Fc silenced antibody drug conjugates (ADCS) and uses thereof

Abstract

To provide antibodies containing Fc regions with altered effector functions, and antigen-binding portions thereof, and to provide antibody drug conjugates, compositions, and methods using the antibodies.SOLUTION: Antibodies containing an Fc region with altered effector function as a result of one or more amino acid substitutions in the Fc region are used. The antibodies and antibody-drug conjugates described herein are useful, inter alia, for depleting cells and treating various hematopoietic diseases, metabolic disorders, cancers (e.g., acute myeloid leukemia (AML)), and autoimmune diseases.SELECTED DRAWING: Figure 11E

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application is a continuation of U.S. Provisional Application No. 62 / 749,662, filed October 23, 2018, and U.S. Provisional Application No. 62 / 749,662, filed November 30, 2018. No. 62 / 773,839, filed on February 19, 2019, and U.S. Provisional Application No. 62 / Priority is claimed to US Pat. No. 6,807,363, the contents of each priority application being incorporated herein by reference. It is inserted.

[0002] [Technical field] The present invention provides antibodies that have enhanced effector function as a result of one or more amino acid substitutions in the Fc region. The present invention relates to the field of antibodies or antibody-drug conjugates thereof, which comprise an altered Fc region. Furthermore, administration of antibodies or antibody drug conjugates (ADCs) with modified Fc regions may This has led to a variety of pathological conditions (e.g., hematological disorders, metabolic disorders, cancer, and autoimmune diseases, among others). for the treatment of a patient suffering from the disease, wherein the antibody or ADC inhibits hematopoietic cells (e.g., hematopoietic The antigens can bind to antigens expressed by stem cells or cells of the host immune system. [Background technology]

[0003] The Fc region of an antibody controls the cytotoxic activity of the antibody and influences the serum half-life of the antibody. However, in a therapeutic context, the cytotoxic effects of antibodies The target function is often undesirable and may also increase the risk of infection by activating the host's immune defenses. Some of the Fc region may cause safety concerns and unwanted side effects. Some amino acid changes may silence or reduce the effector function of the antibody. In fact, previous studies have shown that antibodies have a specific ability to bind to Fc receptors. Amino acid positions within the Fc region of antibodies that affect the ATP-dependent ATP synthesis have been identified (e.g., Wang et al. (201 8) Protein Cell. 2018 Jan; 9(1): 63-73. For example, the Fc mutations S239D and I332E has been described in the literature as enhancing ADCC function (e.g., Lazar et al. (2006) ) Engineered antibody Fc variants with enhanced effector function. Proc Natl Aca d Sci USA. 103:4005-4010). Other mutations are associated with reduced FcγR and C1q binding. (e.g., amino acid changes L234A / L235A in IgG1, or amino acid changes F 234A / L235A)(Xu et al. In vitro characterization of five humanized OKT3 effecto r function variant antibodies. Cell Immunol. 2000; 200:16-26). However, Fc mutations How this can affect antibody drug conjugates (ADCs) (particularly toxicities) (when the enzyme binds to an antibody or Fc containing fragments within the Fc region), This is a very important thing.

[0004] Despite advances in medical technology, pathologies of the hematopoietic system (e.g., specific blood There is still a need to treat diseases such as cellular diseases, metabolic disorders, cancer, and autoimmune conditions. Although hematopoietic stem cells have great therapeutic potential, there are several obstacles hindering their clinical use. A drawback is the difficulty involved in ensuring engraftment of hematopoietic stem cell (HSC) transplants in the host. In particular, antibodies targeting cell surface antigens on endogenous HSCs have been developed. Cell therapy has been shown to have undesirable immune stimulatory and immune-modulating functions that prevent exogenous HSC transplants from engrafting. Currently, the engraftment of exogenous hematopoietic stem cell grafts is difficult. This is because the multipotency and hematopoietic function of these cells are preserved after transplantation. Therefore, there is a need for compositions and methods for promoting cytokine secretion. To reduce the possibility of side effects and side effects, There is also a need for improved ADCs that can be used for screening. Summary of the Invention [Problem to be solved by the invention]

[0005] Fc with altered effector function as a result of one or more amino acid substitutions in the Fc region Antibodies containing the region, and antigen-binding portions thereof, and antibody-drug conjugates using said antibodies Gates, compositions, and methods are described herein, particularly antibodies with modified Fc regions. or antibody drug conjugates (ADCs) are provided herein, wherein the antibody or ADC is , antigens expressed by hematopoietic cells (e.g., hematopoietic stem cells) or mature immune cells (e.g., T cells) Furthermore, it provides a conjugation site for toxins and can bind to effectors. ADCs containing Fc mutations that reduce receptor function as well as provide stability are described herein. Thus, the present disclosure provides unique combinations of Fc mutations for ADCs. . [Means for solving the problem]

[0006] In one aspect, provided herein is an antibody comprising an Fc region, wherein the Fc region comprises , amino acid substitutions at positions L234 and L235 (EU index), and amino acid substitution D265C ( and the antibody is an intact IgG antibody. In one embodiment, the Fc region comprises amino acid substitutions at positions L234 and L235 (EU index), and and wherein said antibody comprises the amino acid substitution D265A (EU index) at position 1 of said antibody. In one embodiment, the amino acid substitution at L234 is L234A. In an embodiment, the amino acid substitution at L235 is L235A.

[0007] In some embodiments, the Fc region comprises the amino acid at position H435 (EU index): In one embodiment, the amino acid substitution at H435 is H435A. In this embodiment, an antibody containing the amino acid substitution H435A has the same intact Fc region as an antibody containing the unmodified Fc region. It has a reduced half-life compared to other IgG antibodies.

[0008] In another embodiment, the amino acid sequence consists essentially of the amino acid substitutions L234A, L235A, and D265C (EU index). Antibodies comprising an Fc region having an amino acid substitution of wherein the antibody is an intact IgG antibody. In one embodiment, the antibody is having amino acid substitutions consisting essentially of the amino acid substitutions L234A, L235A, and D265A (EU index) and wherein the antibody is an intact IgG antibody.

[0009] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises L234, L235 (EU index), and D265 (EU index). In one embodiment, the amino acid substitution at D265 is D265C or D 265A (EU index). In another embodiment, the amino acid substitution at L234 is L234A or L23 In another embodiment, the amino acid substitution at L235 is L235A. In another embodiment, the Fc region further comprises an amino acid substitution at position N297 (EU index). In some embodiments, the amino acid substitution at N297 is selected from the group consisting of N297A, N297G, and N297Q (EU index). In another embodiment, the Fc region is selected from the group consisting of: In another embodiment, the amino acid substitution at E233 is E233P (EU index In another embodiment, the Fc region further comprises a deletion of G236 (EU index). In another embodiment, the Fc region comprises an amino acid substitution at position P331 (EU index): In another embodiment, the amino acid substitution at P331 is P331G. In another embodiment, the Fc region does not comprise a substitution at position P331 (EU index). In another embodiment, the Fc region further comprises an amino acid substitution at position P329 (EU index). In one embodiment, the amino acid substitution at P329 is P329G. In another embodiment, the Fc region does not contain a substitution at position I253. (EU index). In another embodiment, the amino acid substitution at position I253 is In another embodiment, the Fc region has a substitution of H310 (EU index) at 1253A. In another embodiment, the amino acid substitution at position H310 is H310A. be.

[0010] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises amino acid substitutions at positions N297 and D265 (EU index). In one embodiment, the amino acid substitution at position D265 is D265C or D265A (EU index). In another embodiment, the amino acid substitutions at N297 are N297A, N297G, and N297Q (EU index). In another embodiment, the Fc region is selected from the group consisting of L234 and L23 5 (EU index). In another embodiment, the L235 amino acid substitution is L235A or L234V. In another embodiment, the Fc region comprises an amino acid substitution at position E233 (EU index). In another embodiment, the amino acid substitution at E233 is E233P (EU index). In another embodiment, the Fc region further comprises a deletion of G236 (EU index). In some embodiments, the Fc region further comprises an amino acid substitution at position P331 (EU index). In another embodiment, the amino acid substitution at P331 is P331G. In another embodiment, the Fc region does not contain a substitution at position P331 (EU index). The region further comprises an amino acid substitution at position P329 (EU index). The amino acid substitution in P329 is P329G. In another embodiment, the Fc region comprises P329 (EU index). In another embodiment, the Fc region does not include a substitution at position I253 (EU index). In another embodiment, the amino acid substitution at position I253 is In another embodiment, the Fc region has an amino acid sequence at position H310 (EU index) and an amino acid sequence at position H311 (EU index) and an amino acid sequence at position H312 (EU index). In another embodiment, the amino acid substitution at H310 is H310A.

[0011] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises: Amino acid substitutions and deletions at G236 (EU index) and D265 (EU index) In one embodiment, the amino acid substitution at D265 is D265C or D265A (EU amino acid substitution). In another embodiment, the amino acid substitution at L234 is L234A or L234V. In another embodiment, the amino acid substitution at L235 is L235A. In another embodiment, the Fc region has the amino acid substitution N29 In another embodiment, the amino acid substitution at position N297 is The amino acid substitution is selected from the group consisting of N297A, N297G and N297Q (EU index). In embodiments, the Fc region further comprises an amino acid substitution at position P331 (EU index). In another embodiment, the amino acid substitution at P331 is P331G. In another embodiment, the Fc region does not contain a substitution at position P331 (EU index). In another embodiment, the region further comprises an amino acid substitution at position P329 (EU index). In another embodiment, the Fc region comprises the amino acid substitution P329 (EU index 29). In another embodiment, the Fc region does not include a substitution at position I253 (EU index). In another embodiment, the amino acid substitution at position I253 is 3A. In another embodiment, the Fc region comprises the amino acid at position H310 (EU index): In another embodiment, the amino acid substitution for H310 is H310A.

[0012] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises amino acid substitutions at positions H435 and D265 (EU index). In one embodiment, the amino acid substitution at position D265 is D265C or D265A (EU index). In another embodiment, the amino acid substitution at H435 is H435A. wherein the Fc region further comprises an amino acid substitution at position N297 (EU index). In this embodiment, the amino acid substitution at N297 is selected from the group consisting of N297A, N297G, and N297Q (EU index). In another embodiment, the Fc region is selected from the amino acid sequence at positions L234 and L235 (EU index). In another embodiment, the amino acid substitution at L234 is L234A or L23 In another embodiment, the amino acid substitution at L235 is L235A. wherein the Fc region further comprises an amino acid substitution at position E233 (EU index). In another embodiment, the amino acid substitution at E233 is E233P (EU index). The Fc region further comprises a deletion of G236 (EU index). In another embodiment, the amino acid sequence of the present invention is a sequence of the present invention, further comprising an amino acid substitution at position P331 (EU index). In another embodiment, the Fc region has an amino acid substitution of P331 (EU index). In another embodiment, the Fc region does not include a substitution at position P329. In another embodiment, the amino acid substitution at P329 is P329G. In another embodiment, the Fc region does not comprise a substitution at position P329 (EU index). In another embodiment, the Fc region comprises an amino acid substitution at position 1253 (EU index). In another embodiment, the amino acid substitution at I253 is I253A. wherein the Fc region further comprises an amino acid substitution at position H310 (EU index). In this embodiment, the amino acid substitution for H310 is H310A.

[0013] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises amino acid substitutions at positions L234 and L235 (EU index), as well as and the amino acid substitution P329 (EU index). In one embodiment, the amino acid substitution L234 is L234A or L234V. In another embodiment, the amino acid substitution at L235 is L235A. In one embodiment, the Fc region further comprises an amino acid substitution at position D265 (EU index). In one embodiment, the amino acid substitution at D265 is D265C or D265A (EU index). In one embodiment, the Fc region comprises an amino acid substitution at position N297 (EU index). In one embodiment, the amino acid substitutions at N297 are N297A, N297G, and N297Q (EU index). In one embodiment, the Fc region is selected from the group consisting of E233 (EU index). In one embodiment, the amino acid substitution at E233 further comprises an amino acid substitution at position E In one embodiment, the Fc region is G236 (EU index). In one embodiment, the Fc region further comprises a deletion of In one embodiment, the amino acid substitution in P331 is P331G. In embodiments, the Fc region does not comprise a substitution at position P331 (EU index). In embodiments, the Fc region further comprises an amino acid substitution at position 1253 (EU index). In one embodiment, the amino acid substitution at I253 is I253A. The region further comprises an amino acid substitution at position H310 (EU index). The 10 amino acid substitution is H310A.

[0014] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises amino acid substitutions at positions L234 and L235 (EU index), as well as and the amino acid substitution P331 (EU index). In one embodiment, the amino acid substitution L234 is L234A or L234V. In one embodiment, the amino acid substitution at L235 is L235A. In one embodiment, the Fc region further comprises an amino acid substitution at position D265 (EU index). In one embodiment, the amino acid substitution at D265 is D265C or D265A (EU index). In one embodiment, the Fc region comprises an amino acid substitution at position N297 (EU index). In one embodiment, the amino acid substitutions at N297 are N297A, N297G, and N297Q (EU index). In one embodiment, the Fc region is selected from the group consisting of E233 (EU index). In one embodiment, the amino acid substitution at E233 further comprises an amino acid substitution at position E In one embodiment, the Fc region is G236 (EU index). In one embodiment, the Fc region further comprises a deletion of In one embodiment, the amino acid substitution at P329 is P329G. In some embodiments, the Fc region does not comprise a substitution at position P329 (EU index). In embodiments, the Fc region further comprises an amino acid substitution at position 1253 (EU index). In one embodiment, the amino acid substitution at I253 is I253A. The region further comprises an amino acid substitution at position H310 (EU index). The amino acid substitution for H310 is H310A.

[0015] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises amino acids at positions E233, L234, and L235 (EU index). In one embodiment, the amino acid substitution at L234 is a deletion of G236 (EU index). , L234A, or L234V. In one embodiment, the amino acid substitution at L235 is L235A. In some embodiments, the amino acid substitution at E233 is E233P (EU index). In one embodiment, the Fc region further comprises an amino acid substitution at position H435 (EU index). In one embodiment, the amino acid substitution at H435 is H435A. In one embodiment, the amino acid substitution at position N297 (EU index) is The amino acid substitution is selected from the group consisting of N297A, N297G and N297Q (EU index). In one embodiment, the Fc region further comprises an amino acid substitution at position P331 (EU index). In one embodiment, the amino acid substitution in P331 is P331G. The Fc region does not contain a substitution at position P331 (EU index). The region further comprises an amino acid substitution at position P329 (EU index). The amino acid substitution in P329 is P329G. In one embodiment, the Fc region is In one embodiment, the Fc region does not include a substitution at position I253 (EU index). In one embodiment, the amino acid substitution at position I253 is In one embodiment, the Fc region has an amino acid sequence at position H310 (EU index) of 253A. In one embodiment, the amino acid substitution at H310 is H310A.

[0016] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises amino acid substitutions at positions 1253, H310, and H345 (EU index). In one embodiment, the amino acid substitution at I253 is I253A. In another embodiment, the amino acid substitution at H435 is H43A. 5A. In another embodiment, the Fc region comprises the amino acid at position N297 (EU index): In another embodiment, the amino acid substitutions at N297 are N297A, N297G, and N297Q (EU In another embodiment, the Fc region is selected from the group consisting of: In another embodiment, the amino acid substitution at position D265 is In another embodiment, the Fc region is E233C or D265A (EU index). (EU index). In another embodiment, the amino acid substitution at amino acid E233 is In another embodiment, the Fc region has a substitution of G236 (EU index). In another embodiment, the Fc region further comprises a deletion of an amino acid at position P329. In another embodiment, the amino acid substitution at P329 is In another embodiment, the Fc region comprises a substitution at position P329 (EU index). In another embodiment, the Fc region comprises an amino acid substitution at position P331 (EU index). In another embodiment, the amino acid substitution in P331 is P331G. In the Fc region does not contain a substitution at position P329 (EU index).

[0017] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises an amino acid substitution at position N297 (EU index). In embodiments, the Fc region comprises amino acid substitutions at positions L234 and L235 (EU index). In another embodiment, the amino acid substitution at L234 is L234A or L234V. In one embodiment, the amino acid substitution at L235 is L235A. In another embodiment, the amino acid sequence of N297 is substituted with an amino acid sequence of N4. In another embodiment, the amino acid substitution is selected from the group consisting of N297A, N297G, and N297Q. The Fc region further comprises an amino acid substitution at position E233 (EU index). In another embodiment, the amino acid substitution at E233 is E233P (EU index). In another embodiment, the Fc region further comprises a deletion of G236 (EU index). In another embodiment, the amino acid sequence of P331 is further comprised of an amino acid substitution at position 31 (EU index). In another embodiment, the Fc region has a substitution of P331 (EU index). In another embodiment, the Fc region does not include an amino acid substitution at position P329. In another embodiment, the amino acid substitution at P329 is P329G. In another embodiment, the Fc region does not comprise a substitution at position P329 (EU index). In another embodiment, the Fc region further comprises an amino acid substitution at position 1253 (EU index). In another embodiment, the amino acid substitution at I253 is I253A. The Fc region further comprises an amino acid substitution at position H310 (EU index). In the example, the amino acid substitution for H310 is H310A.

[0018] In some embodiments, the antibody, or antigen-binding portion thereof, comprises an Fc domain as described herein. Any combination of substitutions for the regions is included.

[0019] In some embodiments, the antibody, or antigen-binding portion thereof, has an S239 (EU index) In one embodiment, the amino acid substitution at S239 is S239C is.

[0020] In some embodiments, the antibody, or antigen-binding portion thereof, has an H435 (EU index) In one embodiment, the amino acid substitution at H435 is H435A In another embodiment, the antibody comprises the amino acid substitution H435A and has an unmodified Fc The half-life is reduced compared to the same intact IgG antibody containing the region.

[0021] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises the amino acid substitutions L234A, L235A, S239C and D265A (EU index). (including s

[0022] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises the amino acid substitutions L234A, L235A, S239C and D265C (EU index). (including s

[0023] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region consists of the amino acid substitutions L234A, L235A and D265C (EU index). Contains essentially amino acid substitutions.

[0024] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region consists of the amino acid substitutions L234A, L235A, and D265A (EU index). Contains essentially amino acid substitutions.

[0025] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region comprises the amino acid substitutions L234A, L235A, S239C and D265A (EU index). It includes amino acid substitutions consisting essentially of (a) .

[0026] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region contains the amino acid substitutions H435A, L234A, L235A, and D265C (EU index). It includes amino acid substitutions consisting essentially of (A).

[0027] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region consists essentially of the amino acid substitutions N297A and D265C (EU index). The amino acid substitutions include:

[0028] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein said Fc region consists essentially of the amino acid substitutions N297G and D265C (EU index). The amino acid substitutions include:

[0029] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region consists essentially of the amino acid substitutions N297Q and D265C (EU index). The amino acid substitutions include:

[0030] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein said Fc region consists essentially of the amino acid substitutions N297A and D265A (EU index). The amino acid substitutions include:

[0031] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein the Fc region consists essentially of the amino acid substitutions N297G and D265A (EU index). The amino acid substitutions include:

[0032] In another aspect, an antibody, or antigen-binding portion thereof, comprising an Fc region is provided herein. wherein said Fc region consists essentially of the amino acid substitutions N297Q and D265A (EU index). The amino acid substitutions include:

[0033] In another aspect, an antibody, or antigen-binding portion thereof, is provided herein, wherein the The antibody binds to the Fc gamma receptor (FcγR) in a manner similar to that of the same antibody containing an unmodified Fc region. a decrease in effector function, defined as a decrease in binding to the FcγR, compared to when In some embodiments, the decrease in binding is greater than or equal to the antibody binding to an FcγR of an unmodified FcγR. at least a 70% reduction, at least at least 80% reduction, at least 90% reduction, at least 95% reduction, at least 98% reduction, at least In another embodiment, the antibody binds to the FcγR at least 99% reduced, or 100% reduced. In another embodiment, antibody binding to the FcγR is determined by biochemical analysis. In another embodiment, antibody binding to the FcγR is assessed by blue-band interferometry (BLI). In another embodiment, the FcγR is assessed using an assay known to those skilled in the art. In another embodiment, the FcγR receptor is an FcγR2 receptor or an Fc In another embodiment, the FcγR2 receptor is FcγR2A, FcγR2B, or FcγR3 receptor. In another embodiment, the FcγR3 receptor is FcγR3A or FcγR3 In another embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the FcγR receptor is an FcγR2A 167R receptor. In another embodiment, the FcγR receptor is an FcγR3A 176V receptor. The receptor is the FcγR3A 176F receptor.

[0034] In another aspect, an antibody, or antigen-binding portion thereof, is provided herein, wherein the The antibody exhibits the same cytokine release activity as the antibody containing the unmodified Fc region in an in vitro cytokine release assay. with at least a 50% reduction in cytokine release compared to that of the antibody In one embodiment, the reduction in cytokine release is Cytokine release compared to the same antibody containing an unmodified Fc region At least 60% reduction, at least 70% reduction, at least 80% reduction, at least 90% reduction a reduction of at least 95%, a reduction of at least 98%, a reduction of at least 99%, or a reduction of 100% In another embodiment, the antibody does not exhibit detectable cytokine release. In some embodiments, the in vitro cytokine release assay comprises a meso-scale detector. Meso Scale Discovery (MSD) tissue culture (TC) pro-inflammatory assay In another embodiment, the in vitro cytokine release assay is In another embodiment, the antibody is assessed using an in vitro mast cell deconjugation assay. In a granulation assay, compared to mast cell degranulation of the same antibody containing an unmodified Fc region , reduces mast cell degranulation, with a reduction in mast cell degranulation of at least 50%. In another embodiment, the reduction in mast cell degranulation is achieved by the same antibody comprising an unmodified Fc region. At least a 60% reduction in mast cell degranulation, at least 7% reduction in mast cell degranulation compared to mast cell degranulation in 0% reduction, at least 80% reduction, at least 90% reduction, at least 95% reduction, at least In another embodiment, the reduction is at least 98%, at least 99%, or 100%. The antibody does not exhibit detectable mast cell degranulation. The mast cell degranulation assay is a beta-hexosaminidase-based method for measuring mast cell degranulation. It is an assay.

[0035] In some embodiments, the IgG isotype is an IgG1 isotype, an IgG2 isotype, or an IgG3 isotype. In another embodiment, the antibody is an IgG1 isotype, an IgG2 isotype, or an IgG3 isotype, or an IgG4 isotype. In yet another embodiment, the antibody is a biotinylated antibody. In another embodiment, the antibody is a monospecific antibody. In another embodiment, the antibody is an intact IgG antibody. In another embodiment, the antibody is specific for CD117, CD45, CD2, CD5, CD137, or CD252. Combine.

[0036] In another aspect, an antibody drug comprising an antibody, or an antigen-binding portion thereof, described herein. Conjugates (ADCs) are provided herein, wherein the antibody, or its antigen-binding The binding moiety is conjugated to the cytotoxin via a linker. The cytotoxin is an RNA polymerase inhibitor. The inhibitor is an amatoxin.

[0037] In another embodiment, the amatoxin is represented by formula (III): [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 together with the oxygen atoms to which they are attached, are optionally substituted forming a 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R5, R 6、 and R7 are each independently H, OH, OR C, OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 hetero alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 hetero alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heptyl cycloalkyl, optionally substituted aryl, or optionally substituted hexachloro It is a teloaryl; L is an optionally substituted C1-C6 alkylene, an optionally substituted C1-C6 hetero C2-C6 alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2- C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally optionally substituted heterocycloalkylene, optionally substituted arylene, optionally optionally substituted heteroarylenes, peptides, dipeptides, -(C=O)-, disulfides, drazone, or a combination thereof; and Z is a reactive substituent present on L and a group present in the antibody or antigen-binding fragment thereof. It is a chemical substructure formed from a coupling reaction between an existing reactive substituent , where Am is exactly one R C Contains substituents.

[0038] In another embodiment, the amatoxin has the 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 together with the oxygen atoms to which they are attached, are optionally substituted forming a 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R5, R 6、 and R7 are each independently H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 hetero alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 hetero alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heptyl cycloalkyl, optionally substituted aryl, or optionally substituted hexachloro It is a teloaryl; L is an optionally substituted C1-C6 alkylene, an optionally substituted C1-C6 hetero C2-C6 alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2- C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally optionally substituted heterocycloalkylene, optionally substituted arylene, optionally optionally substituted heteroarylenes, peptides, dipeptides, -(C=O)-, disulfides, drazone, or a combination thereof; and Z is a reactive substituent present on L and a group present in the antibody or antigen-binding fragment thereof. It is a chemical substructure formed from a coupling reaction between an existing reactive substituent , where Am is exactly one R C Contains substituents.

[0039] In another embodiment, the RNA polymerase inhibitor is amanitin. So, the amanitin is α-amanitin, β-amanitin, γ-amanitin, ε-amanitin. Amanulin, amanine, amaninamide, amanulin, amanuric acid, and proamanuline. In another embodiment, the cytotoxin is selected from the group consisting of Pseudomonas exotoxin A, de Bouganin, diphtheria toxin, saporin, maytansine, maytansinoids, aurista cin, anthracycline, calicheamicin, irinotecan, SN-38, duocalmycin Synthon, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine benzodiazepines, and indolinobenzodiazepine dimers. In another embodiment, the auristatin is MMAE or MMAF. The antigen-binding portion is an interchain conjugate to the native hinge cysteine. In another embodiment, the antibody or The antigen-binding portion binds to the cell by a cysteine ​​residue in the Fc domain of the antibody. In another embodiment, the cysteine ​​residue is conjugated to a cytotoxin. In another embodiment, the antibody is introduced by an amino acid substitution in the Fc domain of the antibody. In another embodiment, the amino acid substitution is S239C.

[0040] In another aspect, an antibody or ADC according to any one of claims 1 to 218 and a pharmaceutically acceptable salt thereof Pharmaceutical compositions comprising an acceptable carrier are provided herein.

[0041] In another aspect, a method for reducing the population of hematopoietic stem cells (HSCs) in a human patient is provided herein. The method further comprises administering to the patient an effective amount of an antibody described herein. or ADC. In one embodiment, the method comprises administering to the patient hematopoietic stem cells. In one embodiment, the transplant is an allogeneic In one embodiment, the transplant is autologous.

[0042] In another aspect, a method comprising administering to a human patient a transplant comprising hematopoietic stem cells is provided herein. The present invention provides a method for treating a hematopoietic stem cell deficiency in a subject, comprising administering to said subject a therapeutically effective amount of hematopoietic stem cells. In one embodiment, the patient has previously been administered an antibody or ADC described herein in an amount sufficient to In one embodiment, the hematopoietic stem cells are CD117+ or CD45+ cells. The patient has a blood disorder, metabolic disorder, cancer, or autoimmune disease, or severe combined immunodeficiency disorder. (severe combined immunodeficiency disease) (SCID).

[0043] In another aspect, provided herein is a method of treating leukemia in a human patient, comprising: wherein the method comprises administering an antibody or ADC described herein to said human having leukemia. administering the compound to a patient.

[0044] In another aspect, a method comprising administering to a human patient a transplant comprising hematopoietic stem cells is provided herein. The present invention provides a method for reducing a population of immune cells in a patient, the method comprising: The patient has previously been administered an antibody or ADC described herein in an amount sufficient to In another embodiment, the immune cells are CD137+, CD2+, or CD5+ cells. The immune cells are T cells.

[0045] In another aspect, a composition comprising an antibody or ADC described herein is provided herein. wherein the composition contains less than 25% hydrophobic degradants after heat stress. In one embodiment, the composition contains less than 20% hydrophobic degradants after heat stress. In an embodiment, the composition contains less than 15% hydrophobic degradants after heat stress. In some embodiments, the composition contains less than 10% hydrophobic degradants after heat stress. In some embodiments, the composition contains less than 5% hydrophobic degradants after heat stress.

[0046] In another aspect, provided herein is a method of treating a stem cell disorder in a human patient. wherein the method comprises administering to the patient a therapeutically effective amount of an antibody, The method includes administering an antigen-binding fragment, or ADC, of ​​the antibody.

[0047] In another aspect, provided herein is a method of treating an immunodeficiency disorder in a human patient. wherein the method comprises administering to the patient a therapeutically effective amount of an antibody, In one embodiment, the immunosuppressant comprises administering an antigen-binding fragment thereof, or an ADC thereof. The immune deficiency disorder may be a congenital immune deficiency or an acquired immune deficiency.

[0048] In another aspect, provided herein is a method of treating a metabolic disorder in a human patient. wherein the method comprises administering to the patient a therapeutically effective amount of an antibody, In one embodiment, the metabolic disorder comprises administering an antigen-binding fragment thereof, or an ADC thereof. Harmful effects include glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidosis, and metachromatic leukodystrophy.

[0049] In another aspect, provided herein is a method of treating an autoimmune disorder in a human patient. wherein the method comprises administering to the patient a therapeutically effective amount of an antibody, In some embodiments, the method further comprises administering to the patient an antigen-binding fragment thereof, or an ADC thereof. The autoimmune disorders include multiple sclerosis, human systemic lupus erythematosus, rheumatoid arthritis, and inflammatory Intestinal diseases, treatment of psoriasis, type 1 diabetes, acute disseminated encephalomyelitis, Addison's disease, alopecia universalis, severe Orthodontic spondylitis, antiphospholipid syndrome, aplastic anemia, autoimmune hemolytic anemia, autoimmune Hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune oophoritis, Barrow's disease Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, chronic fatigue immune deficiency syndrome , chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac disease coeliac sprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythematosus, autonomic neuropathy, endometriosis, essential mixed type Cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture syndrome, Graves' disease , Guillain-Barré syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenia Purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki Disease, lichen planus, Lyme disease, Meniere's disease, mixed connective tissue disease, myasthenia gravis, neuromyelitis Otonia, opsoclonus-myoclonus syndrome, optic neuritis, Ordo's thyroiditis thyroiditis), pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, Primary biliary cirrhosis, polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica, primary Primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome group, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff person Syndrome, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, vulva and Wegener's granulomatosis.

[0050] In another aspect, provided herein is a method of treating cancer in a human patient, comprising administering to said patient a therapeutically effective amount of a compound selected from the group consisting of ribozymes, cyclosporine ... wherein the method comprises administering to the patient a therapeutically effective amount of an antibody, an antigen thereof, or a combination thereof, as described herein. In some embodiments, the cancer is The cancer is selected from the group consisting of leukemia, lymphoma, multiple myeloma, and neuroblastoma.

[0051] In some embodiments of any of the above aspects, the antibody comprises an unmodified Fc region. Decreased binding to Fc gamma receptors (FcγRs) compared to the binding of the same antibody to FcγRs In some embodiments, the reduced binding is antibody binding to said FcγR compared to binding to the FcγR of the same antibody comprising an unmodified Fc region. Compared to, at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, at least At least a 95% reduction, at least a 98% reduction, at least a 99% reduction, or a 100% reduction. In certain embodiments, the antibody does not detectably bind to the FcγR. In some embodiments, antibody binding to the FcγR is assessed by biolayer interferometry (BLI). In some embodiments, the FcγR is an FcγR1 receptor, an FcγR2 receptor, or is an FcγR3 receptor. In some embodiments, the FcγR1 receptor is FcγR1 In some embodiments, the FcγR1 receptor is FcγR1A, FcγR1B, or FcγR1C. In some embodiments, the FcγR1 receptor is FcγR2A, FcγR2B, or FcγR2C. In some embodiments, the Fc receptor is human FcγR3A or FcγR3B. It is a receptor.

[0052] In some embodiments of any of the above aspects, the IgG isotype is an IgG1 isotype. The isotype may be an IgG2 isotype, an IgG3 isotype, or an IgG4 isotype.

[0053] In some embodiments of any of the above aspects, the antibody is a human antibody.

[0054] In some embodiments of any of the above aspects, the antibody is a chimeric or humanized antibody. do.

[0055] In some embodiments of any of the above aspects, the antibody is a monoclonal antibody. .

[0056] In some embodiments of any of the above aspects, the antibody is selected from the group consisting of CD117, CD45, CD2, CD 5, CD137, or CD252.

[0057] In another aspect, an antibody drug conjugate (ADC) comprising any of the antibodies herein is Provided herein is a method for treating a cytotoxin-associated leukemia, wherein the antibody is conjugated to a cytotoxin via a linker. It is being done.

[0058] In some embodiments of the conjugates herein, the cytotoxin inhibits RNA polymerase In some embodiments, the RNA polymerase inhibitor is a hydroxybenzoate. It's Kishin.

[0059] In some embodiments, the amatoxin 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 together with the oxygen atoms to which they are attached, are optionally substituted forming a 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R5, R 6、 and R7 are each independently H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 hetero alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 hetero alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heptyl cycloalkyl, optionally substituted aryl, or optionally substituted hexachloro It is a teloaryl; L is an optionally substituted C1-C6 alkylene, an optionally substituted C1-C6 hetero C2-C6 alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2- C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally is an optionally substituted heteroarylene; and Z is a reactive substituent present on L and a group present in the antibody or antigen-binding fragment thereof. It is a chemical substructure formed from a coupling reaction between an existing reactive substituent , where Am is exactly one R C Contains substituents.

[0060] In some embodiments, the amatoxin 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 together with the oxygen atoms to which they are attached, are optionally substituted forming a 5-membered heterocycloalkyl group; R3 is H, R C , or RD is; R4, R5, R 6、 and R7 are each independently H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 hetero alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 hetero alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heptyl cycloalkyl, optionally substituted aryl, or optionally substituted hexachloro It is a teloaryl; L is an optionally substituted C1-C6 alkylene, an optionally substituted C1-C6 hetero C2-C6 alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2- C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally is an optionally substituted heteroarylene; and Z is a reactive substituent present on L and a group present in the antibody or antigen-binding fragment thereof. It is a chemical substructure formed from a coupling reaction between an existing reactive substituent , where Am is exactly one R C Contains substituents.

[0061] In some embodiments, the RNA polymerase inhibitor is amanitin. In some embodiments, the amanitin is α-amanitin, β-amanitin, or γ-amanitin. , ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, and proa manulin.

[0062] In some embodiments, the cytotoxin is Pseudomonas exotoxin A, de bouganin, Diphtheria toxin, saporin, maytansine, maytansinoids, auristatin, ant Lacycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolo Benzodiazepines, pyrrolobenzodiazepine dimers, indolinobenzodiazepines, and In some embodiments, the compound is selected from the group consisting of indolinobenzodiazepine dimers. The auristatin is MMAE or MMAF.

[0063] In some embodiments of the conjugates herein, the antibody comprises an Fc domain of the antibody. It is conjugated to the toxin by a cysteine ​​residue in the main chain. In this embodiment, the cysteine ​​residue is an amino acid substitution in the Fc domain of the antibody. In some embodiments, the amino acid substitution is D265C.

[0064] In another aspect, a method for the preparation of a composition comprising an antibody or ADC described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions are provided herein.

[0065] In yet another aspect, a method for reducing the population of hematopoietic stem cells (HSCs) in a human patient comprises the steps of: Provided herein, wherein the method comprises administering to the patient an effective amount of a compound described herein. The method includes administering an antibody or ADC.

[0066] In some embodiments of the methods described herein, the method further comprises administering to the patient hematopoietic stem cells. In some embodiments, the implant further comprises administering an allogeneic implant comprising: In some embodiments, the transplant is autologous.

[0067] In another aspect, a method comprising administering to a human patient a transplant comprising hematopoietic stem cells is provided herein. The present invention provides a method for treating a hematopoietic stem cell deficiency in a subject, comprising administering to said subject a therapeutically effective amount of hematopoietic stem cells. The patient has previously been administered an antibody or ADC described herein in an amount sufficient to

[0068] In some embodiments of the methods described herein, the patient has a blood disorder, a metabolic disorder, Cancer, autoimmune disease, or severe combined immunodeficiency disease have SCID.

[0069] In a further aspect, provided herein is a method of treating leukemia in a human patient. wherein the method comprises administering an antibody or ADC described herein to said human patient having leukemia. This includes administering the compound to a subject.

[0070] In one embodiment, a method for reducing the population of CD117+ cells (in a human patient in need thereof) is provided. provided herein are methods comprising administering to the patient an effective amount of an anti-CD117 antibody drug. and administering an antibody drug conjugate (ADC) to the patient, wherein the antibody drug conjugate (ADC) comprises an anti-CD117 antibody conjugated to an amatoxin via a linker, and has the formula Ab -ZL-Am, where Ab has a H435A mutation (EU index) in the Fc region of the antibody. wherein L is a linker; and Z is a chemical moiety. and Am is an amatoxin. In one embodiment, the ADC is administered to a transplant comprising hematopoietic stem cells. In another embodiment, the ADC is administered to a subject receiving a transplant comprising hematopoietic stem cells. and simultaneously administering the same to the patient.

[0071] In another embodiment, a human patient is administered an anti-CD117 antibody in an amount sufficient to reduce the population of CD117+ cells. A method for administering an ADC to a patient in need thereof is provided. provided herein, wherein the antibody drug conjugate (ADC) comprises a linker and by the formula Ab-ZL-Am. wherein the Ab contains a H435A mutation (EU index) in the Fc region of the antibody. is an anti-CD117 antibody, L is a linker, Z is a chemical moiety, and Am is an amato and thereafter administering to the patient a transplant comprising hematopoietic stem cells. In embodiments, the transplant comprising hematopoietic stem cells is administered to a patient in a manner that substantially reduces the concentration of the ADC in the patient's blood. In another embodiment, the hematopoietic stem cells or The progeny have the potential for hematopoietic stem cell function at least two days after transplantation of the hematopoietic stem cells into the patient. In yet another embodiment, the hematopoietic stem cells or their progeny maintain the potential. and localizing the hematopoietic stem cells to hematopoietic tissues and re-establishing hematopoiesis after transplantation of the hematopoietic stem cells into a human subject. In a further embodiment, the patient has a disease selected from the group consisting of: Adenosine deaminase deficiency and severe combined immunodeficiency disease immunodeficiency disease, hyperimmunoglobulin M syndrome , Chediak-Higashi disease, hereditary lymphohistiocytosis lymphohistiocytosis), osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major ( thalassemia major), systemic sclerosis, systemic lupus erythematosus ( systemic lupus erythematosus), multiple sclerosis, and juvenile rheumatoid arthritis. In another embodiment, the patient has an autoimmune disorder or a hematological cancer. The immune disorder is selected from the group consisting of: multiple sclerosis, human systemic lupus erythematosus Treatment of rheumatoid arthritis, inflammatory bowel disease, psoriasis, type 1 diabetes, acute disseminated encephalomyelitis, and Asian Son's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid syndrome, aplastic anemia, autoimmune Epidemic hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, Autoimmune oophoritis, Baro's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease , chronic fatigue immune deficiency syndrome, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial Pemphigoid, Celiac sprue-dermatitis herpetiformis s), cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythematosus, autonomic neuropathy, Endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasti Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic acute and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial bladder disease Cystitis, juvenile arthritis, Kawasaki disease, lichen planus, Lyme disease, Meniere's disease, mixed connective tissue disease, Myasthenia gravis, neuromyotonia, opsoclonus-myoclonus syndrome, optic nerve thyroiditis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyxitis Myositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyendocrine syndrome, Polymyalgia umatica, primary agammaglobulinemia, Inno's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's disease syndrome, stiff-person syndrome, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveal tract infection inflammation, vasculitis, vitiligo, vulvodynia, and Wegener's granulomatosis.

[0072] In another embodiment, a human subject with a hematological cancer is administered an effective amount of an anti-CD117 antibody drug conjugate. A method of treating a human subject with a hematological cancer comprising administering an anticoagulant (ADC) to a subject is described herein. wherein the antibody drug conjugate (ADC) is linked to an amino acid sequence via a linker. and is represented by the formula Ab-ZL-Am. wherein the Ab is an anti-CD117 antibody containing the H435A mutation (EU index) in the Fc region of said antibody. L is a linker, Z is a chemical moiety, and Am is an amatoxin. In one embodiment, the hematological cancer is leukemia. In another embodiment, the anti-CD117 In yet another embodiment, the Fc region of the antibody comprises a D265C mutation (EU index). The 117 antibody comprises a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:8, a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:9, a CDR4 domain comprising the amino acid sequence set forth in SEQ ID NO:10, a CDR5 domain comprising the amino acid sequence set forth in SEQ ID NO:11, a CDR6 domain comprising the amino acid sequence set forth in SEQ ID NO: (SEQ ID NO): 8, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 9. a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 12. In another embodiment, the anti-CD117 antibody comprises a light chain variable region comprising a CDR3 domain comprising a CDR1 domain. The antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13, and a In another embodiment, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14. The ADC, after administration to the patient, is activated by cancer cells, autoimmune cells, or hematopoietic stem cells. and is taken inside.

[0073] In another embodiment, the Am-LZ is represented by formula (I): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached, forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R5, R 6、 and R7 are each independently H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 hetero alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 hetero alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heptyl cycloalkyl, optionally substituted aryl, or optionally substituted hexachloro It is a teloaryl; L is an optionally substituted C1-C6 alkylene, an optionally substituted C1-C6 hetero C2-C6 alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2- C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally optionally substituted heterocycloalkylene, optionally substituted arylene, optionally optionally substituted heteroarylene, dipeptide, -C(=O)-, peptide, or combinations thereof; and, Z is a reactive substituent present on L and a group present in the antibody or antigen-binding fragment thereof. It is a chemical substructure formed from a coupling reaction between an existing reactive substituent , where Am is exactly one R C Contains substituents.

[0074] In another embodiment, the 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, R5, R 6、 and R7 are each independently H, OH, OR C , OR D , RC , 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 an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 hetero alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 hetero alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heptyl cycloalkyl, optionally substituted aryl, or optionally substituted hexachloro It is a teloaryl; L is an optionally substituted C1-C6 alkylene, an optionally substituted C1-C6 hetero C2-C6 alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2- C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally optionally substituted heterocycloalkylene, optionally substituted arylene, optionally optionally substituted heteroarylene, dipeptide, -C(=O)-, peptide, or combinations thereof; and, Z is a reactive substituent present on L and a group present in the antibody or antigen-binding fragment thereof. It is a chemical substructure formed from a coupling reaction between an existing reactive substituent , where Am is exactly one R C In another embodiment, the ADC is administered to the human patient. The subject is administered a dose of about 0.1 mg / kg to about 0.3 mg / kg. [Brief explanation of the drawings]

[0075] [Figure 1] Figures 1A-1E graphically illustrate the results of standard biolayer interferometry (BLI) assays to assess the binding responses of antibodies with the indicated amino acid substitutions to the Fc gamma receptor, as indicated in the legend. The binding response of each anti-CD117 antibody variant, normalized relative to WT IgG1 binding, is shown in Figure 1A, and the quantified normalized binding responses of each anti-CD117 antibody variant are shown in Figure 1B. Figure 1C shows the quantified binding responses of anti-CD45 antibody variants, normalized relative to WT anti-CD45 antibody binding (Figure 1C; as used herein, "ic" refers to interchain conjugates to native hinge cysteines). The binding responses of additional anti-CD117 antibody variants, normalized relative to WT IgG1 (isotype control) binding, are shown in Figure 1D, and the quantified normalized binding responses of each anti-CD117 antibody variant are shown in Figure 1E. [Figure 2] Figure 2 graphically illustrates the results of a mast cell degranulation assay, in which beta-hexosaminidase was measured after incubation of mast cells with the indicated antibodies. Beta-hexosaminidase release from mast cells was measured by monitoring the production of para-nitrophenol from the substrate 4-nitrophenyl N-acetyl-β-D-glucosaminide and is shown on the y-axis as the absorbance of para-nitrophenol at 405 nm. [Figure 3]Figures 3A, 3B, 3C, 3D, and 3E graphically show the results of in vitro cytokine release assays measuring the release of IL-6 (Figure 3A), IL-8 (Figure 3B), TNFα (Figure 3C), IL-1B (Figure 3D), and GM-CSF (Figure 3E) from human peripheral blood mononuclear cells (PBMCs) after incubation with the indicated antibodies. Human PBMCs in Figures 3A-3D were isolated from one of four donors, as indicated in the legends. [Figure 4] Figures 4A and 4B illustrate the results of an in vitro antibody-dependent cellular phagocytosis (ADCP) assay, demonstrating that Fc effector silencing in certain Fc variants reduces ADCP activity compared to controls. Figure 4A illustrates the results of flow cytometry analysis of co-expression of CFSE and CD134 staining. Figure 4B illustrates the results of incubation of MDMs with Kasumi-1 cells (1:2 molar ratio) with increasing concentrations of the indicated antibodies for 2 hours at 37°C. [Figure 5] 5A, 5B, and 5C each illustrate a chromatogram showing the results of a thermal stability study assessing the melting point temperature of the indicated antibodies. [Figure 6] Figures 6A and 6B are tables showing the Tm 1 (CH2 unfolding) and Tm2 (fab / CH3 unfolding) melting temperatures for each of the indicated antibodies, respectively, as measured in the thermal stability assays depicted in Figure 5A (see Figure 6A) and Figures 5B and 5C (see Figure 6B). [Figure 7]7A and 7B depict chromatograms showing the elution profiles of the indicated antibodies analyzed by hydrophobic interaction chromatography (HIC) at time = 0 at room temperature (non-stressed conditions) or after a 30-minute incubation at 60°C (stressed conditions). Figure 7A depicts chromatograms showing the elution profiles of the Ab1 antibody and certain Ab1 Fc variants analyzed by hydrophobic interaction chromatography (HIC) at time = 0 at room temperature (non-stressed conditions; Figure 7A [lower panel chromatogram]) or after a 30-minute incubation at 60°C (stressed conditions; Figure 7A [upper panel chromatogram]). Figure 7B depicts chromatograms showing the elution profiles of the Ab1 antibody and certain Ab1 Fc variants analyzed by hydrophobic interaction chromatography (HIC) at time = 0 at room temperature (non-stressed conditions) or after a 30 minute incubation at 60°C (stressed conditions). [Figure 8] Figures 8A-8E graphically illustrate the results of the HIC assays of Figures 7A and 7B, showing the area percentages of the antibody monomer (i.e., "main") peak (Figures 8A and 8D) or hydrophobic degradation peaks (Figures 8B, 8C, and 8E) for the indicated antibodies after exposure to stressed or non-stressed conditions. [Figure 9] Figures 9A-9E illustrate the results of size exclusion chromatography assays, in which the area fraction of the antibody monomer peak (Figures 9A and 9D) or polymer aggregate peak (Figures 9B, 9C, and 9E) was measured for the indicated antibodies after exposure to time = 0 at room temperature (unstressed conditions) or after a 30-minute incubation at 60°C (stressed conditions). [Figure 10]Figures 10A and 10B graphically illustrate the results of a non-human primate pharmacokinetic assay, depicting the concentration (ng / mL) of various doses (0.1 mg / kg and 0.3 mg / kg) of an engineered fast half-life anti-CD-117-amatoxin ADC against time (i.e., hours post-dose; x-axis) and Figure 10B illustrates that the timing of ADC-mediated decline and clearance results in a transplant conditioning post-dose window. [Figure 11] Figures 11A-11E show the results of flow cytometry assays to phenotypically detect the reduction of hematopoietic stem cells (i.e., CD34+ CD90+ CD45RA- HSCs) (Figures 11A and 11C) or assessment of colony-forming units (CFUs) derived from bone marrow aspirates (Figures 11B and 11D), plotted against the x-axis (x-axis) and various doses of ADC1 antibody-drug conjugate (ADC) compared to the control (i.e., PBS). Figures 11C and 11D also show data corresponding to unconjugated anti-CD117 antibody ("anti-CD117"). Figure 11E shows phenotypic analysis of bone marrow hematopoietic stem cells (treated vs. untreated) (day 7 post-treatment) using flow cytometry. [Figure 12] Figures 12A-12C graphically show the results of assays detecting neutrophil counts (10 / mL) (Figure 12A) and lymphocyte counts (10 / mL) (Figures 12B and 12C), with the horizontal axis representing the number of days after administration of various doses of the ADC1 antibody drug conjugate (ADC), compared to a control (i.e., PBS). Figure 12C further shows data corresponding to lymphocyte counts in cynomolgus monkeys administered unconjugated anti-CD117 antibody ("anti-CD117"). [Figure 13]Figures 13A-13C graphically show the results of assays detecting plasma alanine aminotransaminase levels (ALT; U / mL) (Figures 13A and 13C) and plasma bilirubin levels (U / mL) (Figure 13B), with the horizontal axis representing the number of days after administration of various doses of the ADC1 antibody drug conjugate (ADC), compared to a control (i.e., PBS). Figure 13C further shows data corresponding to plasma levels of ALT in cynomolgus monkeys administered unconjugated anti-CD117 antibody ("anti-CD117"). [Figure 14] FIG. 14 shows images of liver and kidney tissue isolated from cynomolgus monkeys 35 days after administration of ADC1 (0.3 mg / kg) or control (PBS). [Figure 15] Figure 15 graphically depicts the results of an assay detecting reticulocyte counts (10 / mL) versus days after administration of various doses (0.1 mg / kg or 0.3 mg / kg) of the ADC1 antibody drug conjugate (ADC) compared to control (i.e., PBS) or unconjugated anti-CD117 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0076] [Detailed explanation] Antibodies with modified Fc regions and conjugates thereof (antibody drug conjugates; ADCs) ) are disclosed herein, wherein said modification reduces the effector function of the antibody. The modifications to the Fc region may further enhance or substantially eliminate the antibody-drug conjugate. This may result in increased production of antibodies and / or decreased half-life of the antibody. The interaction of these antibodies with cells of the immune system results in antibody-dependent cell-mediated cytotoxicity (ADC). ependent cell-mediated cytotoxicity (ADCC)) and complement-dependent cytotoxicity (complement d It can affect various responses, including dependent cytotoxicity (CDC). The binding of the Fc region of an antibody to Fc receptors on the surface of the body initiates many biological responses (e.g., For example, antibody-coated particles are phagocytosed and destroyed, and immune complexes are cleared. The antibody-coated target cells are lysed by killer cells [i.e. ADCC, release of inflammatory mediators, placental transfer and immunoglobulin This may cause a decrease in antibody effector function. By eliminating or substantially eliminating the need for antibodies of the present disclosure, the antibodies of the present disclosure may be used to, for example, prevent or substantially eliminate the need for certain therapies (e.g., For example, hematopoietic stem cell therapy (e.g., hematopoietic stem cell transplantation therapy) and hematopoietic cell depletion therapy and [e.g., blood cancers, diseases and disorders of the immune system, autoimmune diseases, graft versus host disease (GVHD)]. Various immune system reactions that can be detrimental in the treatment of diseases such as erythrocyte sequestration (e.g., erythrocyte thrombocytopenia) and erythrocyte thrombocytopenia (e.g., erythrocyte thrombocytopenia). In this case, it is possible to preferably avoid causing a reaction (e.g., release of cytokines (Avoidance of vasoconstriction, or mast cell degranulation is avoided).

[0077] Thus, therapeutically useful anti-hematopoietic cell antibodies with altered Fc regions are Antibody (anti-HC antibody) is included herein. For example, the antibodies or ADCs are used as a conditioning procedure (for patients receiving hematopoietic stem cell-containing transplants). Such procedures are useful in the preparation of hematopoietic stem cell transplants. According to the methods described herein, in some embodiments, cells (e.g., hematopoietic stem cells, e.g., hematopoietic stem cells and / or mature immune cells [e.g., T cells antigens expressed by the GNNK+ cells (e.g., CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137) ADCs, antibodies, or antigens thereof capable of binding to CD252, CD253, and combinations thereof By administering the binding fragment to a patient, the patient is able to (e.g., undergo hematopoietic stem cell transplantation) Conditioning (for transplant therapy or to reset the immune system) In some embodiments, the antibodies or ADCs contemplated herein are used to treat diseases of the hematopoietic system or For example, in some embodiments, the compounds described herein may be used to treat disorders. The antibodies or ADCs contemplated herein may be used to treat hematologic cancers. In a typical example, an antibody or ADC contemplated herein can be used to treat graft-versus-host disease (GVHD). In certain embodiments, the method may be used to treat graft-versus-host disease (GvHD). The antibodies or ADCs contemplated herein can be used to treat T-cell mediated diseases or disorders. As described herein, the antibody may be administered as an antibody drug conjugate (ADC). The compound may be covalently conjugated to a cytotoxin to form one or more The ADC, antibody, or antigen-binding fragment thereof capable of binding to the antigen is administered to a patient receiving hematopoietic Administration to a patient in need of stem cell transplantation therapy may, for example, select endogenous hematopoietic stem cells. by gradually reducing the number of hematopoietic stem cells (which can be compensated for by transplantation of exogenous hematopoietic stem cells). This may promote the engraftment of hematopoietic stem cell grafts.

[0078] In certain embodiments, the present invention provides an isolated antibody that binds to the ectodomain of human CD117. Anti-CD117 antibodies, particularly isolated human anti-CD117 antibodies, are provided, wherein said isolated The anti-CD117 antibody has an altered Fc region, where the alteration alters the effector functions of the antibody. The isolated anti-CD117 antibodies identified herein reduce or substantially eliminate the ability to The combined regions are listed below.

[0079] The following sections discuss how to best support the patient (e.g., cancer) in the treatment of hematopoietic stem cell grafts to promote engraftment. or patients suffering from autoimmune diseases, or patients requiring hematopoietic stem cell transplantation therapy. A description of the antibody or conjugate thereof, which may be administered to a subject (e.g., instructions on how to administer such therapeutic agents to a patient (prior to hematopoietic stem cell transplantation); do.

[0080] definition As used herein, the term "about" means within 5% of the stated value or Indicates a low value.

[0081] As used herein, the term "allogeneic" when used in the context of transplantation , transferred from a donor to a genetically distinct recipient of the same species. The term "allogeneic" is used to define the cells (or tissues or organs) to be transplanted. "Cells" are types of cells that are genetically different between two individuals but belong to the same species (e.g., humans). Typically, the term "allogeneic" refers to a transplant from a donor without a blood relative of the same species. used to define the cells (e.g., stem cells) to be transplanted into the recipient do.

[0082] 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.

[0083] 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).

[0084] As used herein, the term "immune cells" includes, but is not limited to, hematopoietic cells. It is intended to include cells of origin and cells that play a role in the immune response. Cells 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 include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells. , allogeneic or autologous.

[0085] 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). .

[0086] 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. do not have.

[0087] 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.

[0088] 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.

[0089] 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) 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 are screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, intact immunoglobulins, or other methods. by enzymatic or chemical cleavage of globulin, or in some cases by methods known in the art. In one embodiment, the antibody can be produced by known chemical peptide synthesis procedures. The antigen-binding fragment of comprises the Fc region.

[0090] As used herein, the term "anti-CD117 antibody" or "antibody that binds to CD117" refers to The antibodies may be useful as diagnostic and / or therapeutic agents targeting CD117. refers to an antibody that is capable of binding to CD117 with sufficient affinity.

[0091] 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.

[0092] As used herein, the terms "anti-CD2 antibody" or "antibody that binds to CD2" or "antibody that binds to CD2" An "anti-CD2 ADC" or "CD2-binding ADC" is an ADC that binds to CD2, which is found on the cell surface of cells such as T cells. When used herein, the term refers to an antibody or ADC that specifically binds to human CD2.

[0093] As used herein, the terms "anti-CD5 antibody" or "antibody that binds to CD5" or "antibody that binds to CD5" An "anti-CD5 ADC" or "CD5-binding ADC" refers to an ADC that binds to CD5, which is found on the cell surface of cells such as T cells. When used herein, the term refers to an antibody or ADC that specifically binds to human CD5.

[0094] As used herein, the term "anti-CD137 antibody" or "antibody that binds to CD137" refers to The antibodies may be useful as diagnostic and / or therapeutic agents targeting CD137. refers to an antibody that can bind to CD137 with sufficient affinity.

[0095] As used herein, the term "anti-CD252 antibody" or "antibody that binds to CD252" refers to The antibodies may be useful as diagnostic and / or therapeutic agents targeting CD252. In a preferred embodiment, the term "antibody" refers to an antibody that is capable of binding to CD252 with sufficient affinity to The antibody specifically binds to human CD252 (hCD252). CD252 is found on antigen-presenting cells. can be.

[0096] 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 which can be directed against a hematopoietic stem cell surface antigen, CD117 (e.g., GNNK+ CD117); The other is a receptor or receptors involved in signal transduction pathways that specifically enhance cell proliferation. Subunits specific for various hematopoietic stem cell surface antigens or other cell surface proteins In some embodiments, the binding specificities can be linked to the same target. may be directed against unique, non-overlapping epitopes on the target antigen (i.e., bypass biparatopic antibody).

[0097] As used herein, an "intact" or "full length" antibody is one that is Two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by peptide bonds Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain variable region (HVR). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The H and VL regions can be further subdivided into regions of hypervariability (called complementarity-determining regions (CDRs)). Each VH and VL has a sequence of: It consists of three CDRs and four FRs, and from the amino terminus to the carboxyl terminus, it is as follows: The heavy and light chain variable regions are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region of the antibody contains the binding domain that interacts with the antigen. Phosphorus binds to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q, etc.) .

[0098] 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).

[0099] As used herein, the term "heat stress" refers to the damage caused to a molecule (e.g., an antibody, its antigen, Stress caused by any temperature change on the Fc containing binding fragment, or ADC In one embodiment, the heat stress is directed against an antibody, an Fc containing an antigen-binding fragment thereof, Alternatively, the ADC may be incubated at 60° C. for 30 minutes.

[0100] 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., CD45, CD2, CD5, CD137, or CD252]), wherein the antibody -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 - 8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less (less than 10 -12 A number less than means, for example, 10-13 ) for the target D specifically binds if In one embodiment, the term "specifically binds" means to bind to a molecule that specifically binds to at least about 1 x 10 -6 Medium, 1x10 -7 M, 1x10 -8 Medium, 1x10 -9 Medium, 1x10 -10 Medium, 1 x 10 -11 Medium, 1x10 -12 Antibodies with Kd of M or greater binds to the original antigen and / or has an affinity at least two-fold greater than its affinity for a nonspecific antigen. K refers to the ability of an antibody to bind to an antigen with high affinity. D , standard The measurement is performed according to Bio-Layer Interferometry (BLI). It is understood that the antibody can specifically bind to two or more antigens that are related in sequence. For example, in certain embodiments, the antibody is directed against an antigen (e.g., CD117 [GNNK+ CD117], Human and non-human (e.g., murine or non-human) CD45, CD2, CD5, CD137, or CD252 It may bind specifically to both primate (human) orthologs.

[0101] 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. Pat. Nos. 5,807,715; 4,816,567; and 4,816 See ,397.

[0102] The terms "Fc," "Fc region," and "Fc domain," as used herein, refer to the IgG Immunoglobulin (IgG) crystallizable fragments obtained by papain digestion of the molecule. The Fc region refers to the two nucleotides of an IgG molecule linked by disulfide bonds. It contains the C-terminal half of the heavy chain of the antibody, which does not have antigen-binding activity but contains the sugar moiety and the complement. They contain binding sites for Fc receptors, including the FcRn receptor and the like (see below). For example, the Fc region comprises a second constant domain CH2 (e.g., residues 231-340 of EU positions of human IgG1) and The antibody comprises the constant domain CH3 (e.g., residues 341 to 447 of EU position of human IgG1). When used, the Fc region may refer to a "lower hinge region" (e.g., I Residues at EU positions 233-239 of gG1).

[0103] Fc refers to this region in isolation, or to an antibody, antibody fragment, or Fc fusion protein. This region may be referred to as "the region in the context of the EU region." It has been observed at many positions within the Fc domain, including positions 270, 272, 312, 315, 356, and 358. Therefore, there is a significant difference between the sequences presented in this application and those known in the art. Minor differences may exist. Therefore, the term "wild-type IgG Fc domain" or "WT IgG Fc" is used. "c domain" refers to any naturally occurring IgG Fc region (i.e., any allele). The sequences of the heavy chains of IgG1, IgG2, IgG3, and IgG4 can be found in many sequence databases. For example, in the Uniprot database (www.uniprot.org), the accession numbers are P01857(IGHG1_human), P01859(IGHG2_human), P01860(IGHG3_human) and P01861(IGHG1_hu An example of a "WT" Fc region is SEQ ID NO: 15 (heavy chain constant region containing the Fc region). (provided).

[0104] 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 domain is essential for The antibody containing the amino acid (e.g., antibody, fusion protein, or conjugate) may contain one or more amino acids. does not contain amino acid substitutions, deletions, insertions, or modifications (e.g., amino acids naturally occurring at the corresponding positions in the Fc region) an unaltered Fc region containing the amino acid residues present in the Fc region, which otherwise has the same amino acid sequence at least one or more Fc ligands (e.g., Fc gamma R) compared to the corresponding antibody The binding affinity for

[0105] 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.

[0106] The term "Fc gamma receptor" or "Fc gamma R" as used herein refers to an Fc gamma receptor. Binds to the Fc region of a IgG antibody and is a protein encoded by the Fc gamma R gene In humans, this family is not limited to However, Fc.gamma.RI (CD64) (e.g., isoforms Fc.gamma.RIa, Fc.gamma. Fc.gamma.RIb, and Fc.gamma.RIc; Fc.gamma.RII (CD32) (e.g., the isoform Fc.gamma.RIb) Fc.gamma.RIIa [e.g., allotypes H131 and R131], Fc.gamma.RIIb [e.g., Fc.gamma.R IIb-1 and Fc.gamma.RIIb-2], and Fc.gamma.RIIc, etc.); and Fc.gamma.RIII (CD16) (e.g., isoform Fc.gamma.RIIIa [e.g., allotypes V158 and F158] and Fc.gamma.RIIIb [e.g., allotypes Fc.gamma.RIIIb-NA1 and Fc.gamma.RIIIb- NA2], as well as any undiscovered human Fc.gamma.R or Fc.gamma.R isoforms or Fc gamma R includes allotypes. Fc gamma R is a protein that can be expressed in any organism (for example, but not limited to, However, they can be derived from other species (e.g., humans, mice, rats, rabbits, and monkeys). Mouse Fc. Gamma R includes, but is not limited to, Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) , Fc.gamma.RIII (CD16), and Fc.gamma.RIII-2 (CD16-2), as well as any unidentified mouse Fc.gamma.R or an isoform or allotype of Fc.gamma.R.

[0107] 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).

[0108] 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 silencing is 99%, or 100%. ed) antibodies do not exhibit detectable binding to FcγR. FcγR of antibodies with altered Fc regions Binding to the ATP can be determined by various techniques known in the art, including, but not limited to, equilibrium Methods [e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analy tical Biochemistry, Vol. 373:52-60, 2008; radioimmunoassay (RIA)], or surface Plasmon resonance assays or other mechanism-based kinetic assays [e.g., BIAC ORE® 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 Measurements can be made using electrophoresis and chromatography (e.g., gel filtration). 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, The binding affinity may be determined by using a label (e.g., a fluorescent label, a luminescent label, or an isotopic label). and dynamics, Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott t-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. An example of a competitive binding assay involves binding a labeled antigen to an antibody of interest in a non- -incubating in the presence of increasing amounts of labeled antigen, and and detecting the antibody bound to the labeled antigen. From the data, Scatchard plot analysis can be used to identify the parental antibody of interest against a specific antigen. The binding affinity and binding off-rate may be determined. Immunoassays may also be used to measure the antigen, in which case the antigen is reacted with a labeled compound. The antibody of interest conjugated to a 100-kDa antibody and the presence of increasing amounts of an unlabeled second antibody were analyzed. Incubate under

[0109] 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.

[0110] 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 peptides containing an Fc domain that have the ability to specifically bind to antigen-bearing target cells. Proteins (e.g., Fc fusion proteins and Fc conjugate proteins) are used to inhibit cell-mediated cytotoxicity. It is intended to cause harm.

[0111] For simplicity, we will assume that cell-mediated cellular responses result from the activity of polypeptides containing an Fc domain. Cellular damage is also referred to herein as ADCC activity. The ability to mediate lysis of target cells by ADCC can be assayed. ADCC activity To assess the effect of a polypeptide of interest (e.g., an antibody), the polypeptide is co-transfected with immune effector cells. The target cells are then added to the target cells, resulting in cytolysis of the target cells. Solutions are typically extracted from lysed cells using a label (e.g., a radioactive substrate, a fluorescent dye, or a natural The release of intracellular proteins is detected by the Effector 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. Immunol. Methods 184:29 (1995). Alternatively, or in addition, The ADCC activity of an antibody can be measured in vivo (see, e.g., Clynes et al., Proc. Natl. Acad. Sci. USA 9 5:652 (1998)).

[0112] 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) This refers to the process of preparing the hematopoietic stem cells for transplantation. 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, CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137, or CD252, etc. The ADC, antibody, or antigen-binding fragment thereof that produces the hematopoietic stem cells is administered to a patient. The patient may be conditioned for cell transplant therapy. The antibody is covalently conjugated to a cytotoxin to form an ADC. Antibodies, antigen-binding fragments thereof, capable of binding to one or more of the above antigens may be used. Administration of a medicament or ADC to a patient in need of hematopoietic stem cell transplantation therapy may be beneficial, e.g., selectively depleting endogenous hematopoietic stem cells, thereby reducing the number of exogenous hematopoietic stem cells transplanted promotes hematopoietic stem cell graft survival by creating vacancies that can be filled by Sometimes I do.

[0113] 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.

[0114] 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.

[0115] 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 host 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.)

[0116] "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 same 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; US Pat. Nos. 5,530,101; 5,585,089; 5 ,693,761; 5,693,762; and 6,180,370 to Queen et al.; EP239400; PCT Publication WO 91 / 099 67; US Pat. No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28 :489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Pr See US Pat. No. 5,565,332; and US Pat. No. 5,565,332.

[0117] 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 taken up in tissues where the cells have homed, colonized, or engrafted. or progression of the disease, hematopoietic stem and progenitor cell production, and assessing the progress 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 measured by measuring the recovery rate of bone marrow cells from donor cells in bone marrow aspirate samples. It can also be evaluated by

[0118] 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 produce differentiated progeny more quickly.

[0119] 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., For example, CD45, CD2, CD5, CD137, or CD252).

[0120] 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.

[0121] As used herein, the terms "subject" and "patient" refer to a subject, as described herein. refers to a living organism, such as a human, receiving treatment 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.

[0122] 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 offspring of the human or animal. , for example, may be a population of hematopoietic stem cells.

[0123] 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 allows each The domain pairs with a complementary domain on another polypeptide chain to form a homodimeric structure. Thus, the term "triabody" refers to a trivalent antibody containing three peptide chains, Each 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 H Domain 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).

[0124] 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, myeoblast, basophil leukocytes, neutrophils, eosinophils, microglial cells, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages such as phage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte This describes substances such as hematopoietic lineage cells.

[0125] 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 may be autologous, syngeneic, or allogeneic cells.

[0126] 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).

[0127] 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).

[0128] As used herein, the phrase "substantially cleared from the blood" refers to a therapeutic After administering an agent (e.g., an anti-CD117 antibody, or an antigen-binding fragment thereof) to a patient wherein the concentration of the therapeutic agent in a blood sample isolated from the patient is If the agent is such that it cannot be detected by conventional methods (e.g., the therapeutic agent above the noise threshold of the device or assay used to detect the therapeutic agent. Various techniques known in the art (e.g., For example, ELISA-based detection assays known in the art or described herein. Detect antibodies, antibody fragments, and protein ligands using immunoassays, such as immunoassays. Additional assays that can be used to detect antibodies, or antibody fragments These include immunoprecipitation and immunoblot assays, among others, which are well known in the art. can be.

[0129] 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.

[0130] 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 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, the cell number or relative concentration of hematopoietic stem cells is increased. Beneficial results of the therapies described herein also include an increase in the number of conditions. After hematopoietic stem cell transplantation and subsequent hematopoietic stem cell transplantation, one or more cells of the hematopoietic lineage ( Megakaryocyte, thrombocyte, platelet, red blood cell, mast cell, myeloblast, eosinophil Basophils, neutrophils, eosinophils, microglial cells, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, or B lymphocytes, etc. This may include an increase in cell number or relative concentration. Beneficial results may include the suppression of cancer cells (e.g., CD117+ leukemia cells) or autoimmune cells (e.g., autoimmune cells). CD117+ autoimmune lymphocytes, such as CD117+ T-cells that express T-cell receptors that cross-react with antigens. This may include a reduction in the amount of disease-causing cell populations, such as the number of lymphocytes (leukocytes) To the extent that the methods of the present disclosure are directed to preventing a disorder, the term "prevent" refers to the prevention of a disease state. It is understood that the term "anti-inflammatory" does not necessarily mean that the reaction is completely prevented. When used in combination with other drugs, the term prevent refers to identifying a population susceptible to a disorder and preventing the disease before it develops. The term refers to a person skilled in the art who is capable of administering a compound of the present disclosure to a patient in need thereof, or to a disease state. This does not mean that the situation will be completely avoided.

[0131] 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 Jar, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile Rheumatoid Arthritis), and "Bone Marrow Transplantation for Non-Malignant Disease", A SH Education Book, 1:319-338 (2000) (This disclosure is for the purpose of prescribing hematopoietic stem cell transplantation therapy. and the like, which are incorporated herein by reference in their entirety. suffer from or are affected by any of the diseases or disorders listed in Additionally or alternatively, a patient "in need" of a hematopoietic stem cell transplant may be Patients with or without one of the following conditions (nevertheless, nucleomegaly) thrombocyte, platelet, red blood cell, mast cell, myeloblast, basophil leukocytes, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages Hematopoietic lineages such as phages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes Decreased levels of one or more endogenous cell types in an otherwise healthy subject [e.g., Some of those skilled in the art may, for example, Flow cytometry and fluorescence-activated cell sorting (FACS), among other procedures known in the art, are used to identify the cells. levels of one or more of the above cell types or other blood cell types by the ACS method. It can be easily determined whether the IL-10 receptor agonist activity is decreased relative to otherwise healthy subjects. Cut.

[0132] 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.

[0133] 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 endogenous hematopoietic stem cell populations or hematopoietic progenitors from the subject's bone marrow tissue) by removing a cell population) and / or engrafting stem cells into a target tissue of interest. Any disease, disorder, or condition that can be treated or cured by transplanting or implanting a For example, it has been shown that type 1 diabetes can be cured by hematopoietic stem cell transplantation. and benefit from conditioning in accordance with the compositions and methods described herein. Additional disorders that can be treated using the compositions and methods described herein include: Harms include, but are not limited to, sickle cell anemia, thalassemia, Fanconi anemia, recurrent Aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV / AIDS, metachromatic leukodystrophy Strophy, Diamond-Blackfan Anemia, and Shwachman-Diamond The patient conditioning and / or hematopoietic stem cell syndromes described herein are included. Additional diseases that can be treated using transplantation methods include inherited blood disorders (e.g., sickle cell disease). cytic anemia) and autoimmune diseases such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease The conditioning and / or transplantation methods described herein can be used to treat diseases such as: Further diseases that may be treated include malignant tumors (e.g., neuroblastoma), or hematological cancers (e.g., For example, the cancer may be acute myeloid leukemia, , acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large intestine large B-cell lymphoma, or non-Hodgkin's lymphoma Treatment using the conditioning and / or transplantation methods described herein may Further diseases for which treatment is possible include myelodysplastic syndromes. The subject has or is affected by a metabolic storage disorder. For example, the subject has a glomerular filtration rate of 2000 or more. Colagenosis, mucopolysaccharidosis, Gaucher disease, Hurler disease, sphingolipidosis, metachromatic white matter dystrophies, or a metabolic disorder selected from the group consisting of the treatments and and any other disease or disorder that can benefit from therapy, including but not limited to However, severe combined immunodeficiency, Wiskott-Aldrich syndrome, and hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage Thalassemia, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus (systemic lupus erythematosus), multiple sclerosis, juvenile rheumatoid arthritis), and "Bone "Marrow Transplantation for Non-Malignant disease", ASH Education Book, 1:319-338 (2000) (This disclosure is intended to provide a basis for the treatment of patients with HIV who may be treated by prescribing hematopoietic stem cell transplant therapy. and is incorporated herein by reference in its entirety. may be suffering from or affected by a disease or disorder.

[0134] 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 translation rate of these genes or to improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include polynucleotide sequences that improve the transfer of the expression vector. It contains 5' and 3' untranslated regions and polyadenylation that allow efficient transcription of the gene on the target. The expression vectors described herein may also contain such a transcription signal site. It contains a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers include ampicillin, chloramphenicol, kanamycin, and Encodes resistance to antibiotics such as thrombin, thrombin, and nourseothricin Contains genes.

[0135] 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 The reactive functional groups of a molecule, such as the cytotoxin binding fragments, can be linked to the cytotoxin binding fragments described herein. Conjugates are formed by chemical bonding with a suitable reactive functional group on another molecule, such as a The carboxylate may contain a linker between the two molecules that are attached to each other (e.g., between an antibody and a cytotoxin). Examples of linkers that can be used to form conjugates include peptides. containing linkers (e.g., naturally occurring or non-naturally occurring amino acids, e.g., D-amino acids Linkers include those described herein and in the art. They may be prepared using a variety of strategies known in the art. Depending on the functional moiety, the linker can be hydrolyzed by, for example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, etc. Decomposition, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage Cleavage can occur due to cleavage (e.g., Leriche et al., Bioorg. Med. Chem., 20:57 1-582, 2012).

[0136] 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. (such as those described herein or known in the art), vinblastine, vin Blastine sulfate, vincristine, vincristine sulfate, vindesine, and vinorelbine Vinca alkaloids such as docetaxel and paclitaxel, taxanes such as discode Macrolides such as lumolide, colchicine, and epothilones, and their derivatives Examples of suitable anti-inflammatory drugs include, but are not limited to, steroid derivatives (such as epothilone B or its derivatives).

[0137] 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.

[0138] The term "acyl," as used herein, refers to -C(=O)R, as defined herein. As shown, R is hydrogen (an "aldehyde"), C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 a Alkynyl, C3-C7 carbocyclyl, C6-C 20 aryl, 5-10 membered heteroaryl, or 5-10 membered Non-limiting examples include formyl, acetyl, propanoyl, Benzoyl and acryloyl are included.

[0139] As used herein, the term "C1-C 12 "Alkyl" means a straight or refers to branched saturated hydrocarbons. Typical C1-C 12 The alkyl group is not limited to However, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl are On the other hand, the branch C1-C 12 Alkyl includes, but is not limited to, -isopropyl. -propyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl Examples include C1-C 12 The alkyl group may be unsubstituted or substituted. be.

[0140] As used herein, the term "alkenyl" refers to an alkyl group having at least one site of unsaturation, i.e., carbon-carbon, sp 2 C2-C containing normal, secondary, or tertiary carbon atoms with double bonds 12 Refers to hydrocarbons. Examples include, but are not limited to: ethylene vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl , -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl The alkenyl group may be unsubstituted or substituted. There is.

[0141] As used herein, "alkynyl" refers to an alkyl group having at least one site of unsaturation, i.e., a carbon- C2-C containing normal, secondary, or tertiary carbon atoms with carbon, sp triple bonds 12 Carbonated water Examples include, but are not limited to, acetylene and propargyl. An alkynyl group can be unsubstituted or substituted.

[0142] As used herein, "aryl" refers to a C-C 20 Refers to a carbocyclic aromatic group. Examples of aryl groups Examples include, but are not limited to, phenyl, naphthyl, and anthracenyl. The aryl group can be unsubstituted or substituted.

[0143] As used herein, "arylalkyl" refers to an alkyl group having a carbon atom (typically a terminal or sp 3 charcoal Acyclic aryl groups in which one of the hydrogen atoms bonded to the aryl group is replaced by an aryl radical. Typical arylalkyl groups include, but are not limited to: benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthopheny The arylalkyl group may be substituted or unsubstituted, and may be substituted or unsubstituted. For example, the alkyl portion of an arylalkyl group (e.g., alkanyl, alkenyl, or alkenyl) The alkyl group has 1 to 6 carbon atoms, and the aryl moiety has 5 to 14 carbon atoms. The alkaryl group may be unsubstituted or substituted.

[0144] As used herein, "cycloalkyl" refers to a saturated carbocyclic radical, which is a monocyclic The cycloalkyl group may be a monocyclic or bicyclic group having 3 to 7 carbon atoms. Examples of the monocyclic ring include a ring having 7 to 12 carbon atoms, and a bicyclic ring having 7 to 12 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups include cyclohexyl, cycloheptyl, and cyclooctyl. may not be present or may have been replaced.

[0145] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic radical, which is a single The cycloalkenyl group may be cyclic or bicyclic. Examples of the monocyclic ring include a ring having a carbon atom, and a bicyclic ring having 7 to 12 carbon atoms. Examples of cycloalkenyl groups include 1-cyclopent-1-enyl and 1-cyclopent-2-enyl. , 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and and 1-cyclohex-3-enyl. Cycloalkenyl groups include unsubstituted, Or may be substituted.

[0146] 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).

[0147] As used herein, "heteroaryl" and "heterocycloalkyl" refer to, respectively, refers to an aromatic or non-aromatic ring system in which one or more ring atoms may be heteroatoms, e.g. Nitrogen, oxygen, and sulfur. Heteroaryl or heterocycloalkyl radicals are It contains 2 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. The aryl or heterocycloalkyl has 3 to 7 ring members (2 to 6 carbon atoms and N, O, P). and 1 to 3 heteroatoms selected from S), or ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S); Bicyclic rings with a ring structure (e.g., bicyclic [4,5], [5,5], [5,6], or [6,6] systems) Heteroaryl and heterocycloalkyl may be unsubstituted or substituted. This sometimes happens.

[0148] 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.

[0149] Examples of heteroaryl groups include, but are not limited to, pyridyl, thiazolinone ... Zolyl, tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyridinyl Imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indole benzoyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, isoxazolyl , pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-isoindolyl Ndazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalyl nyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, Phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl , phenothiazinyl, furazanil, phenoxazinyl, isochromanyl, chromanyl, i Midazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, benzotriazolyl , benzisoxazolyl, and isatinoyl.

[0150] Examples of heterocycloalkyl include, but are not limited to, dihydropyridyl. Lysyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, piperidinyl , 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydro tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroiso Quinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinunyl Examples include clidinyl, and morpholinyl.

[0151] 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:

[0152] 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.

[0153] As used herein and as described above, alkyl, alkenyl, alkynyl, aryl, Suitable for any of arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, etc. As used herein, "substituted" means that one or more hydrogen atoms are each independently replaced with a substituent. Unless otherwise constrained by the definitions of the individual substituents, the above Chemical substructures such as "alkyl", "heteroalkyl", "alkenyl", heteroalkenyl," "alkynyl," "heteroalkynyl," "cycloalkyl," "Heterocycloalkyl," "aryl," and "heteroaryl" groups, etc., are optionally to, for example, alkyl, alkynyl, cycloalkyl, heterocycloalkyl, alkyl Aryl, alkyl, heteroaryl, alkyl, cycloalkyl, alkyl, hetero Cycloalkyl, amino, ammonium, acyl, acyloxy, acylamino, amino Carbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl aryl, sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy, methyl, cyano, hydroxy, mercapto, nitro, etc. Typical substituents include, but are not limited to, 1 to 5 substituents. but not -X, -R, -OH, -OR, -SH, -SR, NH2, -NHR, -N(R)2, -N + (R)3, -CX3, -CN, - OCN, -SCN, -NCO, -NCS, -NO, -NO2, -N3, -NC(=O)H, -NC(=O)R, -C(=O)H, -C(=O)R, -C (=O)NH2, -C(=O)N(R)2, -SO3-, -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NH 2, -S(=O)2N( R)2, -S(=O)R, -OP(=O)(OH) 2, -OP(=O)(OR)2, -P(=O)(OR)2, -PO3, -PO3H2, -C(=O)X, -C (=S)R, -CO2H, -CO2R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NH 2, -C(=O)N(R)2 , -C(=S)NH 2, -C(=S)N(R)2, -C(=NH)NH 2, and —C(═NR)N(R)2; each X is independently selected in each occurrence from F, Cl, Br, and I; and each R is , C1-C 12 Alkyl, C6-C 20 Aryl, C3-C 14 heterocycloalkyl or heteroaryl, The protecting group and the prodrug moiety are independently selected in each case. Wherever "optionally substituted" is described, the group shall be Independently in each case, each of the groups may be substituted with one or more of the above substituents. Adjacent substituents undergo ring closure, e.g., ring closure of adjacent functional substituents, e.g., la Actams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals, amina This includes situations where a hemiaminal is formed, for example, to provide a protecting group. That's fine too.

[0154] 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".

[0155] 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 Reactions involving the reaction of a substituent with a nucleophilic / electrophilic pair ( 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.

[0156] 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.

[0157] As used herein, "drug-to-antibody ratio" or "DAR" " refers to the number of drugs (e.g., amatoxins) attached to the antibodies of the ADC. The DAR of an ADC may range from 1 to 8, although higher numbers are possible depending on the number of In certain embodiments, the ADCs described herein comprise 1, 2, 3, 4, 5, 6, 7, or It has a DAR of 8.

[0158] 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.

[0159] Fc-engineered antibodies The present disclosure provides a method for the production of antibodies to hematopoietic cells that have Fc modifications that allow for Fc silencing and that respond to antigens expressed by hematopoietic cells. Antibodies, or antigen-binding fragments thereof, capable of binding to the For example, the present disclosure provides methods for Fc silencing. and Fc modifications that allow binding to antigens expressed by hematopoietic cells (e.g., but not limited to, but not CD117 [e.g., GNNK+ CD117], or CD45); or mature immune cells (e.g., , T-cells) expressing antigens (e.g., CD45, CD2, CD5, CD137, or CD252). The antibody, or antigen-binding fragment thereof, can be administered in combination with a therapeutic agent, such as (i) a CD11 7+ [e.g., GNNK+ CD117], or CD45+ hematopoietic stem cells; or CD45+, CD2+, CD5+, CD137 + or CD252+ immune cells [e.g., T-cells]. and (ii) transplanted hematopoietic stem cells in patients in need of transplant therapy. as "naked" antibodies or ADCs to promote engraftment of These therapeutic activities are based in part on the discovery that they can be used to treat, for example, blood cells (e.g. hematopoietic stem cells, leukocytes, immune cells [e.g. mature immune cells (e.g. T cells) ]) expressed antigens (e.g., CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137 anti-hematopoietic cell (HC)-antibodies (e.g., anti-CD252, etc.) 117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, and anti-CD252 antibody, etc. ) or an antigen-binding fragment thereof is used to target, for example, cancer cells, autoimmune cells, or hematopoietic stem cells. by binding to the ATP-dependent ATPases and subsequently inducing cell death. By reducing endogenous hematopoietic stem cells, transplanted hematopoietic stem cells can be may be provided with a niche in which they can home in and then become productive. In this way, the transplanted hematopoietic stem cells can be used to treat the patient (e.g., Successful engraftment in a human patient (e.g., a human patient suffering from a stem cell disorder as described herein). can be done.

[0160] The antibodies, or antigen-binding fragments thereof, described herein may be prepared by administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof, Modifications and / or mutations that change the properties of the fragment (e.g., increasing half-life or The ADCC vector may contain modifications and / or mutations that increase or decrease ADCC.

[0161] In one embodiment, an antibody is provided that comprises one or more radiolabeled amino acids. The recognized antibodies can be used for both diagnostic and therapeutic purposes (as an alternative, radioimmunoassay). (It is possible to conjugate to a radioactively labeled molecule.) Non-limiting examples include 3H, 14C, 15N, 35S, 90Y, 99Tc and 125I, 131I and 186Re. Radiolabeled amino acids and related peptide derivatives include, but are not limited to: Methods for preparation are known in the art (see, e.g., Junghans et al., Cancer Chemistry, 2004). Motherapy and Biotherapy 655-686, 2nd ed., Chafner and Longo, eds., Lippincott Raven (19 96) and U.S. Patent Nos. 4,681,581, 4,735,210, 5,101,827, U.S. Patent No. 5,102,990 [US RE35,500], U.S. Patent No. 5,648,471, U.S. Patent No. 5,697,902 For example, radioisotopes can be conjugated by the chloramine T method. This can sometimes happen.

[0162] In certain embodiments, an anti-HC antibody (e.g., an anti-CD117 antibody, an anti-CD45 antibody, an anti-CD2 antibody, an anti- anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody), or an antigen-binding fragment thereof, and a modified Fc region, wherein the modified Fc region has at least and one amino acid modification such that the molecule has increased affinity for Fc gamma R (FcγR) or Certain amino acid positions within the Fc region affect binding to FcγRs upon direct contact. Specifically, amino acids 234-239 (hinge region), amino acid amino acids 265-269 (B / C loop), amino acids 297-299 (C' / E loop), and amino acids 327-332 (F / G loop) (See Sondermann et al., 2000 Nature, 406: 267-273.) Some implementations In some embodiments, the antibodies described herein are capable of binding to FcγRs based on structural and crystallographic analysis. and a variant Fc region in which at least one residue that makes direct contact with In some embodiments, an anti-HC antibody (e.g., an anti-CD117 antibody, an anti-CD45 antibody, an anti-CD2 antibody) is used. , anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody), or an antigen-binding fragment thereof The Fc region of is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th E d. Public Health Service, NH1, MD (1991) (expressly incorporated herein by reference) ) contains an amino acid substitution at amino acid 265 according to the EU index as found in "Kabat The "EU index" refers to the numbering of human IgG1 EU antibodies. The Fc region comprises a D265A mutation. In one embodiment, the Fc region comprises a D265C mutation. In some embodiments, the Fc region of the antibody (or fragment thereof) is In one embodiment, the amino acid substitution at amino acid 234 according to the EU index is as follows: In some embodiments, the Fc region comprises a L234A mutation. CD117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody The Fc region of the antibody, or an antigen-binding fragment thereof, may be identified by the EU index, e.g., as in Kabat. In one embodiment, the Fc region comprises an amino acid substitution at amino acid 235 with L235A In yet another embodiment, the Fc region comprises the L234A and L235A mutations (referred to herein as "L234A and L235A"). In another embodiment, the Fc region comprises L 234A and L235A mutations, wherein the Fc region does not include a P329G mutation. In one embodiment, the Fc region comprises a D265C, a L234A, and a L235A mutation (referred to herein as "D265C.L234A.L235A"). In another embodiment, the Fc region comprises D265C, L234A, and L235A mutations. In yet a further embodiment, the Fc region does not comprise a P329G mutation. The Fc region contains D265C, L234A, L235A, and H435A mutations (referred to herein as "D265C.L234A.L235A"). In another embodiment, the Fc region comprises D265C, L234A, L235A, and and H435A mutations, wherein the Fc region does not comprise a P329G mutation. The Fc region contains a D265C and H435A mutation (also referred to herein as "D265C.H435A"). In yet another embodiment, the Fc region comprises D265A, S239C, L234A, and L235A mutations (as described herein). In yet another embodiment, the method includes the steps of: The Fc region comprises D265A, S239C, L234A, and L235A mutations, wherein the Fc region comprises P329G In another embodiment, the Fc region does not contain the D265C, N297G, and H435A mutations (Honmei et al., 2002). In another embodiment, the Fc region comprises: Includes the D265C, N297Q, and H435A mutations (also referred to herein as "D265C.N297Q.H435A"). In another embodiment, the Fc region comprises E233P, L234V, L235A, and delG236 (deletion of 236) mutations ( Also referred to in the specification as "E233P.L234V.L235A.delG236" or "EPLVLAdelG"). In embodiments, the Fc region comprises E233P, L234V, L235A, and delG236 (deletion of 236) mutations. In another embodiment, the Fc region does not comprise a P329G mutation. 3P, L234V, L235A, delG236 (deletion of 236), and H435A mutations (referred to herein as "E233P.L234V.L2 In another embodiment, the nucleotide sequence of the present invention is a nucleotide sequence of the present invention. The Fc region comprises E233P, L234V, L235A, delG236 (deletion of 236), and H435A mutations, wherein: In another embodiment, the Fc region does not contain the P329G mutation. In another embodiment, the Fc region comprises the mutations L234A, L235A, S239C, and D265A. and D265A mutations, wherein the Fc region does not comprise a P329G mutation. In another embodiment, the Fc region comprises H435A, L234A, L235A, and D265C mutations. The Fc region comprises H435A, L234A, L235A, and D265C mutations, wherein the Fc region comprises P329G Does not contain mutations.

[0163] 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 effector binding compared to the binding of the same antibody to the FcR without an Fc region In some embodiments, the antibody has an altered Fc region, which results in a decreased function. As a result, the antibody binds to the Fc gamma receptor (F Binding to an FcγR) of an antibody containing an unmodified Fc region is compared to binding to said FcγR of the same antibody containing an unmodified Fc region. In some embodiments, the FcγR is an Fc 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 another embodiment, the FcγR is FcγR3B. In another embodiment, the decrease in binding is due to an increase in antibody binding to the FcγR. at least at least 70% reduction, at least 80% reduction, at least 90% reduction, at least 95% reduction, at least In other embodiments, the result is at least a 98% reduction, at least a 99% reduction, or a 100% reduction. The decrease in binding of the antibody to an FcγR is due to the increased affinity of the antibody to said FcγR compared to the antibody containing the unmodified Fc region. At least 70% to 100% reduction, at least 80% to 100% reduction, at least A reduction of at least 90% to 100%, a reduction of at least 95% to 100%, or a reduction of at least 98% to 100% do.

[0164] In some embodiments, the antibody has an altered Fc region such that the antibody compared with the same antibody containing an unmodified Fc region 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 due to altered At least a 50% reduction in cytokine release compared to the cytokine release 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 In some embodiments, the reduction is at least a 98% reduction, at least a 99% reduction, or a 100% reduction. The reduction in cytokine release was greater than that of the same antibody containing an unmodified Fc region. At least 70%-100% reduction in cytokine release compared to IL-16, at least 80%-100% reduction in cytokine release compared to IL-16 A reduction of at least 90% to 100%, a reduction of at least 95% to 100%. In this state, cytokine release is by immune cells.

[0165] In some embodiments, the antibody has an altered Fc region such that the antibody compared with the same antibody containing an unmodified Fc region in an in vitro mast cell degranulation assay. Compared to mast cell degranulation, mast cell degranulation was reduced by at least 50%. In some embodiments, the reduction in mast cell degranulation is due to altered At least 100% of mast cell degranulation was observed compared to 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 In some embodiments, the reduction is at least a 98% reduction, at least a 99% reduction, or a 100% reduction. 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 compared to non-human leukocytes, at least 80%-100% reduction in mast cell degranulation compared to non-human leukocytes A reduction of at least 90% to 100% or a reduction of at least 95% to 100%.

[0166] 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 discovery (ADCP) assay compared to an ADCP assay of the same antibody containing an unmodified Fc region. and 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 Fc Compared with the antibody-dependent cellular phagocytosis of the same antibody containing the At least a 70% reduction, at least an 80% reduction, or at least a 90% reduction in agocytosis , at least a 95% reduction, at least a 98% reduction, at least a 99% reduction, or a 100% reduction is.

[0167] In some embodiments, the anti-HC antibodies described herein (e.g., anti-CD117 antibodies, anti- -CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody) The Fc region contains one or a combination of the following modifications: D265A, D265C, D265C / H43 5A, D265C / LALA, D265C / LALA / H435A, D265A / S239C / L234A / L235A / H435A, D 265A / S239C / L234A / L235A, D265C / N297G, D265C / N297G / H435A, D265C (EPLVL AdelG *), D265C (EPLVLAdelG ) / H435A, D265C / N297Q / H435A, D265C / N297Q, EPL VLAdelG / H435A, EPLVLAdelG / D265C, EPLVLAdelG / D265A, N297A, N297G, or N297 Q.

[0168] 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. Labels on one or more of the components being evaluated may be utilized and / or various assays may be performed. detection methods (for example, but not limited to, chromogenic labels, fluorescent labels, luminescent labels, or For binding affinity and kinetics, see Paul, W. E. ., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999)( This is described in detail in (focusing on antibody-immunogen interactions). An example of a binding assay involves combining a labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen. and detecting the antibody bound to the labeled antigen. From the data, Scatchard-P. Lot analysis determines the affinity and binding off-rate of the antibody of interest for a particular antigen. Competition with a second antibody may be measured using a radioimmunoassay. In this case, the antigen may be conjugated to a target antibody conjugated to a labeling compound. and in the presence of increasing amounts of an unlabeled second antibody.

[0169] 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 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction a reduction of at least 95%, a reduction of at least 98%, a reduction of at least 99%, or a reduction of 100% The number of nuclei is small (e.g., assessed by Bio-Layer Interferometry (BLI)). (e.g., as described in Example 1).

[0170] 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. the ability to induce granulocyte proliferation (e.g., as described in Example 2), or its (e.g., human granulocyte proliferation) the ability to induce cytokine release by peripheral blood mononuclear cells (e.g., as described in Example 3) The following may be assayed:

[0171] Thus, in one embodiment, the Fc region is (e.g., an unmodified Fc region). Antibodies with short half-lives include mutations that result in a decreased half-life (compared to the antibody). In certain instances (e.g., previous studies) where antibodies are expected to function as short-lived therapeutic agents, The conditioning described herein involves administering the antibody followed by administering HSCs. Ideally, HSC (also commonly referred to as Get antigens [e.g., CD117 (e.g., GNNK+ CD117), CD45, CD2, CD5, CD137, or CD252] ], but unlike endogenous stem cells, express the anti-HC antibody [e.g., anti-CD117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody] The antibodies should be substantially cleared before administering the drug (not the anticoagulant). In embodiments, the Fc region comprises a mutation at position 435 (EU index according to Kabat). In one embodiment, the mutation is an H435A mutation.

[0172] In certain embodiments, the anti-HC antibodies described herein (e.g., anti-CD117 antibodies, anti-CD45 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody) has a cytotoxicity of about 24 h (e.g., in humans). hours or less, about 23 hours or less, about 22 hours or less, about 21 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, about 15 hours or less, about 14 hours or less, about 13 hours or less The half-life may be about 12 hours or less, or about 11 hours or less.

[0173] In certain embodiments, the anti-HC antibodies described herein (e.g., anti-CD117 antibodies, anti-CD45 The antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody) may be administered to a subject (e.g., a human). 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 It has a reduction life.

[0174] 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.

[0175] 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. In some embodiments, the system is conjugated to a ribosomal protein (e.g., an amatoxin). The amino acid residues are provided by mutations in the Fc domain of the antibody, or antigen-binding fragment thereof. For example, the cysteine ​​residues are Cys118, Cys239, and Cys265. In one embodiment, the anti-HC antibody (e.g., anti-CD117 antibody) may be selected from the group consisting of: antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody), or The Fc region of the antigen-binding fragment of In one embodiment, the Fc region comprises an amino acid substitution at position 65. In one embodiment, the Fc region comprises a D265C mutation. In one embodiment, the Fc region comprises a D265C and a H435A mutation. In one embodiment, the Fc region comprises D265C, L234A, and L235A mutations. In one embodiment, the anti-HC antibody (e.g., anti-CD 117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody), or the Fc region of an antigen-binding fragment thereof, In one embodiment, the Fc region comprises an amino acid substitution at amino acid 239. In one embodiment, the Fc region comprises a S239C mutation. In one embodiment, the Fc region comprises an L234A mutation, an L235A mutation, an S239C mutation, and a D265A mutation. In another embodiment, the Fc region comprises S239C and H435A mutations. In yet another embodiment, the Fc region comprises a L234A mutation, a L235A mutation, and a S239C mutation. In another embodiment, the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, and a S239C mutation. In embodiments, the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, a S239C mutation, and a D265A mutation. Includes differences.

[0176] In particular, Fc amino acid positions refer to the EU numbering index unless otherwise indicated. do.

[0177] Methods for engineering antibodies to contain any of the Fc modifications herein are well known in the art. These methods include, but are not limited to, the antibody or at least the antibody Site-specific (or oligonucleotide) binding to the prepared DNA molecule encoding the constant region Examples of mutagenesis include preparation by PCR-mediated mutagenesis, PCR mutagenesis, and cassette mutagenesis. Site-directed mutagenesis is well known in the art (see, e.g., Carter et al., Nucleic Acids ds Res., 13:4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA, 82: 488 (1987). PCR mutagenesis also allows the generation of amino acid sequence variants of a starting polypeptide. Higuchi, in PCR Protocols, pp. 177-183 (Academic Press , 1990); and Vallette et al., Nuc. Acids Res. 17:723-733 (1989). Another method for preparing ants (cassette mutagenesis) is described by Wells et al., Gene, 34:315 -323 (1985).

[0178] Anti-CD117 antibody The present disclosure also provides antibodies capable of binding to CD117, such as GNNK+ CD117, or an antigen thereof. The binding fragments may be used as the sole therapeutic agent or as an ADC to: Based in part on the discovery that CD117+ cells can be used to treat cancers characterized by CD117+ cells (e.g., To treat autoimmune diseases (e.g., acute myeloid leukemia or myelodysplastic syndromes) and and (ii) promoting the engraftment of transplanted hematopoietic stem cells in patients requiring transplantation therapy. These therapeutic activities may be achieved by targeting cells such as cancer cells, autoimmune cells, or hematopoietic stem cells. CD117 expressed on the surface of target cells (e.g., GNNK+ CD117) is detected by an anti-CD117 antibody or its anti-CD117 antibody. by binding of the protozoan binding fragment and subsequently inducing cell death. It can cause a decrease in endogenous hematopoietic stem cells, which can lead to a decline in transplantation. This may provide a niche to which the isolated hematopoietic stem cells can home, and After this, productive hematopoiesis can be established. In this way, the transplanted hematopoietic stem cells In a subject (e.g., a human patient suffering from a stem cell disorder described herein), Can be easily engrafted

[0179] Human CD117 (also known as c-Kit, mRNA NCBI reference sequence: NM_000222.2, protein NCBI reference sequence: Antibodies and antigen-binding fragments (GNNK+ CD1) capable of binding to the antibody (reference sequence: NP_000213.1) 17) and antibodies and antigen-binding fragments capable of binding to the antigens. and the compositions and methods described herein to condition patients for the procedure. These may be used in combination. Polymorphisms affecting the IL-1 domain or extracellular domain are currently not used for non-oncology indications. At least four isoforms of CD117 have been identified, and they are involved in tumorigenesis. Additional isoforms may be expressed in tumor cells. Two of these are located in the intracellular domain of the protein and two in its outer juxtamembrane region. There are two extracellular isoforms (GNNK+ and GNNK-) that share a four amino acid sequence (G These isoforms differ in that they are either NNK+ or absent (GNNK-). Although it has been reported that the ligand binding to the GNNK-isoform is It has been reported that the incorporation into the cell and degradation of GNNK+ isoforms increases when the incorporation of GNNK+ isoforms into the cell is accelerated. The form is shown in Figure 1, where antibodies raised against this isoform identify GNNK+ and GNNK- proteins. Because it contains CD117, it may be used as an immunogen to generate antibodies capable of binding to CD117.

[0180] In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO:1 a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 14; The light chain variable region is shown in the column.

[0181] In another embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises the heavy chain variable region (VH) of Ab85. and three CDR sequences of the light chain variable region (LH) amino acid sequence. nothing.

[0182] In another embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is Ab85 (referred to herein as Ab2 The heavy chain variable region (VH) amino acid sequence and the light chain variable region (LH) amino acid sequence of Arrays, including

[0183] The heavy chain variable region (VH) amino acid sequence is shown below as SEQ ID NO: 13. Ab85 The VH CDR amino acid sequences of NYWIG (VH CDR1; SEQ ID NO: IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 8); and HGRGYEGYE GAFDI (VH CDR3; SEQ ID NO:9).

[0184] Ab85 VH sequence [ka]

[0185] The light chain variable region (VL) amino acid sequence of Ab85 is shown below as SEQ ID NO: 14. The VL CDR amino acid sequence of Ab85 is underlined and is as follows: RSSQGIRSDLG (VL CDR1; SEQ ID NO: 10); DASNLET (VL CDR2; SEQ ID NO: 11; and QQANGF PLT (VL CDR3; SEQ ID NO: 12).

[0186] Ab85 VL sequence [ka]

[0187] In another embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is Ab249 (referred to herein as Ab 3) and the heavy chain variable region (VH) amino acid sequence of the light chain variable region (LH) amino acid sequence of the Arrays, including

[0188] The heavy chain variable region (VH) amino acid sequence of Ab249 is shown below as SEQ ID NO: 346. The VH CDR amino acid sequences of Ab249 are underlined and are as follows: TSWIG (VH CDR1; sequence IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 341); and HGLGYNGYEGAFDI (VH CDR3; SEQ ID NO: 342).

[0189] Ab249 VH sequence [ka]

[0190] The light chain variable region (VL) amino acid sequence of Ab249 is shown below as SEQ ID NO: 347. The VL CDR amino acid sequence of Ab249 is underlined and is as follows: RASQGIGSALA (VL CDR 1; SEQ ID NO: 343); DASNLET (VL CDR2; SEQ ID NO: 344); and QQLNGYPLT (VL CDR3; SEQ ID NO:345).

[0191] Ab249 VL sequence [ka]

[0192] 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.)

[0193] Thus, in certain embodiments, the anti-CD117 antibody is selected from the group consisting of SEQ ID Nos: 7, 8, and and a heavy chain comprising the CDR set (CDR1, CDR2, and CDR3) set forth in SEQ ID Nos. and a light chain comprising the CDRs set forth in: 10, 11, and 12. In other embodiments, the anti-CD117 antibody , CDR sets (CDR1, CDR2, and CDR3) set forth in SEQ ID Nos: 340, 341, and 342 and a heavy chain comprising CDRs set forth in SEQ ID Nos: 343, 344, and 345. The light chain includes

[0194] In another embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, is Ab67 (neutral antibody). antibody; herein interchangeably referred to as Ab1), and a light chain variable region (LH) amino acid sequence.

[0195] The heavy chain variable region (VH) amino acid sequence of Ab67 is shown below as SEQ ID NO: 354. The VH CDR amino acid sequence of Ab67 is underlined and is as follows: FTFSDADMD (VH CDR1; sequence SEQ ID NO: 348); RTRNKAGSYTTEYAASVKG (VH CDR2; SEQ ID NO: 349) and AREPKYWIDFDL (VH CDR3; SEQ ID NO: 350).

[0196] Ab67 VH sequence [ka]

[0197] The light chain variable region (VL) amino acid sequence of Ab67 is shown below as SEQ ID NO: 355. The VL CDR amino acid sequence of Ab67 is underlined below and is as follows: RASQSISSYLN (VL CDR1; SEQ ID NO: 351); AASSLQS (VL CDR2; SEQ ID NO: 352); and and QQSYIAPYT (VL CDR3; SEQ ID NO: 353).

[0198] Ab67 VL sequence [ka]

[0199] Thus, in certain embodiments, the anti-CD117 antibody is selected from the group consisting of SEQ ID Nos: 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 350, and a heavy chain comprising the CDR set (CDR1, CDR2, and CDR3) set forth in SEQ ID NO: (S and a light chain comprising the CDRs set forth in EQ ID Nos): 351, 352, and 353.

[0200] Additional sequences of the anti-CD117 antibodies or binding fragments described herein are provided in Table 5. do.

[0201] The anti-CD117 antibodies or binding fragments described herein may also be used in combination with other antibodies known in the art. Thus, modifications and / or mutations that change the properties of the antibody and / or fragment (e.g., Increased half-life, increased or decreased ADCC, modifications and / or mutations, etc. It may include.

[0202] In one embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, is a variant and a variant Fc region, wherein the variant Fc region has at least one The amino acid modifications result in the molecule having altered affinity for Fc gamma R. Certain amino acid positions within the Fc region have been identified by crystallographic studies in direct contact with FcγR. Specifically, amino acids 234-239 (hinge region) and 265-269 (B / C loop) The amino acids 297-299 (C' / E loop), and 327-332 (F / G loop) are shown in Fig. (See, e.g., Hörmann et al., 2000 Nature, 406: 267-273). For example, amino acid positions 234 and 235 of the Fc region The amino acid substitution at 235 affects binding to Fc receptors, particularly Fc gamma receptors (FcγRs). In one embodiment, the antibody has been identified as decreasing the affinity of IgG antibodies. The anti-CD117 antibodies described herein may contain amino acid substitutions at L234 and / or L235 (e.g., L234 The antibodies described herein contain an Fc region containing L235A and L235A (EU index). - The CD117 antibody is known to directly contact FcγR based on structural and crystallographic analysis, at least In one embodiment, the variant Fc region may comprise a variant Fc region in which at least one residue is altered. The Fc region of the anti-CD117 antibody (or a fragment thereof containing the Fc region) may be determined by the method described in Kabat et al., Sequence s of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991) (expressly incorporated herein by reference) Contains an amino acid substitution at amino acid 265. "Index" refers to the numbering of human IgG1 EU antibodies unless otherwise specified, and is used herein to refer to Fc Used to refer to amino acid positions.

[0203] In one embodiment, the Fc region comprises a D265A mutation. contains the D265C mutation.

[0204] In some embodiments, the Fc region of the anti-CD117 antibody (or fragment thereof) is In one embodiment, the amino acid substitution at amino acid 234 according to the EU index as in In some embodiments, the Fc region comprises a L234A mutation. The Fc region of the IgG1 gene (or fragment thereof) is amino acid 235 according to the EU index as per Kabat. In one embodiment, the Fc region comprises an amino acid substitution at L235A. In one embodiment, the Fc region comprises the L234A and L235A mutations. The Fc region contains the D265C, L234A, and L235A mutations.

[0205] In certain embodiments, the variant IgG Fc domain does not comprise one or more amino acid substitutions. Decreased binding affinity to Fc gamma R and / or C1q compared to wild-type Fc domains The Fc binding interaction may be achieved by various methods, including one or more amino acid substitutions that result in the Fc binding interaction being eliminated or eliminated. Various effector functions and downstream signaling events (antibody-dependent cell-mediated cytotoxicity) (antibody dependent cell-mediated cytotoxicity (ADCC)) and complement dependent cytotoxicity (com (including, but not limited to, replenishment dependent cytotoxicity (CDC)) Thus, in certain embodiments, antibodies comprising modified Fc regions (e.g., L234A, L235A, L236A, L237A, L238A, L239A, L240A, L241A, L242A, L243A, L244A, L245A, L246A, L247A, L248A, L249A, L250A, L251A, L and / or containing the D265C mutation) have substantially reduced or eliminated effector function. It is missing.

[0206] Affinity for the Fc region can be determined using various techniques known in the art (e.g., limited However, equilibrium methods [e.g., enzyme-linked immunosorbent assay (ELISA)] are not bent assay (ELISA));KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 3 73:52-60, 2008; radioimmunoassay (RIA)], or surface plasmon ion resonance assays or other mechanism-based kinetic assays [e.g., BIACORE TM Solution Analysis or Octet TM analysis (forteBIO)], as well as other methods [e.g., indirect binding assays, Competitive binding assay, fluorescence resonance energy transfer r (FRET)), gel electrophoresis and chromatography (e.g., gel filtration, etc.). These and other methods can be used to measure one or more of the components being evaluated. Labels may be used and / or various detection methods (e.g., but not limited to) may be used. However, chromogenic, fluorescent, luminescent, or isotopic labels may be used. Regarding binding affinity and kinetics, see Paul, WE, ed., Fundamental Immunology, 4t h Ed., Lippincott-Raven, Philadelphia (1999) (which focuses on antibody-immunogen interactions) An example of a competitive binding assay is a method in which a labeled antigen and a target antigen are bound to each other. Incubation of the target antibody in the presence of increasing amounts of unlabeled antigen and detecting the antibody bound to the labeled antigen. From the data, Scatchard plot analysis revealed the presence of specific antigens. The affinity and binding off-rate of the antibody of interest may be determined. Competition with the antibody may also be measured using radioimmunoassays. The starting material is mixed with the antibody of interest conjugated to a labeled compound and increasing amounts of an unlabeled second antibody. The mixture is incubated in the presence of

[0207] In some embodiments, the anti-CD117 antibodies described herein comprise the amino acid sequence of L235A, L235A, and D265C ( The antibodies of the present invention may further be engineered to contain an Fc region comprising a nucleotide sequence similar to that of the Dall'Ace (EU index). cqua et al. (2006) J Biol Chem 281: 23514-24), (Zalevsky et al. (2010) Nat Biotechno l 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), (Petkov a et al. (2006) Int Immunol 18: 1759-69), (Datta-Mannan et al. (2007) Drug Metab Dis pos 35: 86-94), (Vaccaro et al. (2005) Nat Biotechnol 23: 1283-8), (Yeung et al. 2010) Cancer Res 70: 3269-77) and (Kim et al. (1999) Eur J Immunol 29: 2819-25) The half-life of the antibody can be further tuned by introducing additional Fc mutations, such as the Fc mutations described above. and positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434 and 435 Exemplary mutations that may be made alone or in combination include T250Q, M252Y , 1253A, S254T, T256E, P2571, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N The mutations are 434H, N434F, H435A and H435R.

[0208] Thus, in one embodiment, the Fc region comprises a mutation that results in a decreased half-life. An antibody with a half-life (also referred to herein as a "fast" half-life) is one that has a short-lived In certain instances (e.g., administering an antibody, (In the conditioning step described herein, followed by administration of HSCs) Ideally, unlike endogenous stem cells, they generally express CD117. The antibody is administered prior to administration to HSCs that have been treated with anti-CD117 antibodies but are not the target of the antibodies. In one embodiment, the Fc region comprises a nucleotide sequence at position 435. (EU index according to Kabat). In one embodiment, the mutation is a H435A mutation. In another embodiment, the mutation is a D265C mutation. The mutations are H435A and D265C.

[0209] In certain embodiments, the anti-CD117 antibodies described herein are administered for 24 hours or less, for 22 hours or less, 20 hours or less, 18 hours or less, 16 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less Half of the following: 10 hours or less, 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 half-life of the antibody is 5 to 7 hours, 5 to 9 hours, 15 to 20 hours, or 25 to 30 hours. Hours - 11 hours, 5 hours - 13 hours, 5 hours - 15 hours, 5 hours - 20 hours, 5 hours - 24 hours, 7 hours ~24 hours, 9 hours to 24 hours, 11 hours to 24 hours, 12 hours to 22 hours, 10 hours to 20 hours, 8 hours or more 18 hours, or 14 to 24 hours.

[0210] Antibodies that may be used in conjunction with the methods of patient conditioning described herein Anti-CD117 antibodies include those described, for example, in U.S. Pat. No. 5,489,516 (the disclosure of which relates to anti-CD117 antibodies). ATCC Accession Nos. 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 No. 10716 (deposited as BA7.3C.9) R-1 antibody, etc.

[0211] 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).

[0212] 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 disclosed herein) (which includes, for example, the anti-CD117 antibody CK6 (herein: Ab4): CDR-H1 having the amino acid sequence SYWIG (SEQ ID NO: 1); CDR-H2 having the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 2); CDR-H3 having the amino acid sequence HGRGYNGYEGAFDI (SEQ ID NO:3); CDR-L1 having the amino acid sequence RASQGISSALA (SEQ ID NO: 4); CDR-L2 having the amino acid sequence DASSLES (SEQ ID NO: 5); and CDR-L3 having the amino acid sequence CQQFNSYPLT (SEQ ID NO: 6) ) are listed.

[0213] The heavy chain variable region amino acid sequence of CK6 is provided in SEQ ID NO: 27: [ka]

[0214] The light chain amino acid variable sequence of CK6 is provided in SEQ ID NO: 28: [ka]

[0215] 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.

[0216] The disclosure of each of the foregoing publications relates to anti-CD117 antibodies and is incorporated herein by reference. Antibodies and antigens that can be used in conjunction with the compositions and methods described herein Binding fragments include the above-mentioned antibodies and antigen-binding fragments thereof, as well as the above-mentioned non-antibodies. - humanized variants of human antibodies and antigen-binding fragments, and (e.g., competitive CD117 Antibodies or antigens that bind to the same epitopes as those listed above (as assessed by binding assays) Binding fragments are included.

[0217] Exemplary antigen-binding fragments of the antibody include, inter alia, bivariable immunoglobulins (BIVIGs), dual-variable immunoglobulin domain, single-chain Fv molecule (scFv), diabodies Dibodies, triabodies, nanobodies, antibody-like protein scaffolds, Fv fragments , Fab fragments, F(ab')2 molecules, and tandem di-scFv.

[0218] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, the anti-CD117 antibodies described herein may be produced using Such nucleic acids are provided which encode the amino acid sequence comprising the VL of the antibody. The amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or the heavy chain of the antibody) In a further embodiment, one or more vectors containing such nucleic acids may be used. In a further embodiment, a nucleic acid (e.g., an expression vector) is provided. In some such embodiments, the host cell comprises (e.g., For example, it has been transformed with: (1) an amino acid sequence containing the VL of the present antibody and the VH of the present antibody (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 a sequence and a second vector containing a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic organism, e.g., a mammalian cell, e.g., a mammalian cell. Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells) In one embodiment, a method of making an anti-CLL-1 antibody is provided, wherein the method comprises: a host cell comprising nucleic acid encoding an antibody as provided above, suitable for expression of said antibody; and optionally culturing said host cells (or host cell culture medium) under the conditions specified above. and recovering the antibody from the culture medium.

[0219] To recombinantly produce an anti-CD117 antibody, a nucleic acid encoding the antibody (e.g., as described above) is prepared. Such nucleic acids are isolated and further cloned and / or expressed in host cells. Such nucleic acids can be inserted into one or more vectors using conventional procedures (e.g., For example, oligonucleotides capable of specifically binding to the genes encoding the heavy and light chains of the antibody. can be easily isolated and sequenced (by using probes) This can be done.

[0220] Suitable host cells for cloning or expressing antibody-encoding vectors include Examples include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in various cultures, particularly in When glycosylation and Fc effector function are not required, it can be produced in bacteria. For the expression of antibody fragments and polypeptides in bacteria, see See, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (Charlton, Met. hods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 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. It may be isolated as a soluble fraction and further purified.

[0221] 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 Human cervical cancer cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat Liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse breast cancer (MMT 06056 2); TRI cells (described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982) Other useful mammalian host cell lines include: Chinese hamster ovary (CHO) cells (DHFR-CHO cells (Urlaub et al., Proc. Natl. A cad. Sci. USA 77:4216 (1980)); and myeloma cell lines (e.g., Y0, NS0, and Sp2 / 0, etc.) are included in the review of certain mammalian host cell lines suitable for antibody production. For more information, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BK C Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0222] In one embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, is Amino acid sequences that are at least 95%, 96%, 97% or 99% identical to the disclosed SEQ ID Nos. or the anti-CD117 antibody, or antigen-binding fragment thereof, comprises a variable region having a sequence. is at least 95%, 96%, 97% or 99% identical to a SEQ ID No. disclosed herein. The present invention provides a method for the preparation of a medicament ... The CDRs contain the SEQ ID Nos. disclosed herein.

[0223] 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. wherein the anti-CD117 antibody, or antigen-binding fragment thereof, is an amino acid sequence disclosed herein. In yet another embodiment, the light chain comprises a light chain variable region and a light chain constant region having the sequence The anti-CD117 antibody, or antigen-binding fragment thereof, has the amino acid sequence disclosed herein. The antibody comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region, each of which corresponds to a specific region.

[0224] Further anti-CD117 antibodies are described in US 2019 / 0153114 A1 and US 2019 / 0144558 A1, both of which are incorporated herein by reference. The contents of this application are expressly incorporated herein by reference in their entirety.

[0225] The anti-CD117 antibodies and ADCs described herein are useful for, among other things, targeting cell types in the hematopoietic lineage. for treating various disorders such as cancer, autoimmune diseases, metabolic disorders, and stem cell disorders The compositions and methods described herein may be used in methods for (i) treating cancer cells Pathological conditions, such as populations of cells (e.g., leukemia cells) and autoimmune cells (e.g., autoreactive T-cells) and / or (ii) directly reduce the population of cells that cause the transplanted cells. by providing a niche to which the cells can home. The endogenous hematopoietic stem cell population can be reduced so that engraftment is promoted. The activity is directed against antigens expressed by disease-causing endogenous cells, autoimmune cells, or hematopoietic stem cells. by administering an ADC, antibody, or antigen-binding fragment thereof capable of binding to the When directly treating a disease, this administration reduces the amount of cells that cause the pathology of interest. In cases where patients are being prepared for hematopoietic stem cell transplantation, This administration selectively reduces the endogenous hematopoietic stem cell population, thereby A vacancy is created in the hematopoietic tissue, such as bone marrow, which is then filled by the transplanted exogenous hematopoietic cells. The present invention relates to a method for detecting GNNK+ CD117-binding proteins, which may be filled with blood stem cells. The ADC, antibody, or antigen-binding fragment thereof is administered to a patient to achieve both of the above activities. This is based in part on the discovery that ADCs, antibodies, or The antigen-binding fragments are used to directly reduce the population of cancerous or autoimmune cells. It may be administered to patients with cancer or autoimmune diseases, as well as to patients undergoing transplantation. To promote the survival and engraftment of transplanted hematopoietic stem cells, hematopoietic stem cell transplantation therapy is recommended. It may also be administered to patients in need.

[0226] Hematopoietic stem cell transplantation by administration of anti-CD117 ADC, antibody, or antigen-binding fragment thereof The engraftment of the cells can be demonstrated in various experimental measurements. The engraftment of transplanted hematopoietic stem cells is confirmed by the use of ADCs, antibodies, or their derivatives that can bind to CD117. and administering the antigen-binding fragment and subsequently administering a hematopoietic stem cell transplant. The amount of competitive repopulating units (CRUs) present in the bone marrow Furthermore, the donor's hematopoietic stem cells can be transferred to the The vector to be injected catalyzes a chemical reaction that produces a fluorescent, colored, or luminescent product. A reporter gene for an enzyme that encodes a reporter gene for the gene is then inserted into the bone marrow or other tissues. The migration of hematopoietic stem cells can be monitored by monitoring the hematopoietic stem cells in the tissue to which they have homed. It can be observed that the plants take root. By fluorescence activated cell sorting (FACS) analysis method By assessing the quantity and viability of hematopoietic stem and progenitor cells, It is also possible to observe the engraftment of hematopoietic stem cells. Engraftment can also be observed during the post-transplant period. by measuring white blood cell counts in peripheral blood and / or donor bone marrow aspirate samples. This can also be measured by measuring the recovery rate of bone marrow cells by the erythrocytes.

[0227] Anti-CD2 antibody Human CD2 is a T cell surface antigen, T11 / Leu-5, T11, CD2 antigen (p50), and sheep red blood cell receptor. CD2 is also called the Sheep Red Blood Cell Receptor (SRBC). Two isoforms of human CD2 have been identified. Isoform 1 has 351 amino acids. Seed, B. et al. (1987) 84: 3365-69 (Sewell et al. (1986) 83: 8718-22 See also ) and below (NCBI reference sequence: NP_001758.2). [ka]

[0228] The second isoform of CD2 is 377 amino acids and is referred to herein as the NCBI reference sequence: NP_0013 Identified as 15538.1.

[0229] In some embodiments, anti-cancer drugs that may be used in conjunction with the compositions and methods described herein include: -CD2 antibodies include those with one or more, or all, of the following CDRs: a. CDR-H1 having the amino acid sequence EYYMY (SEQ ID NO: 30); b. CDR-H2 having the amino acid sequence RIDPEDGSIDYVEKFKK (SEQ ID NO: 31); c. CDR-H3 having the amino acid sequence GKFNYRFAY (SEQ ID NO: 32); d. CDR-L1 having the amino acid sequence RSSQSLLHSSGNTYLN (SEQ ID NO: 33); e. CDR-L2 having the amino acid sequence LVSKLES (SEQ ID NO: 34); and f. CDR-L3 having the amino acid sequence MQFTHYPYT (SEQ ID NO: 35).

[0230] In one embodiment, the anti-CD2 antibody, or antigen-binding portion thereof, has the sequence set forth in SEQ ID NO: 36 and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 37. The light chain variable region includes the following:

[0231] In some embodiments, anti-cancer drugs that may be used in conjunction with the compositions and methods described herein include: -CD2 antibodies include those with one or more, or all, of the following CDRs: a. CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 38); b. CDR-H2 having the amino acid sequence SGGGF (SEQ ID NO: 39); c. CDR-H3 having the amino acid sequence SSYGEIMDY (SEQ ID NO: 40); d. CDR-L1 having the amino acid sequence RASQRIGTSIH (SEQ ID NO: 42); e. CDR-L2 having the amino acid sequence YASESIS (SEQ ID NO: 43); and f. CDR-L3 having the amino acid sequence QQSHGWPFTF (SEQ ID NO: 44).

[0232] In one embodiment, the anti-CD2 antibody, or antigen-binding portion thereof, has the sequence set forth in SEQ ID NO: 45 and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 47. The light chain variable region includes the following:

[0233] In another embodiment, it can be used in conjunction with the compositions and methods described herein. Anti-CD2 antibodies include those having one or more, or all, of the following CDRs: a. CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 38); b. CDR-H2 having the amino acid sequence SGGGF (SEQ ID NO: 39); c. CDR-H3 having the amino acid sequence SSYGELMDY (SEQ ID NO: 41); d. CDR-L1 having the amino acid sequence RASQRIGTSIH (SEQ ID NO: 42); e. CDR-L2 having the amino acid sequence YASESIS (SEQ ID NO: 43); and f. CDR-L3 having the amino acid sequence QQSHGWPFTF (SEQ ID NO: 44).

[0234] In one embodiment, the anti-CD2 antibody, or antigen-binding portion thereof, has the sequence set forth in SEQ ID NO: 46 and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 47. The light chain variable region includes the following:

[0235] Antibodies and antigen-binding fragments thereof comprising the above CDR sequences are described, for example, in U.S. Pat. No. 6,849 ,258, which relates to anti-CD2 antibodies and antigen-binding fragments thereof. No. 6,239,999, the disclosure of which is incorporated herein by reference.

[0236] Furthermore, in certain embodiments, the anti-CD2 ADCs are characterized by serum CD4+ levels in human subjects for 3 days or less. It has a half-life.

[0237] Additional sequences for the anti-CD2 antibodies or binding fragments described herein are listed in Table 5. Write it down.

[0238] Additional anti-CD2 antibodies that may be used in the compositions and methods described herein, including Antigen-binding fragments of the antibodies, or ADCs, can be produced by techniques known in the art, such as hybridoma production. Hybridomas may be prepared using a mouse system. Protocol for isolating spleen cells for immunization and subsequent fusion Fusion partners and procedures for producing hybridomas are known in the art. Alternatively, HuMAb-Mouse 登録商標 or XenoMouse TM Using anti-CD2 In producing further anti-CD2 antibodies, the CD2 antigen may be isolated and and / or purified. The CD2 antigen is a fragment of CD2 derived from the extracellular domain of CD2. Immunization of animals can be carried out by any method known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring See Harbour Press, 1990. Mice, rats, sheep, goats, pigs, cattle and Methods for immunizing animals, such as horses, are well known in the art. See, e.g., Harlow and See Lane, supra, and U.S. Patent No. 5,994,619. The CD2 antigen may be used to stimulate an immune response. In order to achieve this, the vaccine may be administered with an adjuvant. Complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or IS COM (immunostimulating complex). After immunizing animals with CD2 antigen, they are isolated from the immunized animals. Immortalized antibody-producing cell lines are prepared from the immunized cells. After immunization, the animals are sacrificed and Lymph node and / or splenic B cells can be isolated by methods known in the art (e.g., cancer gene transfer, cancer Viral transduction, exposure to carcinogenic or mutagenic compounds, immortalized cells (e.g., bone marrow) They are immortalized by fusion with myeloma cells and inactivation of tumor suppressor genes. See, e.g., Harlow and Lane, supra. Hybridomas are cultured to identify desired characteristics (e.g., robust growth, Select for specific characteristics, including high growth, high antibody production, and desirable antibody properties, may be subjected to further screening.

[0239] Anti-CD2 antibodies for use in the anti-CD2 ADCs described herein also bind to CD2. A library of antibodies or antibody fragments is screened in high-throughput screening for potential molecules. Such methods include those known in the art. In vitro display techniques known in the art, such as, inter alia, phage display , bacterial display, yeast display, mammalian cell display, ribosomal These include RNA display, mRNA display, and cDNA display. To isolate antibodies, antigen-binding fragments, or ligands that bind to biologically relevant molecules, The use of phage display for this purpose has been described, for example, by Felici et al., Biotechnol. A nnual Rev. 1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4:1-20, 1998. Each of these disclosures is incorporated herein by reference as it relates to in vitro display technology. Kay, Perspective. Drug Discovery Des. 2:251-268, 1995, incorporated herein by reference. and randomized as described in Kay et al., Mol. Divers. 1:139-140, 1996. Combinatorial peptide libraries are constructed to identify polypeptides that bind to cell surface antigens. Each of these disclosures has been directed to the discovery of antigen-binding molecules. The present invention is incorporated by reference herein as it relates thereto. Phage display of functional molecules (e.g., endothelial proteins) has been successfully achieved. (e.g., EP 0349578; EP 4527839; and EP 0589877, and Chiswell and M See Cafferty, Trends Biotechnol. 10:80-84 1992, each of which discloses antigen binding. The present specification relates to the use of in vitro display technology to discover synthetic molecules. In addition, functional antibody fragments (e.g., Fab and scFv fragments) can be used. It has also been demonstrated that the expression of these proteins in an in vitro display format (e.g., McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-62 8, 1991. Each of these disclosures describes in vitro detection of antigen-binding molecules. (These terms are incorporated herein by reference as they relate to the Play Platform) .

[0240] In addition to in vitro display techniques, procedures described in, for example, US 2013 / 0288373 may be used. While using computational modeling techniques, anti-CD2 antibodies or antibody fragments were analyzed in silico (The disclosure provides molecular methods for identifying anti-CD2 antibodies.) (These are incorporated herein as modeling methods.) For example, computational modeling Using genomic DNA techniques, one skilled in the art can identify specific epitopes on CD2, such as extracellular epitopes of CD2. A library of antibodies or antibody fragments is screened in search of molecules that can bind to the antibody. Screening may be done in silico.

[0241] In some embodiments, the anti-CD2 antibodies used in the ADCs described herein are capable of expressing CD2 in cells. When identifying anti-CD2 antibodies (or fragments thereof), further The technique is used to bind to CD2 on the surface of cells (e.g., T cells) and further bind to receptors such as antibodies or antigens that can be taken up by the cells by antibody-mediated endocytosis For example, the in vitro display techniques described above may be used to identify binding fragments. The antibody or its antigen-binding fragment that binds to CD2 on the surface of hematopoietic stem cells and subsequently is internalized. Phage detection is often used to screen for specific fragments. Play techniques such as these may be used in conjunction with this screening paradigm This technique is representative of anti-CD2 antibodies that bind to CD2 and are subsequently internalized into CD2+ cells. To identify the fragment, one skilled in the art can use the method of Williams et al., Leukemia 19 :1432-1438, 2005 (the phage display technology described in the disclosure of which is incorporated herein by reference in its entirety).

[0242] The amount of anti-CD2 antibody or fragment thereof that can be incorporated into a given antibody can be determined, for example, by methods known in the art. For example, the radionuclide internalization assays described herein may be used to assess the radionuclide uptake. Anti-CD2 antibodies described herein or identified using in vitro display techniques known in the art. or fragments thereof, can be converted to radioisotopes (e.g., 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I , 125 I, 129 I, 131 I, 211 At, 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu, 177 Lu, 7 7 As, 72 As, 86 Y, 90 Y, 89 Zr, 212 Bi, 213 Bi, or 225 By incorporating Ac), For example, radioactive halogens (e.g., 18 F, 75 Br, 77 Br, 122 I, 12 3 I, 124 I, 125 I, 129 I, 131 I, 211 At) onto beads containing electrophilic halogen reagents (e.g. polystyrene beads) (e.g., iodinated beads, Thermo Fisher Scientific, Inc. ., Cambridge, MA) to incorporate into antibodies, their fragments, or ligands. Radiolabeled antibodies or their fragments may be incorporated into The antibody may be incubated with hematopoietic stem cells for a sufficient period of time. or a fragment thereof, and detecting radiation (e.g., gamma rays) emitted from the obtained hematopoietic stem cells. The radiation emitted from the washed buffer is compared with that emitted from the washed buffer (e.g., gamma rays). The internalization assays described above may also be used to characterize ADCs.

[0243] In some embodiments, the anti-CD2 antibody (or fragment thereof) is a defined serum For example, an anti-CD2 antibody (or fragment thereof) has a half-life of about ADCs containing such anti-CD2 antibodies may also be used, e.g., in combination with anti-CD2 antibodies, which may have a serum half-life of 1 to 24 hours. For example, it may have a serum half-life of about 1 to 24 hours in human patients. Pharmacokinetic analysis by measuring the amount of hydroxybenzoates may be performed by assays known in the art. .

[0244] To recombinantly produce an anti-CD2 antibody, nucleic acid encoding the antibody (e.g., as described above) is prepared. (such as) are isolated and further cloned and / or expressed in host cells. Such nucleic acids can be inserted into one or more vectors using conventional procedures (e.g., and an oligonucleotide capable of specifically binding to genes encoding the heavy and light chains of the antibody. Can be easily isolated and sequenced (by using probes) can be done.

[0245] Suitable host cells for cloning or expressing antibody-encoding vectors include Examples include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in various cultures, particularly in When glycosylation and Fc effector function are not required, it can be produced in bacteria. For the expression of antibody fragments and polypeptides in bacteria, see See, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (Charlton, Met. hods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 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. It may be isolated as a soluble fraction and further purified.

[0246] 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 Human cervical cancer cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat Liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse breast cancer (MMT 06056 2); TRI cells (described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982) Other useful mammalian host cell lines include: Chinese hamster ovary (CHO) cells (DHFR-CHO cells (Urlaub et al., Proc. Natl. A cad. Sci. USA 77:4216 (1980)); and myeloma cell lines (e.g., Y0, NS0, and Sp2 / 0, etc.) are included in the review of certain mammalian host cell lines suitable for antibody production. For more information, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BK C See, for example, Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell. or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0247] Anti-CD5 antibody Human CD5 is also known as lymphocyte antigen T1, T1, Leu-1, and LEU1. CD5 is expressed on human T cells. Two isoforms of human CD5 have been identified. Isoform 1 is , containing 495 amino acids, Gladkikh et al (2017) Cancer Med.6(12):2984 and Jones et al. (1986) Nature 323 (6086): 346). The sequence is shown below (NCBI reference sequence: NP_055022.2): mpmgslqpla tlyllgmlva sclgrlswyd pdfqarltrs nskcqgqlev ylkdgwhmvc sqswgrsskq wedpsqaskv cqrlncgvpl slgpflvtyt pqssiicygq lgsfsncshs rndmchslgl tclepqkttp pttrpppttt peptapprlq lvaqsggqhc agvvefysgs lggtisyeaq dktqdlenfl cnnlqcgsfl khlpeteagr aqdpgepreh qplpiqwkiq nssctslehc frkikpqksg rvlallcsgf qpkvqsrlvg gssicegtve vrqgaqwaal cdsssarssl rweevcreqq cgsvnsyrvl dagdptsrgl fcphqklsqc helwernsyc kkvfvtcqdp npaglaagtv asiilalvll vvllvvcgpl aykklvkkfr qkkqrqwigp tgmnqnmsfh rnhtatvrsh aenptashvd neysqpprns hlsaypaleg alhrssmqpd nssdsdydlh gaqrl (SEQ ID NO: 48) (SEQ ID NO: 48).

[0248] The second isoform of human CD5 (SEQ ID NO: 399) is 438 amino acids (see above). (See underlined portion) and identified as NCBI reference sequence: NP_00133385.1. Isoform 1 Unlike CD5, CD5 isoform 2 is an intracellular protein. Compared to isoform 1, it contains a different 5' UTR and has an identical 5' coding region. The resulting isoform 2 lacks the N-terminal in-frame region compared to isoform 1. CD5 isoform 2 has a shorter leader peptide than isoform 1. and intracellular isoforms found in a subset of B lymphocytes. The ADC is specific for human CD5 isoform 1, which represents the extracellular version of human CD5.

[0249] In some embodiments, an anti-inflammatory drug may be used in the methods and compositions described herein. The CD5 antibody is antibody 5D7v (Ab5D7v). The heavy chain variable region (VH) amino acid sequence of Ab5D7v is as follows: Shown in sequence number (SEQ ID NO): 49. [ka]

[0250] The VH CDR amino acid sequence of Ab5D7v is underlined above and is as follows: FSLSTSGMG(VH CDR DR1; SEQ ID NO: 51); WWDDD (VH CDR2; SEQ ID NO: 52); and RRATG TGFDY (VH CDR3; SEQ ID NO: 53).

[0251] The light chain variable region (VL) amino acid sequence of Ab5D7v is provided below as SEQ ID NO: 50. Provide. [ka]

[0252] The VL CDR amino acid sequence of Ab5D7v is underlined above and is as follows: QDVGTA(VL CDR1 WTSTRHT (VL CDR2; SEQ ID NO: 55); and YNSYN T(VL CDR3; SEQ ID NO: 56).

[0253] In one embodiment, the anti-CD5 ADC comprises the amino acid sequence set forth in SEQ ID NO: 51. a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 52; and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 52. and a heavy chain comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 53; and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 54, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 55, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 56; and a light chain comprising a CDR3 domain comprising the amino acid sequence set forth in The antibody is conjugated to a cytotoxin via a linker.

[0254] In one embodiment, the anti-CD5 ADC comprises the amino acid sequence set forth in SEQ ID NO: 49. and a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 50. and a light chain comprising a variable region, wherein the antibody is linked to a cell via a linker. Conjugated to a cytotoxin.

[0255] In another embodiment, the anti-CD5 antibody used in the ADCs described herein is the 5D7 antibody. (See, e.g., US 20080254027, the disclosure of which is incorporated herein by reference. In another embodiment, the compounds used in the methods and compositions (including ADCs) described herein are Anti-CD5 antibodies that may be used are variants of the 5D7 antibody (see, e.g., US 20080254027, the disclosure of which is incorporated herein by reference).

[0256] Furthermore, in certain embodiments, the anti-CD5 ADCs are characterized by serum CD5+ levels in human subjects for 3 days or less. It has a half-life.

[0257] Additional sequences for the anti-CD5 antibodies or binding fragments described herein are listed in Table 5. Write it down.

[0258] Additional anti-CD5 antibodies that may be used in the ADCs described herein include hybrid The antibody may be identified using techniques known in the art, such as hybridoma production. may be prepared using the mouse system for immunization and subsequent fusion. Protocols for isolating splenocytes are known in the art. Fusion partners and procedures for this purpose are also known. 登録商標 or XenoMouse TM may be used to generate anti-CD5 antibodies. In preparing the antibody, the CD5 antigen is isolated and / or purified. The immunization of animals can be carried out using methods known in the art. This can be done by any method, see, for example, Harlow and Lane, Antibodies: A Labora See the Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing animals such as rats, sheep, goats, pigs, cattle and horses are well known in the art. See, e.g., Harlow and Lane, supra, and U.S. Pat. No. 5,994,619. The CD5 antigen may be administered with an adjuvant to stimulate the immune response. Adjuvants known in the art include complete and incomplete Freund's adjuvant. , RIBI (muramyl dipeptide) or ISCOM (immunostimulating complex). After immunization, antibody-producing immortalized cell lines are prepared from cells isolated from the immunized animal. After vaccination, the animals are sacrificed and lymph node and / or splenic B cells are isolated using methods known in the art. methods (e.g., oncogene transduction, oncogenic virus transduction, carcinogenesis or mutagenesis) Exposure to compounds, fusion with immortalized cells (e.g., myeloma cells), and the expression of tumor suppressor genes See, e.g., Harlow and Lane, supra. The cells are cultured to produce the desired characteristics (e.g., robust growth, high antibody production, and desired antibody characteristics). The resulting clones may be selected, cloned and further screened.

[0259] Anti-CD5 antibodies for use in the anti-CD5 ADCs described herein also bind to CD5. A library of antibodies or antibody fragments is screened in high-throughput screening for potential molecules. Such methods include those known in the art. In vitro display techniques known in the art, such as, inter alia, phage display , bacterial display, yeast display, mammalian cell display, ribosomal These include RNA display, mRNA display, and cDNA display. To isolate antibodies, antigen-binding fragments, or ligands that bind to biologically relevant molecules, The use of phage display for this purpose has been described, for example, by Felici et al., Biotechnol. A nnual Rev. 1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4:1-20, 1998. Each of these disclosures is incorporated herein by reference as it relates to in vitro display technology. Kay, Perspective. Drug Discovery Des. 2:251-268, 1995, incorporated herein by reference. and randomized as described in Kay et al., Mol. Divers. 1:139-140, 1996. Combinatorial peptide libraries are constructed to identify polypeptides that bind to cell surface antigens. Each of these disclosures has been directed to the discovery of antigen-binding molecules. The present invention is incorporated by reference herein as it relates thereto. Phage display of functional molecules (e.g., endothelial proteins) has been successfully achieved. (e.g., EP 0349578; EP 4527839; and EP 0589877, and Chiswell and M See Cafferty, Trends Biotechnol. 10:80-84 1992, each of which discloses antigen binding. The present specification relates to the use of in vitro display technology to discover synthetic molecules. In addition, functional antibody fragments (e.g., Fab and scFv fragments) can be used. It has also been demonstrated that the expression of these proteins in an in vitro display format (e.g., McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-62 8, 1991, each of which discloses in vitro display methods for the discovery of antigen-binding molecules. (The same is incorporated herein by reference as it relates to the platform.)

[0260] In addition to in vitro display techniques, procedures described in, for example, US 2013 / 0288373 may be used. While using computational modeling techniques, anti-CD5 antibodies or antibody fragments were analyzed in silico (The disclosure provides a molecular design for identifying anti-CD5 antibodies.) (These are incorporated herein by reference as they relate to modeling methods.) For example, computational modeling Using genomic DNA techniques, one skilled in the art can identify specific epitopes on CD5, such as extracellular epitopes of CD5. A library of antibodies or antibody fragments is screened in search of molecules that can bind to the antibody. Screening may be done in silico.

[0261] In some embodiments, the anti-CD5 antibodies used in the ADCs described herein are capable of inhibiting intracellular When identifying anti-CD5 antibodies (or fragments thereof), further The technique is used to bind to CD5 on the surface of cells (e.g., T cells) and further bind to receptors such as antibodies or antigens that can be taken up by the cells by antibody-mediated endocytosis For example, the in vitro display techniques described above may be used to identify binding fragments. The antibody or its antigen-binding fragment that binds to CD5 on the surface of hematopoietic stem cells and subsequently is internalized. Phage detection is often used to screen for specific fragments. Play techniques such as these may be used in conjunction with this screening paradigm This technique is representative of anti-CD5 antibodies that bind to CD5 and are subsequently internalized into CD5+ cells. To identify the fragment, one skilled in the art can use the method of Williams et al., Leukemia 19 :1432-1438, 2005 (the phage display technology described in the disclosure of which is incorporated herein by reference in its entirety).

[0262] The amount of anti-CD5 antibody or fragment thereof that can be incorporated into a given antibody can be determined, for example, by methods known in the art. For example, the radionuclide internalization assays described herein may be used to assess the radionuclide uptake. Anti-CD5 antibodies described herein or identified using in vitro display techniques known in the art. or fragments thereof, can be converted to radioisotopes (e.g., 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I , 125 I, 129 I, 131 I, 211 At, 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu, 177 Lu, 7 7 As, 72 As, 86 Y, 90 Y, 89 Zr, 212 Bi, 213 Bi, or 225 By incorporating Ac), For example, radioactive halogens (e.g., 18 F, 75 Br, 77 Br, 122 I, 12 3 I, 124 I, 125 I, 129 I, 131 I, 211At) onto beads containing electrophilic halogen reagents (e.g. polystyrene beads) (e.g., iodinated beads, Thermo Fisher Scientific, Inc. ., Cambridge, MA) to incorporate into antibodies, their fragments, or ligands. Radiolabeled antibodies or their fragments may be incorporated into The antibody may be incubated with hematopoietic stem cells for a sufficient period of time. or a fragment thereof, and detecting radiation (e.g., gamma rays) emitted from the obtained hematopoietic stem cells. The radiation emitted from the washed buffer is compared with that emitted from the washed buffer (e.g., gamma rays). The internalization assays described above may also be used to characterize ADCs.

[0263] In some embodiments, the anti-CD5 antibody (or fragment thereof) is a defined serum For example, an anti-CD5 antibody (or fragment thereof) has a half-life of about ADCs containing such anti-CD5 antibodies may also be used, e.g., in combination with anti-CD5 antibodies, which may have a serum half-life of 1 to 24 hours. For example, it may have a serum half-life of about 1 to 24 hours in human patients. Pharmacokinetic analysis by measuring the amount of hydroxybenzoates may be performed by assays known in the art. .

[0264] To recombinantly produce an anti-CD5 antibody, nucleic acid encoding the antibody (e.g., the nucleic acid sequences described above) can be prepared. (such as) are isolated and further cloned and / or expressed in host cells. Such nucleic acids can be inserted into one or more vectors using conventional procedures (e.g., and an oligonucleotide capable of specifically binding to genes encoding the heavy and light chains of the antibody. Can be easily isolated and sequenced (by using probes) can be done.

[0265] Suitable host cells for cloning or expressing antibody-encoding vectors include Examples include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in various cultures, particularly in When glycosylation and Fc effector function are not required, it can be produced in bacteria. For the expression of antibody fragments and polypeptides in bacteria, see See, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (Charlton, Met. hods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 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. It may be isolated as a soluble fraction and further purified.

[0266] 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 Human cervical cancer cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat Liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse breast cancer (MMT 06056 2); TRI cells (described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982) Other useful mammalian host cell lines include: Chinese hamster ovary (CHO) cells (DHFR-CHO cells (Urlaub et al., Proc. Natl. A cad. Sci. USA 77:4216 (1980)); and myeloma cell lines (e.g., Y0, NS0, and Sp2 / 0, etc.) are included in the review of certain mammalian host cell lines suitable for antibody production. For more information, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BK C See, for example, Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell. or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0267] In some embodiments, it may be used in conjunction with the compositions and methods described herein. Some anti-CD5 antibodies include CDR-H1, CDR-H2, CDR-H3, and CDR-L1 listed in Tables 1 and 2 below. , CDR-L1, and CDR-L2 regions.

[0268] [Table 1-1] [Table 1-2] [Table 1-3]

[0269] [Table 2-1] [Table 2-2]

[0270] Anti-CD137 antibody CD137 is also known as CDw137, TNFRSF9, 4-1BB, and ILA. The recombinant fragments and their ADCs can be used as therapeutic agents to treat GVHD or autoimmune diseases. Preventing hematopoietic stem cell-mediated GVHD in patients suffering from or at risk of having the disease. Furthermore, ligands that bind to CD137 (e.g., human CD137L) may be used for treatment. ) as a therapeutic agent to prevent and treat patients suffering from or at risk of GVHD. These ligands (e.g., soluble human CD137) can be used to prevent and treat For example, to promote antibody-dependent cell-mediated cytotoxicity (ADCC), effector domains The antibody may be covalently linked to a polypeptide (e.g., an Fc domain).

[0271] T cells have been shown to express CD137, an antigen that binds to the transmembrane TNF receptor, a costimulatory molecule. It is a member of the Receptor superfamily and is expressed on various hematopoietic cells and activates T cells. promotes activation and regulates T cell proliferation and survival (see, e.g., Cannons et al., J.I. See, e.g., Munol. 167:1313-1324, 2001. This disclosure relates to the expression of CD137 by T cells. (The disclosure of which is incorporated herein by reference.) Antibodies and antigen-binding fragments thereof The antibody can be administered by techniques known in the art and described herein (e.g., immunization, computer modeling techniques, and in vitro selection methods (e.g., phage selection described below) display and cell-based display platforms) Therefore, identification may be possible.

[0272] The methods disclosed herein can be used to prevent and treat GVHD or autoimmune diseases. Anti-CD137 antibodies that may be used for this purpose include those having one or more or all of the following CDRs: These include: a. CDR-H1 having the amino acid sequence STYWIS (SEQ ID NO: 278); b. CDR-H2 having the amino acid sequence KIYPGDSYTNYSPSFQG (SEQ ID NO: 279); c. CDR-H3 having the amino acid sequence RGYGIFDY (SEQ ID NO: 280); d. CDR-L1 having the amino acid sequence SGDNIGDQYAH (SEQ ID NO: 281); e. CDR-L2 having the amino acid sequence QDKNRPS (SEQ ID NO: 282); and f. CDR-L3 having the amino acid sequence ATYTGFGSLAV (SEQ ID NO: 283).

[0273] The methods disclosed herein can be used to prevent and treat GVHD or autoimmune diseases. Additional anti-CD137 antibodies that may be used for this purpose include those having one or more or all of the following CDRs: Some of the features include: a. CDR-H1 having the amino acid sequence STYWIS (SEQ ID NO: 278); b. CDR-H2 having the amino acid sequence KIYPGDSYTNYSPSFQG (SEQ ID NO: 279); c. CDR-H3 having the amino acid sequence RGYGIFDY (SEQ ID NO: 280); d. CDR-L1 having the amino acid sequence SGDNIGDQYAH (SEQ ID NO: 281); e. CDR-L2 having the amino acid sequence QDKNRPS (SEQ ID NO: 282); and f. CDR-L3 having the amino acid sequence STYTFVGFTTV (SEQ ID NO: 284).

[0274] Additional anti-CD137 antibodies include those having one or more or all of the following CDRs: R: a. CDR-H1 having the amino acid sequence NSYAIS (SEQ ID NO: 285); b. CDR-H2 having the amino acid sequence GIIPGFGTANYAQKFQG (SEQ ID NO: 286); c. CDR-H3 having the amino acid sequence RKNEEDGGFDH (SEQ ID NO: 287); d. CDR-L1 having the amino acid sequence SGDNLGDYYAS (SEQ ID NO: 288); e. CDR-L2 having the amino acid sequence DDSNRPS (SEQ ID NO: 289); and f. CDR-L3 having the amino acid sequence QTWDGTLHFV (SEQ ID NO: 290).

[0275] Further anti-CD137 antibodies or ADCs include those having one or more or all of the following CDRs: Some examples include: a. CDR-H1 having the amino acid sequence SDYYMH (SEQ ID NO: 291); b. CDR-H2 having the amino acid sequence VISGSGSNTYYADSVKG (SEQ ID NO: 292); c. CDR-H3 having the amino acid sequence RLYAQFEGDF (SEQ ID NO: 293); d. CDR-L1 having the amino acid sequence SGDNIGSKYVS (SEQ ID NO: 294); e. CDR-L2 having the amino acid sequence SDSERPS (SEQ ID NO: 295); and f. CDR-L3 having the amino acid sequence QSWDGSISRV (SEQ ID NO: 296).

[0276] Such antibodies are described, for example, in U.S. Pat. No. 9,468,678, and these disclosures are incorporated herein by reference. - CD137 antibodies and antigen-binding fragments thereof, which are incorporated herein by reference. The antibodies and fragments thereof disclosed in U.S. Patent No. 9,468,678 are incorporated herein. It may be used in conjunction with the methods disclosed herein.

[0277] In another embodiment, an anti- The CD137 antibody was the mouse anti-CD137 antibody BBK2 (Thermo Fisher; MS621PABX) or the BBK2 antibody The BBK2 antibody (BBK-2 antibody or anti-CD137 antibody) is an anti-CD137 antibody containing an antigen-binding region corresponding to The human 4-1BB recombinant protein (also sometimes called BB antibody) is a 4-1BB antibody that binds to CD137. A mouse monoclonal antibody (IgG1, also known as C1) that binds to the ectodomain of In certain embodiments, the methods and compositions of the disclosure comprise a binding domain of a BBK2 antibody. In another embodiment, the methods and compositions of the disclosure include an anti-CD137 antibody comprising a CDR. The composition includes an antibody that competitively inhibits the binding of the BBK2 antibody to its epitope on CD137. In certain embodiments, the anti-CD137 antibody is humanized BBK2 or chimeric BBK2.

[0278] In some embodiments, the methods and compositions described herein comprise a method for targeting a BBK2 variable heavy chain region and a variable In certain embodiments, the antibody comprises a chimeric anti-CD137 (ch-BBK2) antibody comprising a light chain region. The chimeric BBK2 antibody is an IgG1 antibody containing a human constant region. The heavy chain amino acid sequence of ch-BBK2 is , set forth in SEQ ID NO: 297, and the light chain amino acid sequence of ch-BBK2 is set forth in the sequence The CDR regions (CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19 ...20, CDR21, CDR22, CDR23, CDR24, C and CDR3) are written below in bold, with variable regions in italics.

[0279] [ka]

[0280] [ka]

[0281] The aforementioned CDR regions (and the BBK2 antibody) are described in Lee et al. (2002) European Journal of Immunogenetic s 29(5):449-452. Thus, in one embodiment, the anti-CD137 antibody BBK2 (ch- The VH CDR amino acid sequences of the VH CDRs ... (SEQ ID NO: 299); NIYPSDSYT (VH CDR2; SEQ ID NO: 300) and TRNGVEGYPH YYAME (VH CDR3; SEQ ID NO: 301). VH of the anti-CD137 antibody BBK2 (including ch-BBK2). The amino acid sequence of the L CDR is as follows: SQDLSNH (VL CDR1; SEQ ID NO: 30 2); YYTS (VL CDR2; SEQ ID NO: 303) and CQQGYTLPY (VL CDR3; SEQ ID NO: 304) EQ ID NO):304).

[0282] Alternatively, the CDR regions of BBK2 may be defined by Kabat numbering. The CDRs defined by are set forth below for each of the heavy and light chain sequences (in bold below): The variable regions of BBK2 are in italics.

[0283] [ka]

[0284] [ka]

[0285] Thus, in one embodiment, the VH CDR amino acid sequences of the anti-CD137 antibody BBK2 (including ch-BBK2) are The columns are as follows: SYWIN (VH CDR1; SEQ ID NO: 305); NIYPSDSYTNYN QKFKD (VH CDR2; SEQ ID NO: 306) and NGVEGYPHYYAMEY (VH CDR3; SEQ ID NO: SEQ ID NO: 307), and the VL CDR amino acid sequence of the anti-CD137 antibody BBK2 (including ch-BBK2) is They are as follows: RASQDLSNHLY (VL CDR1; SEQ ID NO: 308); YTSRLHS (VL CDR2; SEQ ID NO: 309) and QQGYTLPYT (VL CDR3; SEQ ID NO: 310) ).

[0286] The heavy chain variable region of BBK2 is set forth in SEQ ID NO:311 as follows: QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSNTVY MQLNSPTSEDSAVYYCTRNGVEGYPHYYAMEYWGQGTSVTVSS . The light chain variable region of BBK2 is set forth in SEQ ID NO:312 as follows: DIQMTQTTSALSASLGDRVTIGCRASQDLSNHLYWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQ EDVATYFCQQGYTLPYTFGGGTKLEIK . Anti-CD137 antibodies (including anti-CD137 ADCs) are as set forth in SEQ ID Nos: 311 and 312. The heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence may each be do.

[0287] In one embodiment, an anti-CD137 antibody, such as a chimeric (ch-BBK2) antibody or a humanized BBK2 antibody, The antibody has a CDR1 comprising the amino acid sequence of SEQ ID NO: 305, a CDR2 comprising the amino acid sequence of SEQ ID NO: 306, and CDR3 comprising the amino acid sequence of SEQ ID NO: 307. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 308; and a CDR1 comprising the amino acid sequence of SEQ ID NO: 309. CDR2 comprising the amino acid sequence of SEQ ID NO: 309, and CDR3 comprising the amino acid sequence of SEQ ID NO: 310. and a light chain variable region comprising a CDR3 comprising the amino acid sequence.

[0288] In one embodiment, an anti-CD137 antibody, such as a chimeric (ch-BBK2) antibody or a humanized BBK2 antibody, The antibody has a CDR1 comprising the amino acid sequence of SEQ ID NO: 299, a CDR2 comprising the amino acid sequence of SEQ ID NO: 300, and CDR3 comprising the amino acid sequence of SEQ ID NO: 301. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 302; and a CDR1 comprising the amino acid sequence of SEQ ID NO: 303. CDR2 comprising the amino acid sequence of SEQ ID NO: 303, and CDR3 comprising the amino acid sequence of SEQ ID NO: 304. and a light chain variable region comprising a CDR3 comprising the amino acid sequence.

[0289] Therefore, BBK2, humanized BBK2, or chimeric BBK2 antibodies can be used in combination with the anti-CD137 ADCs described herein. Each of these antibodies may be used in methods known in the art. and conjugated to any of the cytotoxins described below using the methods described herein. This may cause the device to malfunction.

[0290] Additional sequences for the anti-CD137 antibodies or binding fragments described herein are listed in Table 5. Please write in.

[0291] Other anti-CD137 antibodies that may be used in conjunction with the cytotoxins described herein are described in the art. Techniques known in the art (e.g., hybridoma production) may be used to identify the hybridoma. Primoma may be prepared using a mouse system. Immunization and subsequent fusion Protocols for isolating splenocytes for hybridization are known in the art. Fusion partners and procedures for producing HuMAb-Mouse are also known. 登録商標 or XenoMouse TM To generate human anti-CD137 antibodies, In this case, the CD137 antigen is isolated and / or purified. The immunization of animals can be carried out using methods known in the art. For example, see Harlow and Lane, Antibodies: A Lab Oratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing animals such as rats, sheep, goats, pigs, cattle and horses are well known in the art. See, e.g., Harlow and Lane, supra, and U.S. Pat. No. 5,994,619. The CD137 antigen may be administered with an adjuvant to stimulate an immune response. Adjuvants known in the art include complete and incomplete Freund's ajuva Examples include RIBI (muramyl dipeptide) and ISCOM (immunostimulating complexes). After immunizing animals with the antibody, antibody-producing immortalized cell lines are prepared from cells isolated from the immunized animals. After immunization, the animal is sacrificed and lymph node and / or splenic B cells are isolated using methods known in the art. known methods (e.g., oncogene transduction, oncogenic virus transduction, oncogenic or mutagenic Exposure to virulent compounds, fusion with immortalized cells (e.g., myeloma cells), and tumor suppressors Immortalized by gene inactivation. See, for example, Harlow and Lane, supra. The clones are selected from clones that exhibit the desired characteristics (e.g., robust growth, high antibody production, and desired antibody characteristics). The resulting clones may be selected, cloned, and further screened. do.

[0292] Anti-CD137 antibodies can be prepared by the use of nucleic acids encoding the amino acid sequences of the CD137 binding molecules provided by the present invention. The nucleotide sequence may be generated from an isolated nucleic acid molecule containing the nucleotide sequence. The amino acid sequence to be encoded may be any part of an antibody (e.g., a CDR, including one, two, or three CDRs). The sequence may be a full-length heavy chain or a full-length light chain variable region. The nucleic acids of the present disclosure can be, for example, DNA or RNA, and can contain intron sequences. Typically, the nucleic acid is a cDNA molecule.

[0293] In addition to antibodies and antigen-binding fragments, water-soluble CD137 ligands, such as human CD137 ligand, are also available. and administering the compound to a patient according to the methods described herein to treat the patient prior to hematopoietic stem cell transplantation therapy. For example, CD137 ligands such as human CD137 ligand may be used to condition the patient. Conjugation of the antibody to another effector molecule, such as a cytotoxin or an Fc domain (e.g., according to methods described below or known in the art). Maytansine cytotoxins for use in conjunction with the methods described herein include, for example, maytansine cytotoxins. Human CD137 ligand-IgG1 Fc conjugate, human CD137 ligand-IgG2 Fc conjugate, Human CD137 ligand-IgG3 Fc conjugate, human CD137 ligand-IgG4 Fc conjugate , Human CD137 Ligand-IgA Fc Conjugate, Human CD137 Ligand-IgE Fc Conjugate , human CD137 ligand-IgM Fc conjugate, and human CD137 ligand-IgD Fc conjugate Examples include:

[0294] Antibodies and ligands for use in conjunction with the compositions and methods described herein include: , variants of these antibodies as described above, for example, antibody fragments containing or lacking an Fc domain. and humanized variants of the non-human antibodies described herein, and the antibodies described herein. of the CDRs or equivalent regions thereof of the antibody, antibody fragment, or soluble ligand to be used , one or more or all of the antibody-like protein scaffolds (e.g., 10 Fn3 domain) , etc.

[0295] Anti-CD252 antibody The present invention also provides the protein CD252 (also known as OX40 Ligand (OX40L)) from the NCBI Reference Sequence: NP_003317.1; Uniprot Accession Number: P23510; SEQ ID NO: 313 or 314 ) and used as a therapeutic agent for preventing and treating GVHD. The present invention provides an antibody, or antigen-binding fragment thereof, which may It may be used alone or conjugated to a cytotoxin as an antibody drug conjugate (ADC). There may be a gate.

[0296] In some embodiments, the methods and compositions (e.g., ADCs) described herein include The heavy and light chain amino acid sequences of the IgG1A ... In one embodiment, the anti-CD252 antibody, or antigen-binding portion thereof, , a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 315, and a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 1 In one embodiment, the anti-CD252 antibody comprises a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 316. The antibody, or antigen-binding portion thereof, comprises the CDRs set forth in the amino acid sequence of SEQ ID NO: 315. and a heavy chain variable region comprising the CDRs set forth in the amino acid sequence of SEQ ID NO: 316. The amino acid sequences of SEQ ID NOs: 315 and 316 are shown below: .

[0297] In certain embodiments, the anti-CD252 antibody, or antigen-binding portion thereof, is selected from the group consisting of SEQ ID NO: NO): A heavy chain variable region comprising the CDRs set forth in the amino acid sequence of 317 to 319, and SEQ ID NO: A light chain variable region comprising the CDRs set forth in the amino acid sequence of SEQ ID NO: 320 to 322. The amino acid sequences of ):3 to 8 are shown below.

[0298] Anti-CD252 VH amino acid sequence (CDR sequences below are defined by IMGT) [ka]

[0299] Anti-CD252 VL amino acid sequence (CDR sequences below are defined by IMGT) [ka]

[0300] In certain embodiments, the anti-CD252 antibodies used in the methods and compositions disclosed herein comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 315, and a ID NO): 316. In embodiments, the anti-CD252 antibody is engineered to have a short half-life.

[0301] In one embodiment, the compounds used in the methods and compositions (including ADCs) described herein Anti-CD252 antibodies available include 11C3.1 (Biolegend, catalog #326302), 159403 (R&D Syst ems, Catalog #MAB10541), 159408 (R&D Systems, Catalog #MAB1054), MM0505-8S23 (N Novus, Catalog #NBP2-11969), and oxelumab (Novus Catalog #NBP2-52 687-0.1).

[0302] In one embodiment, the compounds used in the methods and compositions (including ADCs) described herein The anti-CD252 antibody may be the mouse monoclonal anti-CD252 antibody 11C3.1, or 11C3 11C3.1 (Biolegend Cat No. 32) is an anti-CD252 antibody containing the antigen-binding region corresponding to the 11C3.1 antibody. It is available from 6302 (February 27, 2019).

[0303] In one embodiment, the anti-CD252 antibody comprises CDR1, CDR2, and CDR3 of the anti-CD252 antibody 11C3.1 The antibody comprises a heavy chain and a light chain variable region comprising CDR1, CDR2, and CDR3 of the anti-CD252 antibody 11C3.1. In embodiments, the anti-CD252 antibodies used in the compositions and methods disclosed herein are human The antibody is a synthetic 11C3.1 antibody.

[0304] In one embodiment, the compounds used in the methods and compositions (including ADCs) described herein The anti-CD252 antibody may be the mouse monoclonal anti-CD252 antibody 159403 or 1594 It is an anti-CD252 antibody containing the antigen-binding region corresponding to antibody 03. 159403 (sold by R&D Systems) Catalog #MAB10541 (February 27, 2019).

[0305] In one embodiment, the anti-CD252 antibody comprises CDR1, CDR2, and CDR3 of the anti-CD252 antibody 159403. The heavy chain and light chain variable region includes CDR1, CDR2, and CDR3 of the anti-CD252 antibody 159403. In embodiments, the anti-CD252 antibodies used in the compositions and methods disclosed herein are human It is a 159403 antibody.

[0306] In one embodiment, the compounds used in the methods and compositions (including ADCs) described herein The anti-CD252 antibody may be the mouse monoclonal anti-CD252 antibody 159408 or 1594 It is an anti-CD252 antibody containing the antigen-binding domain corresponding to antibody 08. Catalog #MAB1054 (February 27, 2019).

[0307] In one embodiment, the anti-CD252 antibody comprises CDR1, CDR2, and CDR3 of the anti-CD252 antibody 159408. The heavy chain and light chain variable region includes CDR1, CDR2, and CDR3 of the anti-CD252 antibody 159408. In embodiments, the anti-CD252 antibodies used in the compositions and methods disclosed herein are human It is a 159408 antibody.

[0308] In one embodiment, the compounds used in the methods and compositions (including ADCs) described herein The anti-CD252 antibody used is the mouse monoclonal anti-CD252 antibody MM0505-8S23, or This is an anti-CD252 antibody containing the antigen-binding region corresponding to the MM0505-8S23 antibody. US, catalog #NBP2-11969 (February 27, 2019). This antibody is Domas (fused with splenocytes from mice immunized with human TNFSF4 (also known as OX40 ligand) It was produced from a mouse myeloma.

[0309] In one embodiment, the anti-CD252 antibody comprises CDR1, CDR2, and CDR3 of the anti-CD252 antibody MM0505-8S23. and a light chain variable region comprising CDR1, CDR2, and CDR3 of the anti-CD252 antibody MM0505-8S23. In another embodiment, the anti-CD252 antibody used in the compositions and methods disclosed herein The antibody is the humanized MM0505-8S23 antibody.

[0310] In one embodiment, the compounds used in the methods and compositions (including ADCs) described herein The anti-CD252 antibody is the rabbit monoclonal anti-CD252 antibody oxelumab, or It is an anti-CD252 antibody containing an antigen-binding region corresponding to the oxelumab antibody. Sold by ovus, catalog #NBP2-52687-0.1 (February 27, 2019).

[0311] In one embodiment, the anti-CD252 antibody comprises CDR1, CDR2, and CDR3 of the anti-CD252 antibody oxelumab. and a light chain variable region comprising CDR1, CDR2, and CDR3 of the anti-CD252 antibody oxelumab. In another embodiment, the antibodies used in the compositions and methods disclosed herein include In some embodiments, the anti-CD252 antibody is a humanized oxelumab antibody. The antibody, or antigen-binding portion thereof, may comprise a heavy chain having the amino acid sequence set forth in SEQ ID NO: 323. and a light chain set forth in the amino acid sequence of SEQ ID NO: 324. In one embodiment, the anti-CD252 antibody, or antigen-binding portion thereof, comprises an amino acid sequence similar to that of SEQ ID NO: 331. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 332. In one embodiment, the anti-CD252 antibody, or antigen-binding portion thereof, comprises a CD252 antibody having SEQ ID NO: A heavy chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 325 to 327, and SEQ ID NOs: In one embodiment, the CDRs are selected from the group consisting of a light chain variable region, ... In one embodiment, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 331. and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 332. The amino acid sequences of SEQ ID NOs: 323-330 are shown below.

[0312] Oxelumab full-length heavy chain sequence (the CDR sequences below are defined by IMGT; heavy chain variable region Region (underlined in SEQ ID NO: 331): [ka]

[0313] Oxelumab full-length light chain sequence (CDR sequences below are defined by IMGT; light chain variable region Region (underlined in SEQ ID NO: 332): [ka]

[0314] The anti-CD252 antibodies or binding fragments described herein are also known in the art. Thus, modifications and / or mutations (e.g., halving) that change the properties of the antibody and / or fragment may be used. (e.g., increasing the ATPase activity, increasing or decreasing ADCC, modifications and / or mutations) This sometimes happens.

[0315] In certain embodiments, the anti-CD252 antibodies or the binding fragment comprises a variant Fc region, wherein the variant Fc region , and at least one amino acid modification relative to the wild-type Fc region, such that the molecule Certain amino acid positions within the Fc region alter the affinity for FcγR. This is known from direct contact crystallographic studies. amino acids 265-269 (B / C loop), amino acids 297-299 (C' / E loop), and amino acids The acid 327-332 (F / G) loop (Sondermann et al., 2000 Nature, 406: 267-273). Thus, the anti-CD252 antibodies described herein have been shown to bind to Fcγ The Fc region may comprise a variant Fc region in which at least one residue in direct contact with R is altered. In one embodiment, the Fc region of the anti-CD252 antibody (or fragment thereof) comprises the Fc region of the anti-CD252 antibody (or fragment thereof) described in Kabat et al. al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Ser. Vice, NH1, MD (1991) (expressly incorporated herein by reference) Contains an amino acid substitution at amino acid 265 according to the index. The "EU index" refers to the numbering of human IgG1 EU antibodies. In one embodiment, the Fc region comprises a D265A mutation. In another embodiment, the Fc region comprises a D265C mutation. In this embodiment, the Fc region of the anti-CD252 antibody (or fragment thereof) comprises a nucleotide sequence similar to that of Kabat's In one embodiment, the amino acid substitution at amino acid 234 according to the EU index is: In some embodiments, the anti-CD252 antibody (or its Fc region) comprises a L234A mutation. The Fc region of the IgG fragment is the amino acid at amino acid 235 according to the EU index as given in Kabat. In one embodiment, the Fc region comprises a L235A mutation. In a further embodiment, the Fc region comprises the L234A and L235A mutations. , D265C, L234A, and L235A mutations.

[0316] In certain embodiments, the variant IgG Fc domain does not comprise one or more amino acid substitutions. Decreased binding affinity to Fc gamma R and / or C1q compared to wild-type Fc domains The Fc binding interaction may be achieved by various methods, including one or more amino acid substitutions that result in the Fc binding interaction being eliminated or eliminated. Various effector functions and downstream signaling events (antibody-dependent cell-mediated cytotoxicity) (antibody dependent cell-mediated cytotoxicity (ADCC)) and complement dependent cytotoxicity (com (including, but not limited to, replenishment dependent cytotoxicity (CDC)) Thus, in certain embodiments, an anti-CD252 antibody comprising an altered Fc region (e.g., L234A , L235A, and D265C mutations) have substantially reduced effector function, or It has disappeared.

[0317] Affinity for the Fc region can be determined using various techniques known in the art (e.g., limited However, equilibrium methods [e.g., enzyme-linked immunosorbent assay (ELISA)] are not bent assay (ELISA));KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 3 73:52-60, 2008; radioimmunoassay (RIA)], or surface plasmon ion resonance assays or other mechanism-based kinetic assays [e.g., BIACORE TM Solution Analysis or Octet TM analysis (forteBIO)], as well as other methods [e.g., indirect binding assays, Competitive binding assay, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration), etc. These and other methods can be used to measure the amount of a target substance present on one or more components being evaluated. may utilize identification and / or various detection methods (e.g., but not limited to) However, chromogenic, fluorescent, luminescent, or isotopic labels may be used. Regarding binding affinity and kinetics, see Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999) (which focuses on antibody-immunogen interactions) An example of a competitive binding assay is a method in which a labeled antigen and a target antigen are bound to each other. The antibody is incubated in the presence of increasing amounts of unlabeled antigen. and detecting antibodies bound to said labeled antigen. From the data, Scatchard plot analysis revealed the specific antigen The affinity and binding off-rate of the antibody of interest may be determined. Competition for the antigen may also be measured using radioimmunoassays. The antibody of interest conjugated to a labeled compound and increasing amounts of an unlabeled second antibody were added. The mixture is incubated in the presence of the .

[0318] The antibodies of the present invention can be further modified to produce antibodies, for example, 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.

[0319] Thus, in one embodiment, the Fc region comprises a mutation that results in a decreased half-life. Antibodies with half-lives are particularly useful in certain situations where the antibody is expected to function as a short-lived therapeutic agent. In certain instances (e.g., the methods described herein of administering an antibody followed by administering HSCs), In the conditioning step), it can be beneficial. Ideally, endogenous stem cells Unlike cells, which also generally express CD252, they are not the target of the anti-CD252 antibody. The antibodies should be substantially cleared before administering the new HSCs. In some embodiments, the Fc region comprises a mutation at position 435 (EU index according to Kabat). In embodiments, the mutation is an H435A mutation.

[0320] In certain embodiments, the anti-CD252 antibodies described herein are administered for about 14 hours or less, for about 13 hours or less, In some embodiments, the half-life of the agonist described herein is about 12 hours or less, or about 11 hours or less. The anti-CD252 antibody has a half-life of about 24 hours or less, a half-life of about 22 hours or less, a half-life of about 20 hours or less Half-life of about 18 hours or less, Half-life of about 16 hours or less, About 14 hours or less, About 13 hours or less, About 12 hours or less In one embodiment, the antibody has a half-life of about 11 hours or less. , about 1 hour to about 20 hours, about 2 hours to about 18 hours, about 4 hours to about 16 hours, about 6 hours to about 14 hours, about 8 hours to 12 hours, 11 hours to 12 hours, 11 hours to 24 hours, 12 hours to 22 hours, 1 hour 0 hours to approximately 20 hours, approximately 8 hours to approximately 18 hours, approximately 1 hour to approximately 6 hours, approximately 2 hours to approximately 5 hours, approximately 3 hours about 14 hours to about 24 hours.

[0321] In some embodiments, the Fc region confers a reduced half-life and enhances the efficacy of the antibody. Contains two or more mutations that significantly reduce or completely eliminate the function of the receptor. In this embodiment, the Fc region contains mutations that result in decreased half-life, and (e.g., structural and at least one residue that can make direct contact with FcγR (based on crystallographic analysis). In one embodiment, the Fc region comprises a H435A mutation, a L234A mutation, and a L235A mutation. In one embodiment, the Fc region comprises the mutations H435A and D265C. In embodiments, the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, and a D265C mutation.

[0322] In some embodiments, the antibody, or antigen-binding fragment thereof, via cysteine ​​residues in the Fc domain of its antigen-binding fragment, In some embodiments, the cysteine ​​residue is introduced by mutation in the Fc domain of the antibody, or antigen-binding fragment thereof. For example, the cysteine ​​residue is selected from the group consisting of Cys118, Cys239, and Cys265. In one embodiment, the anti-CD252 antibody (or fragment thereof) The Fc region contains an amino acid substitution at amino acid 265 according to the EU index as per Kabat In one embodiment, the Fc region comprises a D265C mutation. In one embodiment, the Fc region comprises D265C, L234A, and L235A mutations. In one embodiment, the Fc region comprises D265C, L234A, L235A, and H435A mutations. Includes differences.

[0323] In some embodiments of these aspects, the cysteine ​​residue is It is naturally present in the Fc domain of the antigen-binding fragment. For example, the Fc domain , an IgG Fc domain, such as a human IgG1 Fc domain, wherein the cysteine ​​residue is Cys26 1, Csy321, Cys367, and Cys425.

[0324] 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. Please note that

[0325] In one embodiment, the anti-CD252 antibody, or antigen-binding fragment thereof, is Amino acid sequences that are at least 95%, 96%, 97% or 99% identical to the disclosed SEQ ID Nos. or the anti-CD252 antibody, or antigen-binding fragment thereof, comprises a variable region having a sequence. are at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein. The present invention provides a method for the preparation of a medicament ... The CDRs comprise the SEQ ID Nos disclosed herein.

[0326] In certain embodiments, the anti-CD252 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-CD252 antibody, in certain embodiments, is directed against human CD252 and / or rhesus CD252. 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 CD252 (e.g., human CD252 and / or rhesus CD2 52). In some embodiments, the antibody or antigen-binding fragment thereof binds to: As measured by Bio-Layer Interferometry (BLI) assay , about 90 nM to 100 nM, about 80 nM to 90 nM, about 70 nM to 80 nM, about 60 nM to 70 nM, about 50 nM to 60 nM M, approx. 40 nM~50 nM, approx. 30 nM~40 nM, approx. 20 nM~30 nM, approx. 10 nM~20 nM, approx. 8 nM~10 nM , about 6 nM to 8 nM, about 4 nM to 6 nM, about 2 nM to 4 nM, about 1 nM to 2 nM, or about 1 nM or less D in, Binds to CD252 (e.g., human CD252 and / or rhesus CD252).

[0327] The antibodies, and binding fragments thereof, disclosed herein can be used in combination with other antibodies, as described in detail below. , may be used in conjugates.

[0328] Exemplary antigen-binding fragments of the aforementioned antibodies include, inter alia, bivariable immunoglobulins (BIGs), domain (dual-variable immunoglobulin domain), single-chain Fv molecule (scFv), diabody antibodies, triabodies, nanobodies, antibody-like protein scaffolds, Fv fragments, These include Fab fragments, F(ab')2 molecules, and tandem di-scFvs. Anti-CD252 antibodies available include full-length antibodies, bispecific antibodies, and bispecific antibodies. Variable domain antibodies, multi-chain or single-chain antibodies, and / or antibodies that specifically bind to human CD252 It may be in the form of fragments, including Fab, Fab', (Fab')2, Fv, scFv (single chain Fv), v), surrobodies (including surrogate light chain constructs), single domain antibodies, camelized antibodies These include, but are not limited to, antibodies, such as IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM, etc. In some embodiments, the anti-CD252 antibody is derived from any isotype. , IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0329] In one embodiment, the anti-CD252 antibody, or antigen-binding fragment thereof, is Amino acid sequences that are at least 95%, 96%, 97% or 99% identical to the disclosed SEQ ID Nos. or the anti-CD252 antibody, or antigen-binding fragment thereof, comprises a variable region having a sequence. are at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein. The present invention provides a method for the preparation of a medicament ... The CDRs comprise the SEQ ID Nos disclosed herein.

[0330] 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 engraftment 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): 336 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 3 below. It is suspected.

[0331] [Table 3]

[0332] 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.

[0333] CD45RO is currently the only known CD45 isoform expressed on hematopoietic stem cells. RA and CD45RABC are either not detected or are excluded from the hematopoietic stem cell phenotype. RB 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 a key regulator of IL-1 expression in Asian populations. The polymorphism of exon 6 of CD45RC is highly prevalent in the Japanese population. This polymorphism results in high expression of CD45RO and low expression of CD45RA, CD45RB, and CD45RB. Furthermore, CD45RA variants (e.g., CD45RAB and CD45 RAC etc.) indicates a polymorphism in exon 4 that is associated with autoimmune disease.

[0334] 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.

[0335] 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.

[0336] 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. Met. d. 169:1179-1184, 1989, and this disclosure relates to anti-CD45 antibodies. and incorporated herein by reference), as well as humanized variants thereof. Additional anti-inflammatory drugs that may be used in conjunction with the patient conditioning methods described herein include: - CD45 antibodies include monoclonal antibodies AHN-12.1, AHN-12, AHN-12.2, AHN-12.3, and AHN-1 2.4, HLe-1, and KC56(T200) (which are described, e.g., in Harvath et al., J. Immunol. 146:9 49-957, 1991, the disclosure of which is incorporated herein by reference as it relates to anti-CD45 antibodies. and humanized variants thereof.

[0337] 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, Among the clones, the anti-CD45 antibodies 39E11, 16C9, and 1G10 are specifically described. Anti-CD45 antibodies; U.S. Patent No. 7,160,987 (which are described, e.g., in ATCC Accession No. HB-118 73 produces and releases anti-CD45 antibodies [e.g., monoclonal antibody 6G3, etc.]. ) and U.S. Patent No. 6,099,838 (which, e.g., discloses anti-CD4 5 antibody MT3, and ATCC accession numbers HB220 [also called MB23G2] and HB223 produced and anti-CD45 antibodies described in US 2004 / 00969, which describe antibodies that induce and release CD45. 01 and US 2008 / 0003224 (which are, for example, those produced by ATCC Accession No. PTA-7339) and releasing anti-CD45 antibodies [e.g., monoclonal antibody 17.1, etc.] and anti-CD45 antibodies that have been reported (these disclosures relate to anti-CD45 antibodies). and incorporated herein by reference).

[0338] 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 their human antibodies Affinity maturation can be performed, for example, by using the methods described herein. or by in vitro display techniques known in the art (e.g., see Example 6 below). This may be performed using phage display (as described in).

[0339] 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).

[0340] 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. 10 (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).

[0341] 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:337. The light chain variable region (VL) amino acid sequence of apamistaumab is set forth in SEQ ID NO: 338. In other embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, has the sequence identified as SEQ ID NO: a variable heavy chain comprising the amino acid residues set forth in SEQ ID NO: 337, and a variable heavy chain comprising the amino acid residues set forth in SEQ ID NO: 338 In one embodiment, the anti-CD45 antibody comprises the CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR19, CDR11, CDR12, CDR13, CDR14, CDR1 A heavy chain variable region containing CDR1, CDR2, and CDR3, and a light chain variable region containing CDR1, CDR2, and CDR3 of apamistaumab Includes the area.

[0342] In one embodiment, the anti-CD45 antibody comprises a heavy chain of an anti-CD45 antibody described herein and a 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 .

[0343] In another embodiment, the antibody, or antigen-binding fragment thereof, is an anti-CD4 5 antibodies, (e.g., at least 95% identity to the anti-CD45 antibodies herein), a fragment containing an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity In certain embodiments, the antibody comprises a HC variable region of an anti-CD45 antibody herein. The variant comprises a modified heavy chain (HC) variable region containing a variable 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 (iii) differs from an antibody in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions. and / or (iv) is at least about 75%, 80%, 85%, or 90% different from said anti-CD45 antibody; %, 90%, 95%, 96%, 97%, 98% or 99% identical to (i)-(iv), In any of the above, the amino acid substitutions may be conservative or non-conservative. and wherein the modified heavy chain variable region binds to CD45 of an anti-CD45 antibody. Enhanced biological activity compared to the heavy chain variable region of the anti-CD45 antibody while retaining its specificity. may have therapeutic activity.

[0344] The disclosure of each of the above publications is incorporated herein by reference in its entirety. Antibodies and antigen-binding fragments that may be used in conjunction with the compositions and methods described herein The antibodies and antigen-binding fragments thereof described above, as well as the non-human antibodies and Humanized variants of antigen-binding fragments and their functional groups can be used to identify antigen-binding fragments, e.g., by competitive CD45 binding assays. When evaluated by the above method, it is possible to detect an antibody that binds to the same epitope as the above antibody or antigen-binding fragment. The antibody or antigen-binding fragment thereof may be used.

[0345] Methods for identifying antibodies 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., CD2, CD5, CD137, or Libraries of antibodies or antibody fragments were searched for molecules capable of binding to CD252. Using methods for high-throughput screening of cancer, autoimmune diseases, To treat and administer to a patient in need of hematopoietic stem cell therapy as described herein (e.g., Identifying and affinity-composing useful antibodies for conditioning human subjects (e.g., human patients) Such methods include in vitro display methods known in the art. IgM technologies, such as phage display, bacterial display, yeast display, among others Display, mammalian cell display, ribosome display, mRNA display Antibodies that bind to biologically relevant molecules. or using phage display to isolate antigen-binding fragments , for example, Felici et al., Biotechnol. Annual Rev. 1:149-183, 1995; Katz, Annual R ev. Biophys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechn. The disclosures of each of these have been reviewed in J. Immunol. 4:1-20, 1998, and are based on in vitro The present application is incorporated by reference as it relates to spray technology. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, A randomized combinatorial peptide library was constructed as described in 1996. It has also been attempted to construct a polypeptide that binds to a cell surface antigen by selecting the polypeptide. The disclosures of each of these publications are incorporated herein by reference as they relate to the discovery of antigen-binding molecules. Proteins (e.g., multimeric proteins) are used as functional molecules. The display has been successfully implemented (e.g., EP 0349578; EP 4527839; and EP 0589877, and Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992 See, e.g., U.S. Pat. No. 6,229,999, each of which discloses in vitro display techniques for discovering antigen-binding molecules. (These techniques are incorporated herein by reference in their entirety as they relate to the use of the techniques.) Functional antibody fragments (e.g., Fab and scFv fragments) can be generated by in vitro display Expression in various formats has also been attempted (e.g., McCafferty et al., Nature re 348:552- 554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1 991; and Clackson et al., Nature 352:624-628, 1991, the disclosures of each of which are incorporated herein by reference. Related in vitro display platforms for discovering antigen-binding molecules (The disclosure of which is incorporated herein by reference in its entirety.) Human anti-HC antibodies (e.g., anti-CD117 antibodies) , anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody) are also used, e.g. For example, HuMAb-mouse 登録商標 or XenoMouse TM In particular, These techniques can be used to identify antigens expressed by hematopoietic stem cells (e.g., CD117 [e.g., GNNK+ C D117], or CD45), or antigens expressed by mature immune cells (e.g., T-cells) (e.g., , CD2, CD5, CD137, or CD252), and then hematopoietic stem cell transplantation. To reduce endogenous hematopoietic stem cells in a patient (e.g., a human patient) requiring transplantation therapy. identify the affinity of antibodies, antibodies, or fragments, which can be used to There are things that can be improved.

[0346] In addition to in vitro display techniques, computational modeling techniques are used to identify the genes that cause hematopoietic stem cells to develop. antigens expressed by mature immune cells (e.g., CD117 [e.g., GNNK+ CD117], or CD45), or A cell (e.g., a T-cell) binds to an antigen (e.g., CD2, CD5, CD137, or CD252) expressed by the cell. In silico, we design and identify antibodies or antibody fragments that can bind to the antibody. For example, using computational modeling techniques, one skilled in the art may be able to determine whether hematopoietic stem cells antigens expressed by mature immune cells (e.g., CD117 [e.g., GNNK+ CD117], or CD45), or mature immune cells. An antigen (e.g., CD2, CD5, CD137, or CD252) expressed by immune cells (e.g., T-cells) , binding to a specific epitope (e.g., an extracellular epitope of the antigen) on Libraries of antibodies or antibody fragments are screened in silico for molecules that can Antibodies or antibodies identified by these computer techniques may be used. The antigen-binding fragments can be administered in a variety of therapeutic methods described herein (e.g., as described herein). and methods of treating cancer and autoimmune diseases, as well as methods of treating patients with the conjugates described herein. It may be used in combination with a conditioning procedure.

[0347] Further techniques can be used to identify antigens expressed by hematopoietic stem cells (e.g., CD117 [e.g., GNNK+ C D117], or CD45), or antigens expressed by mature immune cells (e.g., T-cells) (e.g., , CD2, CD5, CD137, or CD252), and can bind to, for example, a receptor an antibody that can be taken up by the cell by antibody-mediated endocytosis, or For example, the in vitro display techniques described above may be used to identify antibody fragments. 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., CD2, CD5, CD137, or antibodies, or antibody fragments thereof, that bind to and subsequently become internalized in the target cell (e.g., CD252) Phage display may be adapted to screening for specific targets. One such technique that may be used in conjunction with this screening paradigm is Anti-HC antibodies (e.g., anti-CD117 antibodies, anti-CD45 antibodies, anti-CD2 antibodies, anti-CD5 to identify antibodies, anti-CD137 antibodies, or anti-CD252 antibodies) or antibody fragments, and To identify the subsequent uptake into hematopoietic stem cells (or immune cells), one skilled in the art Those skilled in the art will appreciate that the phage display described in Williams et al., Leukemia 19:1432-1438, 2005 play technology (the disclosure of which is incorporated herein by reference in its entirety). For example, mutagenesis methods known in the art can be used to modify antibodies, antibody fragments, and fragments, Fab fragments, diabodies, tribodies, among others. and 10 Fn3 domain), or ligand (which contains a randomized amino acid cassette [ For example, one or more CDRs or equivalent regions thereof, or an antibody or antibody fragment, It is possible to generate recombinant phage libraries encoding (in many cases) 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. It may be designed to be non-immunogenic in humans by making it do.

[0348] 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. Libraries can be analyzed using antibodies against antigens (e.g., CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD13 7, or CD252) (e.g., first to remove non-specific protein binding To remove phages encoding antibodies or antibody fragments that exhibit binding, and To remove phages encoding antibodies or fragments thereof that bind to the target, The phage library was incubated with a blocking agent [e.g., milk protein, bovine serum albumin (BOA)]. and / or IgG, and then expressing, for example, CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137, or CD252. or incubating with a population of hematopoietic stem cells or mature immune cells (e.g., T-cells). Anti-HC antibodies (e.g., anti-CD117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody) or anti-CD252 antibody) or antibody fragment thereof targets a cell surface cognate antigen (e.g., binds to CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137, or CD252], and Subsequently, the cells are incubated for a sufficient time (e.g., 30 minutes at 4°C) for uptake by the hematopoietic stem cells. Incubate the phage library on target cells ( For example, they may be incubated with cells such as cancer cells, autoimmune cells, or hematopoietic stem cells. This can be followed by washing with, for example, cold (4°C) 0.1 M glycine buffer, pH 2.8. and bind to target cells (e.g., cancer cells, autoimmune cells, or hematopoietic stem cells). and to be internalized by the target cell, the antigen (CD117 [e.g. , GNNK+ CD117], CD45, CD2, CD5, CD137, or CD252) The target cells (e.g., antibodies, or phages containing antibody fragments) may be removed. For example, antibodies or antibodies thereof taken up by cancer cells, autoimmune cells, or hematopoietic stem cells Phage bound to the fragment can be isolated, for example, by lysing the cells and extracting them from the cell culture medium. The phage that has been incorporated into the phage may then be recovered and identified. For example, bacterial cells can be transfected with harvested phage using methods known in the art. The phage is amplified in bacterial cells by incubation in T medium. The phage recovered from this medium may then be subjected to, for example, By determining the nucleic acid sequence of the gene encoding the antibody or antibody fragment inserted into The encoded antibody or antibody fragment thereof may then be identified. and by chemical synthesis (e.g., antibody fragments thereof, e.g., scFv fragments) or recombinant production. Antibodies may be prepared de novo by the use of existing antibodies (e.g., full-length antibodies).

[0349] The amount of the prepared antibody or antibody fragment thereof that can be incorporated into the 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. Identified using in vitro display techniques described herein or known in the art Anti-HC antibodies (e.g., anti-CD117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody) or anti-CD252 antibody) or antibody fragment thereof, is irradiated with a radioisotope (e.g., 18 F, 7 5 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, 67 Ga, 111 In, 99 Tc, 169 Yb, 1 86 Re, 64 Cu, 67 Cu, 177 Lu, 77 As, 72 As, 86 Y, 90 Y, 89 Zr, 212 Bi, 213 Bi, or 225 Ac) For example, radioactive halogens (e.g. , 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131I, 211 At, etc.) to electrophilic halo Beads (e.g., polystyrene beads) containing the iodine reagent (e.g., iodinated beads, The Antibodies or antibody fragments were isolated using rmo (Fisher Scientific, Inc., Cambridge, MA). The radiolabeled antibody, its fragment, or ADC may be incorporated into a Allow the target to incubate for a sufficient period of time (e.g., 30 minutes to 6 hours at 4°C, 1 hour at 4°C, etc.). Incubate with host cells (e.g., cancer cells, autoimmune cells, or hematopoietic stem cells) Then, add chilled (4°C) 0.1 M glycine buffer at pH 2.8. 4. Wash the cells (using a lyophilized antibody) to remove any antibodies or fragments thereof that were not incorporated into the cells. The antibody or antibody fragment thereof taken up into the target cells is then removed. Radiation emitted from cells (e.g., cancer cells, autoimmune cells, or hematopoietic stem cells) The radiation emitted from the collected washing buffer (e.g., gamma radiation) is detected and compared with the radiation emitted from the collected washing buffer (e.g., gamma radiation). The internal uptake assay described above may be used to identify ADCs. Sometimes it is indicated.

[0350] 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., anti-CD117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody The present invention provides an isolated nucleic acid encoding an antibody (VL) comprising the VL of the antibody. and / or an amino acid sequence comprising VH (e.g., the light chain and / or the heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., In a further embodiment, a host cell containing such a nucleic acid is provided. In some such embodiments, the host cell comprises (e.g., (1) an amino acid sequence comprising the VL of the antibody and the VH of the antibody; (2) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody; a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody; and a...

Claims

1. An antibody comprising an altered Fc region, wherein said Fc region comprises the amino acid sequence of SEQ ID NO: 15, with substitutions consisting essentially of amino acid substitutions H435A, L234A, L235A, and D265C (EU index).

2. The antibody of claim 1, wherein the antibody comprises the amino acid sequence of SEQ ID NO:

20.

3. 2. The antibody of claim 1, wherein the antibody does not detectably bind to an FcγR, wherein the FcγR is human Fc gamma RI, human Fc gamma RIIA 167R, human Fc gamma RIIA 167H, or human Fc gamma RIIIA 176V, and antibody binding to the FcγR is assessed by biolayer interferometry (BLI).

4. The antibody of claim 1, wherein the antibody does not exhibit detectable cytokine release.

5. The antibody of claim 4, wherein the in vitro cytokine release assay is a Meso Scale Discovery (MSD) tissue culture (TC) pro-inflammatory assay.

6. 10. The antibody of claim 1, wherein the antibody does not exhibit detectable mast cell degranulation.

7. The antibody of claim 6, wherein the in vitro mast cell degranulation assay is a beta-hexosaminidase-based mast cell degranulation assay.

8. The antibody according to any one of claims 1 to 7, wherein (a) the antibody is a bispecific antibody; (b) the antibody is a monoclonal antibody; or (c) the antibody specifically binds to CD117, CD45, CD2, CD5, CD137, or CD252.

9. 9. An antibody drug conjugate (ADC) comprising the antibody, or an antigen-binding portion thereof, of any one of claims 1 to 8, wherein the antibody is conjugated to a cytotoxin via a linker.

10. 10. The ADC of claim 9, wherein the cytotoxin is a DNA alkylating agent.

11. 10. The ADC of claim 9, wherein the RNA polymerase inhibitor is an amatoxin.

12. 10. The ADC of claim 9, wherein the cytotoxin is selected from the group consisting of Pseudomonas exotoxin A, de bouganin, diphtheria toxin, saporin, maytansine, maytansinoid, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, and indolinobenzodiazepine dimer.

13. A pharmaceutical composition comprising the antibody or ADC of any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

14. 13. The antibody or ADC of any one of claims 1 to 12 for use in a method for reducing the population of hematopoietic stem cells (HSCs) in a human patient.

15. 15. The antibody or ADC of claim 14, wherein: (a) the method further comprises administering to the patient a transplant comprising hematopoietic stem cells; (b) the antibody, or antigen-binding portion thereof, is administered to the human patient prior to administration of a transplant comprising hematopoietic stem cells; and / or (c) the patient has a blood disease, metabolic disorder, cancer, or autoimmune disease, or severe combined immunodeficiency disease (SCID).

16. 16. The antibody or ADC of claim 15, wherein the transplant is allogeneic or autologous.

17. 16. The antibody or ADC of claim 15, wherein the hematopoietic stem cells are CD117+ or CD45+ cells.

18. 13. The antibody or ADC of any one of claims 1 to 12 for use in treating leukemia in a human patient.

19. 15. The antibody or ADC of any one of claims 1 to 14 for use in a method for reducing a population of immune cells in a human patient, the method comprising administering the antibody, or an antigen-binding portion thereof, to the human patient prior to administration of a transplant comprising hematopoietic stem cells.

20. 20. The antibody or ADC of claim 19, wherein the immune cell is a CD137+, CD2+, or CD5+ cell, and / or the immune cell is a T cell.

21. 10. A composition comprising the antibody of claim 1, wherein the composition contains less than 15% hydrophobic degradants after heat stress.

22. The antibody or ADC of any one of claims 1 to 12 for treating a stem cell disorder, an immunodeficiency disorder, a metabolic disorder, an autoimmune disorder, or cancer.

23. 23. The antibody or ADC of claim 22, wherein: (a) the immunodeficiency disorder is a congenital immunodeficiency disorder or an acquired immunodeficiency disorder; (b) the metabolic disorder is selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidosis, and metachromatic leukodystrophy; (c) The autoimmune disorder is multiple sclerosis, human systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, psoriasis, type 1 diabetes, acute disseminated encephalomyelitis, Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid syndrome, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune oophoritis, Barrow's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, chronic fatigue immune deficiency syndrome, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac sprue-dermatitis herpetiformis), cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythematosus, autonomic neuropathy, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, Meniere's disease, mixed connective tissue disease, myasthenia gravis, neuromyotonia, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis thyroiditis), pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-person syndrome, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis; or (d) the cancer is selected from the group consisting of leukemia, lymphoma, multiple myeloma, and neuroblastoma.

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