Fc-silent antibody-drug conjugate (ADC) and its use

Abstract

Disclosed are antibodies and antibody-drug conjugates having Fc regions with substitutions that result in essentially inactive Fc regions. 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. The compositions and methods described herein can be used to directly treat disorders, for example, by reducing populations of CD45+ or CD117+ cancer cells or CD45+ autoimmune cells. The compositions and methods described herein can also be used to prepare patients for hematopoietic stem cell transplantation therapy and improve hematopoietic stem cell transplant graft survival by selectively depleting endogenous hematopoietic stem cells prior to the transplant procedure.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims priority to U.S. Provisional Application No. 62 / 749,662, filed on October 23, 2018; U.S. Provisional Application No. 62 / 773,839, filed on November 30, 2018; and U.S. Provisional Application No. 62 / 807,363, filed on February 19, 2019. The content of each priority application is incorporated herein by reference.

[0002] [Technical Field] The present invention relates to the field of antibodies or antibody - drug conjugates comprising an Fc region with altered effector function as a result of one or more amino acid substitutions in the Fc region. The present invention further relates to the treatment of patients suffering from various pathologies (e.g., inter alia, blood diseases, metabolic disorders, cancer, and autoimmune diseases) by administering an antibody or antibody - drug conjugate (ADC) having a modified Fc region, wherein the antibody or ADC is capable of binding to an antigen expressed by hematopoietic cells (e.g., hematopoietic stem cells or cells of the host immune system).

Background Art

[0003] The Fc region of an antibody can control the cytotoxic activity of the antibody and affect the serum half-life of the antibody. However, in a therapeutic context, the cytotoxic effector function of an antibody is often undesirable and can also cause safety concerns and unwanted side effects by activating the host's immune defense. Some amino acid changes in the Fc region have been reported to silence or reduce the effector function of an antibody. In fact, previous studies have identified amino acid positions within the Fc region of an antibody that affect the ability of the antibody to bind to Fc receptors (see, for example, Wang et al. (2018) Protein Cell. 2018 Jan; 9(1): 63-73). For example, the Fc mutations S239D and I332E have been described in the literature as enhancing the ADCC function (see, for example, Lazar et al. (2006) Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA. 103:4005-4010). Other mutations are associated with a decrease in FcγR and C1q binding (e.g., the amino acid changes L234A / L235A in IgG1 or F234A / L235A in IgG4) (Xu et al. In vitro characterization of five humanized OKT3 effector function variant antibodies. Cell Immunol. 2000; 200:16-26). However, little is known about how Fc mutations can affect antibody-drug conjugates (ADCs), especially when the toxin binds to an antibody or an Fc containing a fragment within the Fc region).

[0004] Despite the progress of medical technology, there is still a need to treat disorders of the hematopoietic system (e.g., in particular, diseases of certain blood cells, metabolic disorders, cancers, and autoimmune conditions, etc.). While hematopoietic stem cells have great therapeutic potential, a drawback that has hindered their clinical use has been the difficulty in ensuring engraftment of hematopoietic stem cell (HSC) transplants in the host. In particular, hematopoietic stem cell therapies, including antibodies that target cell surface antigens on endogenous HSCs, can induce undesirable immune-stimulatory and effector functions that prevent engraftment of exogenous HSC transplants. Currently, there is a need for compositions and methods to promote engraftment of exogenous hematopoietic stem cell transplants such that the multi-potency and hematopoietic function of these cells are preserved after transplantation. For example, improved ADCs that can be used for conditioning with reduced effector function to reduce the potential for cytokine secretion and side effects are also needed.

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0005] Antibodies comprising an Fc region with altered effector function as a result of one or more amino acid substitutions in the Fc region, and antigen-binding portions thereof, and antibody-drug conjugates, compositions, and methods using said antibodies are described herein. In particular, antibodies or antibody-drug conjugates (ADCs) having a modified Fc region are provided herein, wherein said antibody or ADC can bind to an antigen expressed by hematopoietic cells (e.g., hematopoietic stem cells) or mature immune cells (e.g., T cells). Further provided herein are ADCs comprising Fc mutations that provide a conjugation site for a toxin, reduce effector function, and provide stability. Accordingly, the present disclosure provides a unique combination of Fc mutations for ADCs.

MEANS FOR SOLVING THE PROBLEMS

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

[0007] In some embodiments, the Fc region further comprises an amino acid substitution at position H435 (EU index). In certain embodiments, the amino acid substitution at H435 is H435A. In certain embodiments, an antibody comprising the amino acid substitution H435A has a reduced half-life compared to the same intact IgG antibody comprising an unmodified Fc region.

[0008] In another aspect, an antibody comprising an Fc region having amino acid substitutions consisting essentially of L234A, L235A, and D265C (EU index) is provided herein, and wherein the antibody is an intact IgG antibody. In certain embodiments, the antibody comprises an Fc region having amino acid substitutions consisting essentially of L234A, L235A, and D265A (EU index), and wherein the antibody is an intact IgG antibody.

[0009] In another aspect, an antibody comprising an Fc region, or an antigen-binding portion thereof, is provided herein, wherein the Fc region comprises amino acid substitutions at positions L234, L235 (EU index), and D265 (EU index). In certain embodiments, the amino acid substitution at D265 is D265C or D265A (EU index). 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 further comprises an amino acid substitution at position N297 (EU index). In another 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 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 at G236 (EU index). In another embodiment, 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 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 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 I253 (EU index). In another embodiment, the amino acid substitution at I253 is I253A. In another embodiment, the Fc region further comprises an amino acid substitution at position H310 (EU index). In another embodiment, the amino acid substitution at H310 is H310A.

[0010] In another aspect, an antibody, or an antigen-binding portion thereof, that includes an Fc region is provided herein, where the Fc region includes amino acid substitutions at positions N297 and D265 (EU index). In certain embodiments, the amino acid substitution at position D265 is D265C or D265A (EU index). In another 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 further includes amino acid substitutions at positions L234 and L235 (EU index). In another embodiment, the amino acid substitution at L234 is L234A or L234V. In another embodiment, the L235 amino acid substitution is L235A. In another embodiment, the Fc region further includes 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 includes a deletion at position G236 (EU index). In another embodiment, the Fc region further includes 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 include a substitution at position P331 (EU index). In another embodiment, the Fc region further includes an amino acid substitution at position P329 (EU index). In another embodiment, the amino acid substitution at P329 is P329G. In another embodiment, the Fc region does not include a substitution at position P329 (EU index). In another embodiment, the Fc region further includes an amino acid substitution at position I253 (EU index). In another embodiment, the amino acid substitution at I253 is I253A. In another embodiment, the Fc region further includes an amino acid substitution at position H310 (EU index). In another embodiment, the amino acid substitution at H310 is H310A.

[0011] In another aspect, an antibody comprising an Fc region, or an antigen-binding portion thereof, is provided herein, wherein the Fc region comprises amino acid substitutions at positions E233, L234, L235, and D265 (EU index), a deletion at G236 (EU index), and an amino acid substitution at D265 (EU index). In certain embodiments, the amino acid substitution at D265 is D265C or D265A (EU index). 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 amino acid substitution at E233 is E233P (EU index). In another embodiment, the Fc region further comprises an amino acid substitution at position N297 (EU index). In another 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 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 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 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 I253 (EU index). In another embodiment, the amino acid substitution at I253 is I253A. In another embodiment, the Fc region further comprises an amino acid substitution at position H310 (EU index). In another embodiment, the amino acid substitution at H310 is H310A.

[0012] In another aspect, an antibody comprising an Fc region, or an antigen-binding portion thereof, is provided herein, wherein the Fc region comprises amino acid substitutions at positions H435 and D265 (EU index). In certain embodiments, the amino acid substitution at position D265 is D265C or D265A (EU index). In another embodiment, the amino acid substitution at H435 is H435A. In another embodiment, the Fc region further comprises an amino acid substitution at position N297 (EU index). In another 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 further 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 another embodiment, the amino acid substitution at L235 is L235A. In another embodiment, 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 at position G236 (EU index). In another embodiment, 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 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 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 I253 (EU index). In another embodiment, the amino acid substitution at I253 is I253A. In another embodiment, the Fc region further comprises an amino acid substitution at position H310 (EU index). In another embodiment, the amino acid substitution at H310 is H310A.

[0013] In another aspect, an antibody comprising an Fc region, or an antigen-binding portion thereof, is provided herein, wherein the Fc region comprises amino acid substitutions at positions L234 and L235 (EU index), and amino acid substitution P329 (EU index). In certain embodiments, the amino acid substitution at L234 is L234A or L234V. In another embodiment, the amino acid substitution at L235 is L235A. In certain embodiments, the Fc region further comprises an amino acid substitution at position D265 (EU index). In certain embodiments, the amino acid substitution at D265 is D265C or D265A (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position N297 (EU index). In certain embodiments, the amino acid substitution at N297 is selected from the group consisting of N297A, N297G, and N297Q (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position E233 (EU index). In certain embodiments, the amino acid substitution at E233 is E233P (EU index). In certain embodiments, the Fc region further comprises a deletion at position G236 (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position P331 (EU index). In certain embodiments, the amino acid substitution at P331 is P331G. In certain embodiments, the Fc region does not comprise a substitution at position P331 (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position I253 (EU index). In certain embodiments, the amino acid substitution at I253 is I253A. In certain embodiments, the Fc region further comprises an amino acid substitution at position H310 (EU index). In certain embodiments, the amino acid substitution at H310 is H310A.

[0014] In another aspect, an antibody comprising an Fc region, or an antigen-binding portion thereof, is provided herein, wherein the Fc region comprises amino acid substitutions at positions L234 and L235 (EU index), and amino acid substitution P331 (EU index). In certain embodiments, the amino acid substitution at L234 is L234A or L234V. In certain embodiments, the amino acid substitution at L235 is L235A. In certain embodiments, the Fc region further comprises an amino acid substitution at position D265 (EU index). In certain embodiments, the amino acid substitution at D265 is D265C or D265A (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position N297 (EU index). In certain embodiments, the amino acid substitution at N297 is selected from the group consisting of N297A, N297G, and N297Q (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position E233 (EU index). In certain embodiments, the amino acid substitution at E233 is E233P (EU index). In certain embodiments, the Fc region further comprises a deletion at position G236 (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position P329 (EU index). In certain embodiments, the amino acid substitution at P329 is P329G. In certain embodiments, the Fc region does not include a substitution at position P329 (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position I253 (EU index). In certain embodiments, the amino acid substitution at I253 is I253A. In certain embodiments, the Fc region further comprises an amino acid substitution at position H310 (EU index). In certain embodiments, the amino acid substitution at H310 is H310A.

[0015] In another aspect, an antibody comprising an Fc region, or an antigen-binding portion thereof, is provided herein, wherein the Fc region comprises amino acid substitutions at positions E233, L234, and L235 (EU index), and a deletion of G236 (EU index). In certain embodiments, the amino acid substitution at L234 is L234A or L234V. In certain embodiments, the amino acid substitution at L235 is L235A. In certain embodiments, the amino acid substitution at E233 is E233P (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position H435 (EU index). In certain embodiments, the amino acid substitution at H435 is H435A. In certain embodiments, the Fc region further comprises an amino acid substitution at position N297 (EU index). In certain embodiments, the amino acid substitution at N297 is selected from the group consisting of N297A, N297G, and N297Q (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position P331 (EU index). In certain embodiments, the amino acid substitution at P331 is P331G. In certain embodiments, the Fc region does not comprise a substitution at position P331 (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position P329 (EU index). In certain embodiments, the amino acid substitution at P329 is P329G. In certain embodiments, the Fc region does not comprise a substitution at position P329 (EU index). In certain embodiments, the Fc region further comprises an amino acid substitution at position I253 (EU index). In certain embodiments, the amino acid substitution at I253 is I253A. In certain embodiments, the Fc region further comprises an amino acid substitution at position H310 (EU index). In certain embodiments, the amino acid substitution at H310 is H310A.

[0016] In another aspect, an antibody or an antigen-binding portion thereof that includes an Fc region is provided herein, where the Fc region includes amino acid substitutions at positions I253, H310, and H345 (EU index). In certain embodiments, the amino acid substitution at I253 is I253A. In another embodiment, the amino acid substitution at H310 is H310A. In another embodiment, the amino acid substitution at H435 is H435A. In another embodiment, the Fc region further includes an amino acid substitution at position N297 (EU index). In another 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 further includes an amino acid substitution at position D265 (EU index). In another embodiment, the amino acid substitution at D265 is D265C or D265A (EU index). In another embodiment, the Fc region further includes 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 includes a deletion at position G236 (EU index). In another embodiment, the Fc region further includes an amino acid substitution at position P329 (EU index). In another embodiment, the amino acid substitution at P329 is P329G. In another embodiment, the Fc region does not include a substitution at position P329 (EU index). In another embodiment, the Fc region further includes 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 include a substitution at position P329 (EU index).

[0017] In another aspect, an antibody, or an antigen-binding portion thereof, comprising an Fc region is provided herein, where the Fc region comprises an amino acid substitution at the position of N297 (EU index). In certain embodiments, the Fc region further comprises amino acid substitutions at the positions of L234 and L235 (EU index). In another embodiment, the amino acid substitution of L234 is L234A or L234V. In another embodiment, the amino acid substitution of L235 is L235A. In another embodiment, the Fc region does not comprise substitutions at the positions of L234 and L235 (EU index). In another embodiment, the amino acid substitution of N297 is selected from the group consisting of N297A, N297G, and N297Q. In another embodiment, the Fc region further comprises an amino acid substitution at the position of E233 (EU index). In another embodiment, the amino acid substitution of 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 further comprises an amino acid substitution at the position of P331 (EU index). In another embodiment, the amino acid substitution of P331 is P331G. In another embodiment, the Fc region does not comprise a substitution at the position of P331 (EU index). In another embodiment, the Fc region further comprises an amino acid substitution at the position of P329 (EU index). In another embodiment, the amino acid substitution of P329 is P329G. In another embodiment, the Fc region does not comprise a substitution at the position of P329 (EU index). In another embodiment, the Fc region further comprises an amino acid substitution at the position of I253 (EU index). In another embodiment, the amino acid substitution of I253 is I253A. In another embodiment, the Fc region further comprises an amino acid substitution at the position of H310 (EU index). In another embodiment, the amino acid substitution of H310 is H310A.

[0018] In some embodiments, the antibody, or an antigen-binding portion thereof, comprises any combination of substitutions to the Fc region described herein.

[0019] In some embodiments, the antibody, or antigen-binding portion thereof, further comprises an amino acid substitution at the position of S239 (EU index). In certain embodiments, the amino acid substitution of S239 is S239C.

[0020] In some embodiments, the antibody, or antigen-binding portion thereof, further comprises an amino acid substitution at the position of H435 (EU index). In certain embodiments, the amino acid substitution of H435 is H435A. In another embodiment, the antibody comprises the amino acid substitution H435A and has a reduced half-life compared to an otherwise identical intact IgG antibody comprising an unmodified Fc region.

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

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

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

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

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

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

[0027] In another aspect, an antibody, or an antigen-binding portion thereof, that includes an Fc region is provided herein, where the Fc region includes amino acid substitutions that consist essentially of the amino acid substitutions N297A and D265C (EU index).

[0028] In another aspect, an antibody, or an antigen-binding portion thereof, that includes an Fc region is provided herein, where the Fc region includes amino acid substitutions that consist essentially of the amino acid substitutions N297G and D265C (EU index).

[0029] In another aspect, an antibody, or an antigen-binding portion thereof, that includes an Fc region is provided herein, where the Fc region includes amino acid substitutions that consist essentially of the amino acid substitutions N297Q and D265C (EU index).

[0030] In another aspect, an antibody, or an antigen-binding portion thereof, that includes an Fc region is provided herein, where the Fc region includes amino acid substitutions that consist essentially of the amino acid substitutions N297A and D265A (EU index).

[0031] In another aspect, an antibody, or an antigen-binding portion thereof, that includes an Fc region is provided herein, where the Fc region includes amino acid substitutions that consist essentially of the amino acid substitutions N297G and D265A (EU index).

[0032] In another aspect, an antibody, or an antigen-binding portion thereof, that includes an Fc region is provided herein, wherein the Fc region includes amino acid substitutions that consist essentially of the amino acid substitutions N297Q and D265A (EU index).

[0033] In another aspect, an antibody, or an antigen-binding portion thereof, is provided herein, wherein the antibody has a reduction in effector function, defined as a decrease in binding to the Fc gamma receptor (FcγR) as compared to binding of the same antibody that includes an unmodified Fc region to the FcγR. In some embodiments, the decrease in binding is at least a 70% decrease, at least an 80% decrease, at least a 90% decrease, at least a 95% decrease, at least a 98% decrease, at least a 99% decrease, or a 100% decrease in antibody binding to the FcγR as compared to binding of the same antibody that includes an unmodified Fc region to the FcγR. In another embodiment, the antibody does not bind to the FcγR to a detectable extent. In another embodiment, the antibody binding to the FcγR is evaluated by biolayer interferometry (BLI). In another embodiment, the antibody binding to the FcγR is evaluated using an assay known to those of skill in the art. In another embodiment, the FcγR is the FcγR1 receptor. In another embodiment, the FcγR receptor is the FcγR2 receptor or the FcγR3 receptor. In another embodiment, the FcγR2 receptor is FcγR2A, FcγR2B, or FcγR2C. In another embodiment, the FcγR3 receptor is FcγR3A or FcγR3B. In another embodiment, the Fc receptor is a human Fc receptor. In other embodiments, the FcγR receptor is the FcγR2A 167R receptor. In other embodiments, the FcγR receptor is the FcγR3A 176V receptor. In other embodiments, the FcγR receptor is the FcγR3A 176F receptor.

[0034] In another aspect, the antibody, or antigen-binding portion thereof, is provided herein, where the antibody reduces cytokine release in an in vitro cytokine release assay with at least a 50% decrease in cytokine release as compared to cytokine release of the same antibody containing an unmodified Fc region. In certain embodiments, the decrease in cytokine release is at least a 60% decrease, at least a 70% decrease, at least an 80% decrease, at least a 90% decrease, at least a 95% decrease, at least a 98% decrease, at least a 99% decrease, or a 100% decrease in cytokine release as compared to cytokine release of the same antibody containing an unmodified Fc region. In another embodiment, the antibody does not exhibit detectable cytokine release. In another embodiment, the in vitro cytokine release assay is a Meso Scale Discovery (MSD) tissue culture (TC) inflammation assay. In another embodiment, the in vitro cytokine release assay is evaluated using an assay known to those of skill in the art. In another embodiment, the antibody reduces mast cell degranulation in an in vitro mast cell degranulation assay with at least a 50% decrease in mast cell degranulation as compared to mast cell degranulation of the same antibody containing an unmodified Fc region. In another embodiment, the decrease in mast cell degranulation is at least a 60% decrease, at least a 70% decrease, at least an 80% decrease, at least a 90% decrease, at least a 95% decrease, at least a 98% decrease, at least a 99% decrease, or a 100% decrease in mast cell degranulation as compared to mast cell degranulation of the same antibody containing an unmodified Fc region. In another embodiment, the antibody does not exhibit detectable mast cell degranulation. In another embodiment, the in vitro mast cell degranulation assay is a beta-hexosaminidase-based mast cell degranulation assay.

[0035] In some embodiments, the IgG isotype is the IgG1 isotype, the IgG2 isotype, the IgG3 isotype, or the IgG4 isotype. In another embodiment, the antibody is a human antibody, a chimeric or humanized antibody. In yet another embodiment, the antibody is a bispecific antibody. In another embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is an intact IgG antibody. In another embodiment, the antibody specifically binds to CD117, CD45, CD2, CD5, CD137, or CD252.

[0036] In another aspect, an antibody-drug conjugate (ADC) comprising an antibody, or an antigen-binding portion thereof, described herein is provided herein, wherein the antibody, or an antigen-binding portion thereof, is conjugated to a cytotoxin via a linker. In one embodiment, the cytotoxin is an RNA polymerase inhibitor. In another embodiment, the RNA polymerase inhibitor is amatoxin.

[0037] In another embodiment, the amatoxin is represented by formula (III),

Chemical formula

[0038] In another embodiment, the amatoxin is represented by formula (IB), [Chemical] Here, R1 is H, OH, OR A , or OR C ; R2 is H, OH, OR B , or OR C ; R A and R B together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D ; R4, R5, R 6、 and R7 are each independently H, OH, OR C , OR D , R C , or R D ; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D ; R9 is H, OH, OR C , or OR D ; X is -S-, -S(O)-, or -SO2-; R C is -L-Z; R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is optionally substituted C1-C6 alkylene, optionally substituted C1-C6 heteroalkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, peptide, dipeptide, -(C=O)-, disulfide, hydrazone, or a combination thereof; and Z is a chemical substructure formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within said antibody, or an antigen-binding fragment thereof, where Am contains exactly one R C substituent.

[0039] In another embodiment, the RNA polymerase inhibitor is amanitin. In another embodiment, the amanitin is selected from the group consisting of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amanine amide, amanullin, amanullinic acid, and proamanullin. In another embodiment, the cytotoxin is selected from the group consisting of Pseudomonas exotoxin A, debuganin, diphtheria toxin, saporin, maytansine, maytansinoid, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, and indolinobenzodiazepine dimer. In another embodiment, the auristatin is MMAE or MMAF. In another embodiment, the antibody, or an antigen-binding portion thereof, is conjugated to the cytotoxin via an interchain conjugate to native hinge cysteine. In another embodiment, the antibody, or an antigen-binding portion thereof, is conjugated to the cytotoxin by a cysteine residue in the Fc domain of the antibody. In another embodiment, the cysteine residue is introduced by an amino acid substitution in the Fc domain of the antibody. In another embodiment, the amino acid substitution is D265C. In another embodiment, the amino acid substitution is S239C.

[0040] In another aspect, provided herein is a pharmaceutical composition comprising an antibody or ADC according to any one of claims 1 to 218 and a pharmaceutically acceptable carrier.

[0041] In another aspect, provided herein is a method of reducing a population of hematopoietic stem cells (HSCs) in a human patient, wherein the method comprises administering to the patient an effective amount of an antibody or ADC described herein. In certain embodiments, the method further comprises administering to the patient a graft comprising hematopoietic stem cells. In certain embodiments, the graft is allogeneic. In certain embodiments, the graft is autologous.

[0042] In another aspect, methods are provided herein that include administering to a human patient a graft comprising hematopoietic stem cells, where the patient has been pre-administered an antibody or ADC as described herein in an amount sufficient to reduce the population of hematopoietic stem cells in the patient. In certain embodiments, the hematopoietic stem cells are CD117+ or CD45+ cells. In another embodiment, the patient has a blood disorder, a metabolic disorder, cancer, or an autoimmune disease, or a severe combined immunodeficiency disease (SCID).

[0043] In another aspect, methods are provided herein for treating leukemia in a human patient, where the method includes administering an antibody or ADC as described herein to the human patient having leukemia.

[0044] In another aspect, methods are provided herein that include administering to a human patient a graft comprising hematopoietic stem cells, where the patient has been pre-administered an antibody or ADC as described herein in an amount sufficient to reduce the population of immune cells in the patient. In certain embodiments, the immune cells are CD137+, CD2+, or CD5+ cells. In another embodiment, the immune cells are T cells.

[0045] In another aspect, compositions are provided herein that include an antibody or ADC as described herein, where the composition comprises less than 25% hydrophobic degradation products after heat stress. In certain embodiments, the composition comprises less than 20% hydrophobic degradation products after heat stress. In another embodiment, the composition comprises less than 15% hydrophobic degradation products after heat stress. In another embodiment, the composition comprises less than 10% hydrophobic degradation products after heat stress. In another embodiment, the composition comprises less than 5% hydrophobic degradation products after heat stress.

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

[0047] In another aspect, a method of treating an immunodeficiency disorder in a human patient is provided herein, where the method comprises administering to the patient a therapeutically effective amount of an antibody, an antigen-binding fragment thereof, or an ADC as described herein. In certain embodiments, the immunodeficiency disorder is a primary immunodeficiency or a secondary immunodeficiency.

[0048] In another aspect, a method of treating a metabolic disorder in a human patient is provided herein, where the method comprises administering to the patient a therapeutically effective amount of an antibody, an antigen-binding fragment thereof, or an ADC as described herein. In certain embodiments, the metabolic disorder is selected from the group consisting of glycogen storage disease, mucopolysaccharidosis, Gaucher disease, Hurler disease, sphingolipidosis, and metachromatic leukodystrophy.

[0049] In another aspect, a method of treating an autoimmune disorder in a human patient is provided herein, where the method comprises administering to the patient a therapeutically effective amount of an antibody, an antigen-binding fragment thereof, or an ADC as described herein. In some embodiments, the autoimmune disorder is selected from the group consisting of multiple sclerosis, human systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, psoriasis treatment, type 1 diabetes, acute disseminated encephalomyelitis, Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune oophoritis, Baló disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythematosus, dysautonomia, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture 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, Lyme disease, Ménière's disease, mixed connective tissue disease, myasthenia gravis, neuromyotonia, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-person syndrome, Takayasu arteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia, and Wegener's granulomatosis.

[0050] In another aspect, a method of treating cancer in a human patient is provided herein, where the method comprises administering to the patient a therapeutically effective amount of an antibody, an antigen-binding fragment thereof, or an ADC as described herein. In some embodiments, 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 has a reduction in effector function defined as a reduction in binding to an Fc gamma receptor (FcγR) of the same antibody containing an unmodified Fc region, compared to binding to the FcγR. In certain embodiments, the reduction in binding is at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, at least a 95% reduction, at least a 98% reduction, at least a 99% reduction, or a 100% reduction in antibody binding to the FcγR compared to binding of the same antibody containing an unmodified Fc region to the FcγR. In certain specific embodiments, the antibody does not bind to the FcγR to a detectable extent. In some embodiments, the antibody binding to the FcγR is evaluated by biolayer interferometry (BLI). In some embodiments, the FcγR is an FcγR1 receptor, an FcγR2 receptor, or an FcγR3 receptor. In some embodiments, the FcγR1 receptor is FcγR1A, FcγR1B, or FcγR1C. In some embodiments, the FcγR2 receptor is FcγR2A, FcγR2B, or FcγR2C. In some embodiments, the FcγR3 receptor is FcγR3A or FcγR3B. In some embodiments, the Fc receptor is a human Fc receptor.

[0052] In some embodiments of any of the above aspects, its IgG isotype is an IgG1 isotype, 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.

[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 specifically binds to CD117, CD45, CD2, CD5, CD137, or CD252.

[0057] In another aspect, an antibody-drug conjugate (ADC) comprising any of the antibodies herein is provided herein, wherein the antibody is conjugated to a cytotoxin via a linker.

[0058] In some embodiments of the conjugates herein, the cytotoxin is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is amatoxin.

[0059] In some embodiments, the amatoxin is represented by formula (IA)

Chemical Formula

[0060] In some embodiments, the amatoxin is represented by formula (IB) [Chemical] Here, R1 is H, OH, OR A or OR C ; R2 is H, OH, OR B or OR C ; R A and R B together with the oxygen atom to which they are attached optionally form a substituted 5-membered heterocycloalkyl group; R3 is H, R C or R D ; R4, R5, R 6、 and R7 are each independently H, OH, OR C or OR D R C or R D ; R8 is OH, NH2, OR C or OR D NHR C or NR C R D ; R9 is H, OH, OR C or OR D ; X is -S-, -S(O)- or -SO2-; R C is -L-Z; R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is optionally substituted C1-C6 alkylene, optionally substituted C1-C6 heteroalkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene; and Z is a chemical substructure formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within the antibody, or an antigen-binding fragment thereof, where Am contains exactly one R C substituent.

[0061] In some embodiments, the RNA polymerase inhibitor is amanitin. In some embodiments, the amanitin is selected from the group consisting of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amanine amide, amanullin, amanullinic acid, and proamanullin.

[0062] In some embodiments, the cytotoxin is selected from the group consisting of Pseudomonas exotoxin A, debuganin, diphtheria toxin, saporin, maytansine, maytansinoid, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, and indolinobenzodiazepine dimer. In some embodiments, the auristatin is MMAE or MMAF.

[0063] In some embodiments of the conjugates herein, the antibody is conjugated to the toxin by a cysteine residue in the Fc domain of the antibody. In some embodiments, the cysteine residue is introduced by an amino acid substitution in the Fc domain of the antibody. In some embodiments, the amino acid substitution is D265C.

[0064] In another aspect, provided herein is a pharmaceutical composition comprising an antibody or ADC as described herein and a pharmaceutically acceptable carrier.

[0065] In yet another aspect, provided herein is a method of reducing a population of hematopoietic stem cells (HSCs) in a human patient, wherein the method comprises administering to the patient an effective amount of an antibody or ADC as described herein.

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

[0067] In another aspect, provided herein is a method comprising administering to a human patient a graft comprising hematopoietic stem cells, wherein the patient has been previously administered an antibody or ADC as described herein in an amount sufficient to reduce the population of hematopoietic stem cells in the patient.

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

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

[0070] In one aspect, a method of reducing a population of CD117+ cells (in a human patient in need thereof) is provided herein, the method comprising administering to the patient an effective amount of an anti-CD117 antibody-drug conjugate (ADC), wherein the antibody-drug conjugate (ADC) comprises an anti-CD117 antibody conjugated to amatoxin via a linker, and is represented by the formula Ab-Z-L-Am, wherein Ab is an anti-CD117 antibody comprising an H435A mutation (EU index) in the Fc region of the antibody, L is a linker, Z is a chemical substructure, and Am is amatoxin. In certain embodiments, the ADC is administered prior to receiving a graft comprising hematopoietic stem cells. In another embodiment, the ADC is administered to the patient simultaneously with receiving a graft comprising hematopoietic stem cells.

[0071] In another aspect, provided herein is a method for administering to a human patient in need thereof an amount of an anti-CD117 antibody-drug conjugate (ADC) sufficient to reduce the population of CD117+ cells, wherein the antibody-drug conjugate (ADC) comprises an anti-CD117 antibody conjugated to amatoxin via a linker and is represented by the formula Ab-Z-L-Am, where Ab is an anti-CD117 antibody comprising an H435A mutation (EU index) in the Fc region of the antibody, L is a linker, Z is a chemical substructure, and Am is amatoxin; and thereafter administering to the patient a graft comprising hematopoietic stem cells. In certain embodiments, the graft comprising hematopoietic stem cells is administered to the patient after the concentration of the ADC has been substantially cleared from the patient's blood. In another embodiment, the hematopoietic stem cells or their progeny maintain the potential for hematopoietic stem cell function more than 2 days after transplantation of the hematopoietic stem cells into the patient. In yet another embodiment, the hematopoietic stem cells or their progeny localize to hematopoietic tissue and can re-establish 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, hyper immunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis. In another embodiment, the patient has an autoimmune disorder or a blood cancer.In another embodiment, the autoimmune disorder is selected from the group consisting of: multiple sclerosis, human systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, psoriasis treatment, type 1 diabetes, acute disseminated encephalomyelitis, Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune oophoritis, Barlow's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythematosus, dysautonomia, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture 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, Lyme disease, Meniere's disease, mixed connective tissue disease, myasthenia gravis, neuromyotonia, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-person syndrome, Takayasu arteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia, and Wegener's granulomatosis.

[0072] In another aspect, provided herein is a method of treating a human subject having a blood cancer, comprising administering to the human subject having a blood cancer an effective amount of an anti-CD117 antibody-drug conjugate (ADC), wherein the antibody-drug conjugate (ADC) comprises an anti-CD117 antibody conjugated to amatoxin via a linker and is represented by the formula Ab-Z-L-Am, where Ab is an anti-CD117 antibody comprising an H435A mutation (EU index) in the Fc region of the antibody, L is a linker, Z is a chemical substructure, and Am is amatoxin. In certain embodiments, the blood cancer is leukemia. In another embodiment, the Fc region of the anti-CD117 antibody comprises a D265C mutation (EU index). In yet another embodiment, the anti-CD117 antibody comprises a heavy chain variable region comprising 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, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:9; and a light chain variable region comprising a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:10, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:11, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:12. In another embodiment, the anti-CD117 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14. In another embodiment, the ADC is internalized by cancer cells, autoreactive cells, or hematopoietic stem cells after administration to the patient.

[0073] In another embodiment, the Am-L-Z is represented by formula (I)

Chemical formula

[0074] In another embodiment, the Am-L-Z is represented by formula (IB). [Chemical formula] wherein R1 is H, OH, OR A , or OR C ; R2 is H, OH, OR B , or OR C ; R A and R B , when present, together with the oxygen atom to which they are attached, form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D ; R4, R5, R 6、 and R7 are each independently H, OH, OR C , OR D , R C , or R D ; R8 is OH, NH2, OR C , OR D , NHR C , or NRC R D is; R9 is H, OH, OR C or OR D is; X is -S-, -S(O)-, or -SO2-; R C is -L-Z; R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is optionally substituted C1-C6 alkylene, optionally substituted C1-C6 heteroalkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -C(=O)-, peptide, or a combination thereof; and, Z is a chemical substructure formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within the antibody, or an antigen-binding fragment thereof, where Am contains exactly one R C substituent. In another embodiment, the ADC is administered to the human patient at a dose of about 0.1 mg / kg to about 0.3 mg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0075]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Mode for Carrying Out the Invention

[0076] [Detailed Description] Antibodies having a modified Fc region, and conjugates thereof (antibody-drug conjugates; ADCs), are disclosed herein, where said modification reduces or substantially eliminates the effector function of the antibody. Modifications to the Fc region may further enable an antibody-drug conjugate and / or decrease the half-life of the antibody. Interactions of the antibody and antibody-antigen complexes with cells of the immune system can affect various responsiveness including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Binding of the Fc region of the antibody to Fc receptors on the cell surface can trigger many biological responses (e.g., phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells [i.e., ADCC], release of inflammatory mediators, control of placental transfer and immunoglobulin production, etc.). By reducing or substantially eliminating the effector function of the antibody, the antibodies of the present disclosure can preferably avoid triggering various immune system reactions that may be detrimental in certain therapies (e.g., hematopoietic stem cell therapies [e.g., hematopoietic stem cell transplantation therapies] and reduction of hematopoietic cells [e.g., treatment of blood cancers, immune system diseases and disorders, autoimmune diseases, graft versus host disease, etc.]) (e.g., cytokine release can be avoided or mast cell degranulation can be avoided).

[0077] Accordingly, anti-hematopoietic cell antibodies (also referred to as anti-HC antibodies) having a modified Fc region that are useful for treatment are included herein. For example, the antibodies or ADCs herein are useful in conditioning procedures (preparing a patient for receiving a graft containing hematopoietic stem cells). Such procedures facilitate engraftment of the hematopoietic stem cell graft. According to the methods described herein, in some embodiments, antigens expressed by hematopoietic cells (e.g., hematopoietic stem cells, e.g., hematopoietic stem cells and / or mature immune cells [e.g., T cells]) (e.g., CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137, CD252, and combinations thereof) can be bound by an ADC, antibody, or antigen-binding fragment thereof, and by administering the same to a patient, the patient may be conditioned (e.g., for hematopoietic stem cell transplantation therapy or to reset the immune system). In some embodiments, the antibodies or ADCs contemplated herein may be used to treat hematological diseases or disorders. For example, in some embodiments, the antibodies or ADCs contemplated herein may be used to treat blood cancers. In another non-limiting example, the antibodies or ADCs contemplated herein may be used to treat graft-versus-host disease (“GvHD”). In certain embodiments, the antibodies or ADCs contemplated herein may be used to treat T-cell mediated diseases or disorders. As described herein, the antibody may be covalently conjugated to a cytotoxin to form an antibody-drug conjugate (ADC). Administering an ADC, antibody, or antigen-binding fragment thereof that can bind to one or more of the foregoing antigens to a patient in need of hematopoietic stem cell transplantation therapy may, for example, promote engraftment of the hematopoietic stem cell graft by selectively reducing endogenous hematopoietic stem cells (thereby creating a space to be filled by an exogenous hematopoietic stem cell graft).

[0078] In certain embodiments, the invention provides an isolated anti-CD117 antibody, particularly an isolated human anti-CD117 antibody, that binds to the ectodomain of human CD117, wherein the isolated anti-CD117 antibody has a modified Fc region, wherein the modification reduces or substantially eliminates the effector function of the antibody. The binding regions of the isolated anti-CD117 antibodies identified herein are described below.

[0079] The following sections provide descriptions of antibodies or conjugates thereof that may be administered to a patient (e.g., a patient suffering from cancer or an autoimmune disease, or a patient in need of a hematopoietic stem cell transplantation therapy, etc.) to promote engraftment of a hematopoietic stem cell graft, and methods of administering such therapeutic agents to a patient (e.g., prior to a hematopoietic stem cell transplantation).

[0080] Definition As used herein, the term "about" refers to a value that is within 5% of the recited value, either higher or lower.

[0081] As used herein, the term "allogeneic" when used in the context of transplantation is used to define cells (or tissues or organs) transplanted from a donor to a recipient that are of the same species but genetically different. Thus, the term "allogeneic cells" refers to cell types that are genetically different between two individuals but belong to the same species (e.g., human). Typically, the term "allogeneic" is used to define cells (e.g., stem cells) transplanted from a donor to a recipient who is not related by blood of the same species.

[0082] As used herein, the term "autologous" refers to cells or grafts in which the donor and recipient are the same subject.

[0083] As used herein, the term "allogeneic" refers to cells in which the donor and recipient species are different.

[0084] As used herein, the term "immune cell" is intended to include, but is not limited to, cells of hematopoietic origin and cells that play a role in the immune response. Immune cells include, but are not limited to, T cells and natural killer (NK) cells. Natural killer cells are well known in the art. In certain embodiments, cell lines such as NK-92 cells are included as natural killer cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells. Immune cells can be allogeneic or autologous.

[0085] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to a particular antigen or is immunologically reactive with a particular antigen. Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific 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 quadri-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, "isolated" refers to a polypeptide (e.g., an antibody) that has been identified and separated and / or recovered from a cell or cell culture in which it is expressed. Usually, an isolated antibody is prepared by at least one purification step. Thus, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. For example, an isolated antibody that specifically binds to CD117 is substantially free of antibodies that specifically bind to antigens other than CD117.

[0087] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available in the art or known, and is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies useful in the present disclosure can be prepared using a variety of techniques known in the art, including using hybridoma, recombinant, and phage display technologies, or combinations thereof. Unless otherwise specified, the term "monoclonal antibody" (mAb) is meant to include both intact molecules and antibody fragments (e.g., including Fab and F(ab')2 fragments, etc.) that are capable of specifically binding to a target protein. As used herein, Fab and F(ab')2 fragments refer to antibody fragments that lack the Fc fragment of an intact antibody. In certain embodiments, the antibody fragment includes an Fc region.

[0088] Generally, an antibody comprises a heavy chain and a light chain that contain an antigen-binding region. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability (called complementarity-determining regions (CDRs)), interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, and are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, 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 may mediate the binding of the immunoglobulin to host tissues or factors (e.g., various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q), etc.).

[0089] As used herein, the term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of the full-length antibody. The antibody fragment can be, for example, Fab, F(ab')2, scFv, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments included within the term "antigen-binding fragment" of an antibody include, but are not limited to, the following: (i) a monovalent fragment consisting of a Fab fragment, VL, VH, CL, and CH1 domains; (ii) a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of a single arm of an antibody; (v) a dAb containing VH and VL domains; (vi) a dAb fragment consisting of a VH domain (see, for example, Ward et al., Nature 341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) an isolated complementarity-determining region (CDR); and (ix) a combination of two or more (e.g., two, three, four, five, or six) isolated CDRs that may optionally be linked by a synthetic linker. Further, the two domains (VL and VH) of an Fv fragment are encoded by separate genes, but they can be linked by a linker using recombinant methods, resulting in a single protein chain (where the VL and VH regions pair to form a monovalent molecule) (known as single-chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be screened for utility in the same manner as intact antibodies.Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis procedures known in the art. In certain embodiments, the antigen-binding fragment of an antibody comprises an Fc region.

[0090] As used herein, the term "anti-CD117 antibody" or "antibody that binds CD117" refers to an antibody that can bind CD117 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting CD117.

[0091] As used herein, the term "anti-CD45 antibody" or "antibody that binds CD45" refers to an antibody that can bind CD45 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting CD45.

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

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

[0094] As used herein, the term "anti-CD137 antibody" or "antibody that binds CD137" refers to an antibody that can bind CD137 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting CD137.

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

[0096] As used herein, the term "bispecific antibody" refers to a monoclonal, e.g., human or humanized, antibody that can bind to, for example, at least two different antigens or two different epitopes. For example, one of the binding specificities can be directed to a hematopoietic stem cell surface antigen, CD117 (e.g., GNNK+ CD117), and the other can be specifically bound to various hematopoietic stem cell surface antigens or another cell surface protein, such as a receptor or receptor subunit involved in a signal transduction pathway that particularly enhances cell proliferation. In some embodiments, the binding specificities can be directed to unique, non-overlapping epitopes on the same target antigen (i.e., biparatopic antibody).

[0097] As used herein, an "intact" or "full-length" antibody refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability (called complementarity determining regions (CDRs)), interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, and are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors (e.g., 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 "complementary determining region" (CDR) refers to the hypervariable regions found in both the light and heavy chain variable domains of an antibody. The more highly conserved portions of the variable domains are called the framework regions (FR). The positions of the amino acids representing the hypervariable regions of an antibody can vary depending on context and the various definitions known in the art. Some positions within the variable domain may be considered hybrid hypervariable positions, which may be considered within the hypervariable region under one set of criteria, while being considered outside the hypervariable region under another set of criteria. One or more of these positions may also be found in the extended hypervariable region. The antibodies described herein may include modifications at these hybrid hypervariable positions. The variable domains of the native heavy and light chains each predominantly adopt a β-sheet structure and include four framework regions linked by three CDRs (the CDRs form loops that link the β-sheet structure and in some cases form part of it). The CDRs in each chain are grouped together in proximity by the framework regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, together with the CDRs from the other antibody chain, contribute to the formation of the target binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD., 1987). In certain embodiments, the numbering of the amino acid residues of the immunoglobulin is done according to the Kabat et al. system of numbering the amino acid residues of immunoglobulins, unless otherwise specified (however, any antibody numbering scheme may be utilized, including but not limited to IMGT and Chothia).

[0099] As used herein, the term "thermal stress" refers to the stress caused by any temperature change to a molecule (e.g., an antibody, an Fc including its antigen-binding fragment, or an ADC). In certain embodiments, the thermal stress is incubating an antibody, an Fc including its antigen-binding fragment, or an ADC at 60 °C for 30 minutes.

[0100] As used herein, the term "specifically binds" refers to the ability of an antibody (or an ADC) to recognize and bind to a specific protein structure (epitope) rather than proteins in general. If an antibody is specific for epitope "A", in a reaction containing labeled "A" and said antibody, the amount of labeled A that binds to said antibody decreases in the presence of a molecule containing epitope A (or free, unlabeled A). For example, if an antibody (when labeled) can be competitively displaced from its target by the corresponding unlabeled antibody, said antibody "specifically binds" to the target. In certain embodiments, the antibody binds to a target (e.g., an antigen expressed by hematopoietic stem cells [e.g., CD117 (e.g., GNNK+ CD117), or CD45; or an antigen expressed by mature immune cells [e.g., T-cells] [e.g., CD45, CD2, CD5, CD137, or CD252]) with a K -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 means a value less than, e.g., 10 -12 less than the numerical value, e.g., 10 -13 ) for said target, and specifically binds. In certain embodiments, the term "specifically binds" means at least about 1x10 D M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1 x 10-11 M, 1x10 -12 Refers to the ability of an antibody to bind an antigen with a Kd of M or greater and / or to bind an antigen with an affinity at least two-fold greater than its affinity for a non-specific antigen. In certain embodiments, K D is measured according to standard Bio-Layer Interferometry (BLI). However, it should be understood that the antibody can specifically bind two or more antigens with related sequences. For example, in certain embodiments, the antibody may specifically bind both human and non-human (e.g., mouse or non-human primate) orthologs of an antigen (e.g., CD117 [GNNK+ CD117], CD45, CD2, CD5, CD137, or CD252).

[0101] As used herein, the term "chimeric" antibody refers to an antibody having a variable sequence derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region typically selected from a human immunoglobulin template. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(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 4,816,397.

[0102] As used herein, the terms "Fc", "Fc region", and "Fc domain" refer to a portion of an immunoglobulin (IgG molecule) related to the crystalline fragment obtained by papain digestion of the IgG molecule. The Fc region includes the C-terminal halves of the two heavy chains of the IgG molecule, linked by disulfide bonds. This has no antigen-binding activity, but includes a sugar moiety and binding sites for Fc receptors including complement and FcRn receptors (see below). For example, the Fc region includes the second constant domain CH2 (e.g., residues 231-340 of EU position of human IgG1) and the third constant domain CH3 (e.g., residues 341-447 of EU position of human IgG1). As used herein, the Fc region includes the "lower hinge region" (e.g., residues 233-239 of EU position of IgG1).

[0103] Fc may refer to this region in isolation or in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at many positions within the Fc domain, including, but not limited to, EU positions 270, 272, 312, 315, 356, and 358, etc., and thus there may be minor differences between the sequences presented in this application and those known in the art. Thus, "wild-type IgG Fc domain" or "WT IgG Fc domain" refers to any naturally occurring IgG Fc region (i.e., any allele). The sequences of the heavy chains of human 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_human), respectively. An example of a "WT" Fc region is shown in SEQ ID NO: 15, which provides a heavy chain constant region including the Fc region.

[0104] As used herein, the term "modified Fc region" or "variant Fc region" refers to an IgG Fc domain that contains one or more amino acid substitutions, deletions, insertions, or modifications introduced at any position within the Fc region. In certain embodiments, the variant IgG Fc domain contains one or more amino acid substitutions that result in a decreased or abolished binding affinity for Fc gamma R and / or C1q as compared to the wild-type Fc domain that does not contain any amino acid substitutions. Further, Fc binding interactions are essential for various effector functions and downstream signaling events (including, but not limited to, antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)). Thus, in certain embodiments, an antibody that contains a variant Fc domain (e.g., an antibody, fusion protein, or conjugate) may have an altered binding affinity for at least one Fc ligand (e.g., Fc gamma R) as compared to the corresponding antibody that has the same amino acid sequence except that it does not contain one or more amino acid substitutions, deletions, insertions, or modifications (e.g., an unmodified Fc region that contains the amino acid residues that are naturally present at the corresponding positions in the Fc region).

[0105] The variant Fc domains described herein are defined according to the amino acid modifications that compose them. For all amino acid substitutions discussed herein with respect to the Fc region, the numbering always follows the EU index as in Kabat. Thus, for example, D265C is an Fc variant in which the aspartic acid (D) at EU position 265 is replaced with cysteine (C) relative to the parental Fc domain. Similarly, for example, D265C / L234A / L235A defines an Fc variant having substitutions at EU positions 265 (D to C), 234 (L to A), and 235 (L to A) relative to the parental Fc domain. Variants may also be designated according to their final amino acid composition at the mutated EU amino acid positions. For example, the L234A.L235A mutation may be referred to as "LALA". As a further example, the E233P.L234V.L235A.delG236 (deletion of 236) mutation may be referred to as "EPLVLAdelG". As yet another example, the I253A.H310A.H435A mutation may be referred to as "IHH". Note that the order in which substitutions are shown is arbitrary.

[0106] As used herein, the term "Fc gamma receptor" or "Fc gamma R" refers to any member of a family of proteins that bind to the Fc region of IgG antibodies and are encoded by the Fc gamma R genes. In humans, this family includes, but is not limited to, Fc gamma RI (CD64) (e.g., isoforms Fc gamma RIa, Fc gamma RIb, and Fc gamma RIc, etc.); Fc gamma RII (CD32) (e.g., isoforms Fc gamma RIIa [e.g., allotypes H131 and R131], Fc gamma RIIb [e.g., Fc gamma RIIb-1 and Fc gamma RIIb-2], and Fc gamma RIIc, etc.); and Fc gamma RIII (CD16) (e.g., isoforms Fc gamma RIIIa [e.g., allotypes V158 and F158] and Fc gamma RIIIb [e.g., allotypes Fc gamma RIIIb-NA1 and Fc gamma RIIIb-NA2], and any undiscovered human Fc gamma R or isoform or allotype of Fc gamma R). Fc gamma R may be derived from any living organism (e.g., but not limited to, humans, mice, rats, rabbits, and monkeys, etc.). 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), and any undiscovered mouse Fc gamma R or isoform or allotype of Fc gamma R.

[0107] As used herein, the term "effector function" refers to a biochemical event resulting from the interaction between an Fc domain and an Fc receptor. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. As used herein, "effector cell" refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and gamma delta T cells, and are derived from any living organism (e.g., but not limited to, humans, mice, rats, rabbits, and monkeys).

[0108] As used herein, the terms “silent,” “silenced,” or “silencing” refer to an antibody having a modified Fc region described herein, wherein binding to Fc gamma receptor (FcγR) is reduced as compared to binding to FcγR of the same antibody containing an unmodified Fc region (e.g., when measured by BLI, e.g., a reduction in binding to FcγR of at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, as compared to binding to FcγR of the same antibody containing an unmodified Fc region). In some embodiments, the Fc-silenced antibody does not exhibit detectable binding to FcγR. Binding of an antibody having a modified Fc region to FcγR can be measured using a variety of techniques known in the art (e.g., without limitation, equilibrium methods [e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; radioimmunoassay (RIA)], or surface plasmon resonance assays or other assays based on the kinetics of other mechanisms [e.g., BIACORE® analysis or Octet® analysis (forteBIO)], as well as other methods [e.g., indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration), etc.]). These and other methods may utilize labels on one or more components to be evaluated and / or may use various detection methods (e.g., without limitation, chromogenic labels, fluorescent labels, luminescent labels, or isotope labels, etc.). Regarding binding affinity and kinetics, it is described in detail in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999) (which focuses on antibody-immunogen interactions).An example of a competitive binding assay is a radioimmunoassay that involves incubating a labeled antigen and an antibody of interest in the presence of increasing amounts of unlabeled antigen and detecting the antibody bound to the labeled antigen. From that data, the affinity and binding off-rate of the antibody of interest for a particular antigen may be determined by Scatchard plot analysis. Competition with a second antibody may also be measured using a radioimmunoassay. In this case, the antigen is incubated in the presence of an antibody of interest conjugated to a labeled compound and increasing amounts of unlabeled second antibody.

[0109] As used herein, the term “same antibody comprising an unmodified Fc region” refers to an antibody that lacks the recited amino acid substitutions (e.g., D265C, L234A, L235A, and / or H435A) but otherwise has the same amino acid sequence as the antibody with the modified Fc being compared.

[0110] The term “antibody dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which a polypeptide comprising an Fc domain (e.g., an antibody) binds to an Fc receptor (FcR) present on certain cytotoxic cells (e.g., primarily NK cells, neutrophils, and macrophages), and these cytotoxic effector cells become specifically bound to a “target cell” having an antigen, and subsequently the target cell is killed by that cytotoxin (Hogarth et al., Nature review Drug Discovery 2012, 11:313). In addition to antibodies and their fragments, other peptides comprising an Fc domain (e.g., Fc fusion proteins and Fc conjugate proteins) that have the ability to specifically bind to antigen-bearing target cells are contemplated to effect cell-mediated cytotoxicity.

[0111] For simplicity, cell-mediated cytotoxicity resulting from the activity of a polypeptide comprising an Fc domain is also referred to herein as ADCC activity. The ability of any particular polypeptide of the present disclosure to mediate lysis of target cells by ADCC can be assayed. To evaluate ADCC activity, a polypeptide of interest (e.g., an antibody) is added to target cells together with immune effector cells, resulting in lysis of the target cells. Cell lysis is generally detected by release of a label (e.g., a radiolabeled substrate, a fluorescent dye, or a native intracellular protein) from the lysed cells. Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Specific examples of in vitro ADCC assays are 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 additionally, the ADCC activity of an antibody of interest may be evaluated in vivo (e.g., in an animal model as disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA 95:652 (1998)).

[0112] As used herein, the terms "conditioning" and "condition" refer to the process of preparing a patient to receive a graft (e.g., a graft comprising hematopoietic stem cells). Such procedures promote engraftment of hematopoietic stem cell grafts (e.g., as inferred from the sustained increase in the amount of viable hematopoietic stem cells in blood samples isolated from patients after conditioning procedures and subsequent hematopoietic stem cell transplantation). In accordance with the methods described herein, a patient may be conditioned for hematopoietic stem cell transplantation therapy by administering to the patient an ADC, an antibody, or an antigen-binding fragment thereof that can bind to an antigen expressed by hematopoietic stem cells (e.g., CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137, or CD252, etc.). As described herein, the antibody may be covalently conjugated to a cytotoxin to form an ADC. Administering to a patient in need of hematopoietic stem cell transplantation therapy an antibody, an antigen-binding fragment thereof, or an ADC that can bind to one or more of the above antigens may promote engraftment of the hematopoietic stem cell graft, for example, by selectively reducing endogenous hematopoietic stem cells, thereby creating a space to be filled by an exogenous hematopoietic stem cell graft.

[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 an autoimmune disease or cancer.

[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 or 50%. This 50% decrease in serum concentration reflects the amount of drug circulating.

[0115] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. A human antibody may include amino acid residues not encoded by the human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or mutations introduced by gene rearrangement or somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species such as a mouse are grafted onto human framework sequences. Human antibodies may be produced in human cells (e.g., by recombinant expression), or by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. When a human antibody is a single-chain antibody, it may include a linker peptide not found in native human antibodies. For example, an Fv may include a linker peptide such as from about two to about eight glycine or other amino acid residues, which linker peptide links the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin. Human antibodies may be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. Human antibodies may also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes (see, e.g., PCT Publication Nos. WO 1998 / 24893; WO 1992 / 01047; WO 1996 / 34096; WO 1996 / 33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598).

[0116] The "humanized" form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin that contains minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are of human immunoglobulin sequences. The humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. See, for example, Riechmann et al., 1988, Nature 332:323-7; U.S. 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 / 09967; U.S. 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, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat. No. 5,565,332.

[0117] As used herein, the term "engraftment potential" is used to refer to the ability of hematopoietic stem cells and hematopoietic progenitor cells to re-engraft into tissues, regardless of whether such cells are naturally circulating or provided by transplantation. The term encompasses all events surrounding or leading to engraftment, such as cell tissue homing and colony formation of cells within the tissue of interest. The efficiency or rate of engraftment can be evaluated or quantified using any clinically acceptable parameter known to those of skill in the art, for example, by evaluating the competitive repopulating unit (CRU); uptake or expression of a marker in a tissue to which the stem cells have homed, colonized, or engrafted; or by evaluating the progression of the subject by disease progression, survival of hematopoietic stem cells and hematopoietic progenitor cells, or survival of the recipient. Engraftment can also be determined by measuring the white blood cell count in the peripheral blood after transplantation. Engraftment can also be evaluated by measuring the recovery rate of bone marrow cells by donor cells in a bone marrow aspiration sample.

[0118] As used herein, the term "hematopoietic stem cell" ("HSC") refers to an immature blood cell having the ability to self-renew and to differentiate into mature blood cells including, but not limited to, diverse lineages including granulocytes (e.g., myeloblasts, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Such cells may include CD34 + cells. CD34 +Cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are thought to contain a subpopulation of cells with the characteristics of the stem cells defined above, while in mice, HSCs are CD34-. Furthermore, HSCs also refer to long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). LT-HSCs and ST-HSCs are distinguished based on their functional potential and the expression of cell surface markers. For example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A, etc.). In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, CD48-, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra, etc.), while ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra, etc.). In addition, ST-HSCs are less quiescent and more proliferative than LT-HSCs under homeostatic conditions. However, LT-HSCs have a higher self-renewal ability (i.e., LT-HSCs can survive throughout adulthood and be continuously transplanted through successive recipients), while ST-HSCs have a limited self-renewal ability (i.e., ST-HSCs can only survive for a limited period and cannot be continuously transplanted). Any of these HSCs can be used in the methods described herein. ST-HSCs are particularly useful because they are highly proliferative and can therefore give rise to differentiated progeny more rapidly.

[0119] As used herein, the term "anti-hematopoietic cell antibody" or "anti-HC antibody" refers to an antibody that specifically binds to an antigen expressed by hematopoietic stem cells (e.g., CD117 [e.g., GNNK+ CD117], or CD45); or an antigen expressed by mature immune cells (e.g., T-cells) (e.g., CD45, CD2, CD5, CD137, or CD252).

[0120] As used herein, the term "functional potential of hematopoietic stem cells" refers to 1) multi-potency (which refers to the ability to differentiate into multiple diverse blood lineages, including, but not limited to, granulocytes [e.g., myeloblasts, neutrophils, eosinophils, basophils], erythrocytes [e.g., reticulocytes, 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, T cells, and B cells]), 2) self-renewal (which refers to the ability of hematopoietic stem cells to give rise to daughter cells with the same potential as the mother cell, and further, this ability can occur repeatedly throughout an individual's lifespan without exhaustion), and 3) the ability of hematopoietic stem cells or their progeny, when re-introduced into a transplant recipient, to home to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis, including the functional characteristics of hematopoietic stem cells.

[0121] As used herein, the terms "subject" and "patient" refer to a living organism, such as a human, who is undergoing treatment for a specific disease or condition as described herein. For example, a patient, such as a human patient, may undergo treatment prior to hematopoietic stem cell transplantation therapy to facilitate engraftment of exogenous hematopoietic stem cells.

[0122] As used herein, the term "donor" refers to a human or animal from whom one or more cells have been isolated prior to administering the cells or their progeny to a recipient. The one or more cells may be, for example, a population of hematopoietic stem cells.

[0123] As used herein, the term "diabody" refers to a bivalent antibody comprising two polypeptide chains, where each polypeptide chain is a V H and a V L domain linked by a linker that is too short to allow intramolecular association of the V H and V L domains (e.g., a linker consisting of five amino acids), such that each domain pairs with a complementary domain on a separate polypeptide chain to form a homodimeric structure. Accordingly, the term "triabody" refers to a trivalent antibody comprising three peptide chains, each of which is a V H and a V L domain linked by a linker that is too short to allow intramolecular association of the V H domain and a V L domain (e.g., a linker consisting of 1-2 amino acids) within the same peptide chain. The thus-constructed peptides are usually trimerized (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993) such that the V H domain and the V L domain of adjacent peptide chains are spatially close to each other because the peptide folds into its native structure.

[0124] As used herein, the term "endogenous" describes a molecule, cell, tissue, or organ that is naturally found in a particular living organism, such as a human patient (e.g., hematopoietic stem cells, or cells of the hematopoietic lineage such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglial cells, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, or B-lymphocytes).

[0125] As used herein, the term "recipient" refers to a patient who receives a graft such as a graft containing a population of hematopoietic stem cells. The transplanted cells administered to the recipient may be, for example, autologous, syngeneic, or allogeneic cells.

[0126] As used herein, the term "sample" refers to a specimen taken from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or dermis), pancreatic juice, chorionic villus sample, and cells).

[0127] As used herein, the term "scFv" refers to a single-chain Fv antibody in which the variable domain of the heavy chain derived from an antibody and the variable domain of the light chain are joined to form a single chain. The scFv fragment is a single polypeptide chain containing the variable regions (V L ) (e.g., CDR-L1, CDR-L2, and / or CDR-L3) of the antibody light chain and the variable region (V H ) (e.g., CDR-H1, CDR-H2, and / or CDR-H3) of the antibody heavy chain, separated by a linker. The V L region and V HThe linker that connects the domains may be a peptide linker composed of amino acids that make up the protein. Alternative linkers may be used to increase the resistance of the scFv fragment to proteolysis (e.g., a linker containing D-amino acids), to increase the solubility of the scFv fragment (e.g., a hydrophilic linker such as a polyethylene glycol-containing linker or a polypeptide containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (e.g., a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to reduce the immunogenicity of the scFv fragment (e.g., a linker containing a glycosylation site). Those skilled in the art will also understand that the variable regions of the scFv molecules described herein can be modified such that the amino acid sequences vary from the antibody molecules from which they are derived. For example, nucleotide or amino acid substitutions that result in conservative substitutions or changes at amino acid residues (e.g., in the CDRs and / or framework residues) may be made to preserve or enhance the functionality of the scFv (the ability to bind to the antigen recognized by the corresponding antibody).

[0128] As used herein, the phrase "substantially cleared from the blood" refers to a time point after administration of a therapeutic agent (e.g., an anti-CD117 antibody, or an antigen-binding fragment thereof) to a patient, where the concentration of the therapeutic agent in a blood sample isolated from the patient is such that the therapeutic agent cannot be detected by conventional methods (e.g., where the therapeutic agent cannot be detected above the noise threshold of the device or assay used to detect the therapeutic agent). Various techniques known in the art (e.g., ELISA-based detection assays known in the art or described herein) may be used to detect antibodies, antibody fragments, and protein ligands. Additional assays that may be used to detect an antibody, or an antibody fragment, include, among others, immunoprecipitation and immunoblot assays, which are known in the art.

[0129] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

[0130] As used herein, the term "to treat" or "treatment" means reducing the severity and / or frequency of disease symptoms, eliminating the disease symptoms and / or the underlying cause of said symptoms, reducing the frequency or likelihood of the disease symptoms and / or their underlying cause, and improving or repairing damage caused directly or indirectly by the disease, any improvement of any result of the disease (e.g., increased survival period, reduced morbidity and / or reduced side effects which are by-products of alternative treatment modalities, etc.); as will be readily understood in the art, it is preferred but not a requirement of the treatment act to completely eradicate the disease. Beneficial or desired clinical outcomes include, but are not limited to, promoting engraftment of exogenous hematopoietic cells in a patient following antibody conditioning therapy and subsequent hematopoietic stem cell transplantation as described herein. Further beneficial outcomes include, in patients in need of hematopoietic stem cell transplantation, an increase in the number of hematopoietic stem cells or an increase in the relative concentration thereof following conditioning therapy and subsequent administration of an exogenous hematopoietic stem cell graft to said patient. Beneficial outcomes of the therapies described herein may also include an increase in the number of cells or an increase in the relative concentration of one or more cells of the hematopoietic lineage (megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglial cells, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, or B lymphocytes, etc.). Further beneficial outcomes may include a decrease in the amount of a cell population causing disease, such as a population of cancer cells (e.g., CD117+ leukemia cells) or autoreactive cells (e.g., CD117+ autoreactive lymphocytes such as CD117+ T-cells expressing a T-cell receptor that cross-reacts with a self-antigen). As long as the methods of the present disclosure are directed to preventing a disorder, it is understood that the term "prevent" does not require that the disease state be completely blocked.Rather, as used herein, the term "prevent" refers to what one of ordinary skill in the art can do, such as identifying a population susceptible to a disorder and administering a compound of the present disclosure prior to the onset of the disease. The term does not mean that the disease state is completely avoided.

[0131] As used herein, a patient "in need of" hematopoietic stem cell transplantation includes patients who exhibit a defect or deficiency in one or more types of blood cells, as well as patients having a stem cell disorder, an autoimmune disease, cancer, or other conditions described herein. Hematopoietic stem cells generally 1) are multi-potent and thus can differentiate into multiple and diverse blood lineages including, but not limited to, granulocytes (e.g., myeloblasts, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, 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), 2) can self-renew and thus give rise to daughter cells with the potential to be equivalent to the mother cells, and 3) upon re-introduction into a transplant recipient, home to the hematopoietic stem cell niche and have the ability to re-establish productive and sustained hematopoiesis. Thus, hematopoietic stem cells may be administered in vivo to patients with a defect or deficiency in one or more cell types of the hematopoietic lineage to reconstitute the population of defective or deficient cells. For example, the patient may be suffering from cancer, and the deficiency may be caused by administration of a chemotherapeutic agent or other agent that selectively or non-specifically reduces the cancerous cell population. Further, or alternatively, the patient may be suffering from an abnormal hemoglobinopathy (e.g., non-malignant abnormal hemoglobinopathy) such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome. The subject may be suffering from or affected by adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome. The subject may have or be affected by a hereditary blood disorder (e.g., sickle cell anemia) or an autoimmune disease. Further, or alternatively, the subject may have or be 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 lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the subject has myelodysplastic syndrome. In some embodiments, the subject has an autoimmune disease such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, type I diabetes, or another autoimmune condition described herein. In some embodiments, the subject is in need of chimeric antigen receptor T-cell (CART) therapy. In some embodiments, the subject has or is affected by a metabolic storage disorder. The subject may have a metabolic disorder selected from the group consisting of glycogen storage disease, mucopolysaccharidosis, Gaucher disease, Hurler disease, sphingolipidosis, metachromatic leukodystrophy, or any other disease or disorder (including, but not limited to, severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, marble bone disease, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis) that can benefit from the treatments and therapies disclosed herein, as well as diseases or disorders described in "Bone Marrow Transplantation for Non-Malignant disease", ASH Education Book, 1:319-338 (2000) (this disclosure relates to conditions that may be treated by prescribing hematopoietic stem cell transplantation therapy, which is hereby incorporated by reference in its entirety).Additionally or alternatively, a patient "in need of" a hematopoietic stem cell transplant may be a patient who has or has not been affected by one of the aforementioned conditions (nevertheless showing a decrease in the level of one or more endogenous cell types within the hematopoietic lineage, such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes, i.e., a decrease compared to the level in a subject who is otherwise healthy). Some of those skilled in the art can readily determine, for example, by flow cytometry and fluorescence-activated cell sorting (FACS) methods, among others procedures known in the art, whether the level of one or more of the aforementioned cell types or other blood cell types is decreased relative to a subject who is otherwise healthy.

[0132] As used herein, the terms "variant" and "derivative" are used interchangeably and refer to naturally occurring, synthetic, and semi-synthetic analogs of the compounds, peptides, proteins, or other substrates described herein. Variants or derivatives of the compounds, peptides, proteins, or other substances described herein may retain or improve the biological activity of the original substance.

[0133] As used herein, the phrase "stem cell disorder" broadly refers to any disease, disorder, or condition that can be treated or cured by conditioning the subject's target tissue and / or by removing the endogenous stem cell population in the target tissue (e.g., by removing the endogenous hematopoietic stem cell population or hematopoietic progenitor cell population from the subject's bone marrow tissue), and / or by engrafting or transplanting stem cells into the subject's target tissue. For example, type I diabetes has been shown to be cured by hematopoietic stem cell transplantation and can benefit from conditioning according to the compositions and methods described herein. Further disorders that can be treated using the compositions and methods described herein include, but are not limited to, sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome. Further diseases that can be treated using the methods of patient conditioning and / or hematopoietic stem cell transplantation described herein include genetic blood disorders (e.g., sickle cell anemia) and autoimmune diseases such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease. Further diseases that can be treated using the conditioning and / or transplantation methods described herein include malignancies (e.g., neuroblastoma) or blood cancers (e.g., leukemia, lymphoma, and myeloma). For example, the cancer may be acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Further diseases that can be treated using the conditioning and / or transplantation methods described herein include myelodysplastic syndromes. In some embodiments, the subject has or is affected by a metabolic storage disorder.For example, the subject may be a metabolic disorder selected from the group consisting of glycogen storage disease, mucopolysaccharidosis, Gaucher disease, Hurler disease, sphingolipidosis, metachromatic leukodystrophy, or any other disease or disorder (including but not limited to severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis), and diseases or disorders described in "Bone Marrow Transplantation for Non-Malignant disease", ASH Education Book, 1:319-338 (2000) (this disclosure relates to conditions that may be treated by prescribing hematopoietic stem cell transplantation therapy, and is hereby incorporated by reference in its entirety), and may have suffered from or been affected by such diseases or disorders.

[0134] As used herein, the term "vector" includes nucleic acid vectors such as plasmids, DNA vectors, plasmids, RNA vectors, viruses, or other suitable replicons. Expression vectors described herein may include polynucleotide sequences, as well as additional sequence elements used, for example, to express proteins and / or to integrate these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids that contain regulatory sequences such as promoter regions and enhancers that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments include polynucleotide sequences that enhance the translation rate of these genes or improve the stability or cytoplasmic translocation of the mRNA resulting from gene transcription. These sequence elements may include, for example, 5' and 3' untranslated regions and polyadenylation signal sites that serve to efficiently transcribe the genes on the expression vector. Expression vectors described herein may also include polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.

[0135] As used herein, the term "conjugate" or "antibody-drug conjugate" or "ADC" refers to an antibody conjugated to a cytotoxin. An ADC is formed by the chemical coupling of a reactive functional group of an antibody, or a molecule such as an antigen-binding fragment thereof, with a suitable reactive functional group of another molecule such as a cytotoxin described herein. The conjugate may include a linker between the two molecules that are conjugated to each other (e.g., between an antibody and a cytotoxin). Examples of linkers that can be used in the formation of conjugates include peptide-containing linkers (e.g., linkers containing naturally occurring or non-naturally occurring amino acids, such as D-amino acids). The linker may be prepared using various strategies described herein and known in the art. Depending on the reactive components therein, the linker may be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0136] As used herein, the term "microtubule-binding agent" refers to a compound that acts by disrupting the microtubule network that is essential for mitosis and interphase cell functions in cells. Examples of microtubule-binding agents include maytansine, maytansinoids, and their derivatives (such as those described herein or known in the art), vinca alkaloids such as vinblastine, vinblastine sulfate, vincristine, vincristine sulfate, vindesine, and vinorelbine, taxanes such as docetaxel and paclitaxel, macrolides such as discodermolide, colchicine, and epothilone, and their derivatives (such as epothilone B or its derivatives), but are not limited thereto.

[0137] As used herein, the term "amatoxin" refers to a member of the amatoxin family of peptides produced by Amanita phalloides mushrooms, or a variant or derivative thereof (e.g., a variant or derivative thereof that can inhibit RNA polymerase II activity). Amatoxins useful in combination with the compositions and methods described herein include, for example, but are not limited to, compounds of formulas (III), (IIIA), (IIIB), and (IIIC) (each as described herein below) (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amanin amide, amanullin, amanullinic acid, or proamanullin, etc.). As described herein, amatoxins may be conjugated to an antibody, or an antigen-binding fragment thereof, for example, via a linker substructure (L) (thereby forming an ADC). Exemplary methods of conjugating amatoxins and linkers useful in such processes are described below. Exemplary linker-containing amatoxins useful for conjugation to an antibody, or antigen-binding fragment, by the compositions and methods are also described herein.

[0138] As used herein, the term "acyl" refers to -C(=O)R, where, as defined herein, R is hydrogen ("aldehyde"), C1-C 12 alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl, C3-C7 carbocyclic, C6-C 20 aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl. Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, and acryloyl.

[0139] As used herein, the term "C1-C 12 alkyl" refers to a straight or branched saturated hydrocarbon having from 1 to 12 carbon atoms. Typical C1-C 12Examples of the alkyl group include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; on the other hand, branched C1-C 12 Examples of the alkyl include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl. The C1-C 12 alkyl group may be unsubstituted or may be substituted.

[0140] As used herein, the term "alkenyl" refers to an unsaturated hydrocarbon containing at least one site, i.e., a carbon-carbon, sp 2 double bond, and containing normal, secondary, or tertiary carbon atoms, having C2-C 12 Examples include, but are not limited to: ethylene or vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, etc. The alkenyl group may be unsubstituted or may be substituted.

[0141] As used herein, "alkynyl" refers to an unsaturated hydrocarbon containing at least one site, i.e., a carbon-carbon, sp triple bond, and containing normal, secondary, or tertiary carbon atoms, having C2-C 12 Examples include, but are not limited to, acetylene and propargyl. The alkynyl group may be unsubstituted or may be substituted.

[0142] As used herein, "aryl" refers to a C6-C 20 carbocyclic aromatic group. Examples of the aryl group include, but are not limited to, phenyl, naphthyl, and anthracenyl. The aryl group may be unsubstituted or may be substituted.

[0143] As used herein, "arylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms attached to a carbon atom (typically a terminal or sp 3 carbon atom) is replaced by an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like. The arylalkyl group contains 6 to 20 carbon atoms. For example, the alkyl moiety of the arylalkyl group (such as an alkanil, alkenyl, or alkynyl group, etc.) has 1 to 6 carbon atoms, and the aryl moiety has 5 to 14 carbon atoms. The alkyl group may be unsubstituted or may be substituted.

[0144] As used herein, "cycloalkyl" refers to a saturated carbocyclic radical, which may be monocyclic or bicyclic. Examples of cycloalkyl groups include a ring having 3 to 7 carbon atoms as a monocyclic ring or a ring having 7 to 12 carbon atoms as a bicyclic ring. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The cycloalkyl group may be unsubstituted or may be substituted.

[0145] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic radical, which may be monocyclic or bicyclic. Examples of cycloalkenyl groups include a ring having 3 to 6 carbon atoms as a monocyclic ring or a ring having 7 to 12 carbon atoms as a bicyclic ring. Examples of monocyclic cycloalkenyl groups include 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl. The cycloalkenyl group may be unsubstituted or may be substituted.

[0146] As used herein, "heteroalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms attached to a carbon atom (typically a terminal or sp 3 carbon atom) is replaced by a heteroaryl radical. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkyl group contains 6 to 20 carbon atoms. For example, the alkyl portion of the heteroarylalkyl group (including alkanil, alkenyl, or alkynyl groups, etc.) has 1 to 6 carbon atoms, and the heteroaryl portion has 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. The heteroaryl portion of the heteroarylalkyl group may be a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms), or may be a bicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) (for example: bicyclic [4,5], [5,5], [5,6], or [6,6] systems).

[0147] As used herein, "heteroaryl" and "heterocycloalkyl" each refer to an aromatic or non-aromatic ring system, where one or more of the ring atoms are heteroatoms, such as nitrogen, oxygen, and sulfur. A heteroaryl or heterocycloalkyl radical contains 2 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. Heteroaryl or heterocycloalkyl may be unsubstituted or may be substituted.

[0148] Heteroaryl and heterocycloalkyl groups are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), particularly Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.

[0149] Examples of heteroaryl groups include, for example, but not limited to, pyridyl, thiazolyl, tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H - indolyl, 1H - indazolyl, purinyl, 4H - quinolidinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH - carbazolyl, carbazolyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phthalazinyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, benzotriazolyl, benzisoxazolyl, and isatinoyl.

[0150] Examples of heterocycloalkyl include, for example, but not limited to, dihydropyridyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, piperidinyl, 4 - piperidonyl, pyrrolidinyl, 2 - pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, bis - tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinuclidinyl, and morpholinyl.

[0151] By way of example and not limitation, carbon - bonded heteroaryls and heterocycloalkyls are bonded at the 2, 3, 4, 5, or 6 positions of pyridine, the 3, 4, 5, or 6 positions of pyridazine, the 2, 4, 5, or 6 positions of pyrimidine, the 2, 3, 5, or 6 positions of pyrazine, the 2, 3, 4, or 5 positions of furan, tetrahydrofuran, thiophene, thienyl, pyrrole or tetrahydropyrrole, the 2, 4, or 5 positions of oxazole, imidazole or thiazole, the 3, 4, or 5 positions of isoxazole, pyrazole, or isothiazole, the 2 or 3 positions of aziridine, the 2, 3 or 4 positions of azetidine, the 2, 3, 4, 5, 6, 7, or 8 positions of quinoline, or the 1, 3, 4, 5, 6, 7, or 8 positions of isoquinoline. More typically, 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 - pyrimidinyl, 4 - pyrimidinyl, 5 - pyrimidinyl, 6 - pyrimidinyl, 2 - pyrazinyl, 3 - pyrazinyl, 5 - pyrazinyl, 6 - pyrazinyl, 2 - thiazolyl, 4 - thiazolyl, or 5 - thiazolyl.

[0152] By way of example and not limitation, nitrogen-linked heteroaryls and heterocycloalkyls are bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, the 2-position of isoindole or isoindoline, the 4-position of morpholine, and the 9-position of carbazole or beta-carboline. More typically, nitrogen-linked heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0153] As used herein, and as applied to any of the above alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, etc., "substituted" means that one or more hydrogen atoms are each independently replaced by a substituent. Unless otherwise restricted by the definition of an individual substituent, the above chemical moieties, e.g., "alkyl", "heteroalkyl", "alkenyl", "heteroalkenyl", "alkynyl", "heteroalkynyl", "cycloalkyl", "heterocycloalkyl", "aryl", and "heteroaryl" groups, etc., are optionally substituted with 1 to 5 substituents selected from the group consisting of, for example, alkyl, alkynyl, cycloalkyl, heterocycloalkyl, alkyl-aryl, alkyl-heteroaryl, alkyl-cycloalkyl, alkyl-heterocycloalkyl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, nitro, etc. Typical substituents include, but are not limited to, -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; wherein each X is independently selected in each case from F, Cl, Br, and I; and each R is independently selected in each case from C1-C 12 alkyl, C6-C 20 aryl, C3-C 14 heterocycloalkyl or heteroaryl, protecting groups, and prodrug moieties. Whenever a group is described as "optionally substituted", the group may be independently substituted in each case with one or more of the above substituents. The substitution may involve adjacent substituents undergoing ring closure, e.g., ring closure of adjacent functional substituents, to form, for example, lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals, aminals, and hemiaminals, which may provide, for example, protecting groups.

[0154] It is necessary to understand that certain radical naming rules may include either a mono-radical or a di-radical depending on the situation. For example, when a substituent requires the positions of two bonds to the rest of the molecule, the said substituent is understood to be a di-radical. For example, substituents identified as alkyls that require the positions of two bonds include di-radicals such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc. Other radical naming rules clearly indicate that the radical is a di-radical such as "alkylene", "alkenylene", "arylene", "heterocycloalkylene", etc.

[0155] As used herein, the term "coupling reaction" refers to a chemical reaction in which two or more substituents suitable for reacting with each other react to form a chemical substructure in which molecular fragments bonded to each substituent are (e.g., covalently) bonded. Coupling reactions include reactions in which a reactive substituent bonded to a fragment that is a cytotoxin (e.g., a cytotoxin known in the art or described herein) reacts with a suitable reactive substituent bonded to a fragment that is an antibody or an antigen-binding fragment thereof (e.g., an antibody, an antigen-binding fragment thereof that is specific for CD117 (such as GNNK+ CD117) known in the art or described herein). Examples of suitable reactive substituents include nucleophilic / electrophilic pairs (e.g., in particular, thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α, β-unsaturated carbonyl pairs), diene / dienophile pairs (e.g., especially azide / alkyne pairs), etc. Coupling reactions include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine condensation, amidation, esterification, disulfide formation, cycloaddition (e.g., especially [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition), aromatic nucleophilic substitution, aromatic electrophilic substitution, and other reaction modalities known in the art or described herein.

[0156] As used herein, "CRU (competitive repopulating unit)" refers to a unit of measurement for long-term engrafting stem cells that can be detected after in vivo transplantation.

[0157] As used herein, "drug-to-antibody ratio" or "DAR" refers to the number of drugs (e.g., amatoxins) conjugated to the antibody of an ADC. Depending on the number of conjugation sites on the antibody, higher payloads are also possible, but the DAR of an ADC can range from 1 to 8. In certain embodiments, the ADCs described herein have a DAR of 1, 2, 3, 4, 5, 6, 7, or 8.

[0158] Whenever a substituent is shown as a di-radical (i.e., having two positions of attachment to the remainder of the molecule), it should be understood that the substituent can be attached in any directional arrangement, unless otherwise specified.

[0159] Fc-modified antibody The present disclosure is based in part on the discovery that an antibody, or an antigen-binding fragment thereof, having an Fc modification that enables Fc silencing and capable of binding to an antigen expressed by hematopoietic cells can be used as a therapeutic agent. For example, the present disclosure provides an antibody, or an antigen-binding fragment thereof, having an Fc modification that enables Fc silencing and capable of binding to an antigen expressed by hematopoietic cells (e.g., but not limited to, CD117 [e.g., GNNK+ CD117], or CD45); or an antigen expressed by mature immune cells (e.g., T-cells) (e.g., CD45, CD2, CD5, CD137, or CD252), as a therapeutic agent (e.g., (i) for treating cancer and autoimmune diseases characterized by CD117+ [e.g., GNNK+ CD117], or CD45+ hematopoietic stem cells; or CD45+, CD2+, CD5+, CD137+, or CD252+ immune cells [e.g., T-cells], and (ii) for promoting engraftment of transplanted hematopoietic stem cells in patients in need of transplantation therapy, a "naked" antibody or ADC). These therapeutic activities may be caused, for example, by an anti-hematopoietic cell (HC)-antibody (e.g., anti-CD117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, and anti-CD252 antibody, etc.) or an antigen-binding fragment thereof that binds to an antigen expressed by blood cells (e.g., hematopoietic stem cells, leukocytes, immune cells [e.g., mature immune cells (e.g., T cells)]) (e.g., CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137, CD252, etc.), and then inducing cell death. Reduction of endogenous hematopoietic stem cells may provide a niche to which transplanted hematopoietic stem cells can home in, and productive hematopoiesis may then be established. In this way, transplanted hematopoietic stem cells can successfully engraft in a patient (e.g., a human patient suffering from a stem cell disorder described herein).

[0160] The antibodies, or antigen-binding fragments thereof, described herein may include modifications and / or mutations that alter the properties of the antibody and / or fragment (e.g., modifications and / or mutations that increase the half-life, or increase or decrease ADCC).

[0161] In certain embodiments, antibodies comprising one or more radiolabeled amino acids are provided. Radiolabeled antibodies can be used for both diagnostic and therapeutic purposes (alternatively, they can be conjugated to a radiolabeled molecule). Non-limiting examples of labels for polypeptides include, but are not limited to, 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, e.g., Junghans et al., Cancer Chemotherapy and Biotherapy 655-686, 2nd ed., Chafner and Longo eds., Lippincott Raven (1996) and U.S. Patent Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 [U.S. RE35,500], 5,648,471, 5,697,902). For example, the radioisotope may be conjugated by the chloramine T method.

[0162] In certain embodiments, an anti-HC antibody (e.g., an anti-CD117 antibody, an anti-CD45 antibody, an anti-CD2 antibody, an anti-CD5 antibody, an anti-CD137 antibody, or an anti-CD252 antibody), or an antigen-binding fragment thereof, comprises a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification relative to the wild-type Fc region, such that the affinity or binding of the molecule to Fc gamma R (FcγR) is altered. Certain amino acid positions within the Fc region are known from crystallographic studies that directly contact FcγR. Specifically, amino acids 234-239 (hinge region), 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). In some embodiments, the antibodies described herein may comprise a variant Fc region in which at least one residue that directly contacts FcγR has been modified based on structural and crystallographic analysis. In certain embodiments, the Fc region of an anti-HC antibody (e.g., an anti-CD117 antibody, an anti-CD45 antibody, an anti-CD2 antibody, an anti-CD5 antibody, an anti-CD137 antibody, or an anti-CD252 antibody), or an antigen-binding fragment thereof, comprises an amino acid substitution at amino acid 265 according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991) (specifically incorporated herein by reference). "EU index as in Kabat" refers to the numbering of human IgG1 EU antibodies. In certain embodiments, the Fc region comprises a D265A mutation. In certain embodiments, the Fc region comprises a D265C mutation. In some embodiments, the Fc region of the antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index as in Kabat, for example. In certain embodiments, the Fc region comprises an L234A mutation.In some embodiments, the Fc region of an anti-HC antibody (e.g., an anti-CD117 antibody, an anti-CD45 antibody, an anti-CD2 antibody, an anti-CD5 antibody, an anti-CD137 antibody, or an anti-CD252 antibody), or an antigen-binding fragment thereof, comprises an amino acid substitution at amino acid 235 according to the EU index as in Kabat. In one embodiment, the Fc region comprises the L235A mutation. In yet another embodiment, the Fc region comprises the L234A and L235A mutations (also referred to herein as "L234A.L235A" or "LALA"). In another embodiment, the Fc region comprises the L234A and L235A mutations, wherein the Fc region does not comprise the P329G mutation. In a further embodiment, the Fc region comprises the D265C, L234A, and L235A mutations (also referred to herein as "D265C.L234A.L235A"). In another embodiment, the Fc region comprises the D265C, L234A, and L235A mutations, wherein the Fc region does not comprise the P329G mutation. In yet a further embodiment, the Fc region comprises the D265C, L234A, L235A, and H435A mutations (also referred to herein as "D265C.L234A.L235A.H435A"). In another embodiment, the Fc region comprises the D265C, L234A, L235A, and H435A mutations, wherein the Fc region does not comprise the P329G mutation. In a further embodiment, the Fc region comprises the D265C and H435A mutations (also referred to herein as "D265C.H435A"). In yet another embodiment, the Fc region comprises the D265A, S239C, L234A, and L235A mutations (also referred to herein as "D265A.S239C.L234A.L235A"). In yet another embodiment, the Fc region comprises the D265A, S239C, L234A, and L235A mutations, wherein the Fc region does not comprise the P329G mutation. In another embodiment, the Fc region comprises the D265C, N297G, and H435A mutations (also referred to herein as "D265C.N297G.H435A"). In another embodiment, the Fc region comprises the D265C, N297Q, and H435A mutations (also referred to herein as "D265C.N297Q.H435A").In another embodiment, the Fc region comprises the E233P, L234V, L235A, and delG236 (deletion of 236) mutations (also referred to herein as "E233P.L234V.L235A.delG236" or "EPLVLAdelG"). In another embodiment, the Fc region comprises the E233P, L234V, L235A, and delG236 (deletion of 236) mutations, wherein the Fc region does not comprise the P329G mutation. In another embodiment, the Fc region comprises the E233P, L234V, L235A, delG236 (deletion of 236), and H435A mutations (also referred to herein as "E233P.L234V.L235A.delG236.H435A" or "EPLVLAdelG.H435A"). In another embodiment, the Fc region comprises the E233P, L234V, L235A, delG236 (deletion of 236), and H435A mutations, wherein the Fc region does not comprise the P329G mutation. In another embodiment, the Fc region comprises the L234A, L235A, S239C, and D265A mutations. In another embodiment, the Fc region comprises the L234A, L235A, S239C, and D265A mutations, wherein the Fc region does not comprise the P329G mutation. In another embodiment, the Fc region comprises the H435A, L234A, L235A, and D265C mutations. In another embodiment, the Fc region comprises the H435A, L234A, L235A, and D265C mutations, wherein the Fc region does not comprise the P329G mutation.

[0163] In some embodiments, the antibody has a modified Fc region, such that in an in vitro effector function assay, the binding of the antibody to an Fc receptor (FcR) is decreased compared to the binding of the same antibody containing an unmodified Fc region to the FcR, resulting in a decrease in effector function. In some embodiments, the antibody has a modified Fc region, such that in an in vitro effector function assay, the binding of the antibody to an Fc gamma-receptor (FcγR) is decreased compared to the binding of the same antibody containing an unmodified Fc region to the FcγR, resulting in a decrease in effector function. 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 some embodiments, the FcγR is FcγR3B. In other embodiments, the decrease in binding is at least a 70% decrease, at least an 80% decrease, at least a 90% decrease, at least a 95% decrease, at least a 98% decrease, at least a 99% decrease, or a 100% decrease in the antibody binding to the FcγR compared to the binding of the same antibody containing an unmodified Fc region to the FcγR. In other embodiments, the decrease in binding is at least a 70% to 100% decrease, at least an 80% to 100% decrease, at least a 90% to 100% decrease, at least a 95% to 100% decrease, or at least a 98% to 100% decrease in the antibody binding to the FcγR compared to the binding of the same antibody containing an unmodified Fc region to the FcγR.

[0164] In some embodiments, the antibody has a modified Fc region, such that in the in vitro cytokine release assay, the antibody has at least a 50% decrease in cytokine release compared to the cytokine release of the same antibody containing an unmodified Fc region, resulting in a decrease in cytokine release. In some embodiments, the decrease in cytokine release is at least a 70% decrease, at least an 80% decrease, at least a 90% decrease, at least a 95% decrease, at least a 98% decrease, at least a 99% decrease, or a 100% decrease in cytokine release compared to the cytokine release of the same antibody containing an unmodified Fc region. In some embodiments, the decrease in cytokine release is at least a 70% - 100% decrease, at least an 80% - 100% decrease, at least a 90% - 100% decrease, or at least a 95% - 100% decrease in cytokine release compared to the cytokine release of the same antibody containing an unmodified Fc region. In preferred embodiments, cytokine release is by immune cells.

[0165] In some embodiments, the antibody has a modified Fc region, such that in the in vitro mast cell degranulation assay, the antibody has at least a 50% decrease in mast cell degranulation compared to the mast cell degranulation of the same antibody containing an unmodified Fc region, resulting in a decrease in mast cell degranulation. In some embodiments, the decrease in mast cell degranulation is at least a 70% decrease, at least an 80% decrease, at least a 90% decrease, at least a 95% decrease, at least a 98% decrease, at least a 99% decrease, or a 100% decrease in mast cell degranulation compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. In some embodiments, the decrease in mast cell degranulation is at least a 70% - 100% decrease, at least an 80% - 100% decrease, at least a 90% - 100% decrease, or at least a 95% - 100% decrease in mast cell degranulation compared to the mast cell degranulation of the same antibody containing an unmodified Fc region.

[0166] In some embodiments, the antibody has a modified Fc region, such that in the antibody, in an in vitro antibody dependent cell phagocytosis (ADCP) assay, antibody dependent cell phagocytosis (ADCP) is reduced by at least 50%, reduced, or avoided, as compared to the ADCP of the same antibody containing an unmodified Fc region. In some embodiments, the reduction in ADCP is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% reduction in antibody dependent cell phagocytosis as compared to the antibody dependent cell phagocytosis of the same antibody containing an unmodified Fc region.

[0167] 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) comprise an Fc region comprising one or a combination of the following modifications: D265A, D265C, D265C / H435A, D265C / LALA, D265C / LALA / H435A, D265A / S239C / L234A / L235A / H435A, D265A / S239C / L234A / L235A, D265C / N297G, D265C / N297G / H435A, D265C (EPLVLAdelG *), D265C (EPLVLAdelG ) / H435A, D265C / N297Q / H435A, D265C / N297Q, EPLVLAdelG / H435A, EPLVLAdelG / D265C, EPLVLAdelG / D265A, N297A, N297G, or N297Q.

[0168] The binding or affinity between the modified Fc region and the Fc gamma receptor can be measured using various techniques known in the art, such as, but not limited to, equilibrium methods [e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; radioimmunoassay (RIA)], or surface plasmon resonance assays or other assays based on the kinetics of other mechanisms [e.g., BIACORE® analysis or Octet® analysis (forteBIO)], as well as other methods [e.g., indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration), etc.]. These and other methods may utilize labels on one or more of the components being evaluated and / or may use various detection methods (e.g., but not limited to, chromogenic labels, fluorescent labels, luminescent labels, or isotope labels, etc.). Regarding binding affinity and kinetics, it is described in detail in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999) (which focuses on antibody-immunogen interactions). An example of a competitive binding assay is a radioimmunoassay that involves incubating 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, the affinity and binding off-rate of the antibody of interest for a particular antigen may be determined by Scatchard plot analysis. Competition with a second antibody may also be measured using a radioimmunoassay. In this case, the antigen is incubated with the antibody of interest conjugated to a labeled compound in the presence of increasing amounts of unlabeled second antibody.

[0169] In certain embodiments, antibodies having the Fc modifications described herein (e.g., D265C, L234A, L235A, and / or H435A) exhibit at least a 70% decrease, at least a 75% decrease, at least an 80% decrease, at least an 85% decrease, at least a 90% decrease, at least a 95% decrease, at least a 98% decrease, at least a 99% decrease, or a 100% decrease in binding to Fc gamma receptors as compared to binding of the same antibody with an unmodified Fc region to said Fc gamma receptors (e.g., as evaluated by Bio-Layer Interferometry (BLI), such as described in Example 1).

[0170] Without wishing to be bound by any theory, the Fc region binding interaction with Fc gamma receptors is thought to be essential for various effector functions and downstream signaling events (e.g., but not limited to, antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)). Thus, in certain aspects, antibodies comprising a modified Fc region (e.g., comprising the L234A, L235A, and / or D265C mutations) exhibit a substantial decrease or loss of effector function. Effector function may be assayed using various methods known in the art (e.g., by measuring a cellular response [e.g., mast cell degranulation or cytokine release] to the antibody of interest). For example, using standard methods in the art, an Fc-modified antibody may be assayed for its ability to induce in vitro mast cell degranulation (e.g., as described in Example 2), or its ability to induce cytokine release (e.g., by human peripheral blood mononuclear cells, as described in Example 3).

[0171] Thus, in certain embodiments, the Fc region comprises a mutation that results in a decrease in half-life (e.g., as compared to an antibody having an unmodified Fc region). An antibody having a short half-life can be beneficial in certain instances where the antibody is expected to function as a short-lived therapeutic agent (e.g., in the conditioning step described herein where the antibody is administered followed by administration of HSCs). Ideally, the antibody should be substantially cleared before administration of HSCs (which also generally express a target antigen [e.g., CD117 (e.g., GNNK+ CD117), CD45, CD2, CD5, CD137, or CD252] but are not targets of 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] unlike endogenous stem cells). In certain embodiments, the Fc region comprises a mutation at position 435 (EU index according to Kabat). In certain embodiments, the mutation is the H435A mutation.

[0172] In certain embodiments, the anti-HC antibodies described herein (e.g., anti-CD117 antibody, anti-CD45 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody) have a half-life of about 24 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, about 12 hours or less, or about 11 hours or less (e.g., in humans).

[0173] In certain 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) have a half-life of about 1-5 hours, about 5-10 hours, about 10-15 hours, about 15-20 hours, or about 20-25 hours (e.g., in humans).

[0174] In some embodiments, the Fc region comprises two or more mutations that decrease the half-life and reduce the effector function of the antibody. In some embodiments, the Fc region comprises a mutation that results in a decrease in half-life and a mutation of at least one residue that can contact directly with FcγR (e.g., based on structural and crystallographic analysis). In certain embodiments, the Fc region comprises the H435A mutation, the L234A mutation, and the L235A mutation. In certain embodiments, the Fc region comprises the H435A mutation and the D265C mutation. In certain embodiments, the Fc region comprises the H435A mutation, the L234A mutation, the L235A mutation, and the D265C mutation.

[0175] In some embodiments, the antibody, or antigen-binding fragment thereof, is conjugated to a cytotoxin (e.g., amatoxin) via a cysteine residue in the Fc domain of the antibody, or antigen-binding fragment thereof. In some embodiments, the cysteine residue is introduced by a mutation in the Fc domain of the antibody, or antigen-binding fragment thereof. For example, the cysteine residue may be selected from the group consisting of Cys118, Cys239, and Cys265. In one embodiment, the Fc region of 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), or antigen-binding fragment thereof, contains an amino acid substitution at amino acid 265 according to the EU index as in Kabat. In one embodiment, the Fc region contains the D265C mutation. In one embodiment, the Fc region contains the D265C and H435A mutations. In one embodiment, the Fc region contains the D265C, L234A, and L235A mutations. In one embodiment, the Fc region contains the D265C, L234A, L235A, and H435A mutations. In one embodiment, the Fc region of 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), or antigen-binding fragment thereof, contains an amino acid substitution at amino acid 239 according to the EU index as in Kabat. In one embodiment, the Fc region contains the S239C mutation. In one embodiment, the Fc region contains the L234A mutation, L235A mutation, S239C mutation and D265A mutation. In another embodiment, the Fc region contains the S239C and H435A mutations. In another embodiment, the Fc region contains the L234A mutation, L235A mutation, and S239C mutation. In yet another embodiment, the Fc region contains the H435A mutation, L234A mutation, L235A mutation, and S239C mutation. In yet another embodiment, the Fc region contains the H435A mutation, L234A mutation, L235A mutation, S239C mutation and D265A mutation.

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

[0177] Methods of engineering an antibody to incorporate any of the Fc modifications herein are well known in the art. These methods include, but are not limited to, site-specific (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis, with respect to the prepared DNA molecule encoding the antibody or at least the constant region of the antibody. Site-specific mutagenesis is well known in the art (see, for example, Carter et al., Nucleic Acids Res., 13:4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA, 82:488 (1987)). PCR mutagenesis is also suitable for generating amino acid sequence variants of the starting polypeptide. See 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 sequence variants (cassette mutagenesis) is based on the technique described in Wells et al., Gene, 34:315-323 (1985).

[0178] Anti-CD117 antibody The present disclosure is also based in part on the discovery that an antibody capable of binding to CD117, such as GNNK+ CD117, or an antigen-binding fragment thereof, can be used as a single therapeutic agent or as an ADC for the following purposes: (i) to treat cancers characterized by CD117+ cells (e.g., acute myeloid leukemia or myelodysplastic syndromes, etc.) and autoimmune diseases, and (ii) to promote engraftment of transplanted hematopoietic stem cells in patients in need of transplantation therapy. These therapeutic activities may be caused, for example, by binding of an anti-CD117 antibody, or an antigen-binding fragment thereof, to CD117 (e.g., GNNK+ CD117) expressed on the surface of cells such as cancer cells, autoimmune cells, or hematopoietic stem cells, and then inducing cell death. By reducing endogenous hematopoietic stem cells, a niche may be provided to which transplanted hematopoietic stem cells can home, and productive hematopoiesis may then be established. In this way, transplanted hematopoietic stem cells can successfully engraft within a patient (e.g., a human patient suffering from a stem cell disorder as described herein).

[0179] Antibodies and antigen-binding fragments (including antibodies and antigen-binding fragments that can bind to GNNK+ CD117) that can bind to human CD117 (also known as c-Kit, mRNA NCBI reference sequence: NM_000222.2, protein NCBI reference sequence: NP_000213.1) may be used in combination with the compositions and methods described herein to condition patients for hematopoietic stem cell transplantation therapy. Polymorphisms that affect the coding region or extracellular domain of CD117, which are present in a significant proportion of the population, are not currently well known in non-tumor indications. At least four isoforms of CD117 have been identified, and additional isoforms may be expressed in tumor cells. Two of the CD117 isoforms are present in the intracellular domain of the protein, and two are present in the region near the outer membrane. The two extracellular isoforms (GNNK+ and GNNK-) differ in that the 4-amino acid sequence is present (GNNK+) or absent (GNNK-). These isoforms have been reported to have the same affinity for the ligand (SCF), but ligand binding to the GNNK-isoform has been reported to increase internalization and degradation into the cell. The GNNK+ isoform may be used as an immunogen for generating antibodies that can bind to CD117 because antibodies generated against this isoform include both GNNK+ and GNNK-proteins.

[0180] In certain embodiments, the anti-CD117 antibody, or antigen-binding site thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 13, and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 14.

[0181] In another embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises three CDR sequences of the heavy chain variable region (VH) amino acid sequence of Ab85, and three CDR sequences of the light chain variable region (LH) amino acid sequence.

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

[0183] The heavy chain variable region (VH) amino acid sequence is shown below as SEQ ID NO: 13. The VH CDR amino acid sequences of Ab85 are underlined and are as follows: NYWIG (VH CDR1; SEQ ID NO:7); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO:8); and HGRGYEGYEGAFDI (VH CDR3; SEQ ID NO:9).

[0184] Ab85 VH sequence

Chemical Structure

[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 sequences of Ab85 are underlined and are as follows: RSSQGIRSDLG (VL CDR1; SEQ ID NO:10); DASNLET (VL CDR2; SEQ ID NO:11); and QQANGFPLT (VL CDR3; SEQ ID NO:12).

[0186] Ab85 VL sequence

Chemical Structure

[0187] In another embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises the heavy chain variable region (VH) amino acid sequence of Ab249 (also interchangeably referred to herein as Ab3), and the light chain variable region (LH) amino acid sequence.

[0188] The amino acid sequence of the heavy chain variable region (VH) 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; SEQ ID NO: 340); IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 341); and HGLGYNGYEGAFDI (VH CDR3; SEQ ID NO: 342).

[0189] Ab249 VH sequence

Chem.

[0190] The amino acid sequence of the light chain variable region (VL) of Ab249 is shown below as SEQ ID NO: 347. The VL CDR amino acid sequences of Ab249 are underlined and are as follows: RASQGIGSALA (VL CDR1; SEQ ID NO: 343); DASNLET (VL CDR2; SEQ ID NO: 344); and QQLNGYPLT (VL CDR3; SEQ ID NO: 345).

[0191] Ab249 VL sequence

Chem.

[0192] Both human antibodies Ab85 and Ab249 are derived from antibody CK6, an antagonist anti - CD117 antibody. Ab85 and Ab249 have improved properties (e.g., improved binding properties) compared to CK6.

[0193] Accordingly, in certain embodiments, the anti-CD117 antibody comprises a heavy chain comprising the CDR sets (CDR1, CDR2, and CDR3) set forth in SEQ ID Nos: 7, 8, and 9, and a light chain comprising the CDRs set forth in SEQ ID Nos: 10, 11, and 12. In other embodiments, the anti-CD117 antibody comprises a heavy chain comprising the CDR sets (CDR1, CDR2, and CDR3) set forth in SEQ ID Nos: 340, 341, and 342, and a light chain comprising the CDRs set forth in SEQ ID Nos: 343, 344, and 345.

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

[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 sequences of Ab67 are underlined and are as follows: FTFSDADMD (VH CDR1; SEQ ID NO: 348); RTRNKAGSYTTEYAASVKG (VH CDR2; SEQ ID NO: 349); and AREPKYWIDFDL (VH CDR3; SEQ ID NO: 350).

[0196] Ab67 VH sequence

Chemical formula

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

[0198] Ab67 VL sequence

Chemical formula

[0199] Thus, in certain embodiments, the anti-CD117 antibody comprises a heavy chain comprising the CDR set (CDR1, CDR2, and CDR3) set forth in SEQ ID Nos: 348, 349, and 350, and a light chain comprising the CDRs set forth in SEQ ID Nos: 351, 352, and 353.

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

[0201] The anti-CD117 antibodies or binding fragments described herein may also include modifications and / or mutations (e.g., modifications and / or mutations that increase half-life, increase or decrease ADCC, etc.) that alter the properties of the antibody and / or fragment, as are known in the art.

[0202] In certain embodiments, the anti-CD117 antibody, or antigen-binding fragment thereof, comprises a variant Fc region, where the variant Fc region comprises at least one amino acid modification relative to the wild-type Fc region, such that the molecule has an altered affinity for Fc gamma R. Certain amino acid positions within the Fc region are known from crystallographic studies in direct contact with FcγR. Specifically, amino acids 234-239 (hinge region), 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). For example, amino acid substitutions at amino acid positions 234 and 235 of the Fc region have been identified as decreasing the affinity of IgG antibodies with respect to binding to Fc receptors, particularly Fc gamma receptors (FcγR). In certain embodiments, the anti-CD117 antibodies described herein comprise an Fc region comprising an amino acid substitution at L234 and / or L235 (e.g., L234A and L235A) (EU index). Thus, the anti-CD117 antibodies described herein may comprise a variant Fc region in which at least one residue that directly contacts FcγR based on structural and crystallographic analysis is modified. In certain embodiments, the Fc region of the anti-CD117 antibody (or fragment thereof comprising the Fc region) comprises an amino acid substitution at amino acid 265 according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991) (specifically incorporated herein by reference). "EU index as in Kabat" or "EU index" refers to the numbering of human IgG1 EU antibodies, unless otherwise specified, and is used when referring to Fc amino acid positions herein.

[0203] In certain embodiments, the Fc region comprises the D265A mutation. In certain embodiments, the Fc region comprises the D265C mutation.

[0204] In some embodiments, the Fc region of said anti-CD117 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index as in Kabat. In certain embodiments, said Fc region comprises the L234A mutation. In some embodiments, the Fc region of said anti-CD117 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 235 according to the EU index as in Kabat. In certain embodiments, said Fc region comprises the L235A mutation. In yet another embodiment, said Fc region comprises the L234A and L235A mutations. In a further embodiment, said Fc region comprises the D265C, L234A, and L235A mutations.

[0205] In certain aspects, the variant IgG Fc domain comprises one or more amino acid substitutions that result in a decreased or abolished binding affinity for Fc gamma R and / or C1q as compared to the wild-type Fc domain that does not contain any amino acid substitutions. Fc binding interactions are essential for various effector functions and downstream signaling events, including but not limited to antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Thus, in certain aspects, antibodies comprising a modified Fc region (e.g., comprising the L234A, L235A, and / or D265C mutations) have substantially reduced or abolished effector functions.

[0206] The affinity for the Fc region can be determined by various techniques known in the art (e.g., without limitation, equilibrium methods [e.g., enzyme-linked immunoabsorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; radioimmunoassay (RIA)], or assays based on surface plasmon resonance assays or the kinetics of other mechanisms [e.g., BIACORE TM analysis or Octet TMAnalysis (forteBIO), as well as other methods [e.g., indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration), etc.] can be used for measurement. These and other methods may utilize labels on one or more components to be evaluated and / or may use various detection methods (e.g., but not limited to, chromogenic labels, fluorescent labels, luminescent labels, or isotope labels, etc.). Regarding binding affinity and kinetics, it is described in detail in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999) (which focuses on antibody-immunogen interactions). An example of a competitive binding assay is a radioimmunoassay that includes incubating a labeled antigen and an antibody of interest in the presence of increasing amounts of unlabeled antigen and detecting the antibody bound to the labeled antigen. From the data, the affinity and binding off-rate of the antibody of interest for a particular antigen may be determined by Scatchard plot analysis. Competition with a second antibody may also be measured using a radioimmunoassay. In this case, the antigen is incubated in the presence of an antibody of interest conjugated to a labeled compound and increasing amounts of an unlabeled second antibody.

[0207] In certain embodiments, the anti-CD117 antibodies described herein include an Fc region that contains L235A, L235A, and D265C (EU index). The antibodies of the invention may be further engineered to further modulate the half-life of the antibody by further introducing Fc mutations such as those 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: 1759-69), (Datta-Mannan et al. (2007) Drug Metab Dispos 35: 86-94), (Vaccaro et al. (2005) Nat Biotechnol 23: 1283-8), (Yeung et al. (2010) Cancer Res 70: 3269-77) and (Kim et al. (1999) Eur J Immunol 29: 2819-25), and may also include positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434 and 435. Exemplary mutations that may be made alone or in combination are the T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations.

[0208] Thus, in certain embodiments, the Fc region comprises a mutation that results in a reduction in half-life. Antibodies having a short half-life (also referred to herein as a "fast" half-life) may be beneficial in certain instances where the antibody is expected to function as a short-lived therapeutic agent (e.g., in the conditioning step described herein of administering the antibody followed by administering HSCs). Ideally, unlike endogenous stem cells, the antibody would be substantially cleared prior to administration of HSCs that generally express CD117 but are not targets of the anti-CD117 antibody. In certain embodiments, the Fc region comprises a mutation at position 435 (EU index according to Kabat). In certain embodiments, the mutation is the H435A mutation. In another embodiment, the mutation is the D265C mutation. In yet another embodiment, the mutation is the H435A mutation and the D265C mutation.

[0209] In certain embodiments, the anti-CD117 antibodies described herein have a half-life of 24 hours or less, 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, 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 certain embodiments, the half-life of the antibody is 5 to 7 hours, 5 to 9 hours, 15 to 11 hours, 5 to 13 hours, 5 to 15 hours, 5 to 20 hours, 5 to 24 hours, 7 to 24 hours, 9 to 24 hours, 11 to 24 hours, 12 to 22 hours, 10 to 20 hours, 8 to 18 hours, or 14 to 24 hours.

[0210] Examples of anti-CD117 antibodies that may be used in conjunction with the methods of patient conditioning described herein include, for example, antibodies produced and released from ATCC accession number 10716 (deposited as BA7.3C.9) as described in U.S. Patent No. 5,489,516, the disclosure of which is incorporated herein by reference in its entirety as it relates to anti-CD117 antibodies (e.g., the SR-1 antibody).

[0211] In certain embodiments, the anti-CD117 antibodies described herein include an Fc region comprising L235A, L235A, D265C, and H435A (EU index).

[0212] Additional anti-CD117 antibodies that can be used in combination with the methods of conditioning a patient described herein include the anti-CD117 antibodies described in U.S. Patent No. 7,915,391 (which describes, for example, a humanized SR-1 antibody); the anti-CD117 antibodies described in U.S. Patent No. 5,808,002 (which describes, for example, the anti-CD117 A3C6E2 antibody), and, for example, the anti-CD117 antibodies described in WO 2015 / 050959 (which describes anti-CD117 antibodies that bind to an epitope comprising Pro317, Asn320, Glu329, Val331, Asp332, Lus358, Glue360, Glue376, His378, and / or Thr380 of human CD117); and U.S. 2012 / 0288506 (also published as U.S. Patent No. 8,552,157) (which describes, for example, the anti-CD117 antibody CK6 (also interchangeably referred to herein as Ab4) having the following CDR sequences: CDR-H1 having the amino acid sequence SYWIG (SEQ ID NO: 1); CDR-H2 having the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 2); CDR-H3 having the amino acid sequence HGRGYNGYEGAFDI (SEQ ID NO: 3); CDR-L1 having the amino acid sequence RASQGISSALA (SEQ ID NO: 4); CDR-L2 having the amino acid sequence DASSLES (SEQ ID NO: 5); and CDR-L3 having the amino acid sequence CQQFNSYPLT (SEQ ID NO: 6) ) are included.

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

Chem.

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

Chem.

[0215] Additional anti-CD117 antibodies and antigen-binding fragments thereof that can be used in combination with the compositions and methods described herein include those described in US 2015 / 0320880, such as clone 9P3, NEG024, NEG027, NEG085, NEG086, and 20376.

[0216] The disclosure of each of the foregoing publications, which is hereby incorporated by reference herein, relates to anti-CD117 antibodies. Antibodies and antigen-binding fragments that can be used in combination with the compositions and methods described herein include the foregoing antibodies and antigen-binding fragments thereof, as well as humanized variants of the foregoing non-human antibodies and antigen-binding fragments, and antibodies or antigen-binding fragments that bind to the same epitope as the foregoing (e.g., as evaluated by a competitive CD117 binding assay).

[0217] Exemplary antigen-binding fragments of the foregoing antibodies include, inter alia, dual-variable immunoglobulin domain, single-chain Fv molecule (scFv), diabody, triabody, nanobody, antibody-like protein scaffold, Fv fragment, Fab fragment, F(ab')2 molecule, and tandem di-scFv.

[0218] Antibodies may be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In certain embodiments, isolated nucleic acids encoding the anti-CD117 antibodies described herein are provided. Such nucleic acids may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In certain such embodiments, the host cell comprises (e.g., is transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In certain embodiments, the host cell is a eukaryote, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In certain embodiments, a method of making an anti-CLL-1 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody and optionally recovering the antibody from the host cell (or host cell culture medium).

[0219] To recombinantly produce an anti-CD117 antibody, a nucleic acid encoding the antibody (e.g., a nucleic acid as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibody) and sequenced.

[0220] Suitable host cells for cloning or expressing vectors encoding antibodies include the prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expressing antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, which describes expressing antibody fragments in E. coli). After expression, the antibody may be isolated as a soluble fraction from the bacterial cell paste and further purified.

[0221] As a host, vertebrate cells can also be used. For example, mammalian cell lines adapted to grow in suspension may be beneficial. Other examples of useful mammalian host cell lines include monkey kidney CV1 cells transformed with SV40 (COS-7); human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); dog kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), etc.); and myeloma cell lines (e.g., Y0, NS0, and Sp2 / 0, etc.). For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

[0222] In certain embodiments, the anti-CD117 antibody, or antigen-binding fragment thereof, comprises a variable region having an amino acid sequence that is at least 95%, 96%, 97%, or 99% identical to the SEQ ID No disclosed herein. Alternatively, the anti-CD117 antibody, or antigen-binding fragment thereof, comprises CDRs that comprise the SEQ ID No disclosed herein, having a framework region of the variable region described herein, having an amino acid sequence that is at least 95%, 96%, 97%, or 99% identical to the SEQ ID No disclosed herein.

[0223] In certain embodiments, the anti-CD117 antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region and a heavy chain constant region having the amino acid sequences disclosed herein. In another embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, comprises a light chain variable region and a light chain constant region having the amino acid sequences disclosed herein. In yet another embodiment, the anti-CD117 antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region having the amino acid sequences disclosed herein.

[0224] Additional anti-CD117 antibodies are described in US 2019 / 0153114 A1 and US 2019 / 0144558 A1, the contents of both applications are hereby expressly incorporated by reference in their entirety.

[0225] The anti-CD117 antibodies and ADCs described herein may be used, inter alia, in methods for treating various disorders such as diseases of cell types in the hematopoietic lineage, cancer, autoimmune diseases, metabolic disorders, and stem cell disorders. By the compositions and methods described herein, (i) populations of cells that cause a pathological condition, such as populations of cancer cells (e.g., leukemia cells) and autoimmune cells (e.g., autoreactive T-cells), can be directly reduced, and / or (ii) the population of endogenous hematopoietic stem cells can be reduced such that engraftment of transplanted hematopoietic stem cells is promoted by providing a niche to which the transplanted cells can home. The above activities are obtained by administering an ADC, antibody, or antigen-binding fragment thereof that can bind to an antigen expressed by endogenous cells, autoimmune cells, or hematopoietic stem cells that cause the disease. When directly treating a disease, this administration can reduce the amount of cells that cause the condition of interest. In cases where a patient is being prepared for hematopoietic stem cell transplantation therapy, this administration selectively reduces the population of endogenous hematopoietic stem cells, thereby creating a space in hematopoietic tissue such as bone marrow, which space may then be filled with transplanted exogenous hematopoietic stem cells. The present invention is based in part on the finding that administration of an ADC, antibody, or antigen-binding fragment thereof that can bind to CD117 (e.g., GNNK+ CD117) to a patient can provide the effects of both of the above activities. An ADC, antibody, or antigen-binding fragment that binds to CD117 may be administered to a patient suffering from cancer or an autoimmune disease in order to directly reduce the population of cancerous or autoimmune cells, and may also be administered to a patient in need of hematopoietic stem cell transplantation therapy in order to promote the likelihood that the transplanted hematopoietic stem cells will survive and engraft.

[0226] The engraftment of hematopoietic stem cell grafts by administration of anti-CD117 ADCs, antibodies, or antigen-binding fragments thereof can be demonstrated in various experimental measurements. For example, engraftment of transplanted hematopoietic stem cells can be evaluated by administering an ADC, antibody, or antigen-binding fragment thereof that can bind to CD117 and subsequently evaluating the amount of competitive repopulating unit (CRU) present in the patient's bone marrow after administration of the hematopoietic stem cell graft. Further, a reporter gene such as an enzyme that catalyzes a chemical reaction to produce a fluorescent product, chromogenic product, or luminescent product can be incorporated into a vector that transfects donor hematopoietic stem cells, and subsequently, the engraftment of the hematopoietic stem cell graft can be observed by monitoring the corresponding signal in tissues such as bone marrow to which the hematopoietic stem cells have homed. Also, for example, engraftment can be observed by evaluating the amount and viability of hematopoietic stem cells and hematopoietic progenitor cells as measured by fluorescence activated cell sorting (FACS) analysis methods known in the art. Engraftment can also be measured by measuring the white blood cell count in peripheral blood and / or the recovery rate of bone marrow cells by donor cells in a bone marrow aspiration sample during the period after transplantation.

[0227] Anti-CD2 antibody Human CD2 is also known as the T cell surface antigen T11 / Leu-5, T11, CD2 antigen (p50), and Sheep Red Blood Cell Receptor (SRBC). CD2 is expressed on T cells. Two isoforms of human CD2 have been identified. Isoform 1 contains 351 amino acids and is described in Seed, B. et al. (1987) 84: 3365-69 (see also Sewell et al. (1986) 83: 8718-22) and below (NCBI reference sequence: NP_001758.2). [Chem.]

[0228] The second isoform of CD2 is 377 amino acids and is identified herein as NCBI Reference Sequence: NP_001315538.1.

[0229] In certain embodiments, anti-CD2 antibodies that may be used in conjunction with the compositions and methods described herein include those having 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 certain embodiments, an anti-CD2 antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 36 and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 37.

[0231] In certain embodiments, anti-CD2 antibodies that may be used in conjunction with the compositions and methods described herein 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. A CDR-H2 having the amino acid sequence SGGGF (SEQ ID NO: 39); c. A CDR-H3 having the amino acid sequence SSYGEIMDY (SEQ ID NO: 40); d. A CDR-L1 having the amino acid sequence RASQRIGTSIH (SEQ ID NO: 42); e. A CDR-L2 having the amino acid sequence YASESIS (SEQ ID NO: 43); and f. A CDR-L3 having the amino acid sequence QQSHGWPFTF (SEQ ID NO: 44).

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

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

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

[0235] Antibodies and antigen-binding fragments thereof that include the CDR sequences described above are, for example, described in U.S. Patent No. 6,849,258, the content of which is related to anti-CD2 antibodies and antigen-binding fragments thereof, the disclosure of which is incorporated herein by reference.

[0236] Furthermore, in certain embodiments, the anti-CD2 ADC has a serum half-life in human subjects of 3 days or less.

[0237] Additional sequences for the anti-CD2 antibodies or binding fragments described herein are set forth in Table 5.

[0238] Additional anti-CD2 antibodies, antigen-binding fragments thereof, or ADCs that may be used in the compositions and methods described herein may be identified using techniques known in the art, such as hybridoma production. Hybridomas may be prepared using a mouse system. Protocols for immunization and isolation of spleen cells for subsequent fusion are known in the art. Fusion partners and procedures for producing hybridomas are also known. Alternatively, HuMAb-Mouse 登録商標 or XenoMouse TMIt may also be used to generate anti-CD2 antibodies. When producing further anti-CD2 antibodies, the CD2 antigen is isolated and / or purified. The CD2 antigen may be 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, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing animals such as mice, rats, sheep, goats, pigs, cows and horses are well known in the art. See, for example, Harlow and Lane, supra and U.S. Patent No. 5,994,619. The CD2 antigen may be administered together with an adjuvant to stimulate an immune response. Adjuvants known in the art include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulating complex). After immunizing an animal with the CD2 antigen, an antibody-producing immortalized cell line is prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph node and / or spleen B cells are immortalized by methods known in the art (e.g., introduction of an oncogene, transduction with an oncogenic virus, exposure to a carcinogenic or mutagenic compound, fusion with immortalized cells (e.g., myeloma cells), and inactivation of a tumor suppressor gene). See, for example, Harlow and Lane, supra. Hybridomas may be selected, cloned and further screened for desired properties (e.g., robust growth, high antibody production, and desired antibody properties).

[0239] In the anti-CD2 ADCs described herein, anti-CD2 antibodies for use may also be identified by using high-throughput screening of a library of antibodies or antibody fragments for molecules capable of binding to CD2. Such methods include in vitro display techniques known in the art, such as, inter alia, phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display. The use of phage display to isolate antibodies, antigen-binding fragments, or ligands that bind to biologically relevant molecules has been reviewed, for example, in Felici et al., Biotechnol. Annual Rev. 1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4:1-20, 1998, each of these disclosures being incorporated herein by reference as related to in vitro display techniques. As described in Kay, Perspect. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, 1996, the construction of randomized combinatorial peptide libraries to select polypeptides that bind to cell surface antigens has also been carried out, each of these disclosures being incorporated herein by reference as related to the discovery of antigen-binding molecules.The phage display of proteins (e.g., multimeric proteins) as functional molecules has been successfully achieved (see, for example, EP 0349578; EP 4527839; and EP 0589877, as well as Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992, the disclosures of each of which are incorporated herein by reference as relating to the use of in vitro display technologies for the discovery of antigen-binding molecules). Furthermore, functional antibody fragments (e.g., Fab and scFv fragments) have also been expressed in in vitro display formats (see, for example, McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, the disclosures of each of which are incorporated herein by reference as relating to in vitro display platforms for the discovery of antigen-binding molecules).

[0240] In addition to in vitro display technologies, anti-CD2 antibodies or antibody fragments may be designed and identified in silico using computational modeling techniques, for example, while using the procedures described in US2013 / 0288373 (the disclosure of which is incorporated herein as relating to molecular modeling methods for identifying anti-CD2 antibodies). For example, using computational modeling techniques, one of ordinary skill in the art may seek molecules that can bind to specific epitopes on CD2, such as extracellular epitopes of CD2, and screen a library of antibodies or antibody fragments in silico.

[0241] In one embodiment, the anti-CD2 antibody used in the ADCs described herein may be taken up into cells. When identifying an anti-CD2 antibody (or fragment thereof), additional techniques may be used to identify an antibody or antigen-binding fragment that binds to CD2 on the surface of a cell (e.g., a T cell) and can be taken up by the cell, for example, by receptor-mediated endocytosis. For example, the in vitro display techniques described above may be adapted to screen for antibodies or antigen-binding fragments thereof that bind to CD2 on the surface of hematopoietic stem cells and are subsequently taken up. Phage display is one representative of such techniques that may be used in combination with this screening paradigm. To identify an anti-CD2 antibody or fragment thereof that binds to CD2 and is subsequently taken up into CD2+ cells, one of ordinary skill in the art may use the phage display technique described in Williams et al., Leukemia 19:1432-1438, 2005 (the disclosure of which is incorporated herein by reference in its entirety).

[0242] The allowable amount of an anti-CD2 antibody or fragment thereof taken up may be evaluated, for example, using a radionuclide internalization assay known in the art. For example, an anti-CD2 antibody or fragment thereof identified using an in vitro display technique described herein or known in the art is labeled with a radioisotope (e.g., 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu, 177 Lu, 77 As,72 As, 86 Y, 90 Y, 89 Zr, 212 Bi, 213 Bi, or 225 Ac) may be incorporated to make it functional. For example, a radioactive halogen (e.g., 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, etc.) may be incorporated into an antibody, its fragment, or a ligand using beads containing an electrophilic halogen reagent (e.g., polystyrene beads) (e.g., iodinated beads, Thermo Fisher Scientific, Inc., Cambridge, MA). A radio-labeled antibody, or its fragment, may be incubated with hematopoietic stem cells for a time sufficient to be taken up therein. The antibody, or its fragment, taken up therein may be detected by the radiation (e.g., γ-rays) released from the obtained hematopoietic stem cells and identified by comparison with the radiation (e.g., γ-rays) released from the recovered wash buffer. The internal uptake assay described above may also be used to characterize an ADC.

[0243] In some embodiments, the anti-CD2 antibody (or its fragment) has a defined serum half-life. For example, the anti-CD2 antibody (or its fragment) may have a serum half-life of about 1 to 24 hours in a human patient. An ADC containing such an anti-CD2 antibody may also, for example, have a serum half-life of about 1 to 24 hours in a human patient. Pharmacokinetic analysis by measuring serum levels may be performed by assays known in the art.

[0244] To recombinantly produce an anti-CD2 antibody, the nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody) using conventional procedures and can be sequenced.

[0245] Suitable host cells for cloning or expressing the vector encoding the antibody include the prokaryotic or eukaryotic cells described herein. For example, the antibody may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expressing antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, which describes expressing antibody fragments in E. coli). After expression, the antibody may be isolated as a soluble fraction from the bacterial cell paste and further purified.

[0246] As a host, vertebrate cells can also be used. For example, mammalian cell lines adapted to grow in suspension may be beneficial. Other examples of useful mammalian host cell lines include simian kidney CV1 cells transformed with SV40 (COS-7); human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); simian kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); dog kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), etc.); and myeloma cell lines (e.g., Y0, NS0, and Sp2 / 0, etc.). For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). In certain embodiments, the host cells are eukaryotic, e.g., Chinese hamster ovary (CHO) cells 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 contains 495 amino acids and is described in Gladkikh et al (2017) Cancer Med.6(12):2984 and Jones et al. (1986) Nature 323 (6086): 346). The amino acid sequence of CD5 (isoform 1) 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) (Sequence ID No. (SEQ ID NO): 48).

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

[0249] In one embodiment, the anti-CD5 antibody that may be used in the methods and compositions described herein is antibody 5D7v (Ab5D7v). The amino acid sequence of the heavy chain variable region (VH) of Ab5D7v is shown in the following Sequence ID No. (SEQ ID NO): 49.

Chemical formula

[0250] The VH CDR amino acid sequences of Ab5D7v are underlined above and are as follows: FSLSTSGMG (VH CDR1; Sequence ID No. (SEQ ID NO): 51); WWDDD (VH CDR2; SEQ ID NO: 52); and RRATGTGFDY (VH CDR3; SEQ ID NO: 53).

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

Chemical formula

[0252] The VL CDR amino acid sequences of Ab5D7v are underlined above and are as follows: QDVGTA (VL CDR1; SEQ ID NO: 54); WTSTRHT (VL CDR2; SEQ ID NO: 55); and YNSYNT (VL CDR3; SEQ ID NO: 56).

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

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

[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 anti-CD5 antibody that may be used in the methods and compositions (including ADCs) described herein is a variant 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 ADC has a serum half-life in a human subject of 3 days or less.

[0257] Additional sequences for the anti-CD5 antibodies or binding fragments described herein are set forth in Table 5.

[0258] Additional anti-CD5 antibodies that may be used in the ADCs described herein may be identified using techniques known in the art such as hybridoma production. Hybridomas may be prepared using a mouse system. Protocols for immunization and subsequent isolation of splenocytes for fusion are known in the art. Fusion partners and procedures for producing hybridomas are also known. Alternatively, HuMAb-Mouse 登録商標 or XenoMouse TM may be used to generate anti-CD5 antibodies. When making additional anti-CD5 antibodies, the CD5 antigen is isolated and / or purified. The CD5 antigen may be a fragment of CD5 derived from the extracellular domain of CD5. Immunization of animals can be performed by any method known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing animals such as mice, rats, sheep, goats, pigs, cows, and horses are well known in the art. See, for example, Harlow and Lane, supra and U.S. Patent No. 5,994,619. The CD5 antigen may be administered with an adjuvant to stimulate an immune response. Adjuvants known in the art include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulating complex). After immunizing an animal with the CD5 antigen, an antibody-producing immortalized cell line is prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph node and / or spleen B cells are immortalized by methods known in the art (e.g., introduction of oncogenes, transduction with oncogenic viruses, exposure to carcinogenic or mutagenic compounds, fusion with immortalized cells (e.g., myeloma cells), and inactivation of tumor suppressor genes). See, for example, Harlow and Lane, supra. Hybridomas may be selected, cloned, and further screened for desired properties (e.g., robust growth, high antibody production, and desired antibody properties).

[0259] The anti-CD5 antibodies for use in the anti-CD5 ADCs described herein may also be identified by using high-throughput screening of a library of antibodies or antibody fragments for molecules capable of binding to CD5. Such methods include in vitro display technologies known in the art, such as, inter alia, phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display. The use of phage display to isolate antibodies, antigen-binding fragments, or ligands that bind to biologically relevant molecules has been reviewed, for example, in Felici et al., Biotechnol. Annual Rev. 1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4:1-20, 1998, and each of these disclosures is incorporated herein by reference as related to in vitro display technologies. As described in Kay, Perspect. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, 1996, the construction of randomized combinatorial peptide libraries to select polypeptides that bind to cell surface antigens has also been carried out, and each of these disclosures is incorporated herein by reference as related to the discovery of antigen-binding molecules.It has been successfully achieved to phage-display proteins (e.g., multimeric proteins) as functional molecules (see, for example, EP 0349578; EP 4527839; and EP 0589877, as well as Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992, the disclosures of each of which are incorporated herein by reference as relating to the use of in vitro display technologies for the discovery of antigen-binding molecules). Furthermore, functional antibody fragments (e.g., Fab and scFv fragments) have also been expressed in an in vitro display format (see, for example, McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, the disclosures of each of which are incorporated herein by reference as relating to in vitro display platforms for the discovery of antigen-binding molecules).

[0260] In addition to in vitro display technologies, for example, while using the procedures described in US2013 / 0288373, computational modeling technologies may be used to design and identify anti-CD5 antibodies or antibody fragments in silico (its disclosure is incorporated herein as relating to molecular modeling methods for identifying anti-CD5 antibodies). For example, using computational modeling technologies, one of ordinary skill in the art may seek molecules that can bind to specific epitopes on CD5, such as extracellular epitopes of CD5, and screen a library of antibodies or antibody fragments in silico.

[0261] In certain embodiments, the anti-CD5 antibodies used in the ADCs described herein may be taken up into cells. When identifying an anti-CD5 antibody (or fragment thereof), additional techniques may be used to identify an antibody or antigen-binding fragment that binds to CD5 on the surface of cells (e.g., T cells) and that can be taken up by the cells, for example, by receptor-mediated endocytosis. For example, the in vitro display techniques described above may be adapted to screen for antibodies or antigen-binding fragments thereof that bind to CD5 on the surface of hematopoietic stem cells and are subsequently taken up into the cells. Phage display is one representative of the techniques that may be used in combination with this screening paradigm. To identify an anti-CD5 antibody or fragment thereof that binds to CD5 and is subsequently taken up into CD5+ cells, one of ordinary skill in the art may use the phage display techniques described in Williams et al., Leukemia 19:1432-1438, 2005 (the disclosure of which is incorporated herein by reference in its entirety).

[0262] The allowable amount of an anti-CD5 antibody or fragment thereof to be taken up may be evaluated, for example, using a radionuclide internalization assay known in the art. For example, an anti-CD5 antibody or fragment thereof identified using in vitro display techniques described herein or known in the art may be labeled with a radioisotope (e.g., 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu, 177 Lu, 77 As,72 As, 86 Y, 90 Y, 89 Zr, 212 Bi, 213 Bi, or 225 Ac) may be incorporated to make it functional. For example, a radioactive halogen (e.g., 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, etc.) may be incorporated into an antibody, its fragment, or a ligand using beads (e.g., polystyrene beads) containing an electrophilic halogen reagent (e.g., iodinated beads, Thermo Fisher Scientific, Inc., Cambridge, MA). A radio-labeled antibody, or its fragment, may be incubated with hematopoietic stem cells for a time sufficient to be taken up therein. An antibody, or its fragment, taken up therein may be detected by the radiation (e.g., γ-rays) emitted from the obtained hematopoietic stem cells and identified by comparison with the radiation (e.g., γ-rays) emitted from the recovered wash buffer. The internal uptake assay described above may also be used to characterize an ADC.

[0263] In some embodiments, the anti-CD5 antibody (or its fragment) has a defined serum half-life. For example, the anti-CD5 antibody (or its fragment) may have a serum half-life of about 1 to 24 hours in a human patient. An ADC containing such an anti-CD5 antibody may also have a serum half-life of about 1 to 24 hours in a human patient, for example. Pharmacokinetic analysis by measuring serum levels may be performed by assays known in the art.

[0264] For the recombinant production of an anti-CD5 antibody, the nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibody) using conventional procedures and can be sequenced.

[0265] Suitable host cells for cloning or expressing the vector encoding the antibody include the prokaryotic or eukaryotic cells described herein. For example, the antibody may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody may be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0266] As a host, vertebrate cells can also be used. For example, mammalian cell lines adapted to grow in suspension may be beneficial. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); dog kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), etc.); and myeloma cell lines (e.g., Y0, NS0, Sp2 / 0, etc.). For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). In certain embodiments, the host cells are eukaryotes, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0267] In some embodiments, anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include those comprising combinations of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions set forth in Tables 1 and 2 below.

[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. Anti-CD137 antibodies, antigen-binding fragments thereof, and ADCs thereof may be used as therapeutic agents to prevent and treat GVHD by hematopoietic stem cells in patients suffering from or at risk of GVHD or autoimmune diseases. Further, ligands that bind to CD137 (e.g., human CD137L, etc.) may be used as therapeutic agents to prevent and treat patients suffering from or at risk of GVHD. These ligands (e.g., soluble human CD137) may be covalently bound to an effector domain (e.g., Fc domain) to promote, for example, antibody-dependent cell-mediated cytotoxicity (ADCC).

[0271] T cells have been shown to express CD137, an antigen that is a transmembrane TNF receptor superfamily of costimulatory molecules, expressed on various hematopoietic cells, promotes T cell activation, and regulates T cell proliferation and survival (see, e.g., Cannons et al., J. Immunol. 167:1313-1324, 2001. This disclosure is incorporated herein by reference as it relates to the expression of CD137 by T cells). Antibodies, and antigen-binding fragments thereof, may be identified by techniques known in the art and described herein (e.g., immunization, computer modeling techniques, and in vitro selection methods such as phage display and cell-based display platforms described below).

[0272] Anti-CD137 antibodies that may be used to prevent and treat GVHD or autoimmune diseases by the methods disclosed herein include those having one or more, or all, of the following CDRs: 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] Additional anti-CD137 antibodies that may be used to prevent and treat GVHD or autoimmune diseases by the methods disclosed herein include those having one or more, or all, of the following CDRs: 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: 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] Examples of additional anti-CD137 antibodies or ADCs include those having one or more, or all, of the following CDRs: 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] The above-mentioned antibodies are described, for example, in U.S. Patent No. 9,468,678, the disclosures of which are incorporated herein by reference as they relate to anti-CD137 antibodies and antigen-binding fragments thereof. The antibodies and fragments thereof disclosed in U.S. Patent No. 9,468,678 may be used in conjunction with the methods disclosed herein.

[0277] In another embodiment, the anti-CD137 antibodies that can be used in the methods and compositions (including ADCs) described herein are mouse anti-CD137 antibody BBK2 (Thermo Fisher; MS621PABX), or anti-CD137 antibodies that contain the antigen-binding region corresponding to the BBK2 antibody. The BBK2 antibody (which may also be referred to as the BBK-2 antibody or anti-4-1BB antibody) is a mouse monoclonal antibody (IgG1, kappa) that binds to the extracellular domain of human 4-1BB recombinant protein (4-1BB is also known as CD137). In certain embodiments, the methods and compositions of the present disclosure include anti-CD137 antibodies that contain the binding region (e.g., CDR) of the BBK2 antibody. In another embodiment, the methods and compositions of the present disclosure include antibodies that competitively inhibit 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 one embodiment, the methods and compositions described herein include a chimeric anti-CD137 (ch-BBK2) antibody that contains the variable heavy chain region and variable light chain region of BBK2. In certain embodiments, the chimeric BBK2 antibody is an IgG1 antibody that contains human constant regions. The heavy chain amino acid sequence of ch-BBK2 is described in SEQ ID NO: 297, and the light chain amino acid sequence of ch-BBK2 is described in SEQ ID NO: 298. The CDR regions (CDR1, CDR2, and CDR3) of each sequence of the heavy chain and light chain are described in bold below. The variable regions are italicized.

[0279]

Chemical formula

[0280]

Chemical formula

[0281] The foregoing CDR regions (and BBK2 antibody) are described in Lee et al. (2002) European J of Immunogenetics 29(5):449-452. Thus, in certain embodiments, the VH CDR amino acid sequences of anti-CD137 antibody BBK2 (including ch-BBK2) are as follows: SGYTFTSYW (VH CDR1; SEQ ID NO: 299); NIYPSDSYT (VH CDR2; SEQ ID NO: 300) and TRNGVEGYPHYYAME (VH CDR3; SEQ ID NO: 301). The VL CDR amino acid sequences of anti-CD137 antibody BBK2 (including ch-BBK2) are as follows: SQDLSNH (VL CDR1; SEQ ID NO: 302); YYTS (VL CDR2; SEQ ID NO: 303) and CQQGYTLPY (VL CDR3; SEQ ID NO: 304).

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

[0283]

Chemical formula

[0284]

Chemical formula

[0285] Accordingly, in one embodiment, the VH CDR amino acid sequences of the anti-CD137 antibody BBK2 (including ch-BBK2) are as follows: SYWIN (VH CDR1; SEQ ID NO: 305); NIYPSDSYTNYNQKFKD (VH CDR2; SEQ ID NO: 306) and NGVEGYPHYYAMEY (VH CDR3; SEQ ID NO: 307), and the VL CDR amino acid sequences of the anti-CD137 antibody BBK2 (including ch-BBK2) 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 described as follows in SEQ ID NO: 311 QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSNTVYMQLNSPTSEDSAVYYCTRNGVEGYPHYYAMEYWGQGTSVTVSS . The light chain variable region of BBK2 is described as follows in SEQ ID NO: 312 DIQMTQTTSALSASLGDRVTIGCRASQDLSNHLYWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDVATYFCQQGYTLPYTFGGGTKLEIK . The anti-CD137 antibody (including anti-CD137 ADC) may include the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence as described in SEQ ID Nos: 311 and 312, respectively.

[0287] In certain embodiments, an anti-CD137 antibody, e.g., a chimeric (ch-BBK2) antibody or a humanized BBK2 antibody, comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 305, a CDR2 having the amino acid sequence of SEQ ID NO: 306, and a CDR3 having the amino acid sequence of SEQ ID NO: 307; and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 308, a CDR2 having the amino acid sequence of SEQ ID NO: 309, and a CDR3 having the amino acid sequence of SEQ ID NO: 310.

[0288] In certain embodiments, an anti-CD137 antibody, e.g., a chimeric (ch-BBK2) antibody or a humanized BBK2 antibody, comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 299, a CDR2 having the amino acid sequence of SEQ ID NO: 300, and a CDR3 having the amino acid sequence of SEQ ID NO: 301; and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 302, a CDR2 having the amino acid sequence of SEQ ID NO: 303, and a CDR3 having the amino acid sequence of SEQ ID NO: 304.

[0289] Accordingly, BBK2, humanized BBK2, or chimeric BBK2 antibodies may be used in the anti-CD137 ADCs and methods described herein. Each of these antibodies may be conjugated to any of the cytotoxins described below using methods known in the art and the methods described herein.

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

[0291] Other anti-CD137 antibodies that may be used in conjunction with the cytotoxins described herein may be identified using techniques known in the art (e.g., hybridoma production). Hybridomas may be prepared using a mouse system. Protocols for immunization and isolation of splenocytes for subsequent fusion are known in the art. Fusion partners and procedures for producing hybridomas are also known. HuMAb-Mouse 登録商標 or XenoMouse TMIn some cases, human anti-CD137 antibodies are generated. When producing anti-CD137 antibodies, the CD137 antigen is isolated and / or purified. The CD137 antigen may be a fragment of CD137 derived from the extracellular domain of CD137. Immunization of animals can be performed by any method known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing animals such as mice, rats, sheep, goats, pigs, cows, and horses are well known in the art. See, for example, Harlow and Lane, supra, and U.S. Patent No. 5,994,619. The CD137 antigen may be administered together with an adjuvant to stimulate an immune response. Adjuvants known in the art include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulating complex). After immunizing the animal with the CD137 antigen, an antibody-producing immortalized cell line is prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph node and / or spleen B cells are immortalized by methods known in the art (e.g., introduction of oncogenes, transduction with oncogenic viruses, exposure to carcinogenic or mutagenic compounds, fusion with immortalized cells (e.g., myeloma cells), and inactivation of tumor suppressor genes). See, for example, Harlow and Lane, supra. Hybridomas may be selected, cloned, and further screened for desired properties (e.g., robust growth, high antibody production, and desired antibody properties).

[0292] An anti-CD137 antibody may be generated from an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of the CD137 binding molecule provided by the present invention. The amino acid sequence encoded by the nucleotide sequence may be any part of the antibody (e.g., a CDR, a sequence containing one, two, or three CDRs, the variable region of the heavy chain, the variable region of the light chain), or the full-length heavy chain or full-length light chain. The nucleic acids of the present disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. Typically, the nucleic acid is a cDNA molecule.

[0293] In addition to antibodies and antigen-binding fragments, a water-soluble CD137 ligand, such as the human CD137 ligand, may be administered to a patient according to the methods described herein to condition the patient prior to hematopoietic stem cell transplantation therapy. For example, a CD137 ligand, such as the human CD137 ligand, may be conjugated to another effector molecule, such as a cytotoxin or an Fc domain (e.g., as described below or according to methods known in the art). Examples of maytansine cytotoxins for use in conjunction with the methods described herein include, for example, 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.

[0294] Antibodies and ligands for use in conjunction with the compositions and methods described herein include variants of those antibodies described above, such as antibody fragments that contain or lack an Fc domain, and humanized variants of the non-human antibodies described herein, and antibody-like protein scaffolds that contain one or more or all of the CDRs or their equivalent regions of the antibodies, antibody fragments, or soluble ligands described herein (e.g.,10 Examples include the Fn3 domain, etc.

[0295] Anti-CD252 antibody The present invention also provides an antibody, or an antigen-binding fragment thereof, that can bind to CD252 (also called OX40 ligand (OX40L), protein NCBI reference sequence: NP_003317.1; Uniprot accession number: P23510; sequence number (SEQ ID NO): 313 or 314), and may be used as a therapeutic agent for preventing and treating GVHD. Such an antibody may be used alone or conjugated to a cytotoxin as an antibody-drug conjugate (ADC).

[0296] In certain embodiments, the methods and compositions (e.g., ADCs) described herein include anti-CD252 antibodies whose heavy and light chain amino acid sequences are set forth in sequence numbers (SEQ ID NO): 315 and 316, respectively. In certain embodiments, the anti-CD252 antibody, or an antigen-binding portion thereof, includes a heavy chain variable region set forth in the amino acid sequence of sequence number (SEQ ID NO): 315, and a light chain variable region set forth in the amino acid sequence of sequence number (SEQ ID NO): 316. In certain embodiments, the anti-CD252 antibody, or an antigen-binding portion thereof, includes a heavy chain variable region that includes the CDRs set forth in the amino acid sequence of sequence number (SEQ ID NO): 315, and a light chain variable region that includes the CDRs set forth in the amino acid sequence of sequence number (SEQ ID NO): 316. The amino acid sequences of sequence numbers (SEQ ID NO): 315 and 316 are shown below.

[0297] In certain specific embodiments, the anti-CD252 antibody, or an antigen-binding portion thereof, includes a heavy chain variable region that includes the CDRs set forth in the amino acid sequences of sequence numbers (SEQ ID NO): 317 - 319, and a light chain variable region that includes the CDRs set forth in the amino acid sequences of sequence numbers (SEQ ID NO): 320 - 322. The amino acid sequences of sequence numbers (SEQ ID NO): 3 - 8 are shown below.

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

Chem.

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

Chem.

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

[0301] In certain embodiments, the anti-CD252 antibody that may be used in the methods and compositions (including ADCs) described herein is 11C3.1 (Biolegend, catalog #326302), 159403 (R&D Systems, catalog #MAB10541), 159408 (R&D Systems, catalog #MAB1054), MM0505-8S23 (Novus, catalog #NBP2-11969), or ozelumab (Novus catalog #NBP2-52687-0.1).

[0302] In certain embodiments, the anti-CD252 antibody that may be used in the methods and compositions (including ADCs) described herein is the mouse monoclonal anti-CD252 antibody 11C3.1, or an anti-CD252 antibody comprising the antigen-binding region corresponding to the 11C3.1 antibody. 11C3.1 (sold by Biolegend CatNo. 326302 (February 27, 2019)).

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

[0304] In certain embodiments, the anti-CD252 antibody that may be used in the methods and compositions (including ADCs) described herein is a murine monoclonal anti-CD252 antibody 159403, or an anti-CD252 antibody comprising an antigen-binding region corresponding to the 159403 antibody. 159403 (Catalog #MAB10541 (February 27, 2019), sold by R&D Systems).

[0305] In certain embodiments, the anti-CD252 antibody comprises a heavy chain comprising CDR1, CDR2, and CDR3 of anti-CD252 antibody 159403, and a light chain variable region comprising CDR1, CDR2, and CDR3 of anti-CD252 antibody 159403. In another embodiment, the anti-CD252 antibody used in the compositions and methods disclosed herein is a humanized 159403 antibody.

[0306] In certain embodiments, the anti-CD252 antibody that may be used in the methods and compositions (including ADCs) described herein is a murine monoclonal anti-CD252 antibody 159408, or an anti-CD252 antibody comprising an antigen-binding region corresponding to the 159408 antibody. 159408 (Catalog #MAB1054 (February 27, 2019), sold by R&D Systems).

[0307] In certain embodiments, the anti-CD252 antibody comprises a heavy chain comprising CDR1, CDR2, and CDR3 of anti-CD252 antibody 159408, and a light chain variable region comprising CDR1, CDR2, and CDR3 of anti-CD252 antibody 159408. In another embodiment, the anti-CD252 antibody used in the compositions and methods disclosed herein is a humanized 159408 antibody.

[0308] In certain embodiments, the anti-CD252 antibody that may be used in the methods and compositions (including ADCs) described herein is a murine monoclonal anti-CD252 antibody MM0505-8S23, or an anti-CD252 antibody comprising an antigen-binding region corresponding to the MM0505-8S23 antibody. MM0505-8S23 (sold by Novus, catalog #NBP2-11969 (February 27, 2019)). This antibody was produced from a hybridoma (a murine myeloma fused with splenocytes from a mouse immunized with human TNFSF4 (also known as OX40 ligand)).

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

[0310] In certain embodiments, the anti-CD252 antibody that may be used in the methods and compositions (including ADCs) described herein is a rabbit monoclonal anti-CD252 antibody orelumab, or an anti-CD252 antibody comprising an antigen-binding region corresponding to the orelumab antibody. Orelumab (sold by Novus, catalog #NBP2-52687-0.1 (February 27, 2019)).

[0311] In certain embodiments, the anti-CD252 antibody comprises a heavy chain comprising the CDR1, CDR2, and CDR3 of the anti-CD252 antibody ocrelizumab, and a light chain variable region comprising the CDR1, CDR2, and CDR3 of the anti-CD252 antibody ocrelizumab. In another embodiment, the anti-CD252 antibody used in the compositions and methods disclosed herein is a humanized ocrelizumab antibody. In some embodiments, the anti-CD252 antibody, or antigen-binding portion thereof, comprises a heavy chain as set forth in the amino acid sequence of SEQ ID NO: 323, and a light chain as set forth in the amino acid sequence of SEQ ID NO: 324. In some embodiments, the anti-CD252 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 331, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 332. In certain embodiments, the anti-CD252 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 325-327, and a light chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 328-330. In certain embodiments, the antibody is an intact antibody comprising a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 331, and a light chain variable region as 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] Full-length ocrelizumab heavy chain sequence (the following CDR sequences are defined by IMGT; the heavy chain variable region (SEQ ID NO: 331) is underlined):

Chemical formula

[0313] Full-length ocrelizumab light chain sequence (the following CDR sequences are defined by IMGT; the light chain variable region (SEQ ID NO: 332) is underlined):

Chemical formula

[0314] The anti-CD252 antibodies or binding fragments described herein may also include modifications and / or mutations (e.g., modifications and / or mutations that increase the half-life, increase or decrease ADCC, etc.) that alter the properties of the antibody and / or fragment, as are known in the art.

[0315] In certain embodiments, the anti-CD252 antibody, or a binding fragment thereof, used in the methods and compositions disclosed herein comprises a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to the wild-type Fc region, such that the molecule has an altered affinity for Fc gamma R. Certain amino acid positions within the Fc region are known from crystallographic studies of direct contact with FcγR. Specifically, amino acids 234-239 (hinge region), 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). Accordingly, the anti-CD252 antibodies described herein may comprise a variant Fc region in which at least one residue that directly contacts FcγR based on structural and crystallographic analysis has been modified. In certain embodiments, the Fc region of the anti-CD252 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991) (which is expressly incorporated herein by reference). "EU index as in Kabat" refers to the numbering of human IgG1 EU antibodies. In certain embodiments, the Fc region comprises the D265A mutation. In certain embodiments, the Fc region comprises the D265C mutation. In some embodiments, the Fc region of the anti-CD252 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index as in Kabat. In certain embodiments, the Fc region comprises the L234A mutation. In some embodiments, the Fc region of the anti-CD252 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 235 according to the EU index as in Kabat. In certain embodiments, the Fc region comprises the L235A mutation. In yet another embodiment, the Fc region comprises the L234A and L235A mutations.In a further embodiment, the Fc region comprises the D265C, L234A, and L235A mutations.

[0316] In certain embodiments, the variant IgG Fc domain comprises one or more amino acid substitutions that result in a decreased or abolished binding affinity for Fc gamma R and / or C1q as compared to the wild-type Fc domain that does not contain any amino acid substitutions. Fc binding interactions are essential for various effector functions and downstream signaling events, including, but not limited to, antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Thus, in certain embodiments, anti-CD252 antibodies that comprise a modified Fc region (e.g., comprising the L234A, L235A, and D265C mutations) have substantially reduced or abolished effector functions.

[0317] Affinity for the Fc region can be determined using a variety of techniques known in the art, such as, but not limited to, equilibrium methods [e.g., enzyme-linked immunoabsorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; radioimmunoassay (RIA)], or surface plasmon resonance assays or other kinetics-based assays [e.g., BIACORE TM analysis or Octet TMIt can be measured using analysis (forteBIO), as well as other methods [e.g., indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration), etc.]. These and other methods may utilize labels on one or more components to be evaluated and / or may use various detection methods (e.g., but not limited to, chromogenic labels, fluorescent labels, luminescent labels, or isotope labels, etc.). Regarding binding affinity and kinetics, it is described in detail in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999) (which focuses on antibody-immunogen interactions). An example of a competitive binding assay is a radioimmunoassay that includes incubating a labeled antigen and an antibody of interest in the presence of increasing amounts of unlabeled antigen and detecting the antibody bound to the labeled antigen. From the data, the affinity and binding off-rate of the antibody of interest for a particular antigen may be determined by Scatchard plot analysis. Competition with a second antibody may also be measured using a radioimmunoassay. In this case, the antigen is incubated in the presence of an antibody of interest conjugated to a labeled compound and increasing amounts of an unlabeled second antibody.

[0318] The antibody of the present invention may be further modified, for example, by further introducing Fc mutations such as those 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: 1759-69), (Datta-Mannan et al. (2007) Drug Metab Dispos 35: 86-94), (Vaccaro et al. (2005) Nat Biotechnol 23: 1283-8), (Yeung et al. (2010) Cancer Res 70: 3269-77) and (Kim et al. (1999) Eur J Immunol 29: 2819-25), and may further include positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434 and 435. Exemplary mutations that can be made alone or in combination are the T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations.

[0319] Accordingly, in certain embodiments, the Fc region comprises a mutation that results in a decrease in half-life. Antibodies with a short half-life can be beneficial in certain instances where the antibody is expected to function as a short-lived therapeutic agent (e.g., in the conditioning step described herein where the antibody is administered followed by administration of HSCs). Ideally, prior to administration of HSCs, which generally express CD252 but are not targets of the anti-CD252 antibody, unlike endogenous stem cells, the antibody should be substantially cleared. In certain embodiments, the Fc region comprises a mutation at position 435 (EU index according to Kabat). In certain embodiments, the mutation is the H435A mutation.

[0320] In certain embodiments, the anti-CD252 antibodies described herein have a half-life of about 14 hours or less, about 13 hours or less, about 12 hours or less, or about 11 hours or less. In certain embodiments, the anti-CD252 antibodies described herein have a half-life of about 24 hours or less, about 22 hours or less, about 20 hours or less, about 18 hours or less, about 16 hours or less, about 14 hours or less, about 13 hours or less, about 12 hours or less, or about 11 hours or less. In certain embodiments, the half-life of the antibody is from about 1 hour to about 20 hours, from about 2 hours to about 18 hours, from about 4 hours to about 16 hours, from about 6 hours to about 14 hours, from about 8 hours to about 12 hours, from about 11 hours to about 12 hours, from about 11 hours to about 24 hours, from about 12 hours to about 22 hours, from about 10 hours to about 20 hours, from about 8 hours to about 18 hours, from about 1 hour to about 6 hours, from about 2 hours to about 5 hours, from about 3 hours to about 4 hours, or from about 14 hours to about 24 hours.

[0321] In some embodiments, the Fc region comprises two or more mutations that confer a reduction in half-life and significantly reduce or completely eliminate the effector function of the antibody. In some embodiments, the Fc region comprises a mutation that results in a reduction in half-life and a mutation of at least one residue that can contact directly with FcγR (e.g., based on structural and crystallographic analyses). In one embodiment, the Fc region comprises the H435A mutation, the L234A mutation, and the L235A mutation. In one embodiment, the Fc region comprises the H435A mutation and the D265C mutation. In one embodiment, the Fc region comprises the H435A mutation, the L234A mutation, the L235A mutation, and the D265C mutation.

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

[0323] In some embodiments of these aspects, the cysteine residue is naturally present in the Fc domain of the antibody or an antigen-binding fragment thereof. For example, the Fc domain may be an IgG Fc domain such as a human IgG1 Fc domain, and the cysteine residue may be selected from the group consisting of Cys261, Csy321, Cys367, and Cys425.

[0324] The variant Fc domains described herein are defined according to the amino acid modifications that constitute them. For all amino acid substitutions discussed herein with respect to the Fc region, the numbering always follows the EU index. Thus, for example, D265C is an Fc variant in which the aspartic acid (D) at EU position 265 is substituted with cysteine (C) relative to the parental Fc domain. Similarly, for example, D265C / L234A / L235A defines an Fc variant having substitutions at EU positions 265 (D to C), 234 (L to A), and 235 (L to A) relative to the parental Fc domain. Variants may also be designated according to their final amino acid composition at the mutated EU amino acid positions. For example, the L234A / L235A mutation may sometimes be referred to as LALA. Note that the order of substitution is arbitrary.

[0325] In certain embodiments, the anti-CD252 antibody, or an antigen-binding fragment thereof, comprises a variable region having an amino acid sequence that is at least 95%, 96%, 97%, or 99% identical to the sequence numbers (SEQ ID Nos) disclosed herein. Alternatively, the anti-CD252 antibody, or an antigen-binding fragment thereof, comprises CDRs that include the sequence numbers (SEQ ID Nos) disclosed herein and have a framework region of the variable region described herein that has an amino acid sequence that is at least 95%, 96%, 97%, or 99% identical to the sequence numbers (SEQ ID Nos) disclosed herein.

[0326] In certain embodiments, the anti-CD252 antibody, or antigen-binding fragment thereof, has a particular dissociation rate that is particularly beneficial when used as part of a conjugate. For example, in certain embodiments, the anti-CD252 antibody has a dissociation rate constant (Koff) of 1×10 -2 ~1×10 -3 、1×10 -3 ~1×10 -4 、1×10 -5 ~1×10 -6 、1×10 -6 ~1×10 -7 or 1×10 -7 ~1×10 -8 when measured by Biolayer Interferometry (BLI) against human CD252 and / or cynomolgus monkey CD252. In some embodiments, the antibody or antigen-binding fragment thereof binds to CD252 (e.g., human CD252 and / or cynomolgus monkey CD252) with a K D of about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nM or less, about 2 nM or less, about 1 nM or less when measured by a Biolayer Interferometry (Bio-Layer Interferometry) (BLI) assay. In some embodiments, the antibody or antigen-binding fragment thereof binds to CD252 (e.g., human CD252 and / or cynomolgus monkey CD252) with a K D of about 90 nM - 100 nM, about 80 nM - 90 nM, about 70 nM - 80 nM, about 60 nM - 70 nM, about 50 nM - 60 nM, about 40 nM - 50 nM, about 30 nM - 40 nM, about 20 nM - 30 nM, about 10 nM - 20 nM, about 8 nM - 10 nM, about 6 nM - 8 nM, about 4 nM - 6 nM, about 2 nM - 4 nM, about 1 nM - 2 nM, or about 1 nM or less when measured by a Biolayer Interferometry (Bio-Layer Interferometry) (BLI) assay.

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

[0328] Exemplary antigen-binding fragments of the antibodies include, inter alia, dual-variable immunoglobulin domain, single-chain Fv molecule (scFv), diabody, triabody, nanobody, antibody-like protein scaffold, Fv fragment, Fab fragment, F(ab')2 molecule, and tandem di-scFv. The anti-CD252 antibodies described herein may be in the form of full-length antibodies, bispecific antibodies, dual-variable domain antibodies, multi-chain or single-chain antibodies, and / or binding fragments that specifically bind to human CD252, including but not limited to Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), surrobodies (including surrogate light chain constructs), single-domain antibodies, camelized antibodies, etc. They may also be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), or IgM. In some embodiments, the anti-CD252 antibody is IgG (e.g., IgG1, IgG2, IgG3 or IgG4).

[0329] In certain embodiments, the anti-CD252 antibody, or antigen-binding fragment thereof, comprises a variable region having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein. Alternatively, the anti-CD252 antibody, or antigen-binding fragment thereof, comprises CDRs that include the SEQ ID Nos disclosed herein and have a framework region of the variable region described herein having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein.

[0330] Anti-CD45 antibody Antibodies and antigen-binding fragments that can bind to human CD45 (mRNA NCBI reference sequence: NM_080921.3, protein NCBI reference sequence: NP_563578.2), including those that can bind to isoform CD45RO, are used in conjunction with the compositions and methods disclosed herein to, for example, promote the engraftment of hematopoietic stem cell grafts in patients in need of hematopoietic stem cell transplantation therapy. In certain embodiments, the compositions and methods disclosed herein include an anti-CD45 antibody or ADC that binds to human CD45RO as set forth in the amino acid sequence of SEQ ID NO: 336. Antibodies that bind to the various isoforms of CD45 disclosed herein are also intended to be used in the methods and compositions disclosed herein. Multiple isoforms of CD45 result from the alternative splicing of 34 exons in the primary transcript. Splicing of exons 4, 5, 6, and 7 (possibly) results in multiple CD45 variations. The expression of the alternative exons is observed in the CD45 isoforms described in Table 3 below.

[0331]

Table 3

[0332] Alternative splicing results in individual exons or combinations of exons that are expressed in various isoforms of the CD45 protein (e.g., CD45RA, CD45RAB, CD45RABC). In contrast, CD45RO lacks the expression of exons 4-6 and is generated from the combination of exons 1-3 and 7-34. There is evidence that exon 7 is also excluded from the protein and that exons 1-3 and 8-34 are spliced together. This protein, named E3-8, is detected at the mRNA level but has not yet been identified by flow cytometry.

[0333] CD45RO is currently the only known CD45 isoform expressed in hematopoietic stem cells. CD45RA and CD45RABC have either not been detected or have been excluded from the phenotype of hematopoietic stem cells. There is evidence from studies in mice that CD45RB is expressed in fetal hematopoietic stem cells but not in adult bone marrow hematopoietic stem cells. In particular, CD45RC has a high proportion of polymorphisms in exon 6 that are observed within the Asian population (the polymorphism in exon 6 of CD45RC is observed in approximately 25% of the Japanese population). This polymorphism results in high expression of CD45RO and decreased levels of CD45RA, CD45RB, and CD45RC. Furthermore, CD45RA variants (such as CD45RAB and CD45RAC, etc.) exhibit polymorphisms in exon 4 that are associated with autoimmune diseases.

[0334] The presence of CD45RO on hematopoietic stem cells and the relatively restricted expression of CD45RO on other immune cells (such as subsets of T and B lymphocytes and various myeloid cells, etc.) make CD45RO a particularly well - suited target for conditioning therapies for patients in need of hematopoietic stem cell transplantation. Since CD45RO lacks only the expression of exons 4, 5, and 6, it is possible to screen for pan - CD45Ab and CD45RO - specific antibodies by using it as an immunogen.

[0335] Anti-CD45 antibodies that may be used in conjunction with the patient conditioning methods described herein include anti-CD45 antibodies and antigen-binding portions thereof. Antigen-binding portions of antibodies are well known in the art and can be readily constructed based on the antigen-binding regions of said antibodies. In an exemplary embodiment, the anti-CD45 antibody used in conjunction with the conditioning methods described herein is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a fully human antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a bispecific variable immunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment, an F(ab')2 molecule, or a tandem di-scFv. Exemplary anti-CD45 antibodies that may be used wholly or in part in the ADCs or methods described herein are provided below.

[0336] In one embodiment, the anti-CD45 antibody is BIOLEGEND (登録商標)Clone HI30, which is available for purchase from (San Diego, California), or is derived therefrom, or is a humanized variant thereof. Humanization of the antibody can be performed by replacing the framework residues and constant region residues of the non-human antibody with those of a germline human antibody according to procedures known in the art (e.g., as described in Example 7 below). Additional anti-CD45 antibodies that may be used in conjunction with the methods described herein include anti-CD45 antibodies ab10558, EP322Y, 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 LT45 (these are available from ABCAM (登録商標) (Cambridge, MA)), or humanized variants thereof. Additional anti-CD45 antibodies that may be used in conjunction with the patient conditioning procedures described herein include anti-CD45 antibody HPA000440 (this is available from SIGMA-ALDRICH (登録商標)(Available for purchase from (St. Louis, MO)), and its humanized variants. Additional anti-CD45 antibodies that may be used in conjunction with the patient conditioning methods described herein include the mouse monoclonal antibody BC8 (which is described, for example, in Matthews et al., Blood 78:1864-1874, 1991, the disclosure of which is incorporated herein by reference as it relates to anti-CD45 antibodies), and its humanized variants. Additional anti-CD45 antibodies that may be used in conjunction with the methods described herein include the monoclonal antibody YAML568 (which is described, for example, in Glatting et al., J. Nucl. Med. 8:1335-1341, 2006, the disclosure of which is incorporated herein by reference as it relates to anti-CD45 antibodies), and its humanized variants. Additional anti-CD45 antibodies that may be used in conjunction with the patient conditioning procedures described herein include the monoclonal antibodies YTH54.12 and YTH25.4 (which are described, for example, in Brenner et al., Ann. N.Y. Acad. Sci. 996:80-88, 2003, the disclosure of which is incorporated herein by reference as it relates to anti-CD45 antibodies), and its humanized variants. Additional anti-CD45 antibodies that may be used in conjunction with the patient conditioning methods described herein include UCHL1, 2H4, SN130, MD4.3, MBI, and MT2 (which are described, for example, in Brown et al., Immunology 64:331-336, 1998, the disclosure of which is incorporated herein by reference as it relates to anti-CD45 antibodies), and its humanized variants.Additional anti-CD45 antibodies that may be used in conjunction with the methods described herein include anti-CD45 antibodies produced and released from American Type Culture Collection (ATCC) accession numbers RA3-6132, RA3-2C2, and TIB122, as well as monoclonal antibodies C363.16A and 13 / 2 (these are described, for example, in Johnson et al., J. Exp. Med. 169:1179-1184, 1989, the disclosure of which is incorporated herein by reference as relating to anti-CD45 antibodies), and humanized variants thereof. Additional anti-CD45 antibodies that may be used in conjunction with the patient conditioning methods described herein include monoclonal antibodies AHN-12.1, AHN-12, AHN-12.2, AHN-12.3, AHN-12.4, HLe-1, and KC56 (T200) (these are described, for example, in Harvath et al., J. Immunol. 146:949-957, 1991, the disclosure of which is incorporated herein by reference as relating to anti-CD45 antibodies), and humanized variants thereof.

[0337] Additional anti-CD45 antibodies that may be used in conjunction with the patient conditioning procedures described herein include, for example, the anti-CD45 antibodies described in U.S. Patent No. 7,265,212 (which describes, among other clones, anti-CD45 antibodies 39E11, 16C9, and 1G10); the anti-CD45 antibodies described in U.S. Patent No. 7,160,987 (which describes, for example, the anti-CD45 antibody produced and secreted by ATCC accession number HB-11873 [such as monoclonal antibody 6G3, etc.]); and the anti-CD45 antibodies described in U.S. Patent No. 6,099,838 (which describes, for example, anti-CD45 antibody MT3, as well as the antibodies produced and secreted by ATCC accession numbers HB220 [also called MB23G2] and HB223), and the anti-CD45 antibodies described in US 2004 / 0096901 and US 2008 / 0003224 (which describe, for example, the anti-CD45 antibody produced and secreted by ATCC accession number PTA-7339 [such as monoclonal antibody 17.1, etc.]) (the disclosures of which are incorporated herein by reference as being related to anti-CD45 antibodies).

[0338] Additional anti-CD45 antibodies that may be used in conjunction with the patient conditioning procedures described herein include antibodies generated and secreted from ATCC accession numbers MB4B4, MB23G2, 14.8, GAP 8.3, 74-9-3, I / 24.D6, 9.4, 4B2, M1 / 9.3.4.HL.2, and their humanized and / or affinity matured variants. Affinity maturation may be carried out, for example, as described herein or using in vitro display techniques known in the art (such as phage display as described in Example 6 below).

[0339] Additional anti-CD45 antibodies that may be used in conjunction with the patient conditioning procedures described herein 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 is incorporated herein by reference as it relates to the anti-CD45 antibody).

[0340] In certain embodiments, the anti-CD45 antibody is selected from apamistamab (or known 90Y-BC8, Iomab-B, BC8; e.g., as described in US20170326259, WO2017155937, and Orozco et al. Blood. 127.3 (2016): 352-359.), or BC8-B10 (e.g., as described in 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 / 0226209, US2016 / 0152733, US9,701,756; US2011 / 0076270, or US7,825,222 (each of which is incorporated by reference).

[0341] For example, in certain embodiments, the anti-CD45 antibody, or antigen-binding fragment thereof, comprises a binding region (e.g., corresponding to a CDR, variable region, binding region of apamistamab). The amino acid sequence of the heavy chain variable region (VH) of apamistamab is set forth in SEQ ID NO: 337. The amino acid sequence of the light chain variable region (VL) of apamistamab is set forth in SEQ ID NO: 338. In other embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a variable heavy chain comprising the amino acid residues set forth in SEQ ID NO: 337, and a light chain variable region comprising the amino acid residues set forth in SEQ ID NO: 338. In certain embodiments, the anti-CD45 antibody comprises a heavy chain comprising CDR1, CDR2, and CDR3 of apamistamab, and a light chain variable region comprising CDR1, CDR2, and CDR3 of apamistamab.

[0342] In certain embodiments, the anti-CD45 antibody comprises the heavy chain of the anti-CD45 antibody described herein, and the light chain variable region of the anti-CD45 antibody described herein. In certain embodiments, the anti-CD45 antibody comprises a heavy chain comprising CDR1, CDR2, and CDR3 of the anti-CD45 antibody described herein, and a light chain variable region comprising CDR1, CDR2, and CDR3 of the anti-CD45 antibody described herein.

[0343] In another embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 95% identical to the anti-CD45 antibody herein (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the anti-CD45 antibody herein). In certain embodiments, the antibody comprises a modified heavy chain (HC) variable region comprising the HC variable domain of the anti-CD45 antibody herein, or a variant thereof, the variant differing from the anti-CD45 antibody in (i) 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differing from the anti-CD45 antibody in at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differing from the anti-CD45 antibody in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions; and / or (iv) comprising an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the anti-CD45 antibody, wherein in any of (i)-(iv), the amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified heavy chain variable region may have enhanced biological activity compared to the heavy chain variable region of the anti-CD45 antibody while retaining the CD45 binding specificity of the anti-CD45 antibody.

[0344] The disclosure of each of the above publications is hereby incorporated by reference in its entirety. Antibodies and antigen-binding fragments that may be used in conjunction with the compositions and methods described herein include the above antibodies and antigen-binding fragments thereof, as well as humanized variants of the above non-human antibodies and antigen-binding fragments, and antibodies or antigen-binding fragments that bind to the same epitope as the above antibodies or antigen-binding fragments, as evaluated, for example, by a competitive CD45 binding assay.

[0345] Method for identifying an antibody To find molecules that can bind to 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 CD252), antibodies, or methods for high-throughput screening of libraries of antibody fragments are used to identify and affinity mature useful antibodies for treating cancer, autoimmune diseases, and conditioning patients (e.g., human patients) in need of hematopoietic stem cell therapy as described herein. Such methods include in vitro display technologies known in the art, such as, inter alia, phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display. The use of phage display to isolate antibodies or antigen-binding fragments that bind to biologically relevant molecules has been reviewed, for example, in Felici et al., Biotechnol. Annual Rev. 1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4:1-20, 1998, and the disclosures of each of these are incorporated herein by reference as related to in vitro display technologies. As described in Kay, Perspect. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, 1996, randomized combinatorial peptide libraries have also been constructed and polypeptides that bind to cell surface antigens have been selected, and the disclosures of each of these are incorporated herein by reference as related to the discovery of antigen-binding molecules.The phage display of proteins (e.g., multimeric proteins) as functional molecules has been successfully accomplished (e.g., see EP 0349578; EP 4527839; and EP 0589877, as well as Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992, the disclosures of each of which are incorporated herein by reference as related to the use of in vitro display technology for discovering antigen-binding molecules). Furthermore, functional antibody fragments (e.g., Fab and scFv fragments) have also been expressed in an in vitro display format (e.g., see McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, the disclosures of each of which are incorporated herein by reference as related to the in vitro display platform for discovering antigen-binding molecules). Human anti-HC antibodies (e.g., anti-CD117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody) may also be generated, for example, in HuMAb-mouse. 登録商標 or XenoMouse TM In particular, these techniques can be used to identify and improve the affinity of antibodies, antibodies, or fragments that can bind to 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 CD252), and can then be used to deplete endogenous hematopoietic stem cells in patients (e.g., human patients) in need of hematopoietic stem cell transplantation therapy.

[0346] In addition to in vitro display technology, computational modeling technology may be used to design and identify in silico antibodies or antibody fragments that can bind to 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 CD252). For example, using computational modeling technology, one of ordinary skill in the art may seek molecules that can bind to a specific epitope (e.g., an extracellular epitope of the antigen) of an antigen expressed by hematopoietic stem cells (e.g., CD117 [e.g., GNNK+ CD117], or CD45), or an antigen expressed by mature immune cells (e.g., T-cells) (e.g., CD2, CD5, CD137, or CD252), and screen a library of antibodies or antibody fragments in silico. Antibodies or antigen-binding fragments thereof identified by these computer technologies may be used in combination with the treatment methods described herein (e.g., methods of treating cancer and autoimmune diseases described herein, and procedures for conditioning patients described herein).

[0347] Using additional techniques, antibodies or antibody fragments can be identified that can bind to 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 CD252), and that can be internalized by said cells, for example, by receptor-mediated endocytosis. For example, the above-described in vitro display techniques can be adapted to screen for antibodies or antibody fragments that bind to 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 CD252) and are subsequently internalized. Phage display is one representative of the techniques that can be used in combination with this screening paradigm. To identify 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 fragments, and subsequently to identify internalization into hematopoietic stem cells (or immune cells), one of ordinary skill in the art may use the phage display technique described in Williams et al., Leukemia 19:1432-1438, 2005 (the disclosure of which is incorporated herein by reference in its entirety). For example, using mutagenesis methods known in the art, antibodies, antibody fragments (e.g., especially scFv fragments, Fab fragments, diabodies, triabodies, and 10A recombinant phage library encoding a Fn3 domain), or a ligand (these contain a randomized amino acid cassette [e.g., in one or more, or all, of the CDRs or their equivalent regions, or an antibody or antibody fragment]) can be produced. The framework region, hinge, Fc domain, and other regions of the antibody or antibody fragment can be designed to be non-immunogenic in humans, for example, by having sequences that differ slightly compared to human germline antibody sequences or human germline antibodies.

[0348] Using phage display techniques described herein or known in the art, a phage library comprising randomized antibodies or antibody fragments covalently attached to phage particles is incubated with an antigen (e.g., CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137, or CD252) (e.g., first, to remove phages encoding antibodies or antibody fragments that show non-specific protein binding and phages encoding antibodies or their fragments that bind to the Fc domain, the phage library is incubated with a blocking agent [e.g., milk protein, bovine serum albumin, and / or IgG, etc.], and then the phage library is incubated with a population of hematopoietic stem cells or mature immune cells [e.g., T-cells] expressing, for example, CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137, or CD252). The phage library may be incubated with target cells (e.g., cancer cells, autoimmune cells, or hematopoietic stem cells, etc.) for a sufficient time (e.g., 30 minutes to 6 hours at 4°C, e.g., 1 hour at 4°C, etc.) for an anti-HC antibody (e.g., anti-CD117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody) or its antibody fragment to bind to the cognate antigen (e.g., CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137, or CD252) on the cell surface and subsequently be taken up by the hematopoietic stem cells. Subsequently, phages containing antibodies or antibody fragments that do not show sufficient affinity for the antigen (CD117 [e.g., GNNK+ CD117], CD45, CD2, CD5, CD137, or CD252) and are bound to and taken up by the target cells (e.g., cancer cells, autoimmune cells, or hematopoietic stem cells, etc.) may be removed, for example, by washing with cold (4°C) 0.1 M glycine buffer at pH 2.8.Phages bound to an antibody or an antibody fragment thereof that have been incorporated into target cells (e.g., cancer cells, autoimmune cells, or hematopoietic stem cells) may be identified, for example, by lysing said cells and recovering the phages incorporated therein from the cell culture medium. Then, for example, the phages may be amplified in bacterial cells by incubating the recovered phages with the bacterial cells in 2×YT medium using methods known in the art. Next, the phages recovered from this medium may be identified, for example, by determining the nucleic acid sequence of the gene encoding the antibody or antibody fragment inserted into the phage genome. The encoded antibody or antibody fragment thereof may subsequently be newly prepared by chemical synthesis (e.g., the antibody fragment, e.g., scFv fragment) or recombinant expression (e.g., full-length antibody).

[0349] The allowable amount of the prepared antibody or antibody fragment thereof incorporated therein may be evaluated, for example, using a radionuclide uptake assay known in the art. For example, an anti-HC antibody (e.g., anti-CD117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody) or an antibody fragment thereof identified using an in vitro display technique described herein or known in the art may be labeled with a radioisotope (e.g., 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu, 177 Lu, 77 As, 72 As, 86 Y, 90 Y,89 Zr, 212 Bi, 213 Bi, or 225 Ac) may be incorporated to make it functional. For example, a radioactive halogen (e.g., 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, etc.) may be incorporated into an antibody or antibody fragment using beads (e.g., polystyrene beads) containing an electrophilic halogen reagent (e.g., iodinated beads, Thermo Fisher Scientific, Inc., Cambridge, MA). The radio-labeled antibody, its fragment or ADC may be incubated with target cells (e.g., cancer cells, autoimmune cells, or hematopoietic stem cells, etc.) for a time sufficient to be incorporated therein (e.g., 30 minutes to 6 hours at 4°C, 1 hour at 4°C, etc.). Then, the cells are washed (e.g., using cold (4°C) 0.1 M glycine buffer at pH 2.8) to remove the antibody or its fragment that was not incorporated. The incorporated antibody or its antibody fragment may be identified by detecting the radiation (e.g., γ-rays) emitted from the obtained target cells (e.g., cancer cells, autoimmune cells, or hematopoietic stem cells, etc.) and comparing it with the radiation (e.g., γ-rays) emitted from the collected wash buffer. The ADC may also be characterized using the internal uptake assay described above.

[0350] Antibodies may be produced using recombinant methods and compositions, such as those described in U.S. Patent 4,816,567. In certain embodiments, isolated nucleic acids encoding the anti-HC antibodies described herein (e.g., anti-CD117 antibodies, anti-CD45 antibodies, anti-CD2 antibodies, anti-CD5 antibodies, anti-CD137 antibodies, or anti-CD252 antibodies) are provided. Such nucleic acids may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH (e.g., the light chain and / or heavy chain of the antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In certain such embodiments, the host cell comprises (e.g., is transformed by): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In certain embodiments, the host cell is a eukaryote, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In certain embodiments, a method of making an anti-CLL-1 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or the culture medium of the host cell).

[0351] To recombinantly produce an anti-HC antibody (e.g., an anti-CD117 antibody, an anti-CD45 antibody, an anti-CD2 antibody, an anti-CD5 antibody, an anti-CD137 antibody, or an anti-CD252 antibody), a nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody) using conventional procedures and can be sequenced.

[0352] Suitable host cells for cloning or expressing a vector encoding an antibody include the prokaryotic or eukaryotic cells described herein. For example, the antibody may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody may be isolated as a soluble fraction from the bacterial cell paste and further purified.

[0353] As a host, vertebrate cells can also be used. For example, mammalian cell lines adapted to grow in suspension may be beneficial. Other examples of useful mammalian host cell lines include the simian kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); simian kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); dog kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), etc.); and myeloma cell lines (e.g., Y0, NS0, and Sp2 / 0, etc.). For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). In certain embodiments, the host cells are eukaryotic, e.g., Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0354] Antibody-drug conjugate (ADC) The antibodies (including anti-CD117 antibodies, etc.) and antigen-binding fragments described in this specification may be conjugated (linked) to cytotoxins via a linker. In some embodiments, the cytotoxic molecule is conjugated to an antibody or an antigen-binding fragment thereof that is taken up into the cell as disclosed herein, such that after the antibody or its fragment is taken up into the cell, the cytotoxin can access its intracellular target and mediate the death of hematopoietic cells. Any number of cytotoxins, for example, 1, 2, 3, 4, 5, 6, 7, or 8, can be conjugated to the anti-CD117 antibody.

[0355] Cytotoxins suitable for use with the compositions and methods described herein include those known in the art, particularly DNA-intercalating agents (e.g., anthracyclines), agents that can disrupt the spindle apparatus (e.g., vinca alkaloids, maytansines, maytansinoids, and their derivatives), RNA polymerase inhibitors (e.g., amatoxins such as α-amanitin and their derivatives), and agents that can disrupt protein biosynthesis (e.g., agents that exhibit rRNA N-glycosidase activity such as saporin and ricin A chain).

[0356] Cytotoxin Various cytotoxins may be conjugated via a linker to an anti-HC antibody (e.g., anti-CD117 antibody, anti-CD45 antibody, anti-CD2 antibody, anti-CD5 antibody, anti-CD137 antibody, or anti-CD252 antibody) for use in the therapies described herein. In particular, an anti-HC ADC (e.g., anti-CD117 ADC, anti-CD45 ADC, anti-CD2 ADC, anti-CD5 ADC, anti-CD137 ADC, or anti-CD252 ADC) comprises an antibody (or antigen-binding fragment thereof) conjugated (i.e., covalently linked by a linker) to a cytotoxic moiety (or cytotoxin). In various embodiments, the cytotoxic moiety exhibits reduced cytotoxicity or no cytotoxicity when conjugated. However, upon cleavage from the linker, it will again exhibit cytotoxicity. In various embodiments, the cytotoxic moiety retains cytotoxicity without being cleaved from the linker. In some embodiments, the cytotoxic molecule conjugates to an antibody, or antigen-binding fragment thereof, that is internalized into a cell as described herein, such that after the antibody, or fragment thereof, is internalized into the cell, the cytotoxin accesses its intracellular target and may, for example, mediate T cell death.

[0357] Accordingly, the ADCs of the disclosure may be of the general formula Ab-(Z-L-D) n wherein the antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently bound) to a linker (L) and, through a chemical substructure (Z), to a cytotoxic moiety ("drug", D), as disclosed herein, respectively.

[0358] Accordingly, the antibody or antigen-binding fragment thereof may be conjugated to a number of drug moieties as indicated by an integer n, where the integer n represents the average number of cytotoxins per antibody, where the integer n may be in the range of, for example, about 1 to about 20. In some embodiments, n is from 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in a preparation of an ADC by a conjugation reaction can be ascertained by conventional means such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the ADC with respect to n can also be determined. In some cases, homogeneous ADCs where n is a particular value can be separated, purified, and characterized from ADCs having other drug loadings by means such as reverse-phase HPLC or electrophoresis.

[0359] For some anti-HC ADCs (e.g., anti-CD117 ADC, anti-CD45 ADC, anti-CD2 ADC, anti-CD5 ADC, anti-CD137 ADC, or anti-CD252 ADC), n may be limited by the number of binding sites on the antibody. For example, where the binding is via a cysteine thiol, the antibody may have only one or several cysteine thiol groups, or only one or several thiol groups with sufficient reactivity for the linker to bind. Generally, antibodies do not contain many free and reactive cysteine groups capable of binding to drug moieties; primarily, the cysteine thiol residues in the antibody exist as disulfide bridges. In certain embodiments, the antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonyl ethylphosphine (TCEP) to generate reactive cysteine thiol groups under partial or full reduction conditions. In certain embodiments, when higher drug loading is achieved (e.g., n > 5), in certain antibody-drug conjugates, aggregation, insolubility, toxicity, or loss of cell permeability may be caused.

[0360] In certain embodiments, during the conjugation reaction, fewer drug moieties than the theoretical maximum conjugate to the antibody. The antibody may, for example, contain lysine residues that do not react with the drug-linker intermediate or linker reagent, as discussed below. Only the most reactive lysine groups are able to react with the amine-reactive linker reagent. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine on the surface.

[0361] The drug loading (drug / antibody ratio) of the ADC may be controlled in various ways, such as (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the time or temperature of the conjugation reaction, (iii) subjecting to partial or limited reducing conditions for thiol modification of cysteine, (iv) manipulating the amino acid sequence of the antibody by recombinant techniques to modify the number and / or position of cysteine residues so as to control the number and / or position of the linker-drug bonds.

[0362] Cytotoxins suitable for use with the compositions and methods described herein include those known in the art, especially DNA-intercalating agents (e.g., anthracyclines), agents capable of disrupting the spindle apparatus (e.g., vinca alkaloids, maytansine, maytansinoids, and their derivatives), RNA polymerase inhibitors (e.g., amatoxins such as α-amanitin, and their derivatives), and agents capable of disrupting protein biosynthesis (e.g., agents exhibiting rRNA N-glycosidase activity such as saporin and ricin A-chain).

[0363] In some embodiments, the cytotoxin is a microtubule-binding agent (e.g., maytansine or a maytansinoid), amatoxin, Pseudomonas exotoxin A, debuganin, diphtheria toxin, saporin, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, indolinobenzodiazepine dimer, indolinobenzodiazepine pseudodimer or variant thereof, or other cytotoxic compounds described herein or known in the art.

[0364] In some embodiments, the cytotoxin of the antibody-drug conjugate is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is amatoxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody-drug conjugate disclosed herein is amatoxin or a derivative thereof (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amanine amide, amanullin, amanullinic acid, proamanullin or a derivative thereof, etc.).

[0365] Further details regarding cytotoxins that may be used in anti-HC ADCs (e.g., anti-CD117 ADC, anti-CD45 ADC, anti-CD2 ADC, anti-CD5 ADC, anti-CD137 ADC, or anti-CD252 ADC) useful in the methods of the present invention are described below.

[0366] Amatoxin The methods and compositions disclosed herein include an ADC comprising an RNA polymerase inhibitor (e.g., amatoxin) as a cytotoxin conjugated to an anti-HC antibody (e.g., an anti-CD117 antibody). In some embodiments, the cytotoxin of the antibody-drug conjugate is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is amatoxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody-drug conjugate disclosed herein is amatoxin or a derivative thereof (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amanine amide, amanullin, amanullinic acid, proamanullin, or a derivative thereof, etc.). Suitable amatoxins are disclosed, for example, in Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459.

[0367] Amatoxins useful in combination with the compositions and methods described herein include, but are not limited to, compounds of formula (III) including α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amanine amide, amanullin, amanullinic acid, proamanullin, etc. Formula (III) is as follows:

Chemical formula

[0368] For example, in certain embodiments, amatoxins useful in combination with the compositions and methods described herein include compounds of formula (IIIA) [Chemical formula] wherein R4, R5, X, and R8 are as defined above, respectively.

[0369] For example, in certain embodiments, amatoxins useful in combination with the compositions and methods described herein include compounds of the following formula (IIIB): [Chemical formula] wherein R1 is H, OH, or ORA is; R2 is H, OH, or OR B is; R A and R B , when present, together with the oxygen atom to which they are attached, form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, or R D is; R4 is H, OH, OR D , or R D is; R5 is H, OH, OR D , or R D is; R6 is H, OH, OR D , or R D is; R7 is H, OH, OR D , or R D is; R8 is OH, NH2, or OR D is; R9 is H, OH, or OR D is; X is -S-, -S(O)-, or -SO2-; and, R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0370] In certain embodiments, amatoxins useful in combination with the compositions and methods described herein include compounds of the following formula (IIIC):

Chemical formula

[0371] In one embodiment, the cytotoxin is amanitin. For example, the antibodies and antigen-binding fragments described herein may bind to amatoxin to form a conjugate represented by the formula Ab-Z-L-Am, where Ab is an antibody or an antigen-binding fragment thereof, L is a linker, Z is a chemical substructure, and Am is amatoxin. Many positions on amatoxin or its derivatives can serve as sites for covalent attachment to the linking moiety L and, thus, can serve as sites for covalent attachment to the antibody or an antigen-binding fragment thereof. Exemplary methods of amatoxin conjugation and linkers useful for such processes are described below. Exemplary linker-containing amatoxins useful for conjugates to antibodies or antigen-binding fragments by the compositions and methods described herein are shown in Structural Formulas (I), (IA), (IB), (II), (IIA), and (IIB) and are recited herein.

[0372] In some embodiments, the amatoxin-linker conjugate Am-L-Z is represented by formula (I)

Chemical formula

Claims

**Claim 1** An IgG1 or IgG4 antibody, or an antigen-binding portion thereof, comprising an Fc region, wherein the Fc region comprises the amino acid substitutions L234A, L235A, D265C, and H435A (EU index), and wherein the IgG1 or IgG4 antibody, or an antigen-binding portion thereof, does not bind detectably to human Fc gamma receptor (R) as determined by biolayer interferometry. **Claim 2** The antibody, or an antigen-binding portion thereof, according to claim 1, wherein the antibody has a decreased half-life compared to the same intact IgG antibody comprising an unmodified Fc region. **Claim 3** The antibody, or an antigen-binding portion thereof, according to claim 2, wherein the half-life is 24 hours or less. **Claim 4** The antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 3, wherein the antibody has a decrease in effector function defined as a decrease in binding to Fc gamma receptor (FcγR) compared to binding of the same antibody comprising an unmodified Fc region to FcγR. **Claim 5** The antibody, or an antigen-binding portion thereof, according to claim 4, wherein: (i) the FcγR is the FcγR1 receptor; (ii) the FcγR receptor is the FcγR2 receptor or the FcγR3 receptor. **Claim 6** The antibody, or an antigen-binding portion thereof, according to claim 5, wherein the FcγR2 receptor is FcγR2A, FcγR2B, or FcγR2C; or the FcγR3 receptor is FcγR3A or FcγR3B. **Claim 7** The antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 6, wherein the antibody decreases cytokine release by at least 50% compared to cytokine release of the same antibody comprising an unmodified Fc region in an in vitro cytokine release assay. **Claim 8** The antibody, or an antigen-binding portion thereof, according to claim 7, wherein: (i) The decrease in cytokine release is at least a 60% decrease, at least a 70% decrease, at least an 80% decrease, at least a 90% decrease, at least a 95% decrease, at least a 98% decrease, at least a 99% decrease, or a 100% decrease in cytokine release compared to the cytokine release of the same antibody containing an unmodified Fc region; or the antibody does not exhibit detectable cytokine release; and / or, (ii) The in vitro cytokine release assay is a tissue culture (TC) inflammation assay from Meso Scale Discovery (MSD). **Claim 9** An antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 6, wherein the antibody reduces mast cell degranulation in an in vitro mast cell degranulation assay with at least a 50% decrease in mast cell degranulation compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. **Claim 10** An antibody, or an antigen-binding portion thereof, according to claim 9, wherein: (i) The decrease in mast cell degranulation is at least a 60% decrease, at least a 70% decrease, at least an 80% decrease, at least a 90% decrease, at least a 95% decrease, at least a 98% decrease, at least a 99% decrease, or a 100% decrease in mast cell degranulation compared to the mast cell degranulation of the same antibody containing an unmodified Fc region; (ii) The antibody does not exhibit detectable mast cell degranulation; and / or, (iii) The in vitro mast cell degranulation assay is a mast cell degranulation assay based on beta-hexosaminidase. **Claim 11** An antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 10, wherein: (i) The IgG isotype is an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, or an IgG4 isotype; (ii) The antibody is a human antibody, a chimeric or humanized antibody; (iii) The antibody is a bispecific antibody; (iv) The antibody is a monoclonal antibody; (v) The antibody is an intact IgG antibody; and / or, (vi) The antibody specifically binds to CD117, CD45, CD2, CD5, CD137, or CD252.

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

13. A pharmaceutical composition comprising the antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 11, and a pharmaceutically acceptable carrier.

14. The antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 11, for use in a method of reducing the population of hematopoietic stem cells (HSCs) in a human patient, wherein the method comprises administering to the patient an effective amount of the antibody, or an antigen-binding portion thereof.

15. The antibody, or an antigen-binding portion thereof, according to claim 14, wherein: (i) the method further comprises administering to the patient a graft comprising hematopoietic stem cells; (ii) the antibody, or an antigen-binding portion thereof, is administered to the human patient before administering the graft comprising hematopoietic stem cells; and / or (iii) the patient has a blood disorder, a metabolic disorder, cancer, an autoimmune disease, or a severe combined immunodeficiency disease (SCID).

16. The antibody, or an antigen-binding portion thereof, according to claim 15, wherein the graft is allogeneic or autologous.

17. The antibody, or an antigen-binding portion thereof, according to claim 15, wherein the hematopoietic stem cells are CD117+ or CD45+ cells.

18. The antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 11, for use in treating leukemia in a human patient.

19. The antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 11, for use in a method of reducing the population of immune cells in a human patient, wherein the method comprises administering to the human patient the antibody, or an antigen-binding portion thereof, before administering a graft comprising hematopoietic stem cells.

20. The antibody, or an antigen-binding portion thereof, according to claim 19, wherein the immune cells are CD137+, CD2+, or CD5+ cells, and / or the immune cells are T cells.

21. A composition comprising an antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 11, wherein the composition comprises less than 25% hydrophobic degradation products after heat stress, less than 20% hydrophobic degradation products after heat stress, less than 15% hydrophobic degradation products after heat stress, less than 10% hydrophobic degradation products after heat stress, or less than 5% hydrophobic degradation products after heat stress.

22. An antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 11, for use in treating a stem cell disorder, an immunodeficiency disorder, a metabolic disorder, an autoimmune disorder, or cancer.

23. An antibody, or an antigen-binding portion thereof, according to claim 22, wherein: (i) the immunodeficiency disorder is a primary immunodeficiency or a secondary immunodeficiency; (ii) the metabolic disorder is selected from the group consisting of glycogen storage disease, mucopolysaccharidosis, Gaucher disease, Hurler disease, sphingolipidosis, and metachromatic leukodystrophy; (iii) The autoimmune disorder is selected from the group consisting of 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 antibody syndrome, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune oophoritis, Baló's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythematosus, autonomic neuropathy, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture 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, Ménière's disease, mixed connective tissue disease, myasthenia gravis, neuromyotonia, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-person syndrome, Takayasu arteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis; or, (iv) The cancer is selected from the group consisting of leukemia, lymphoma, multiple myeloma, and neuroblastoma.

Images