is caused by antibodies) early in the treatment of acute rejection (which may be due to graft-mediated destruction) or in the later stages of treatment, which may be characterized by a sudden decline in graft function. , relatively high doses may be required.

[0183] The immunosuppressant may be administered parenterally and, if desired, by local immunosuppressive therapy, such as intralesional administration. The administration may be by any suitable method. Parenteral administration may include intramuscular, intravenous, or intraarterial administration. Furthermore, the immunosuppressant may be administered by pulse infusion, In particular, immunosuppressive drugs are preferably administered at reduced doses or by continuous infusion.

[0184] In some embodiments, the immunosuppressant is administered prior to stem cell transplantation (i.e., pre-transplant). In some embodiments, the immunosuppressant is administered after stem cell transplantation (i.e., post-transplant). In some embodiments, the immunosuppressant is administered at a dose substantially the same as if the patient were undergoing a transplant. Sometimes, administer.

[0185] After the conditioning regimen is completed, the patient may then, for example, injection of exogenous hematopoietic stem cells (e.g., The physician may administer to the patient an autologous, syngeneic, or allogeneic Injection of allogeneic hematopoietic stem cells, e.g., 1 x 10 3 ~1×10 9 The dose is 100 mg / kg of hematopoietic stem cells. After administering the graft, the physician may, for example, collect blood samples from the patient. By collecting the blood, hematopoietic stem cells or cells of the hematopoietic lineage (e.g., megakaryocytes, microparticles) can be isolated. thrombocytes, platelets, red blood cells, mast cells, myeloblasts, basophils, leukocytes Neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages Increased concentrations of lymphocytes (e.g., dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes) Monitoring the survival of hematopoietic stem cell grafts by measuring the This analysis may be performed, for example, between 1 hour and 6 months after hematopoietic stem cell transplantation therapy, or later (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, approx. 8 hours, approx. 9 hours, approx. 10 hours, approx. 11 hours, approx. 12 hours, approx. 13 hours, approx. 14 hours, approx. 15 hours Hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours Hours, About 24 hours, About 2 days, About 3 days, About 4 days, About 5 days, About 6 days, About 7 days, About 2 weeks, About 3 weeks, About 4 weeks, About 5 weeks, About 6 weeks, About 7 weeks, About 8 weeks, About 9 weeks, About 10 weeks, About 11 weeks, About 12 weeks, About 13 weeks, About 14 weeks, About 15 weeks weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, or The concentration of hematopoietic stem cells or cells of hematopoietic lineage may be increased by the transplantation therapy. After transplantation, the concentration of the corresponding cell type increased (e.g., about 1%, about 2%) compared to the concentration of the corresponding cell type before transplantation. , about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, Increased by approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 200%, approximately 500%, or more If this is observed, anti-HC antibody (e.g., anti-CD117 antibody or anti-CD45 antibody), the antigen Treatment with binding fragments or drug-antibody conjugates improves transplanted hematopoiesis This demonstrates the success of promoting stem cell graft survival.

[0186] Anti-HC antibodies (e.g., anti-CD117 antibodies or anti-CD45 antibodies), antigen-binding fragments thereof In various experiments, we confirmed that administration of thrombin-containing steroids or ADCs resulted in the engraftment of hematopoietic stem cells. For example, the engraftment of transplanted hematopoietic stem cells can be clarified by quantitative measurement. is an antigen expressed by hematopoietic stem cells (e.g., CD117 [e.g., GNNK+ CD117], or CD45) and subsequently administering an antibody, or antigen-binding fragment thereof, capable of binding to After administering the blood stem cell graft, the competitive repopulation units (competitive repopulation units) present in the patient's bone marrow are activated. This can be assessed by assessing the amount of reactive repopulating unit (CRU) Furthermore, the vectors used to transfect donor hematopoietic stem cells contain fluorescent products, chromogenic products, Alternatively, a reporter gene such as an enzyme that catalyzes a chemical reaction that produces a luminescent product can be incorporated. The corresponding signal is then transmitted to the bone marrow or other tissues to which the hematopoietic stem cells have homed. By monitoring the cells, it is possible to observe the survival of the hematopoietic stem cell graft. Alternatively, for example, fluorescence activated cell sorting (FACS) known in the art may be used. Hematopoietic stem cells and hematopoietic progenitor cells, as measured by Fluorescence Acquisition System (FACS) analytical method. By assessing the amount and viability of hematopoietic stem cells, it is also possible to observe the engraftment of hematopoietic stem cells. Engraftment can also be assessed by measuring the number of white blood cells in the peripheral blood during the post-transplant period. and / or to measure bone marrow cell recovery by donor cells in bone marrow aspirate samples. It can also be measured by

[0187] Anti-HC antibody The present disclosure provides antibodies that bind to antigens expressed by hematopoietic cells (e.g., GNNK+ CD117) or CD45. The antibody, or antigen-binding fragment thereof, capable of: (i) CD117+ [e.g., GNNK+ CD1 17], or for treating cancer and autoimmune diseases characterized by CD45+ hematopoietic cells, and (ii) ensuring the engraftment of transplanted hematopoietic stem cells in patients requiring transplantation therapy. Use as a stand-alone therapeutic agent or antibody drug conjugate (ADC) to promote These therapeutic activities are based in part on the discovery that anti-hematogenic (hematogenic) topoietic cell (HC))-antibody (e.g., anti-CD117 antibody or anti-CD45 antibody) or its anti- The original binding fragment may be, for example, a hematopoietic cell (e.g., hematopoietic stem cell), a leukocyte, an immune cell [ For example, mature immune cells (e.g., T cells)]), such as cancer cells, autoimmune cells, or hematopoietic cells Binding to antigens expressed by stem cells (e.g., CD117 [e.g., GNNK+ CD117], or CD45) by binding to ATP and subsequently inducing cell death. By reducing endogenous hematopoietic stem cells, transplanted hematopoietic stem cells may be able to produce more than This may provide a niche where cells can be housed, and then productive hematopoiesis can occur. In this way, the transplanted hematopoietic stem cells may be established in the patient (e.g., and can successfully engraft in human patients suffering from stem cell disorders such as those described in do.

[0188] The anti-HC antibodies described herein (e.g., anti-CD117 antibodies, or anti-CD45 antibodies) are Long antibody, bispecific antibody, dual variable domain antibody, multiple Chain or single-chain antibodies and / or binding fragments that specifically bind to human CD117 or CD45 These may be in the form of Fab, Fab', (Fab')2, Fv, scFv (single chain Fv), saline Examples include surrobodies (including surrogate light chain constructs), single domain antibodies, and camelized antibodies. They also include, but are not limited to, IgA (e.g., IgA1 or IgA2 ), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM, etc. In some embodiments, the anti-HC antibody (e.g., anti-CD11 7 antibody, or anti-CD45 antibody) is an IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0189] Antibodies for use in conjunction with the methods described herein include those containing an Fc domain or and antibody fragments lacking barrier activity, as well as humanized barrier-deficient non-human antibodies as described herein. and the CDRs or equivalent regions of the antibodies or antibody fragments described herein. and antibody-like protein scaffolds (e.g., 10 Fn3dom Exemplary antigen-binding flags of the above antibodies include variants of the above antibodies, such as These include, among others, dual-variable immunoglobulin domains lin domain), single-chain Fv molecules (scFv), diabodies, triabodies, nanobodies, antibody-like Protein scaffolds, Fv fragments, Fab fragments, F(ab')2 molecules, and It includes a di-scFv.

[0190] In certain embodiments, the anti-CD117 antibody, or antigen-binding fragment thereof, is a conjugate. They have specific dissociation rates that are particularly beneficial when used as part of a gate. For example, The anti-CD117 antibody, in certain embodiments, is directed against human CD117 and / or rhesus CD117. and 1×10 as measured by Biolayer Interferometry (BLI). -2 ~1×10 -3 , 1×10 -3 ~1 x10 -4 , 1×10 -5 ~1×10 -6 , 1×10 -6 ~1×10 -7 or 1 x 10 -7 ~1×10 -8 Dissociation rate constant (Kof In some embodiments, the antibody or antigen-binding fragment thereof has Approximately 1% of the total IgG was detected by Bio-Layer Interferometry (BLI) assay. 00 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, about 4 nM or less Bottom, K below about 2 nM, K below about 1 nM D and CD117 (e.g., human CD117 and / or rhesus CD1 17).

[0191] In one embodiment, an anti-HC antibody (e.g., anti-CD117) comprising one or more radiolabeled amino acids is Radiolabeled anti-CD117 antibodies are also provided for diagnostic and therapeutic use. It can be used for both therapeutic and therapeutic purposes (by conjugating to a radiolabeled molecule). (Another feature is that it is possible to label polypeptides.) Non-limiting examples of labeling polypeptides include: Examples include 3H, 14C, 15N, 35S, 90Y, 99Tc, 125I, 131I and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives include, but are not limited to: These methods are well known in the art (see, e.g., Junghans et al., Cancer Chemotherapy and Biotherapy rapy 655-686, 2nd ed., Chafner and Longo, eds., Lippincott Raven (1996) and U.S. Pat. Nos. 4, 6 81,581, U.S. Pat. No. 4,735,210, U.S. Pat. No. 5,101,827, U.S. Pat. No. 5,102,990 [US See U.S. Patent No. RE35,500, U.S. Patent No. 5,648,471, and U.S. Patent No. 5,697,902. For example, radioisotopes may be conjugated by the chloramine T method.

[0192] The anti-HC antibodies described herein (e.g., anti-CD117 or anti-CD45 antibodies), binding fragments, The fragments, or conjugates thereof, may also be used in the preparation of the aforementioned compounds, as known in the art. Modifications and / or mutations that alter the properties of the antibodies and / or fragments (e.g., half-life, and / or mutations that increase ADCC, increase ADCC, or decrease ADCC. There are also.

[0193] In certain embodiments, an anti-HC antibody (e.g., an anti-CD117 or anti-CD45 antibody), or an antigen thereof The binding fragment comprises an altered Fc region, wherein the altered Fc region is a wild-type Fc and at least one amino acid modification to the Fc gamma R (F Certain amino acid positions within the Fc region alter the affinity or binding to Fc This is known from crystallographic studies of direct contact with γR. Specifically, amino acids 234-23 9 (hinge region), amino acids 265 to 269 (B / C loop), amino acids 297 to 299 (C' / E loop), and The amino acid 327-332 (F / G) loop (Sondermann et al., 2000 Nature, 406: 267-273) In some embodiments, the antibodies described herein are characterized by structural and crystallographic Based on the analysis, variants with at least one residue that directly contacts FcγR were identified. In some embodiments, an anti-HC antibody (e.g., anti-CD117 or anti-HC antibody) may comprise an Fc region. The Fc region of the CD45 antibody (or fragment thereof) is as described in Kabat et al., Sequences of Protein ins of Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991) (Book Amino acid 265 according to the EU index as set forth in "EU index as in Kabat" refers to the amino acid substitutions in human IgG1 EU antibodies. In one embodiment, the Fc region comprises a D265A mutation. In some embodiments, the antibody (or its Fc region) comprises a D265C mutation. The Fc region of the IgG fragment is located at amino acid 234 according to the EU index as given in Kabat. In one embodiment, the Fc region comprises an amino acid substitution of L234A. In this embodiment, an anti-HC antibody (e.g., an anti-CD117 or anti-CD45 antibody), (or a flag thereof) The Fc region of the IgG1 gene is identified as amino acid 235 according to the EU index as in, for example, Kabat. In one embodiment, the Fc region comprises a L235A mutation.

[0194] In yet another embodiment, the Fc region comprises an L234A and L235A mutation (referred to herein as "L234A. In another embodiment, the Fc region comprises L234A and L235A (also referred to as "L235A" or "LALA"). In a further embodiment, the Fc region comprises a P35A mutation, and wherein the Fc region does not comprise a P329G mutation. The Fc region contains D265C, L234A, and L235A mutations (also referred to herein as "D265C.L234A.L235A"). In another embodiment, the Fc region comprises D265C, L234A, and L235A mutations. wherein the Fc region does not comprise a P329G mutation. contains D265C, L234A, L235A, and H435A mutations (referred to herein as "D265C.L234A.L235A.H435A"). In another embodiment, the Fc region comprises D265C, L234A, L235A, and H435A. In a further embodiment, the Fc region comprises a P329G mutation, wherein the Fc region does not comprise a P329G mutation. The c region contains the D265C and H435A mutations (also referred to herein as "D265C.H435A"). In another embodiment, the Fc region comprises D265A, S239C, L234A, and L235A mutations (referred to herein as "SEQ ID NO: 1"). In yet another embodiment, the Fc region comprises , D265A, S239C, L234A, and L235A mutations, wherein the Fc region comprises a P329G mutation. In another embodiment, the Fc region contains the D265C, N297G, and H435A mutations (referred to herein as "D265C, N297G, and H435A mutations"). In another embodiment, the Fc region comprises D265C, N297G, H435A. 297Q, and H435A mutations (also referred to herein as "D265C.N297Q.H435A"). In one embodiment, the Fc region contains E233P, L234V, L235A, and delG236 (deletion of 236) mutations (referred to herein as "delG236"). (also referred to as "E233P.L234V.L235A.delG236" or "EPLVLAdelG"). wherein the Fc region comprises E233P, L234V, L235A and delG236 (deletion of 236) mutations; In another embodiment, the Fc region does not comprise the P329G mutation. V, L235A, delG236 (deletion of 236), and H435A mutations (referred to herein as "E233P.L234V.L235A.delG In another embodiment, the Fc region comprises a nucleotide sequence similar to that of the Fc region of the target polypeptide (also referred to as "EPLVLAdelG.H435A" or "EPLVLAdelG.H435A"). contains E233P, L234V, L235A, delG236 (deletion of 236) and H435A mutations, wherein the Fc region In another embodiment, the Fc region does not contain the P329G mutation. In another embodiment, the Fc region comprises the L234A, L235A, S239C and D265A mutations. In another embodiment, the Fc region comprises a P329G mutation, wherein the Fc region does not comprise a P329G mutation. In another embodiment, the Fc region comprises the mutations H435A, L234A, L235A, and D265C. , H435A, L234A, L235A, and D265C mutations, wherein the Fc region comprises a P329G mutation. Not at all.

[0195] In some embodiments, the antibody has an altered Fc region such that the antibody In an in vitro effector function assay, binding to Fc receptors (FcRs) was reduced binding to the FcR compared to the same antibody containing an Fc region that does not contain an Fc domain, In some embodiments, the antibody has an altered Fc region, As a result, the antibody inhibited Fc gamma receptor function in an in vitro effector function assay. Binding to the FcγR of an antibody containing an unmodified Fc region is compared to the binding of the same antibody to the FcγR of the antibody containing an unmodified Fc region. In some embodiments, the FcγR is In some embodiments, the FcγR is FcγR1. In some embodiments, the FcγR is FcγR2A. In some embodiments, the FcγR is FcγR2B. In some embodiments, the FcγR is FcγR2C. In some embodiments, the FcγR is FcγR3A. In one embodiment, the FcγR is FcγR3B. In another embodiment, the decrease in binding is due to a decrease in binding to an FcγR. antibody binding to the FcγR compared to the binding of the same antibody comprising an unmodified Fc region to the FcγR. At least a 70% reduction, at least an 80% reduction, at least a 90% reduction, at least a 95% reduction , at least a 98% reduction, at least a 99% reduction, or a 100% reduction. The decrease in binding is due to antibody binding to FcγR compared to the FcγR of the same antibody containing an unmodified Fc region. At least 70% to 100% reduction, at least 80% to 100% reduction, or at least At least a 90% to 100% reduction, at least a 95% to 100% reduction, or at least a 98% to 100% reduction It is small.

[0196] In some embodiments, the antibody has an altered Fc region such that the antibody The same antibody containing an unmodified Fc region was shown to be significantly superior to the control in an in vitro cytokine release assay. cytokine release was reduced by at least 50% compared to that of the control group. In some embodiments, the reduction in cytokine release is achieved by modifying Reduced cytokine release compared to the cytokine release of the same antibody containing an Fc region without at least 70% reduction, at least 80% reduction, at least 90% reduction, at least 95% reduction, At least a 98% reduction, at least a 99% reduction, or a 100% reduction. In this study, the reduction in cytokine release was observed for the same antibody containing an unmodified Fc region. At least 70%-100% reduction in cytokine release, at least 80%-100% compared to release A reduction of at least 90% to 100% or a reduction of at least 95% to 100%. In embodiments, the cytokine release is by immune cells.

[0197] In some embodiments, the antibody has an altered Fc region such that the antibody compared the same antibody containing an unmodified Fc region in an in vitro mast cell degranulation assay. Compared to mast cell degranulation in HIV-1, mast cell degranulation was reduced by at least 50%. In some embodiments, the reduction in mast cell degranulation is achieved by modifying Less mast cell degranulation compared to the mast cell degranulation of the same antibody containing no Fc region at least 70% reduction, at least 80% reduction, at least 90% reduction, at least 95% reduction, At least a 98% reduction, at least a 99% reduction, or a 100% reduction. In this study, the reduction in mast cell degranulation was comparable to that of the same antibody containing an unmodified Fc region. At least 70%-100% reduction in mast cell degranulation, at least 80%-100% compared to granulocytes a reduction of at least 90% to 100%, a reduction of at least 95% to 100%.

[0198] In some embodiments, the antibody has an altered Fc region such that the antibody In vitro antibody-dependent cell phagocytosis assay (ADC) was performed. In an anticoagulant drug-induced cytosis (ADCP) assay, the antibody was compared with the ADCP of the same antibody containing the unmodified Fc region. Compared with the control group, antibody-dependent cellular phagocytosis (ADCP) was reduced by at least 50%. In some embodiments, the reduction in ADCP is achieved by reducing or avoiding the unmodified ADCP. At least 70% reduction in cytokine release compared to the cytokine release of the same antibody containing the Fc region % reduction, at least 80% reduction, at least 90% reduction, at least 95% reduction, at least The reduction is at least 98%, at least 99%, or 100%.

[0199] In some embodiments, an anti-HC antibody described herein (e.g., an anti-CD117 antibody, or (anti-CD45 antibody) comprises an Fc region containing one of the following modifications or combinations of modifications: :D265A, D265C, D265C / H435A, D265C / LALA, D265C / LALA / H435A, D265A / S239C / L234A / L235A / H435A, D265A / S239C / L234A / L235A, D265C / N297G, D265C / N297G / H435A, D265C (EPLVLAdelG *), D265C (EPLVLAdelG ) / H435A, D265C / N297Q / H435A, D265C / N297Q, EPLVLAdelG / H435A, EPLVLAdelG / D265C, EPLVLAdelG / D26 5A, N297A, N297G, or N297Q.

[0200] The binding or affinity between the modified Fc region and the Fc gamma receptor can be determined using methods known in the art. Various techniques (including, but not limited to, equilibration methods [e.g., enzyme-linked immunosorbent assays]) for ELISA; KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373 :52-60, 2008; radioimmunoassay (RIA)], or surface plasmon resonance assay or Other mechanism-based kinetic assays [e.g., BIACORE® analysis or Oct et® analysis (forteBIO)], as well as other methods [e.g., indirect binding assays, competitive Binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography ( These and other methods may be used to measure the amount of ATP present in the blood. , may utilize labels on one or more of the components being evaluated, and / or various Detection methods (e.g., but not limited to, chromogenic labels, fluorescent labels, luminescent labels, For binding affinity and kinetics, see Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999) (which focuses on antibody-immunogen interactions). An example of a specific binding assay involves combining a labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen. and incubating in the presence of the labeled antigen, and detecting the antibody bound to the labeled antigen. From the data, Scatchard et al. Plot analysis allows the affinity and binding off-rate of the antibody of interest for a particular antigen to be determined. Competition with a second antibody may be determined using a radioimmunoassay. In this case, the antigen is conjugated to a labeled compound. Incubate the antibody in the presence of increasing amounts of an unlabeled second antibody. .

[0201] In some embodiments, the Fc modifications described herein (e.g., D265C, L234A, L235A, and Antibodies with H435A and / or H435B exhibit Fc gamma receptor binding similar to that of the unmodified Fc region. a reduction of at least 70% compared to the binding of the same antibody to said Fc gamma receptor. , at least 80% reduction, at least 90% reduction, at least 95% reduction, at least 98% a reduction, at least a 99% reduction, or a 100% reduction (e.g., biolayer interferometry). When assessed by Blue-Layer Interferometry (BLI).

[0202] Without wishing to be bound by any theory, Fc region binding to Fc gamma receptors The interactions result in a variety of effector functions and downstream signaling events (e.g., limited Although not a known cause of cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADC) is ADCC) and complement dependent cytotoxicity ( Therefore, in certain embodiments, the modified Fc region is believed to be essential for CDC, etc. Antibodies containing the L234A, L235A, and / or D265C mutations (e.g., containing the L234A, L235A, and / or D265C mutations) have effector functions. Effector function is substantially reduced or eliminated. using a variety of methods (e.g., cellular responses to an antibody of interest [e.g., mast cell depletion], These assays may be used to measure the release of cytokines or granules. , using standard methods in the art, Fc-engineered antibodies that induce mast cell degranulation in vitro. their ability to induce granulocytes or cytokine release (e.g., by human peripheral blood mononuclear cells) The ability to induce a phenotype may be assayed.

[0203] The antibodies of the present disclosure can be further modified to produce antibodies, e.g., as described 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. The half-life of the antibody may be further adjusted by inserting May include 3, 254, 256, 257, 307, 376, 380, 428, 434 and 435. Exemplary mutations that can be made together are T250Q, M252Y, 1253A, S254T, T256E, P2571, T3 07A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R mutations is.

[0204] Thus, in one embodiment, the Fc region may be modified to have a reduced half-life (e.g., a reduced half-life compared to an unmodified Fc Antibodies with a short half-life include mutations that result in a shorter half-life compared to antibodies with In certain instances (e.g., antibodies) where the therapeutic agent is expected to function as a short-lived therapeutic agent, The conditioning step described herein involves administering IgG1-associated ... Ideally, unlike endogenous stem cells, they are generally target-specific. They also express target antigens (e.g., CD117 [e.g., GNNK+ CD117] or CD45), but Administering HSCs that are not targeted by anti-HC antibodies (e.g., anti-CD117 antibodies or anti-CD45 antibodies) In one embodiment, the antibody should be substantially cleared before the Fc region is In one embodiment, the region comprises a mutation at position 435 (EU index according to Kabat). The mutation is the H435A mutation.

[0205] In some embodiments, an anti-HC antibody described herein (e.g., an anti-CD117 antibody, or an anti-C D45 antibody) can be expressed for about 24 hours or less, about 23 hours or less, about 22 hours or less, about 21 hours or less (e.g., in humans). hours or less, about 20 hours or less, about 19 hours or less, about 18 hours or less, about 17 hours or less, about 16 hours or less, a half-life of about 15 hours or less, about 14 hours or less, about 13 hours or less, about 12 hours or less, or about 11 hours or less It has.

[0206] In some embodiments, an anti-HC antibody described herein (e.g., an anti-CD117 antibody or an anti-CD4 5 antibody) for about 1 to 5 hours, about 5 to 10 hours, about 10 to 15 hours, about 15 to 20 hours, or about 20 to 25 hours In one embodiment, the anti-HC antibody has a half-life (e.g., in humans). , about 5 to 7 hours; about 5 to 9 hours; about 5 to 11 hours; about 5 to 13 hours; about 5 to 15 hours; about 5 to 20 hours; about 5~24 hours; about 7~24 hours; about 9~24 hours; about 11~24 hours; about 12~22 hours; about 10~20 hours; about 8 to 18 hours; or about 14 to 24 hours.

[0207] In some embodiments, the Fc region reduces the half-life and effector activity of the antibody. In some embodiments, the Fc region comprises two or more mutations that reduce the function of the Fc region. Mutations that result in a decrease in half-life and that can directly contact FcγR (e.g., structure In some embodiments, the mutation of at least one residue is based on quantitative and 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 an H4 mutation, an H435A mutation, and a D265C mutation. These include the 35A mutation, the L234A mutation, the L235A mutation, and the D265C mutation.

[0208] In some embodiments, the antibody, or antigen-binding fragment thereof, is via cysteine ​​residues in the Fc domain of its antigen-binding fragment, to bind to cytotoxins (e.g. For example, amatoxin).

[0209] In some embodiments of these aspects, the cysteine ​​residue is a cysteine ​​residue of the antibody or its The Fc domain of the antigen-binding fragment is naturally present in the Fc domain of the human Fc domain. and the cysteine ​​residues may be Cys261, Cys361, Cys461, Cys561, Cys61, Cys761, Cys861, Cys9 ... It may be selected from the group consisting of sy321, Cys367, and Cys425.

[0210] In some embodiments, the cysteine ​​residue is a cysteine ​​residue of the antibody or its antigen-binding fragment. For example, the cysteine ​​residue is introduced by a mutation in the Fc domain of In some embodiments, the amino acid sequence may be selected from the group consisting of Cys118, Cys239, and Cys265. The anti-HC antibody (e.g., anti-CD117 antibody or anti-CD45 antibody), or its antigen-binding flag The Fc region of this antibody is identified by the amino acid substitution at amino acid 265 according to the EU index as in 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 D265C, L234A, L235A, and L235A mutations. and H435A mutation. In one embodiment, the anti-HC antibody (e.g., an anti-CD117 antibody, or The Fc region of the CD45 antibody, or an antigen-binding fragment thereof, is In one embodiment, the Fc region comprises an amino acid substitution at amino acid 239 with dextrose. In one embodiment, the Fc region comprises an L234A mutation, an L235A mutation, an S239C mutation, and an S239C mutation. In another embodiment, the Fc region comprises a S239C and a H435A mutation. In another embodiment, the Fc region comprises an L234A mutation, an L235A mutation, and an S239C mutation. In another embodiment, the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, and a S239C mutation. In yet another embodiment, the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, an S23 Contains the 9C mutation and the D265A mutation.

[0211] In particular, Fc amino acid positions refer to the EU numbering index unless otherwise indicated. do.

[0212] The variant Fc domains described herein are characterized according to the amino acid modifications that make them up. For all amino acid substitutions discussed herein with respect to the Fc region, the number The designation always follows the EU index. So, for example, D265C is a nucleotide sequence corresponding to the parent Fc domain. and an Fc variant in which the aspartic acid (D) at EU position 265 is substituted with a cysteine ​​(C). Similarly, for example, D265C / L234A / L235A corresponds to the sequence at EU position 265 (D to C) relative to the parent Fc domain, Defines an Fc variant with substitutions at 234 (L to A) and 235 (L to A). The nucleotides are also designated according to their final amino acid composition at the mutated EU amino acid position. For example, the L234A / L235A mutation is sometimes called LALA. The order of substitutions is arbitrary. In particular, amino acid positions in Fc are numbered in EU numbering unless otherwise noted. Refers to the index.

[0213] In some embodiments, the anti-CD117 antibodies or anti-CD45 antibodies herein may be modified with any of the following: or a combination of modifications, including an Fc region containing one of: D265A, D265C, D265C / H435A , D265C / LALA, D265C / LALA / H435A, D265C / N297G, D265C / N297G / H435A, D265 C (IgG2*), D265C (IgG2) / H435A, D265C / N297Q / H435A, D265C / N297Q, EPLVLAdel G / H435A, N297A, N297G, or N297Q.

[0214] The antibodies and binding fragments thereof disclosed herein are described in more detail below. They may be used in conjugates so as to

[0215] 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 or Isolated nucleic acids encoding anti-CD45 antibodies are provided. Such nucleic acids comprise the VL of the antibodies. and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and In a further embodiment, a single molecule comprising such a nucleic acid may encode a nucleotide sequence encoding the nucleotides of the nucleotide sequence of interest (e.g., a nucleotide sequence of the nucleotide sequence of interest and / or a heavy chain). The above vectors (e.g., expression vectors) are provided. In further embodiments, such vectors Host cells comprising the nucleic acid are provided. In some such embodiments, the host cells comprise: (e.g., transformed with): (1) an amino acid sequence comprising the VL of the antibody and the V (2) a vector containing a nucleic acid encoding an amino acid sequence containing the VL of the antibody; A first vector containing a nucleic acid encoding an amino acid sequence containing the VH of the antibody and a second vector containing a nucleic acid encoding an amino acid sequence containing the VH of the antibody In one embodiment, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells) In one embodiment, a method of making an anti-CLL-1 antibody is provided, wherein the method comprises: The method comprises culturing a host cell comprising nucleic acid encoding an antibody as provided above, for expression of said antibody. Culturing, under suitable conditions, and optionally culturing said host cells (or host cell cultures) and recovering the antibody from the culture medium.

[0216] To recombinantly produce an anti-CD117 antibody or an anti-CD45 antibody, nucleic acid encoding the antibody (e.g., The nucleic acid (e.g., as described above) is isolated and further cloned and / or transformed into a host cell. or inserted into one or more vectors for expression. Such nucleic acids can be prepared using conventional procedures. Using (e.g., an antibody capable of specifically binding to the genes encoding the heavy and light chains of the antibody), can be easily isolated (by using oligonucleotide probes), and Sequencing can be done.

[0217] Suitable host cells for cloning or expressing antibody-encoding vectors include Examples of suitable host cells include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be expressed in Produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, See, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (Charl ton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totow a, NJ, 2003), pp. 245-254, which describes the production of antibody fragments in E. coli. After expression, the antibody is transfected into bacterial cells. The soluble fraction may be isolated from the extract and further purified.

[0218] Vertebrate cells can also be used as hosts. For example, cells grown in suspension can be used. 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: MRC 5 cells; and FS4 cells. , Chinese hamster ovary (CHO) cells (DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines (e.g., Y0, NS0, and Sp 2 / 0, etc.) A review of certain mammalian host cell lines suitable for antibody production For details, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BK C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0219] In one embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, or anti-CD117 The D45 antibody, or antigen-binding fragment thereof, can be identified by the sequences set forth in SEQ ID NOS: o) comprising a variable region having an amino acid sequence that is at least 95%, 96%, 97%, or 99% identical to Table 3). Alternatively, the anti-CD117 antibody, or antigen-binding fragment thereof, or anti-CD45 The antibody, or antigen-binding fragment thereof, may be selected from the group consisting of the antibodies, SEQ ID Nos. 95%, 96%, 97% or 99% identical to the amino acid sequence of any of the amino acids described herein. CDRs comprising the SEQ ID Nos. disclosed herein with the framework regions of the variable regions Includes (Table 3).

[0220] In one embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, is In another embodiment, the heavy chain variable region and heavy chain constant region have the disclosed amino acid sequences. The anti-CD117 antibody, or antigen-binding fragment thereof, may have the amino acid sequence disclosed herein. In yet another embodiment, the anti-CD117 comprises a light chain variable region and a light chain constant region having the sequence The antibody, or antigen-binding fragment thereof, may comprise a heavy chain having an amino acid sequence disclosed herein. It comprises a variable region, a light chain variable region, a heavy chain constant region and a light chain constant region.

[0221] In one embodiment, the anti-CD45 antibody, or antigen-binding fragment thereof, is In another embodiment, the heavy chain variable region and heavy chain constant region have the disclosed amino acid sequences. The anti-CD45 antibody, or antigen-binding fragment thereof, may comprise an amino acid sequence as disclosed herein. In yet another embodiment, the anti-C antibody comprises a light chain variable region and a light chain constant region having the sequence The D45 antibody, or antigen-binding fragment thereof, has the amino acid sequence disclosed herein. It comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region.

[0222] Examples of anti-CD117 antibodies and anti-CD45 antibodies are further described herein.

[0223] Anti-CD117 antibody Human CD117 (also known as c-Kit, mRNA NCBI reference sequence: NM_000222.2, protein NCBI reference sequence: Antibodies and antigen-binding fragments (GNNK+CD11) capable of binding to the reference sequence: NP_000213.1 7) and antigen-binding fragments thereof, The compositions and methods described herein for conditioning a patient for It may be used in combination with CD117, which is present in a significant proportion of the population. Polymorphisms affecting the binding region or extracellular domain are currently not used for non-oncology indications. At least four isoforms of CD117 have been identified, Additional isoforms may be expressed in tumor cells. Two of the domains are localized to the intracellular domain of the protein, and two are present in the outer juxtamembrane region. The two extracellular isoforms, GNNK+ and GNNK-, have four amino acid sequences (GNNK+) or These isoforms differ in that they do not react with the same ligand (SCF). Although it has been reported that GNNK-isoforms have affinity for GNNK, ligand binding to GNNK-isoforms is not observed in cells. It has been reported that GNNK+ isoform enhances endocytosis and degradation. This is because antibodies raised against this isoform include GNNK+ and GNNK- proteins. may be used as an immunogen to generate antibodies capable of binding to human CD117. The amino acid sequences of isoforms 1 and 2 are set forth as SEQ ID Nos: 145 and 146, respectively. In certain embodiments, the anti-human CD117 (hCD117) antibodies disclosed herein are described as: The antibody can bind to both isoform 1 and isoform 2 of human CD117. do.

[0224] Examples of anti-CD117 antibodies are described in US 2019 / 0153114 A1 and US 2019 / 0144558 A1; The contents of both applications are expressly incorporated herein by reference in their entirety.

[0225] For example, anti-CD117 antibodies Ab54, Ab55, Ab56, Ab57, Ab58, Ab61, Ab66, Ab67, Ab68, and Ab The amino acid sequences of the 69 different binding regions are listed in Table 3. CD117 antibodies, as well as human anti-CD117 antibodies comprising the variable regions set forth in Table 3, are included in the present disclosure. do.

[0226] In one embodiment, the present invention provides a binding region (e.g., CDRs, possibly Antibody 55 (i.e., antibody 56) or an antigen-binding fragment thereof, comprising the CD117 antibody (i.e., antibody 57) or CD117 antibody fragment (i.e., antibody 58) or CD117 antibody fragment (i.e., antibody 59 ... The amino acid sequence of the heavy chain variable region (VH) of Ab55 is shown in SEQ ID NO: 19 (see Table 3). The amino acid sequences of the VH CDR domains of antibody 55 are shown in SEQ ID NO: 21 (VH CDR1); The light chain of antibody 55 is shown in SEQ ID NO: 22 (VH CDR2) and SEQ ID NO: 23 (VH CDR3). The amino acid sequence of the variable region (VL) is set forth in SEQ ID NO: 20 (see Table 3). The amino acid sequences of the VL CDR domains are shown in SEQ ID NO: 24 (VL CDR1); SEQ ID NO: 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, the anti-CD117 antibody, or antigen-binding portion thereof, has SEQ ID NO: a variable heavy chain CDR set (CDR1, CDR2, and CDR3) according to SEQ ID Nos: 21, 22, and 23; and the light chain variable region CDR sets set forth in SEQ ID Nos: 24, 25, and 26. In other 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. Includes.

[0227] In one embodiment, the present invention provides a binding region (e.g., CDRs, possibly Antibody 54 (i.e., antibody 54), or an antigen-binding fragment thereof, is provided. The amino acid sequence of the heavy chain variable region (VH) of Ab54 is shown in SEQ ID NO: 29 (see Table 3). The amino acid sequences of the VH CDR domains of antibody 54 are shown in SEQ ID NO: 31 (VH CDR1); The light chain of antibody 54 is shown in SEQ ID NO: 32 (VH CDR2) and SEQ ID NO: 33 (VH CDR3). The amino acid sequence of the variable region (VL) is set forth in SEQ ID NO: 30 (see Table 3). The amino acid sequences of the VL CDR domains are shown in SEQ ID NO: 34 (VL CDR1); SEQ ID NO: 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, the anti-CD117 antibody, or antigen-binding portion thereof, has SEQ ID NO: a variable heavy chain CDR set (CDR1, CDR2, and CDR3) according to SEQ ID Nos: 31, 32, and 33; and the light chain variable region CDR sets set forth in SEQ ID Nos: 34, 35, and 36. In other 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. Includes.

[0228] In one embodiment, the present invention provides a binding region (e.g., CDRs, possibly CDRs) corresponding to the binding region of antibody 56. Antibody 56 (i.e., antibody 56), or an antigen-binding fragment thereof, is provided. The amino acid sequence of the heavy chain variable region (VH) of Ab56 is shown in SEQ ID NO: 39 (see Table 3). The amino acid sequences of the VH CDR domains of antibody 56 are shown in SEQ ID NO: 41 (VH CDR1); The light chain of antibody 56 is shown in SEQ ID NO: 42 (VH CDR2) and SEQ ID NO: 43 (VH CDR3). The amino acid sequence of the variable region (VL) is set forth in SEQ ID NO: 40 (see Table 3). The amino acid sequences of the VL CDR domains are shown in SEQ ID NO: 44 (VL CDR1); SEQ ID NO: 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, the anti-CD117 antibody, or antigen-binding portion thereof, has SEQ ID NO: a variable heavy chain CDR set (CDR1, CDR2, and CDR3) according to SEQ ID Nos: 41, 42, and 43; and the light chain variable region CDR sets set forth in SEQ ID Nos: 44, 45, and 46. In other 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. Includes.

[0229] In one embodiment, the present invention provides a binding region (e.g., CDRs, possibly Antibody 57 (i.e., CD117 antibody or antigen-binding fragment thereof) containing the CD117 variant (i.e., CD117 variant) is provided. The amino acid sequence of the heavy chain variable region (VH) of Ab57 is shown in SEQ ID NO: 49 (see Table 3). The amino acid sequences of the VH CDR domains of antibody 57 are shown in SEQ ID NO: 51 (VH CDR1); The light chain of antibody 57 is shown in SEQ ID NO: 52 (VH CDR2) and SEQ ID NO: 53 (VH CDR3). The amino acid sequence of the variable region (VL) is set forth in SEQ ID NO: 50 (see Table 3). The amino acid sequences of the VL CDR domains are shown in SEQ ID NO: 54 (VL CDR1); SEQ ID NO: 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, the anti-CD117 antibody, or antigen-binding portion thereof, has SEQ ID NO: a variable heavy chain CDR set (CDR1, CDR2, and CDR3) according to SEQ ID Nos: 51, 52, and 53; and a set of light chain variable region CDRs set forth in SEQ ID Nos: 54, 55, and 56. In other 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. Includes.

[0230] In one embodiment, the present invention provides a binding region (e.g., CDRs, possibly Antibody 58 (i.e., antibody 59) or an antigen-binding fragment thereof comprising the CD117 antibody ... The amino acid sequence of the heavy chain variable region (VH) of Ab58 is shown in SEQ ID NO: 59 (see Table 3). The amino acid sequences of the VH CDR domains of antibody 58 are shown in SEQ ID NO: 61 (VH CDR1); The light chain of antibody 58 is shown in SEQ ID NO: 62 (VH CDR2) and SEQ ID NO: 63 (VH CDR3). The amino acid sequence of the variable region (VL) is set forth in SEQ ID NO: 60 (see Table 3). The VL CDR domain amino acid sequences are shown 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, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence Variable heavy chain CDR sets (CDR1, CDR2, and CDR3) set forth in SEQ ID Nos: 61, 62, and 63 and a light chain variable region CDR set forth in SEQ ID Nos: 64, 65, and 66. In other embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence identified as SEQ ID NO:60. and a heavy chain variable region set forth in SEQ ID NO: 59. Includes the area.

[0231] In one embodiment, the present invention provides a binding region (e.g., CDRs, possibly Antibody 61 (i.e., a CD117 antibody or antigen-binding fragment thereof) containing the CD117 variant (i.e., a CD117 variant) is provided. The amino acid sequence of the heavy chain variable region (VH) of Ab61 is shown in SEQ ID NO: 69 (see Table 3). The amino acid sequences of the VH CDR domains of antibody 61 are shown in SEQ ID NO: 71 (VH CDR1); The light chain of antibody 61 is shown in SEQ ID NO: 72 (VH CDR2) and SEQ ID NO: 73 (VH CDR3). The amino acid sequence of the variable region (VL) is set forth in SEQ ID NO: 70 (see Table 3). The VL CDR domain amino acid sequences 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, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence Variable heavy chain CDR sets (CDR1, CDR2, and CDR3) set forth in SEQ ID Nos: 71, 72, and 73 and a light chain variable region CDR set forth in SEQ ID Nos: 74, 75, and 76. In other embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence identified as SEQ ID NO:70. and a heavy chain variable region set forth in SEQ ID NO: 69. Includes the area.

[0232] In one embodiment, the present invention provides a binding region (e.g., CDRs, possibly Antibody 66 (i.e., antibody 66A) or an antigen-binding fragment thereof comprising the CD117 antibody (i.e., antibody 66B) or CD117 antibody (i.e., antibody 66C) or an antigen-binding fragment thereof comprising the CD117 antibody (i.e., antibody 66D) or CD117 antibody (i.e., antibody 66E) or CD117 antibody (i.e., antibody 66F) or CD11 The amino acid sequence of the heavy chain variable region (VH) of Ab66 is shown in SEQ ID NO: 79 (see Table 3). The amino acid sequences of the VH CDR domains of antibody 66 are shown in SEQ ID NO: 81 (VH CDR1); The light chain of antibody 66 is shown in SEQ ID NO: 82 (VH CDR2) and SEQ ID NO: 83 (VH CDR3). The amino acid sequence of the variable region (VL) is set forth in SEQ ID NO: 80 (see Table 3). The amino acid sequences of the VL CDR domains are shown in SEQ ID NO: 84 (VL CDR1); SEQ ID NO: 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, the anti-CD117 antibody, or antigen-binding portion thereof, has SEQ ID NO: a variable heavy chain CDR set (CDR1, CDR2, and CDR3) set forth in SEQ ID Nos: 81, 82, and 83; and a set of light chain variable region CDRs set forth in SEQ ID Nos: 84, 85, and 86. In other 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. Includes.

[0233] In one embodiment, the present invention provides a binding region (e.g., CDRs, possibly The present invention provides an anti-CD117 antibody, or an antigen-binding fragment thereof, comprising a nucleotide sequence (a region of the nucleotide sequence) of the antibody 67. The amino acid sequence of the chain variable region (VH) is shown in SEQ ID NO: 9 (see Table 3). The CDR domain amino acid sequences are SEQ ID NO: 11 (VH CDR1); SEQ ID NO: 12 (VH CDR2 The amino acid sequence of the light chain variable region (VL) of antibody 67 is shown in SEQ ID NO: 13 (VH CDR3). The sequence is set forth in SEQ ID NO: 10 (see Table 3). VL CDR domain amino acids of antibody 67 The sequences are SEQ ID NO: 14 (VL CDR1); SEQ ID NO: 15 (VL CDR2) and SEQ ID NO: 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 of antibody 67 is shown in SEQ ID NO: The light chain constant region of antibody 67 is shown in SEQ ID NO: 121. In certain embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO: s): a variable heavy chain CDR set (CDR1, CDR2, and CDR3) according to SEQ ID NOs. 11, 12, and 13, and The present invention also includes a set of light chain variable region CDRs set forth in SEQ ID Nos: 14, 15, and 16. , an anti-CD117 antibody, or an antigen-binding portion thereof, is an amino acid sequence set forth in SEQ ID NO:9. The variable heavy chain comprises an amino acid residue, and a heavy chain variable region set forth in SEQ ID NO: 10. In some embodiments, the anti-CD117 antibody has a heavy chain comprising SEQ ID NO:110 and a nucleotide sequence comprising SEQ ID NO:111. Q ID NO):109.

[0234] In one embodiment, the present invention provides a binding region (e.g., CDRs, possibly Antibody 68 (i.e., antibody 68), or an antigen-binding fragment thereof, is provided. The amino acid sequence of the heavy chain variable region (VH) of Ab68 is shown in SEQ ID NO: 89 (see Table 3). The amino acid sequences of the VH CDR domains of antibody 68 are shown in SEQ ID NO: 91 (VH CDR1); The light chain of antibody 68 is shown in SEQ ID NO: 92 (VH CDR2) and SEQ ID NO: 93 (VH CDR3). The amino acid sequence of the variable region (VL) is set forth in SEQ ID NO: 90 (see Table 3). The VL CDR domain amino acid sequences are shown 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, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence Variable heavy chain CDR sets (CDR1, CDR2, and CDR3) set forth in SEQ ID Nos: 91, 92, and 93 and a light chain variable region CDR set forth in SEQ ID Nos: 94, 95, and 96. In other embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence identified as SEQ ID NO:90. and a heavy chain variable region set forth in SEQ ID NO: 89. Includes the area.

[0235] In one embodiment, the present invention provides a binding region (e.g., CDRs, possibly CDRs) corresponding to the binding region of antibody 69. Antibody 69 (i.e., antibody 69), or an antigen-binding fragment thereof, is provided. The amino acid sequence of the heavy chain variable region (VH) of Ab69 is shown in SEQ ID NO: 99 (see Table 3). The amino acid sequences of the VH CDR domains of antibody 69 are shown in SEQ ID NO: 101 (VH CDR1); The VH CDR2 and VH CDR3 sequences are shown in SEQ ID NO: 102 (VH CDR2) and SEQ ID NO: 103 (VH CDR3), respectively. The amino acid sequence of the light chain variable region (VL) is set forth in SEQ ID NO: 100 (see Table 3). The amino acid sequences of the VL CDR domains of antibody 69 are shown in SEQ ID NO: 104 (VL CDR1); The heavy chain of antibody 69 is shown in SEQ ID NO: 105 (VL CDR2) and SEQ ID NO: 106 (VL CDR3). The constant region is shown in SEQ ID NO: 122. The light chain constant region of antibody 69 is shown in SEQ ID NO: O):121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen-binding portion thereof, The variable heavy chain CDR set (CDR1, CDR2) is set forth in SEQ ID Nos: 101, 102, and 103. , and CDR3), and the light chain variable region CDRs set forth in SEQ ID Nos: 104, 105, and 106. R set. 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 variable light chain comprising the amino acid residues set forth in SEQ ID NO: 99 The heavy chain variable region comprises:

[0236] Certain of the anti-CD117 antibodies described herein are characterized in that the antibodies bind to CD117 on CD117-expressing cells. It is a neutral antibody in that it does not substantially inhibit the activity of the antibody. Neutral antibodies) can be used, for example, in an in vitro stem cell factor (SCF)-dependent cell proliferation assay. In the SCF-dependent cell proliferation assay, the neutral CD117 antibody can be identified as a neutral antibody. Neutral antibodies do not block the binding of SCF to CD117 and inhibit CD117 activity. and will not kill CD34+ cells that divide in an SCF-dependent manner.

[0237] Neutral antibodies are those that can specifically bind to human CD117. In view of this, the cytotoxins described herein 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, as described herein, This is a unique approach to conjugates, and in particular, the conjugates can be used to treat stem cells. This is a technique in which it is used as a conditioning agent before transplantation. The antibody alone or in combination with a cytotoxin as a conjugate may be used in combination with the antibody in addition to the cytotoxin. On the other hand, a neutral anti-CD117 antibody containing Conditioning with conjugates inhibits the activity of the antibody against the effects of the cytotoxin. Secondarily, the antibody's internalization and affinity properties (e.g., dissociation rate) are important factors. Another alternative strategy is to use a cytotoxin containing hydroxybenzoates (HBD) when the degree of hydroxybenzoates is important for effective delivery of the cytotoxin. Present (strategy).

[0238] Examples of neutral anti-CD117 antibodies include Ab58, Ab61, Ab66, Ab67, Ab68, and Ab69. By comparing the amino acid sequences of the CDRs of the neutral anti-CD117 antibodies, the neutral antibodies identified were ) reveals a consensus sequence between the two groups. Ab58 and Ab61 share the same light chain CDR and H C CDR3 is shared, and there are slight differences in HC CDR1 and HC CDR2. The sequences of Ab66, Ab67, Ab68, and Ab69 are set forth in SEQ ID Nos: 133 and 134. Ab66, Ab67, Ab68, and Ab69 are also neutral antibodies. and the same HC CDR3, but these antibodies have different CDRs within their HC CDR1 and HC CDR2 regions. The consensus sequences of these antibodies in the HC CDR1 and HC CDR2 regions are They are set forth in SEQ ID Nos: 139 and 140, respectively.

[0239] For example, in one embodiment, the Fc region of antibody 67 is modified to include a D265C mutation (e.g., For example, SEQ ID NO: 111. In another embodiment, the Fc region of antibody 67 comprises the amino acid sequence D265C, L234A, and modified to include a L235A mutation (e.g., SEQ ID NO: 112). In embodiments, 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 comprises the amino acid sequence D265C, L234A, It is modified to contain the L235A and H435A mutations (eg, SEQ ID NO:114).

[0240] With respect to antibody 55, in one embodiment, the Fc region of antibody 55 is modified to include a D265C mutation. In another embodiment, the Fc region of antibody 55 is selected from the group consisting of D26 5C, L234A, and L235A mutations (e.g., SEQ ID NO: 118). In yet another embodiment, the Fc region of antibody 55 is modified to include D265C and H435A mutations. (e.g., SEQ ID NO: 119). In a further embodiment, the Fc region of antibody 55 comprises D265C , L234A, L235A, and H435A mutations (e.g., SEQ ID NO: 120).

[0241] 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., as in SEQ ID NO: 123). ); D265C, L234A, and L235A mutations (e.g., as in SEQ ID NO: 124); D26 5C and H435A mutations (e.g., as in SEQ ID NO: 125); or D265C, L234A, modified to include L235A and H435A mutations (e.g., as in SEQ ID NO: 126). It can be changed.

[0242] Antagonist antibodies are also described herein, including Ab54, Ab55, Ab56, and Ab57. Ab54, Ab55, Ab56, and Ab57 share the same light chain CDR and the same HC CDR3, but These antibodies have differences within their HC CDR1 and HC CDR2 regions. The consensus sequences of these antibodies in this region are set forth as SEQ ID Nos: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 38, 39, 40, 41, 42, 43, 44, 7 and 128.

[0243] In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence set forth in SEQ ID NO: 1 47, and the amino acid sequence of SEQ ID NO: 148. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region described in is a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147, and ID NO): 149. In one embodiment, the anti-CD117 The antibody, or antigen-binding portion thereof, comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 147. The variable region comprises a light chain variable region and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 150. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence set forth in SEQ ID NO: 147 and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 151. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region as described in , 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: 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: 152. The antibody, or an antigen-binding portion thereof, may comprise a heavy chain polypeptide set forth in the amino acid sequence of SEQ ID NO: 147. The light chain variable region includes a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 153. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence of SEQ ID NO: 147. The heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 154. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region comprising: A heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147, and NO): 155. In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence of or an antigen-binding portion thereof, comprising the heavy chain variable fragment 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: 156. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO: 147. The heavy chain variable region is set forth in the amino acid sequence of SEQ ID NO: 157. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region of the sequence The heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147, and ): 158. 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, comprising a heavy chain variable region 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: 159. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is an amino acid sequence of SEQ ID NO: 147. a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 160; In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region having the sequence The heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147, 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 SEQ ID NO:161. The antigen-binding portion thereof comprises a heavy chain variable region 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: 162. In some embodiments, 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 set forth in the amino acid sequence of SEQ ID NO: 163. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a CD117 antibody having SEQ ID NO: a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 164, and a In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO:65. The antigen-binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 166; and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 167. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises the amino acid sequence of SEQ ID NO: 168. A heavy chain variable region set forth in the sequence, and a light chain set forth in the amino acid sequence of SEQ ID NO: 169. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a variable region selected from the group consisting of SEQ ID NO: The heavy chain variable region set forth in the amino acid sequence of (SEQ ID NO): 170, and SEQ ID NO: 171 In one embodiment, the anti-CD117 antibody or its derivatives comprises a light chain variable region set forth in the amino acid sequence of The antigen-binding portion of the heavy chain variable region is set forth in the amino acid sequence of SEQ ID NO: 172, and and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 173. In the present specification, an anti-CD117 antibody, or an antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO: 174. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 175. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a variant region of SEQ ID NO: (S a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 176, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 177; In one embodiment, the anti-CD117 antibody or its derivatives comprises a light chain variable region set forth in the amino acid sequence set forth in the following paragraphs: The antigen-binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 178, and In one embodiment, the antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 179. The anti-CD117 antibody, or antigen-binding portion thereof, has 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 one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a region set forth in SEQ ID NO: a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 172, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 182; In one embodiment, the anti-CD117 antibody or its anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence set forth in The original binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 183, and a In one embodiment, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 184. The anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence set forth in 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, comprises the sequence identified by SEQ ID NO: a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 187, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 188; In one embodiment, the anti-CD117 antibody or its antigen comprises a light chain variable region set forth in the sequence set forth in the following paragraphs: The binding moiety comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 189, and a heavy chain variable region set forth in the amino acid sequence of the sequence In one embodiment, the antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 190. The anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence set forth in SEQ ID NO: 191. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 192. 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: 193, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 194; In one embodiment, the anti-CD117 antibody, or its antigen binding domain, comprises a light chain variable region set forth in the sequence The conjugate comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 195, and a heavy chain variable region set forth in SEQ ID NO: In one embodiment, the antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 196. - the CD117 antibody, or an antigen-binding portion thereof, is set forth in the amino acid sequence of SEQ ID NO: 197 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 198. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises the sequence set forth in SEQ ID NO: ): 199, and the heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 200 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: 201, and a heavy chain variable region set forth in SEQ ID NO: (SEQ ID NO): 190. In one embodiment, the anti-C The D117 antibody, or an antigen-binding portion thereof, is represented by the amino acid sequence set forth in SEQ ID NO: 202. a heavy chain variable region and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 203; In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence identified as SEQ ID NO: 204, and the heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 205. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region as set forth in the column. The positions are the heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 206, and the heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: EQ ID NO): 207. In one embodiment, the anti-CD1 The antibody 17, or an antigen-binding portion thereof, has the amino acid sequence set forth in SEQ ID NO: 208. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 209. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence set forth in SEQ ID NO: 2 10, and the amino acid sequence of SEQ ID NO: 211. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region described in is a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 212, and ID NO): 213. In one embodiment, the anti-CD117 The antibody, or antigen-binding portion thereof, comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 214. 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: 215. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence set forth in SEQ ID NO: 216 and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 217. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region as described in , 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: 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: 219. The antibody, or an antigen-binding portion thereof, may comprise a heavy chain polypeptide set forth in the amino acid sequence of SEQ ID NO: 220. The light chain variable region comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 221. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence of SEQ ID NO: 222. The heavy 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, comprises a light chain variable region comprising: A heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 224, and NO): 225. In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence of or an antigen-binding portion thereof, comprising the heavy chain variable fragment set forth in the amino acid sequence of SEQ ID NO: 226. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 227. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO: 7. a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 228; In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region having the sequence the heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and the heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 2 In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO:29. The antigen-binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 230. , an anti-CD117 antibody, or an antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO: 7 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 231. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a region set forth in SEQ ID NO: a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 232; In one embodiment, the anti-CD117 antibody or its antigen comprises a light chain variable region set forth in the sequence set forth in the following paragraphs: The binding moiety comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 7, and a In one embodiment, the antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 233. - the CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 7 A heavy chain variable region and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 234. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, has the sequence identified as SEQ ID NO: 7, and the amino acid sequence of SEQ ID NO: 235. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region described in The heavy chain variable region is set forth in the amino acid sequence of SEQ ID NO: 7, and the heavy chain variable region is set forth 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: 236. 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: 7. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 237. In embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO: 7. a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 237; In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a light chain variable region having the sequence The heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 243, 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 SEQ ID NO:244. The antigen-binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 251. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 252. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO: 243. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 256. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a CD117 antibody having SEQ ID NO: a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 259, and a In one embodiment, the anti-CD117 antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 56. The antigen-binding portion comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 260; and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 252. In some embodiments, the anti-CD117 antibody, or antigen-binding portion thereof, comprises the amino acid sequence of SEQ ID NO: 238. A heavy chain variable region set forth in the sequence, and a light chain set forth in the amino acid sequence of SEQ ID NO: 239. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a variable region selected from the group consisting of SEQ ID NO: The heavy chain variable region set forth in the amino acid sequence of (SEQ ID NO): 147, and SEQ ID NO: 239 In one embodiment, the anti-CD117 antibody or its derivatives comprises a light chain variable region set forth in the amino acid sequence of The antigen-binding portion of the heavy chain variable region is set forth in the amino acid sequence of SEQ ID NO: 147, and and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 240. In the present specification, an anti-CD117 antibody, or an antigen-binding portion thereof, has the amino acid sequence of SEQ ID NO: 238. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 241. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a variant region of SEQ ID NO: (S a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 238, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 242 It contains the light chain variable region set forth in the amino acid sequence.

[0244] scFv phage display library for human antibodies as described below Screening for novel anti-CD117 antibodies and their fragments with therapeutic applications In particular, antibodies 85 (Ab85), 86 (Ab86), 87 (Ab87), 88 (Ab88), and 89 (Ab89) were identified. , were identified in this screen.

[0245] The heavy chain variable region (VH) amino acid sequence of Ab85 is shown below as SEQ ID NO: 243. The VH CDR amino acid sequences of Ab85 are underlined and are as follows: NYWIG (VH CDR1; SEQ ID NO: IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 246); and HGRGYEGYEGAFDI (VH CDR3; SEQ ID NO: 247). Ab85 VH sequence [ka] (SEQ ID NO: 243)

[0246] The light chain variable region (VL) amino acid sequence of Ab85 is shown below as SEQ ID NO: 244. The VL CDR amino acid sequence of Ab85 is underlined and is as follows: RSSQGIRSDLG (VL CDR1; SEQ ID NO: 248); DASNLET (VL CDR2; SEQ ID NO: 249); and QQA NGFPLT (VL CDR3; SEQ ID NO: 250). Ab85 VL sequence [ka] (SEQ ID NO: 244)

[0247] Antibody HC-86 / LC-86(Ab86) The heavy chain variable region (VH) amino acid sequence of Ab86 is shown below as SEQ ID NO: 251. The VH CDR amino acid sequence of Ab86 is underlined and is as follows: NYWIG (VH CDR1; SEQ ID NO: (SEQ ID NO: 245); IIYPGDSDIRYSPSLQG (VH CDR2; SEQ ID NO: 253); and H GRGYNGYEGAFDI (VH CDR3; SEQ ID NO: 3). Ab86 VH sequence [ka] (SEQ ID NO: 251)

[0248] The light chain variable region (VL) amino acid sequence of Ab86 is shown below as SEQ ID NO: 252. The VL CDR amino acid sequence of Ab86 is underlined and is as follows: RASQGIGDSLA (VL CDR1; SEQ ID NO: 254); DASNLET (VL CDR2; SEQ ID NO: 249); and QQL NGYPIT (VL CDR3; SEQ ID NO: 255). [ka]

[0249] Antibody HC-87 / LC-87(Ab87) The heavy chain variable region (VH) amino acid sequence of Ab87 is shown below as SEQ ID NO: 243. The VH CDR amino acid sequences of Ab87 are underlined and are as follows: NYWIG (VH CDR1; SEQ ID NO: IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 246); and HGRGYEGYEGAFDI (VH CDR3; SEQ ID NO: 247). Ab87 VH sequence [ka]

[0250] The light chain variable region (VL) amino acid sequence of Ab87 is shown below as SEQ ID NO: 256. The VL CDR amino acid sequence of Ab87 is underlined and is as follows: RASQGIRNDLG (VL CDR1; SEQ ID NO: 257); DASSLES (VL CDR2; SEQ ID NO: 5); and QQLNG YPIT (VL CDR3; SEQ ID NO: 255). Ab87 VL sequence [ka]

[0251] Antibody HC-88 / LC-88(Ab88) The heavy chain variable region (VH) amino acid sequence of Ab88 is shown below as SEQ ID NO: 258. The VH CDR amino acid sequences of Ab88 are underlined and are as follows: NYWIG (VH CDR1; SEQ ID NO: IIYPGDSLTRYSPSFQG (VH CDR2; SEQ ID NO: 259); and HGRGYNGYEGAFDI (VH CDR3; SEQ ID NO: 3). Ab88 VH sequence [ka]

[0252] The light chain variable region (VL) amino acid sequence of Ab88 is shown below as SEQ ID NO: 256. The VL CDR amino acid sequence of Ab88 is underlined and is as follows: RASQGIRNDLG (VL CDR1; SEQ ID NO: 257); DASSLES (VL CDR2; SEQ ID NO: 5); and QQLNG YPIT (VL CDR3; SEQ ID NO: 255). Ab88 VL sequence [ka]

[0253] Antibody HC-89 / LC-89(Ab89) The heavy chain variable region (VH) amino acid sequence of Ab89 is shown below as SEQ ID NO: 260. The VH CDR amino acid sequences of Ab89 are underlined and are as follows: NYWIG (VH CDR1; SEQ ID NO: IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 2); and H GRGYNGYEGAFDI (VH CDR3; SEQ ID NO: 3). Ab89 VH sequence [ka]

[0254] The light chain variable region (VL) amino acid sequence of Ab89 is shown below as SEQ ID NO: 252. The VL CDR amino acid sequence of Ab89 is underlined and is as follows: RASQGIGDSLA (VL CDR1; SEQ ID NO: 254); DASNLET (VL CDR2; SEQ ID NO: 249); and QQL NGYPIT (VL CDR3; SEQ ID NO: 255). Ab89 VL sequence [ka]

[0255] Antibody HC-249 / LC-249(Ab249) The heavy chain variable region (VH) amino acid sequence of Ab249 is shown below as SEQ ID NO: 238. The VH CDR amino acid sequences of Ab249 are underlined and are as follows: TSWIG (VH CDR1; sequence IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 2); and and HGLGYNGYEGAFDI (VH CDR3; SEQ ID NO: 287). Ab249 VH sequence [ka]

[0256] The light chain variable region (VL) amino acid sequence of Ab249 is shown below as SEQ ID NO: 242. The VL CDR amino acid sequence of Ab249 is underlined and is as follows: RASQGIGSALA (VL CDR 1; SEQ ID NO: 288); DASNLET (VL CDR2; SEQ ID NO: 249); and QQLNGYPLT (VL CDR3; SEQ ID NO: 289). Ab249 VL sequence [ka]

[0257] Both human antibodies Ab85 and Ab249 are derived from the antibody CK6, an antagonist anti-CD117 antibody. Ab85 and Ab249 have improved properties compared to CK6 (e.g., improved binding properties). (This is the case.)

[0258] CK6 contains a potential deamidation site within the CDR3 domain of the heavy chain variable region For future antibody production, it is beneficial to remove the asparagine. However, it is very difficult to isolate the potential deamidation site in the Ab85 heavy chain CDR3. If successfully removed, the antibody (with Ab85 heavy and light chain CDRs) then binds to human CD117. It can maintain a high level of specificity for the target molecule and the ability to be taken up into cells. Furthermore, Ab85 exhibited improved dissociation rates compared to its parent.

[0259] Thus, in certain embodiments, the anti-CD117 antibody is selected from the group consisting of SEQ ID Nos: 245, 246, and 247, and a heavy chain comprising a CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID NO: Nos): 248, 249, and 1250, and a light chain comprising the CDRs set forth in 5 x 10 when taken up and measured by BLI -4 s-1 The following k off Has speed.

[0260] Further features that may be used in conjunction with the methods of patient conditioning described herein include: Examples of suitable anti-CD117 antibodies include those described in U.S. Pat. No. 5,489,516 (the disclosure of which is incorporated herein by reference). ATCC Accession No. 10 / 1999, filed on 23 / 1999, which is incorporated herein by reference as it relates to the subject matter. Produced and released from application number 10716 (deposited under BA7.3C.9) ) antibodies (for example, SR-1 antibody, etc.).

[0261] In certain embodiments, the anti-CD117 antibodies described herein comprise the amino acid sequence of L235A, L235A, D265C, and and an Fc region containing H435A (EU index).

[0262] Additional methods that can be used in conjunction with the methods of conditioning a patient described herein include: Suitable anti-CD117 antibodies include those described in U.S. Pat. No. 7,915,391 (which describes, for example, a humanized SR-1 antibody). anti-CD117 antibodies described in U.S. Pat. No. 5,808,002 (which, e.g., describe anti-CD 117 A3C6E2 antibody), as well as anti-CD117 antibodies described, for example, in WO 2015 / 0509 59 (This corresponds to Pro317, Asn320, Glu329, Val331, Asp332, Lus358, Glue360 of human CD117. Anti-CD117 antibodies that bind to epitopes including Glue376, His378, and / or Thr380 are described. ) and the anti-CD117 antibodies described in US 2012 / 0288506 (see U.S. Patent No. 8,552,157); (also published in the same publication) (which describes, for example, the anti-CD117 antibody CK6).

[0263] Additional anti-CD117 antibodies that can be used in combination with the compositions and methods described herein include: The antibodies and their antigen-binding fragments include clones 9P3, NEG024, NEG027, NEG085, and NEG086. and those described in US 2015 / 0320880, such as 20376.

[0264] Anti-CD45 antibody Human CD45 (mRNA NCBI reference sequence: NM_080921.3, protein NCBI reference sequence: NP_563578.2) Antibodies and antigen-binding fragments capable of binding to isoform CD45RO and those that can be used in conjunction with the compositions and methods disclosed herein. For example, promoting hematopoietic stem cell graft survival in patients requiring hematopoietic stem cell transplantation therapy. In some embodiments, the compositions and methods disclosed herein may comprise a sequence similar to that of SEQ ID NO: An anti-CD45 antibody or ADC that binds to human CD45RO as set forth in the amino acid sequence of (SEQ ID NO: 290) The antibodies that bind to the various isoforms of CD45 disclosed herein are also included herein. The multiple isoforms of CD45 are intended for use in the methods and compositions disclosed herein. The form arises from the alternative splicing of 34 exons in the primary transcript. Multiple CD45 variants are generated by splicing sequences 4, 5, 6, and possibly 7. Alternative exon expression is observed in the CD45 isoforms listed in Table 1 below. It is suspected.

[0265] [Table 1]

[0266] Alternative splicing produces various isoforms of the CD45 protein (e.g., CD4 These genes are expressed in CD45RA, CD45RAB, and CD45RABC, resulting in individual exons or combinations of exons. In contrast, CD45RO lacks expression of exons 4-6 and expresses a combination of exons 1-3 and 7-34. Exon 7 is also excluded from the protein, and exons 1-3 and 8-34 are There is evidence that they are spliced ​​together. This protein, named E3-8, Although quality has been detected at the mRNA level, it is currently not possible to detect it by flow cytometry. has not been identified.

[0267] CD45RO is currently the only known CD45 isoform expressed on hematopoietic stem cells. CD45RA and CD45RABC are either not detected or are excluded from the hematopoietic stem cell phenotype. 45RB is expressed in fetal hematopoietic stem cells but is absent in adult bone marrow hematopoietic stem cells. There is evidence from studies conducted in mice. In particular, CD45RC has been shown to be involved in the development of inflammatory cytokines in Asian populations. The polymorphism of exon 6 of CD45RC is observed in a high proportion of cases (the polymorphism of exon 6 of CD45RC is observed in a Japanese population). This polymorphism results in high expression of CD45RO and low expression of CD45RA, CD45RB, and This results in a decrease in the level of CD45RC. In addition, CD45RA variants (e.g., CD45RAB and CD45RAB) 45RAC, etc.) indicate polymorphisms in exon 4 that are associated with autoimmune diseases.

[0268] The presence of CD45RO on hematopoietic stem cells and on other immune cells (e.g., T and B lymphocytes) The relatively limited expression of CD45RO on various myeloid cells (e.g., neutrophils and various myeloid cells) CD45RO is a promising candidate for conditioning therapy in patients requiring hematopoietic stem cell transplantation. CD45RO is a particularly suitable target, as it lacks expression of exons 4, 5, and 6. Therefore, by using it as an immunogen, pan-CD45Ab and CD45RO-specific This makes it possible to screen for antibodies.

[0269] Anti-CD45 antibodies that may be used with the patient conditioning methods described herein The antibodies include anti-CD45 antibodies, and antigen-binding portions thereof. Antigen-binding portions of antibodies are known in the art. These are well known and can be easily constructed based on the antigen-binding regions of the antibodies. In embodiments, anti-CD45 antibodies used in conjunction with the conditioning methods described herein The antibody may be a monoclonal antibody or an antigen-binding fragment thereof, a polyclonal antibody, or and antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, fully human Antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof , a bispecific antibody or an antigen-binding fragment thereof, Dual variable immunoglobulin domains, single-chain Fv molecules (scFv), diabodies, triabodies , nanobodies, antibody-like protein scaffolds, Fv fragments, Fab fragments , F(ab')2 molecules, or tandem di-scFv. Exemplary anti-CD45 antibodies that may be used in whole or in part in the methods are described below. provide.

[0270] In one embodiment, the anti-CD45 antibody is BIOLEGEND (登録商標) (California San The human HIV-1 strain is, or is derived from, clone HI30, available from Antibody humanization involves replacing framework and constant region residues of a non-human antibody with those of the corresponding human antibody. Germline human genomic DNA was isolated according to procedures known in the art (e.g., as described in Example 7 below). This can be done by substituting the antibody of interest. Additional anti-CD45 antibodies that may be used include anti-CD45 antibodies ab10558, EP322Y, and MEM. -28, ab10559, 0.N.125, F10-89-4, HIe-1, 2B11, YTH24.5, PD7 / 26 / 16, F10-89-4, 1B7, ab154885, B-A11, phosphor S1007, ab170444, EP350, Y321, GA90, D3 / 9, X1 6 / 99, and and LT45 (these are ABCAM (登録商標) (Cambridge, MA) or its In conjunction with the patient conditioning procedures described herein, Further anti-CD45 antibodies that may be used in combination include the anti-CD45 antibody HPA000440 (which , SIGMA-ALDRICH (登録商標) (St. Louis, MO)), and its human These include modified variants. An additional anti-CD45 antibody that may be used is the mouse monoclonal antibody BC8 (which is described, for example, in Matthews et al., Blood 78:1864-1874, 1991, and this disclosure are incorporated herein by reference as they relate to anti-CD45 antibodies), and and humanized variants of the nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 3 Further anti-CD45 antibodies include the monoclonal antibody YAML568 (which is used, for example, in Glattinger et al., J. Immunol. 2004; 2005; 2006; 2007; 2009; 2010). et al., J. Nucl. Med. 8:1335-1341, 2006, which disclosure relates to anti-CD45 antibodies. (which are incorporated herein by reference as if fully set forth herein) as well as humanized variants thereof. These may be used in conjunction with the patient conditioning procedures described herein. Further anti-CD45 antibodies include monoclonal antibodies YTH54.12 and YTH25.4 (which , as described, for example, in Brenner et al., Ann. NY Acad. Sci. 996:80-88, 2003; the disclosure of which is incorporated herein by reference as it relates to anti-CD45 antibodies), and humanized variants thereof. Additional anti-CD45 antibodies that may be used in conjunction with the method include UCHL1, 2H4, SN130, MD 4.3, MBI, and MT2 (which are described, for example, in Brown et al., Immunology 64:331-336, 1998) and US Pat. No. 6,299,499, the disclosure of which is incorporated herein by reference as it relates to anti-CD45 antibodies. The methods described herein include the use of the ribozyme A (RI) and the use of the ribozyme B (RI) as a target gene (RI), as well as humanized variants thereof. Additional anti-CD45 antibodies that may be used in conjunction with the American Type Culture Collection are American Type Culture Collection (ATCC) accession number R anti-CD45 antibodies produced and released from A3-6132, RA3-2C2, and TIB122; and monoclonal antibodies C363.16A and 13 / 2 (which are described, for example, in Johnson et al., J. Exp. Med. 169:1179-1184, 1989, which disclosure relates to anti-CD45 antibodies. and humanized variants thereof. may be used in conjunction with the patient conditioning methods described herein. Additional anti-CD45 antibodies include monoclonal antibodies AHN-12.1, AHN-12, AHN-12.2, and AHN-12. 3, AHN-12.4, HLe-1, and KC56(T200) (these are described, for example, in Harvath et al., J. Immuno 146:949-957, 1991, which disclosure is incorporated herein by reference as it relates to anti-CD45 antibodies. , incorporated herein by reference), as well as humanized variants thereof.

[0271] Additional patient conditioning procedures that may be used in conjunction with the patient conditioning procedures described herein include: Anti-CD45 antibodies include those described, for example, in U.S. Pat. No. 7,265,212 (which, for example, The antibodies described herein include, among others, anti-CD45 antibodies 39E11, 16C9, and 1G10. - CD45 antibodies; U.S. Patent 7,160,987 (which is produced, for example, by ATCC Accession No. HB-11873) Describes the generation and release of anti-CD45 antibodies (e.g., monoclonal antibody 6G3, etc.). and U.S. Pat. No. 6,099,838 (which, e.g., describes anti-CD4 5 antibody MT3, and ATCC accession numbers HB220 [also called MB23G2] and HB223 produced and anti-CD45 antibodies described in US 2004 / 0096901 and US 2008 / 0003224 (which are, for example, identified under ATCC Accession No. PTA-733 Anti-CD45 antibodies produced and released by 9 (e.g., monoclonal antibody 17.1, etc.) (These disclosures are directed to anti-CD45 antibodies.) (The text is incorporated herein by reference as it relates to the body.)

[0272] Additional patient conditioning procedures that may be used in conjunction with the patient conditioning procedures described herein include: Anti-CD45 antibodies include those with ATCC accession numbers MB4B4, MB23G2, 14.8, GAP 8.3, and 74-9-3. , I / 24.D6, 9.4, 4B2, M1 / 9.3.4.HL.2, and antibodies produced and released from and humanized and / or affinity matured variants thereof. For example, in vitro display techniques described herein or known in the art (e.g., These may be achieved using techniques such as gene expression analysis (e.g., phage display).

[0273] Additional patient conditioning procedures that may be used in conjunction with the patient conditioning procedures described herein include: Anti-CD45 antibodies include the anti-CD45 antibody T29 / 33, which is described, for example, in Morikawa et al. ., Int. J. Hematol. 54:495-504, 1991 (this disclosure relates to anti-CD45 antibodies). (The text is incorporated herein by reference as if fully set forth herein).

[0274] In certain embodiments, the anti-CD45 antibody is apamistamab (or the known 90Y- BC8, Iomab-B, BC8; see, e.g., US20170326259, WO2017155937, and Orozco et al. Blood. 127.3 (2016): 352-359.), or BC8-B10 (e.g., Li et al. PloS one 13.1 0 (2018): e0205135), each of which is incorporated by reference. Other anti-CD45 antibodies are described, for example, in WO2003 / 048327, WO2016 / 016442, US2017 / 022620 9, US2016 / 0152733, US9,701,756; US2011 / 0076270, or US7,825,222 (each of which is incorporated by reference).

[0275] For example, in one embodiment, the anti-CD45 antibody, or antigen-binding fragment thereof, It contains binding regions (e.g., CDRs, variable regions, corresponding to the binding region of apamistaumab). The heavy chain variable region (VH) amino acid sequence of stamab is set forth in SEQ ID NO: 296 ( See Table 3. The light chain variable region (VL) amino acid sequence of apamistaumab is shown in SEQ ID NO: : 297 (see Table 3). In another embodiment, an anti-CD45 antibody, or an antigen-binding portion thereof, The fragment comprises a variable heavy chain comprising the amino acid residues set forth in SEQ ID NO: 296, and a variable heavy chain comprising the amino acid residues set forth in SEQ ID NO: In one embodiment, the anti-CD45 antibody comprises a light chain variable region set forth in SEQ ID NO: 297. , a heavy chain comprising CDR1, CDR2 and CDR3 of apamistamab, and CDR1, CDR2 of apamistamab and a light chain variable region comprising CDR3.

[0276] In one embodiment, the anti-CD45 antibody comprises a heavy chain of an anti-CD45 antibody described herein, and In one embodiment, the anti-CD45 antibody comprises a light chain variable region of the anti-CD45 antibody described herein. The antibody comprises a heavy chain comprising CDR1, CDR2, and CDR3 of an anti-CD45 antibody described herein, and a CDR1, CDR2, and CDR3 of an anti-CD45 antibody described herein. and a light chain variable region comprising CDR1, CDR2, and CDR3 of the anti-CD45 antibody described in .

[0277] In another embodiment, the antibody, or antigen-binding fragment thereof, is an anti-CD4 5 antibodies (e.g., at least 95% identical to the anti-CD45 antibodies herein). a heavy chain comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the In certain embodiments, the antibody comprises the HC variable region of the anti-CD45 antibodies herein. a modified heavy chain (HC) variable region containing a domain, or a variant thereof, (i) differs from said anti-CD45 antibody in one, two, three, four, or five amino acid substitutions, additions, or deletions. (ii) the anti-CD45 antibody has at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from the preceding one in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions; and / or (iv) at least about 75%, 80%, 85% different from said anti-CD45 antibody; , 90%, 95%, 96%, 97%, 98% or 99% identical to (i)-(iv), In either case, the amino acid substitutions may be conservative or non-conservative. and wherein the modified heavy chain variable region is a CD45 heavy chain variable region of the anti-CD45 antibody. The heavy chain variable region of the anti-CD45 antibody has a higher biological activity than the heavy chain variable region of the anti-CD45 antibody while retaining its binding specificity. is increasing.

[0278] Antibodies and antigen-binding fragments that may be used in conjunction with the compositions and methods described herein The fragments include the above-mentioned antibodies and antigen-binding fragments thereof, and the above-mentioned non-human Humanized variants of antibodies and antigen-binding fragments, as well as, for example, competitive CD45 binding assays. When assessed by ELISA, the antibody or antigen-binding fragment binds to the same epitope as the antibody or antigen-binding fragment described above. and antibodies or antigen-binding fragments thereof.

[0279] Methods for identifying antibodies Binding to antigens expressed by hematopoietic stem cells (e.g., CD117 [e.g., GNNK+ CD117], or CD45) High speed screening of antibody or antibody fragment libraries for molecules that can bind to Treating cancer and autoimmune diseases using methods for high-throughput screening and for patients in need of hematopoietic stem cell therapy as described herein (e.g., Identifying and affinity maturing useful antibodies for conditioning patients Such methods include in vitro display techniques known in the art, For example, phage display, bacterial display, yeast display, among others. ray, mammalian cell display, ribosome display, mRNA display, and cDNA display, etc. Antibodies that bind to biologically relevant molecules, or antibodies Using phage display to isolate original binding fragments, e.g. , Felici et al., Biotechnol. Annual Rev. 1:149-183, 1995; Katz, Annual Rev. Biop hys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4: 1-20, 1998, each of which discloses in vitro displays. The disclosure of which is incorporated herein by reference as it relates to the art. Kay, Perspectives. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, 1996 Construct a randomized combinatorial peptide library as described in Selection of polypeptides that bind to cell surface antigens has also been carried out. The disclosures of which are incorporated herein by reference as they relate to the discovery of antigen-binding molecules. Proteins (e.g., multimeric proteins) can be used as functional molecules in phage digests. Spraying has been successfully achieved (e.g., EP 0349578; EP 4527839; and EP 0 589877, and Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992; Each of these disclosures uses in vitro display technology to discover antigen-binding molecules. (The text of which is incorporated herein by reference as it relates to the present invention.) Furthermore, functional antibodies Fragments (e.g., Fab and scFv fragments) are displayed in vitro using a display format. Expression in vitro has also been attempted (e.g., McCafferty et al., Nature 348:5 52- 554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, each of which discloses antigen binding. Associated with in vitro display platforms for discovering synthetic molecules , which are incorporated herein by reference). Human anti-HC antibodies (e.g., anti-CD117 antibodies, or anti- -CD45 antibody) can also be used, for example, HuMAb-mouse 登録商標 or XenoMouse TM To generate In particular, these techniques can be used to identify antigens expressed by hematopoietic stem cells (e.g., CD4+, CD5+, CD6+, CD8+, CD9+, CD10+, CD12+, CD14+, CD16+, CD18 ... 117 [e.g., GNNK+ CD117], or CD45), which can then bind to hematopoietic stem cells. Depleting endogenous hematopoietic stem cells in a patient (e.g., a human patient) in need of transplant therapy can be used to identify the affinity of antibodies, antibodies, or fragments, and This may result in improvement.

[0280] In addition to in vitro display techniques, computational modeling techniques are used to identify the genesis of hematopoietic stem cells. can bind to antigens expressed by the IL-16 receptor (e.g., CD117 [e.g., GNNK+ CD117], or CD45). Antibodies or antibody fragments that are effective against certain diseases may be designed and identified in silico. For example, using computational modeling techniques, one skilled in the art can determine the antigens ( For example, specific epitopes (e.g., on CD117 [e.g., GNNK+ CD117], or CD45) and searching for molecules capable of binding to the extracellular epitopes of the antigens, such as antibodies or anti- Libraries of antibody fragments may be screened in silico.

[0281] Further techniques can be used to identify antigens expressed by hematopoietic stem cells (e.g., CD117 [e.g., GNNK+ C D117], or CD45), and can bind to, for example, receptor-mediated endocytosis. an antibody, or antibody fragment, that the cell can take up by cytosis. For example, the in vitro display technology described above can be used to identify genes expressed by hematopoietic stem cells. binds to an antigen (e.g., CD117 [e.g., GNNK+ CD117], or CD45), and subsequently By screening for antibodies, or antibody fragments thereof, that are incorporated into the Phage display may be adapted to this screening paradigm. This represents one such technique that may be used in combination with anti-HC antibodies (e.g., To identify and subsequently identify the antibody fragments (anti-CD117 antibody, or anti-CD45 antibody) or antibody fragments. To identify the specific nucleotide sequence that is incorporated into hematopoietic stem cells (or immune cells), a skilled artisan may The authors used the phage display described in Williams et al., Leukemia 19:1432-1438, 2005. Ray technology (the disclosure of which is incorporated herein by reference in its entirety) may be used. For example, antibodies, antibody fragments, etc. may be modified using mutagenesis methods known in the art. (e.g., scFv fragments, Fab fragments, diabodies, triabodies, among others) , and 10 Fn3 domain), or ligands (which contain randomized amino acid cassettes [e.g. For example, one or more CDRs or equivalent regions thereof, or an antibody or antibody fragment, or It is possible to generate a recombinant phage library encoding a phage vector containing all of the phage genes. The framework region, hinge, Fc domain, and other regions of the antibody or antibody fragment can be , e.g., human germline antibody sequences or sequences that are slightly different compared to human germline antibodies. may be designed to be non-immunogenic in humans by .

[0282] using phage display techniques described herein or known in the art and phage containing randomized antibodies or antibody fragments covalently bound to the phage particle. Incubate the library with an antigen (e.g., CD117 [e.g., GNNK+ CD117], or CD45). bate (e.g., first, an antibody that exhibits non-specific protein binding, or an antibody fragment To remove phages encoding the fragments, and to use antibodies or phage library to remove phages encoding the fragments Blocking agents (e.g., milk proteins, bovine serum albumin, and / or IgG) and then incubating the phage library with, for example, CD117 [e.g. hematopoietic stem cells or mature immune cells [e.g., T- Incubate with a population of [cells]. CD45 antibody) or antibody fragment binds to a cell surface cognate antigen (e.g., CD117 [e.g., GNNK+ CD117] or CD45) and subsequently internalized by the hematopoietic stem cells. The pharmacokinetic parameters are then measured and stored at 4°C for a period of time sufficient to achieve the desired results (e.g., 30 minutes to 6 hours at 4°C, e.g., 1 hour at 4°C). The page library may be incubated with the hematopoietic stem cells. Bind hematopoietic stem cells by washing with chilled (4 °C) 0.1 M glycine buffer, pH 2.8 and be taken up by hematopoietic stem cells, NNK+ CD117, or CD45) The phage containing the antibody or its antibody taken up by the hematopoietic stem cells may be removed. Phage bound to the antibody fragments can be isolated, for example, by lysing the cells and extracting the cell culture medium. The phage may then be identified by recovering the phage that has been taken up from the For example, bacterial cells can be cultured together with the recovered phage using methods known in the art. The phage was transfected into bacterial cells by incubation in 2xYT medium at 20°C. The phage recovered from this medium may then be amplified, for example, by phage gel electrophoresis. Determine the nucleic acid sequence of the gene encoding the antibody or antibody fragment inserted into the genome. The encoded antibody or antibody fragment may then be identified. and (e.g., for antibody fragments thereof, e.g., scFv fragments) chemical synthesis. or (e.g., for full-length antibodies) by recombinant expression. It may be prepared differently.

[0283] The amount of antibody or antibody fragment that can be incorporated into the prepared antibody or antibody fragment can be determined, for example, by methods known in the art. Radionuclide internalization assays known in the art may be used to assess the effect of ionizing radiation on the nuclei of the nuclei. For example, the assays described herein may be used to assess the effect of ionizing radiation on the nuclei of .... Anti-H antibodies identified using in vitro display techniques described herein or known in the art. C antibody (e.g., anti-CD117 antibody or anti-CD45 antibody) or antibody fragment, Isotopes (e.g., 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, 67 G a, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu, 177 Lu, 77 As, 72 As, 86 Y, 90 Y, 89 Zr, 212 Bi, 213 Bi, or 225 Ac) can be incorporated to make it functional. For example, , radioactive halogens (e.g., 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 21 1 At, etc.) onto beads (e.g., polystyrene beads) containing electrophilic halogen reagents (e.g., For example, iodized beads (Thermo Fisher Scientific, Inc., Cambridge, MA) were used to detect the antibodies. Radiolabeled antibodies, their fragments, etc. The fragment or ADC is allowed to stand for a sufficient time for incorporation (e.g., 30 minutes to 6 hours at 4°C, e.g., The cells may then be incubated with hematopoietic stem cells (e.g., at 4°C for 1 hour). Wash the cells (e.g., with cold (4 °C) 0.1 M glycine buffer, pH 2.8) , and remove the antibodies or fragments thereof that have not been incorporated into the antibody. or antibody fragments, and irradiating the resulting hematopoietic stem cells with radiation (e.g., gamma rays) emitted by the stem cells. By detecting and comparing with the radiation (e.g., gamma rays) emitted from the collected wash buffer, The internalization assay described above may be used to characterize the ADC. There are also.

[0284] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In some embodiments, the anti-HC antibodies described herein (e.g., The present invention provides isolated nucleic acids encoding such antibodies (anti-CD117 antibodies, or anti-CD45 antibodies). The nucleic acid may comprise an amino acid sequence comprising the VL and / or the VH of the antibody (e.g., In a further embodiment, such a nucleic acid may encode a light chain and / or a heavy chain of the antibody. In a further embodiment, one or more vectors (e.g., expression vectors) containing the nucleic acid are provided. provides a host cell comprising such nucleic acid. In some such embodiments, the host cell comprises (e.g., has been transformed with): (1) a VL of the antibody a vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody; (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody; and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. The host cells may be eukaryotic, such as Chinese hamster ovary (CHO) cells or lymphocytes. In one embodiment, the anti-CLL-1 antibody is produced from a human ovarian or human ovarian lineage cell (e.g., Y0, NS0, Sp20 cells). The present invention provides a method for the preparation of an antibody comprising the steps of: under conditions suitable for expression of said antibody, and optionally and recovering the antibody from the host cell (or the culture medium of the host cell).

[0285] To recombinantly produce anti-HC antibodies (e.g., anti-CD117 antibodies, or anti-CD45 antibodies), by isolating and further growing in a host cell nucleic acid encoding the antibody (e.g., as described above). It is inserted into one or more vectors for cloning and / or expression. Such nucleic acids can be isolated using conventional procedures (e.g., by isolating genes encoding the heavy and light chains of the antibody). (by using oligonucleotide probes that can specifically bind to the target molecule) It can be isolated and sequenced.

[0286] Suitable host cells for cloning or expressing antibody-encoding vectors include Examples of suitable host cells include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be expressed in Produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, See, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, N .J., 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli. After expression, the antibody is extracted from the bacterial cell paste. The soluble fraction may be isolated from the extract and further purified.

[0287] Vertebrate cells can also be used as hosts. For example, cells grown in suspension can be used. 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: MRC 5 cells; and FS4 cells. , Chinese hamster ovary (CHO) cells (DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines (e.g., Y0, NS0, and Sp 2 / 0, etc.) A review of certain mammalian host cell lines suitable for antibody production For details, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BK See C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In embodiments, the host cell is a eukaryote, such as a Chinese hamster ovary (CHO) cell. The cells may be blastocytic or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0288] Antibody-drug conjugates The antibodies and antigen-binding fragments thereof described herein may be linked to a cytotoxic agent via a linker. In some embodiments, the cytotoxic moiety The antibody may be an antibody or its antigen-binding fragment that is internalized by a cell as disclosed herein. The antibody or its fragment is conjugated to a marker, and the antibody or its fragment is taken up into the cell. After the cytotoxin has been released, it can access its intracellular target and mediate the death of the hematopoietic cell. Any number of cytotoxins, e.g., 1, 2, 3, 4, 5, 6, 7, or 8, can be coupled to an anti-HC antibody. It can be conjugated.

[0289] Suitable cytotoxins for use with the compositions and methods described herein include, but are not limited to, DNA-intercalating agents known in the art, among others, such as anthracyclines, cyclins), drugs that can disrupt the spindle apparatus (e.g., vinca alkaloids, maytansine, maytansinoids, and their derivatives), RNA polymerase inhibitors (e.g., amatoxins such as α-amanitin and its derivatives) and disrupt protein biosynthesis Agents that can induce rRNA N-glycosidase activity (e.g., saporin and ricin A chain) Drugs that cause side effects include:

[0290] cytotoxins A variety of cytotoxins can be conjugated via linkers for use in the therapies described herein. It may be conjugated to an anti-HC antibody (e.g., anti-CD117 antibody, anti-CD45 antibody). In addition, the anti-HC ADC (e.g., anti-CD117 ADC or anti-CD45 ADC) may contain a cytotoxic moiety (or an antibody (or its antigen-binding protein) conjugated (i.e., covalently attached by a linker) to a toxin; In various embodiments, the cytotoxic moiety comprises a conjugate. When bound to the cytosine, the cytotoxicity is reduced or absent. After cleavage from the linker, the cytotoxicity is restored. The cytotoxic moiety retains its cytotoxicity without being cleaved from the linker. In some embodiments, the cytotoxic molecule is a cytotoxic molecule that is internalized into a cell, as described herein. or an antigen-binding fragment thereof, such that said antibody, or a fragment thereof is taken up into a cell, after which the cytotoxin targets its intracellular target These cells may access the T-cell network and, for example, mediate T-cell death.

[0291] Thus, the ADCs of the present disclosure have the general formula Ab-(ZLD) n wherein the antibody or its Each antigen-binding fragment (Ab) is linked to a linker (L) as disclosed herein. conjugate (covalently bond) to a cytotoxic moiety ("drug", D) through a chemical moiety (Z) Conjugate.

[0292] Thus, the antibody or antigen-binding fragment thereof may comprise multiple antibodies, as indicated by the integer n. may be conjugated to a number of drug moieties, where n is the integer number of cytotoxins per antibody. where the integer n can range, for example, from about 1 to about 20. In some embodiments, n is 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in the ADC preparations from the ionization reaction was determined by mass spectrometry. This can be demonstrated by conventional means such as photomicroscopy, ELISA assays, and HPLC. The quantitative distribution of ADC with respect to n can also be determined. In some cases, reversed-phase HP By means such as LC or electrophoresis, homogeneous ADCs with specific n values ​​can be prepared, which can be used to separate other drug-loaded ADCs. The ADC can be isolated, purified, and characterized from the resulting ADC.

[0293] For some anti-HC ADCs (e.g., anti-CD117 ADCs, or anti-CD45 ADCs), n is The number of binding sites on the antibody may be limited. For example, If the antibody is a cysteine ​​thiol, the antibody has only one or a few cysteine ​​thiol groups, or They have only one or a few thiol groups that are sufficiently reactive for antibodies to bind to them. The body does not contain many free and reactive cysteine ​​groups available for attachment to drug moieties; Primarily, thiol residues of cysteines in antibodies exist as disulfide bridges. In certain embodiments, the antibody is lysed with dithiothreitol (DTT) or tricarbonylethylphosphite. The reactive cysteines are reduced with a reducing agent such as TCEP under partial or total reducing conditions. In certain embodiments, higher drug loading may be achieved by generating ol groups. (e.g., n>5) can lead to aggregation, insolubility, and toxicity in certain antibody-drug conjugates. This may result in a loss of cell permeability.

[0294] In certain embodiments, during the conjugation reaction, less than the theoretical maximum The drug moiety is conjugated to the antibody. The antibody can be, for example, a drug- May contain lysine residues that do not react with the linker intermediate or linker reagent. Only highly reactive lysine groups can react with amine-reactive linker reagents. In an embodiment, the antibody is subjected to denaturing conditions to remove reactive nucleophilic groups, such as lysine or cysteine. Bring it to the surface.

[0295] The loading (drug / antibody ratio) of the ADC can be controlled in various ways, e.g., (i) by using a drug-linker - limiting the molar excess of intermediates or linker reagents; (ii) limiting the molar excess of intermediates or linker reagents during the conjugation reaction; (iii) limiting the time or temperature for cysteine ​​thiol modification; (iv) controlling the number and / or location of linker-drug bonds; To achieve this, the amino acid sequence of the antibody is modified to alter the number and position of cysteine ​​residues. It can be used to manipulate or control something.

[0296] Suitable cytotoxins for use with the compositions and methods described herein include, but are not limited to, DNA-intercalating agents known in the art, among others, such as anthracyclines, cyclins), drugs that can disrupt the spindle apparatus (e.g., vinca alkaloids, maytansine, maytansinoids, and their derivatives), RNA polymerase inhibitors (e.g., amatoxins such as α-amanitin and its derivatives) and disrupt protein biosynthesis Agents that can induce rRNA N-glycosidase activity (e.g., saporin and ricin A chain) Drugs that cause side effects include:

[0297] In some embodiments, the cytotoxin is a microtubule binding agent (e.g., maytansine or (maytansinoids), amatoxin, Pseudomonas exotoxin A, debouganin, diphtheria riatoxin, saporin, auristatin, anthracycline, calicheamicin, irino Tecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine Indolinobenzodiazepine dimer, indolinobenzodiazepine, indolinobenzodiazepine dimer, indolino a benzodiazepine pseudodimer or variant thereof, or and other cytotoxic compounds described or known in the art.

[0298] In some embodiments, the cytotoxin of the antibody-drug conjugate inhibits RNA polymerase In some embodiments, the RNA polymerase inhibitor is amatoxyl In some embodiments, the antibody-drug complexes disclosed herein are The cytotoxin of the conjugate is amatoxin or its derivative (e.g., α-amanitin, β- Amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, amanuric acid, proamanurin or its derivatives).

[0299] Anti-HC ADCs (e.g., anti-CD117 ADCs, or anti-CD45 ADCs) useful in the methods of the invention include: Further details regarding cytotoxins that may be used in the treatment of cancer are provided below.

[0300] Amatoxins The methods and compositions disclosed herein include conjugating an anti-HC antibody (e.g., an anti-CD117 antibody). ADCs that contain an RNA polymerase inhibitor (e.g., amatoxin) as the gated cytotoxin In some embodiments, the RNA polymerase inhibitor is an amatoxin or In some embodiments, the antibody-drug conjugates disclosed herein are derivatives of The cytotoxin is amatoxin or its derivatives (e.g., α-amanitin, β-amanitin). , γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, amanulin The structures of various natural amatoxins are For example, see Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459. can be.

[0301] Amatoxins useful in conjunction with the compositions and methods described herein include: Examples of amanitin include, but are not limited to, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, Manitin, amanine, amaninamide, amanulin, amanuric acid, proamanulin, etc. Included are compounds according to formula (III), including: Formula (III) is as follows: [ka] where R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; R A and R B When present, together with the oxygen atom to which they are attached, forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D is; R4 is H, OH, OR D , or R D is; R5 is H, OH, OR D , or R D is; R6 is H, OH, OR D , or R D is; R7 is H, OH, OR D , or R D is; R8 is OH, NH2, or OR D is; R9 is H, OH, or OR D is; X is -S-, -S(O)-, or -SO2-; and R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocyclo alkyl, optionally substituted aryl, or optionally substituted heteroaryl It is.

[0302] For example, in some embodiments, in conjunction with the compositions and methods described herein Useful amatoxins include compounds according to formula (IIIA) [ka] where R4, R5, X, and R8 are each defined above.

[0303] For example, in some embodiments, in conjunction with the compositions and methods described herein Useful amatoxins include compounds according to formula (IIIB): [ka] where R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; R A and R B When present, together with the oxygen atom to which they are attached, forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D is; R4 is H, OH, OR D , or R D is; R5 is H, OH, OR D , or R D is; R6 is H, OH, OR D , or R D is; R7 is H, OH, OR D , or R D is; R8 is OH, NH2, or OR D is; R9 is H, OH, or OR D is; X is -S-, -S(O)-, or -SO2-; and R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocyclo alkyl, optionally substituted aryl, or optionally substituted heteroaryl It is.

[0304] In some embodiments, agents useful in conjunction with the compositions and methods described herein include: Matoxins include compounds according to the following formula (IIIC): [ka] where R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; R A and R B When present, together with the oxygen atom to which they are attached, forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D is; R4 is H, OH, OR D , or R D is; R5 is H, OH, OR D , or R D is; R6 is H, OH, OR D , or R D is; R7 is H, OH, OR D , or R D is; R8 is OH, NH2, or OR D is; R9 is H, OH, or OR D is; X is -S-, -S(O)-, or -SO2-; and R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocyclo alkyl, optionally substituted aryl, or optionally substituted heteroaryl It is.

[0305] In one embodiment, the cytotoxin is amanitin.

[0306] For example, the antibodies and antigen-binding fragments described herein can be represented by the formula Ab-ZL-Am. and amatoxin (e.g., a compound of Formula III, IIIA, IIIB, IIIC, IIID, IIIE, IIIF, IIIG, IIIH, IIIH, IIII ... IIB, or IIIC), where Ab refers to an antibody or antigen-binding fragment thereof. L is a linker, Z is a chemical moiety, and Am is an amatoxin. Many positions on the amatoxin or its derivatives are suitable for covalent attachment to the linking moiety L. and therefore, the antibody, or antigen-binding fragment thereof, These can serve as sites for covalent bonding with the cations. Examples of amatoxin conjugations and linkers useful in Exemplary methods are described below. Antibodies, or Exemplary linker-containing amatoxins useful for conjugation to antigen-binding fragments ( Am-LZ) are shown in structural formulas (I), (IA), (IB), (II), (IIA), and (IIB) and are listed herein. do.

[0307] In some embodiments, the amatoxin-linker conjugate Am-LZ has the formula I) [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached, 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 , RC , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocyclo alkyl, optionally substituted aryl, or optionally substituted heteroaryl It is le; L is optionally substituted alkylene (e.g., C1-C6 alkylene), optionally heteroalkylene (e.g., C1-C6 heteroalkylene), optionally substituted optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic alkylene; alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally optionally substituted heteroarylenes, peptides, dipeptides, -(C=O)-, disulfides, a linker, such as a cyclohexyl benzoate, ... Z binds to the reactive substituent present on L and to the target antigen (e.g., CD117). Coupling reactions between reactive groups present in an antibody, or antigen-binding fragment thereof It is a chemical substructure formed from the reaction

[0308] In some embodiments, Am is exactly one R C Contains substituents.

[0309] In some embodiments, LZ is [ka] where S is a nucleotide present in an antibody or antigen-binding fragment thereof that binds to a target antigen. is a sulfur atom representing a reactive substituent present (e.g., derived from the -SH group of a cysteine ​​residue) .

[0310] In some embodiments, LZ is [ka] is.

[0311] In some embodiments, the conjugate Am-LZ-Ab has the formula IV, IVA, or IVB It is represented by one of: [ka] where X is S, SO, or SO2, and Ab is shown to indicate the position of binding to Ab. do.

[0312] In some embodiments, Am-LZ-Ab is [ka] where Ab is shown to indicate the position that binds to Ab.

[0313] In some embodiments, Am-LZ-Ab is [ka] where Ab is shown to indicate the position that binds to Ab.

[0314] In some embodiments, Am-LZ-Ab is [ka] where Ab is shown to indicate the position that binds to Ab.

[0315] In some embodiments, the Am-LZ-Ab precursor, Am-L-Z′, is [ka] where the maleimide reacts with a thiol group on a cysteine ​​in the antibody. .

[0316] In some embodiments, Am-LZ is represented by formula (IA): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached, 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 C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocyclo alkyl, optionally substituted aryl, or optionally substituted heteroaryl It is le; L is optionally substituted alkylene (e.g., C1-C6 alkylene), optionally heteroalkylene (e.g., C1-C6 heteroalkylene), optionally substituted optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic alkylene; alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally optionally substituted heteroarylenes, peptides, dipeptides, -(C=O)-, disulfides, a linker, such as a diazon, a cyclohexyl benzoate ... Z is a group consisting of a reactive substituent present on L and an antibody or antigen-binding fragment thereof that binds to the HC antigen. Antibodies present in the fragment (i.e., anti-HC antibody, e.g., anti-CD117 antibody, or anti-CD45 antibody) is a chemical moiety formed from a coupling reaction between a reactive substituent and where Am is exactly one R C Contains substituents.

[0317] In some embodiments, LZ is [ka] is.

[0318] In some embodiments, LZ is [ka] is.

[0319] In some embodiments, Am-LZ is represented by formula (IB): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached, 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, ORC , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally Substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocyclo alkyl, optionally substituted aryl, or optionally substituted heteroaryl It is le; L is optionally substituted alkylene (e.g., C1-C6 alkylene), optionally heteroalkylene (e.g., C1-C6 heteroalkylene), optionally substituted optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic alkylene; alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally optionally substituted heteroarylenes, peptides, dipeptides, -(C=O)-, disulfides, a linker, such as a diazon, a cyclohexyl benzoate ... Z is a group consisting of a reactive substituent present on L and an antibody or antigen-binding fragment thereof that binds to the HC antigen. Antibodies present in the fragment (i.e., anti-HC antibody, e.g., anti-CD117 antibody, or anti-CD45 antibody) is a chemical moiety formed from a coupling reaction between a reactive substituent and where Am is exactly one R C Contains substituents.

[0320] In some embodiments, LZ is [ka] is.

[0321] In some embodiments, LZ is [ka] is.

[0322] In some embodiments, R A and R B are the oxygen atoms to which they are attached, if present. together to form a 5-membered heterocycloalkyl group of the formula: [ka] Here, Y is -(C=O)-, -(C=S)-, -(C=NR E )-, or -(CR E R E' )-; and R Eand R E' are each independently an optionally substituted C-C alkylene-R C , Ren optionally substituted C1-C6 heteroalkylene-R C , optionally substituted C2-C6 alkene Niren-R C , optionally substituted C2-C6 heteroalkenylene-R C , optionally substituted C2-C6 alkynylene-R C , optionally substituted C2-C6 heteroalkynylene-R C ,any optionally substituted cycloalkylene-R C , optionally substituted heterocycloalkyl Ren-R C、 optionally substituted arylene-R C、 or optionally substituted heteroaromatic groups Liren-R C , is.

[0323] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B When present, together with the oxygen atoms to which they are attached, Form: [ka] R3 is H or R C is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C, OR D , R C , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; X is —S—, —S(O)—, or —SO2—; and where R C and R D are each as defined above.

[0324] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B When present, together with the oxygen atoms to which they are attached, Form: [ka] R3 is H or R C is; R4 and R5 are each independently H, OH, OR C , R C OR D is; R6 and R7 are each H; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; X is —S—, —S(O)—, or —SO2—; and where R C is as defined above.

[0325] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): where R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; R A and R B When present, together with the oxygen atoms to which they are attached, Form: [ka] R3, R4, R6, and R7 are each H; R5 is OR C is; R8 is OH or NH2; R9 is H or OH; X is —S—, —S(O)—, or —SO2—; and where R C Such amatoxin conjugates are as defined above. This is described, for example, in U.S. Patent Application Publication No. 2016 / 0002298, the disclosure of which is incorporated herein by reference. The entirety of which is incorporated herein by reference.

[0326] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): wherein R1 and R2 are each independently H or OH; R3 is R C is; R4, R6, and R7 are each H; R5 is H, OH, or OC1-C6 alkyl; R8 is OH or NH2; R9 is H or OH; X is —S—, —S(O)—, or —SO2—; and where R C Such amatoxin conjugates are as defined above. This is described, for example, in U.S. Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated herein by reference. The entirety of which is incorporated herein by reference.

[0327] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): wherein R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 ...

Claims

1. A method for reducing the population of CD117+ cells in a human patient in need of hematopoietic stem cell transplantation. an effective amount of an anti-CD117 antibody drug prior to the patient receiving an allogeneic hematopoietic stem cell-containing transplant; administering to the patient a polypeptide conjugate and an immunosuppressant.

2. Methods include: a. administering to a human patient an anti-CD117 antibody in an amount sufficient to reduce the population of CD117+ cells in said patient. administering a 117 antibody drug conjugate and an immunosuppressant; and b) thereafter administering to said patient a graft comprising allogeneic hematopoietic stem cells.

3. administering to a human patient a graft comprising allogeneic hematopoietic stem cells, The patient is administered an anti-CD117 antibody drug in an amount sufficient to reduce the population of hematopoietic stem cells in the patient. The patient has previously been administered a vasoconjugate and an immunosuppressant.

4. The method according to any one of claims 1 to 3, wherein said CD117 is GNNK+ CD117.

5. A method for reducing the population of CD45+ cells in a human patient in need of hematopoietic stem cell transplantation. an effective amount of an anti-CD45 antibody drug prior to the patient receiving an allogeneic hematopoietic stem cell-containing transplant; administering to the patient a polypeptide conjugate and an immunosuppressant.

6. Methods include: a. administering to a human patient an anti-CD4 antibody in an amount sufficient to reduce the population of CD45+ cells in said patient; 5. administering an antibody drug conjugate and an immunosuppressant; and b) thereafter administering to said patient a graft comprising allogeneic hematopoietic stem cells.

7. administering to a human patient a graft comprising allogeneic hematopoietic stem cells, The patient is administered an anti-CD45 antibody drug in an amount sufficient to reduce the population of hematopoietic stem cells in the patient. The patient has previously been administered a vasoconjugate and an immunosuppressant.

8. administering to a human patient a graft comprising allogeneic hematopoietic stem cells, The patient is administered an anti-CD45 antibody drug in an amount sufficient to reduce the population of hematopoietic stem cells in the patient. The patient has previously been administered the monoclonal conjugate.

9. and further administering said immunosuppressant to said patient after said patient receives said graft. The method according to any one of claims 1 to 8, comprising:

10. A method for reducing the population of CD117+ cells in a human patient in need of hematopoietic stem cell transplantation. , a method comprising: a. administering to the human patient an amount sufficient to reduce the population of CD117+ cells in the patient; administering an anti-CD117 antibody drug conjugate; b. administering to the human patient a graft comprising allogeneic hematopoietic stem cells; and c) thereafter administering to said patient an immunosuppressant drug.

11. A method for reducing the population of CD45+ cells in a human patient in need of hematopoietic stem cell transplantation. , a method comprising: a. administering to the human patient an anti-CD45 antibody in an amount sufficient to reduce the population of CD45+ cells in the patient; administering a CD45 antibody drug conjugate; b. administering to the human patient a graft comprising allogeneic hematopoietic stem cells; and c) thereafter administering to said patient an immunosuppressant drug.

12. 12. The method according to any one of claims 1 to 11, wherein the graft is a graft that is free of all HLA antigens. The human subject's HLA antigens are matched to allogeneic hematopoietic stem cells.

13. The method according to any one of claims 1 to 11, wherein the graft is a human leukocyte antigen (HLA antigen) of the patient. The allogeneic hematopoietic stem cells contain at least one HLA-mismatch to the donor.

14. 14. The method of claim 13, wherein the allogeneic hematopoietic stem cells are selected from the group consisting of HLA antigens of the patient. It contains at least two HLA-mismatches against

15. 15. The method of claim 14, wherein the allogeneic hematopoietic stem cells are selected from the group consisting of HLA antigens of the patient. Contains at least five HLA-mismatches to

16. 16. The method of claim 15, wherein the allogeneic hematopoietic stem cells are selected from the group consisting of HLA antigens of the patient. , including a complete HLA-mismatch.

17. 17. The method according to any one of claims 1 to 16, wherein the graft is a minor component of the patient. - At least one minor histocompatibility antigen-mismatched against a histocompatibility antigen (miHA) The hematopoietic stem cells include allogeneic hematopoietic stem cells including

18. The method according to any one of claims 1 to 17, wherein the method comprises the steps of: This is effective in establishing donor chimerism.

19. 19. The method of claim 18, wherein the method comprises obtaining at least 85% donor chimerism ( It is effective in establishing donor chimerism.

20. 20. The method of claim 19, wherein the method comprises obtaining at least 90% donor chimerism ( It is effective in establishing donor chimerism.

21. 21. The method of claim 20, wherein the method comprises obtaining at least 95% donor chimerism ( It is effective in establishing donor chimerism.

22. The method according to any one of claims 18 to 21, wherein the donor chimerism Chimerism will be assessed at least 6, 7, 8, 9, or 10 weeks after transplantation.

23. The method according to any one of claims 18 to 22, wherein the donor chimerism Peripheral myeloid chimerism is a type of chimerism that occurs in the bone marrow.

24. The method according to any one of claims 18 to 22, wherein the donor chimerism T-cell chimerism is a type of T-cell chimerism.

25. 25. The method according to any one of claims 1 to 24, wherein the immunosuppressant is cyclophosphamide. It is amide.

26. The method according to any one of claims 1 to 24, wherein the immunosuppressant is a total body irradiation (t otal body irradiation (TBI).

27. 25. The method of any one of claims 1 to 24, wherein the immunosuppressant is be.

28. 25. The method according to any one of claims 1 to 24, wherein the immunosuppressant is an anti-CD8 antibody, anti-CD4 antibodies, or both anti-CD8 and anti-CD4 antibodies.

29. 29. The method according to any one of claims 1 to 28, wherein the immunosuppressant is administered after transplantation. do.

30. The method according to any one of claims 1 to 28, wherein the immunosuppressant is administered prior to transplantation. do.

31. 31. The method of any one of claims 1 to 30, wherein the conjugate is administered to the patient. After administration to the skin, the compound is taken up into the cells by cancer cells, autoimmune cells, or hematopoietic stem cells. do.

32. 32. The method according to any one of claims 1 to 31, wherein the concentration of the conjugate is After the hematopoietic stem cells have been substantially cleared from the patient's blood, a graft containing the hematopoietic stem cells is then administered to the patient. Administer.

33. 33. The method according to any one of claims 1 to 32, wherein said hematopoietic stem cells are transferred into said patient. Two or more days after transplantation, the hematopoietic stem cells or their progeny maintain hematopoietic stem cell functional potential. I have it.

34. 34. The method according to any one of claims 1 to 33, wherein said hematopoietic stem cells are transferred into said patient. After transplantation, the hematopoietic stem cells or their progeny are capable of localizing to hematopoietic tissues; and / or Or hematopoiesis can be re-established.

35. 35. The method of any one of claims 1 to 34, wherein when implanted in the patient, the construct Blood stem cells include megakaryocytes, thrombocytes, platelets, red blood cells, and mast cells. , myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigens Presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes. The method further comprises recovering a population of cells selected from the group consisting of lymphocytes.

36. The method according to any one of claims 1 to 35, wherein the patient suffers from a stem cell disorder. is.

37. 36. The method of any one of claims 1 to 35, wherein the patient has a hemoglobinopathy. The patient is suffering from a chronic kidney disease, autoimmune disorder, myelodysplastic disorder, immunodeficiency disorder, or metabolic disorder.

38. 37. The method of any one of claims 1 to 36, wherein the patient is suffering from cancer. 。

39. The method according to any one of claims 1 to 4 and 14 to 38, wherein the ADC comprises a heavy chain / light chain ( HC / LC) CDR set (CDR1, CDR2, or CDR3), or a HC / LC variable region set as set forth in Table 3; The present invention also includes an anti-CD117 antibody comprising:

40. The method according to any one of claims 1 to 39, wherein the antibody of the conjugate is 1 x 10 as measured by bio-layer interferometry -2 from 1 x 10 -3 , 1 x 10 -3 From 1 x 10 -4 , 1 x 10 -5 From 1 x 10 -6 , 1 x 10 -6 From 1 x 10 -7 , or 1 x 10 -7 From 1 x 10 -8 Dissociation rate (K OFF ).

41. The method according to any one of claims 1 to 40, wherein the antibody of the conjugate is Approximately 100 nM or less as measured by bio-layer interferometry 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 nM Below, K of about 1 nM or less D and binds to CD117.

42. The method according to any one of claims 1 to 40, wherein the antibody of the conjugate is a human It is an antibody.

43. The method according to any one of claims 1 to 40, wherein the antibody of the conjugate is It is an intact antibody.

44. The method according to any one of claims 1 to 43, wherein the antibody of the conjugate is Ig It's G.

45. 45. The method of claim 44, wherein the IgG is an IgG1 isotype, an IgG2 isotype. , IgG3 isotype, or IgG4 isotype.

46. The method according to any one of claims 1 to 45, wherein the antibody is linked to a cell via a linker. Conjugated to a cytotoxin.

47. 47. The method of claim 46, wherein the cytotoxin is an RNA polymerase inhibitor.

48. 48. The method of claim 47, wherein the RNA polymerase inhibitor is an amatoxin. do.

49. 48. The method of claim 47, wherein the RNA polymerase inhibitor is amanitin. 。

50. 50. The method of claim 49, wherein the amanitin is α-amanitin, β-amanitin. , γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanulin acid, and proamanuline.

51. 47. The method of claim 46, wherein the cytotoxin is Pseudomonas exotoxin A, de Bou Ganin, diphtheria toxin, saporin, maytansine, maytansinoids, auristane Chin, anthracycline, calicheamicin, irinotecan, SN-38, duocalmycin Synthin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine Zepines, indolinobenzodiazepine dimers, and indolinobenzodiazepine pseudodimers is selected from the group consisting of:

52. 52. The method of claim 51 , wherein the auristatin is MMAE or MMAF.

53. The method according to any one of claims 46 to 52, wherein the antibody is a fusion protein comprising an Fc domain of the antibody. It is conjugated to the toxin by a cysteine ​​residue in the peptide.

54. 54. The method of claim 53, wherein the cysteine ​​residue is in the Fc domain of the antibody. It is introduced by amino acid substitution.

55. 55. The method of claim 54, wherein the amino acid substitution is D265C.

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