Amatoxin antibody-drug conjugates and uses thereof

Abstract

To provide amatoxins, to provide antibody-drug conjugates (ADCs) comprising an amatoxin, and to provide compositions and methods of using the same.SOLUTION: The present invention provides amatoxins that can be used in ADC for delivery of the amatoxin to a target cell. The present invention further provides particular modifications of antibodies that can be used in antibody drug conjugates for the delivery of the amatoxin to a target cell. The compositions and methods provided herein can be used for cancer therapy. They can also be used to prepare a patient for hematopoietic stem cell transplant therapy and to improve the engraftment of hematopoietic stem cell transplants by selectively depleting endogenous hematopoietic stem cells prior to the transplant procedure. Methods and compositions for the treatment of various hematopoietic diseases, metabolic disorders, cancers, and autoimmune diseases, as well as prevention of graft-versus-host-disease (GVHD), are provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to amatoxins, antibody-drug conjugates (ADCs) comprising amatoxins, compositions comprising such ADCs, and methods of using the same. [Background technology]

[0002] Monoclonal antibodies (mAbs) can be conjugated to therapeutic agents to form antibody-drug conjugates (ADCs). ADCs can show increased efficacy compared to unconjugated antibodies. The linkage of the antibody to the drug can be direct or indirect through a linker. An important aspect of a successful therapeutic ADC is that it is not only effective but also well tolerated. In many cases, cytotoxicity affects both efficacy and tolerability.

[0003] ADCs have been proposed as therapeutic agents for the treatment of cancer. The use of ADCs for the local delivery of cytotoxic or cytostatic agents, i.e., drugs that kill or inhibit tumor cells in the treatment of cancer, would theoretically allow for targeted delivery of the drug moiety to tumors and their intracellular accumulation there, but systemic administration of these unconjugated agents may result in unacceptable levels of toxicity to normal cells as well.

[0004] ADCs have also been proposed as a treatment regimen to prepare patients for transplantation and stem cell therapy. By pretreating patients with cell-specific ADCs, stem cells or immune cells can be selectively depleted while the remaining immune system of the patient remains largely intact. For example, Palchaudhuri et al. (2016) Nat. Biotechnol. 34, 738-745 describe the use of a single-dose anti-CD45 ADC in which the anti-CD45 antibody is conjugated to saporin and its ability to allow engraftment of donor cells for treatment in a sickle cell anemia model. Unlike radiation, CD45-SAP ADCs were reported to avoid neutropenia and anemia and provide rapid recovery of T and B cells with minimal overall toxicity. There remains a need for toxins that can be used for non-genotoxic targeted ADC pretreatment, where the toxin is potent against the target cells while at the same time causing minimal side effects to the patient. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides amatoxins that can be used, for example, in antibody-drug conjugates (ADCs) for the delivery of amatoxins to target cells. The present invention further provides specific modifications of antibodies that can be used in antibody-drug conjugates for the delivery of amatoxins to target cells. The present invention further relates to combinations of said amatoxins and antibodies, which have increased potency, improved in vivo tolerability, and thus a favorable therapeutic window. [Brief description of the drawings]

[0006] [Figure 1] The structures of formulas IV (FIG. 1A), VI (FIG. 1B), and IIa (FIG. 1C) are depicted. "Ab" in FIGS. 1A to 1C represents an antibody. FIGS. 1A, 1B, and 1C represent conjugates A, B, and C, respectively, which are referred to in the Examples. Conjugates A, B, and C are also referred to as ADC A, ADC B, and ADC C.

[0007] [Diagram 2]FIG. 1 illustrates the results of an in vitro cytoxicity assay evaluating the serum stability of the conjugates using Kasumi-1 cells in the presence of the ADC, with preincubation of the ADC in media (A) or 50% human serum (B).

[0008] [Diagram 3] FIG. 1 illustrates the results of an in vitro cytotoxicity assay assessing the difference in kinetics of cytotoxicity between cleavable and non-cleavable conjugates using Kasumi-1 cells in the presence of ADC over repeated incubation times with titrated ADC samples.

[0009] [Figure 4] 4A and 4B illustrate the results of two in vitro cell killing assays using Kasumi-1 cells (4A) and CD34+ cells (4B). The ADC tested was anti-CD117 ADC C.

[0010] [Diagram 5] Illustrates the ability of anti-CD117 ADC C to potently deplete human CD34+ in the bone marrow of humanized NSG mice.

[0011] [Figure 6] 1 is a Kaplan-Meier plot reflecting survival of Kasumi-1 engrafted humanized NSG mice treated with the indicated doses of conjugate or control.

[0012] [Figure 7] Illustrates efficacy of Complex C evaluated in male cynomolgus monkeys. 2.0 mg / kg dose (LALA) 30.2867 Batch issues may have caused a decrease in HSC reactivity.

[0013] [Figure 8] 1 illustrates the tolerability of ADCs containing either Conjugate A or Conjugate C in male cynomolgus monkeys.

[0014] [Figure 9] FIG. 1 illustrates the pharmacokinetic analysis of Conjugate A and Conjugate C administered to male cynomolgus monkeys.

[0015] [Figure 10] FIG. 1 illustrates that anti-CD2 and CD5 ADCs can deplete T cells.

[0016] [Figure 11] Illustrates that anti-CD2 ADCs A and C are saturated by day 5, while some cells still express CD5.

[0017] [Figure 12A-12B] 14A illustrates results showing anti-CD45 ADC A or C (FIG. 14A) or anti-CD45 ADC A or B (FIG. 14B) in an in vitro cell killing assay.

[0018] [Figure 13] 1 illustrates results showing anti-CD45 ADC A or C in an in vitro cell killing assay.

[0019] [Figure 14] 1 illustrates the results of anti-CD45 ADC A or C in an in vivo cell depletion experiment.

[0020] [Figure 15] FIG. 1 illustrates the results of administration of anti-CD45 ADC A or C at several doses to mice. Peripheral lymphocyte, HSC, and lymphocyte levels are shown. All ADCs were administered at 1 mg / kg.

[0021] [Figure 16] 1 illustrates results showing anti-CD137 ADCs A and C in a T cell killing assay.

[0022] [Figure 17]Illustrates cell line serum stability of anti-CD137 ADCs A and C over 48 hours.

[0023] [Figure 18] The structures of amanitin-linker constructs HDP30.2867, 30.0880, 30.2371, and 30.1699 are depicted, respectively.

[0024] [Figure 19] 1 depicts the structures of amanitin-linker constructs HDP30.2115, 30.2060, and 30.2347, respectively.

[0025] [Figure 20] FIG. 1 illustrates the in vitro cytotoxic activity of ADC compounds T-D265C-30.2867 and T-D265C-30.0880 against (A) SKBR-3, (B) NCI-N87, (C) BT474, and (D) JIMT-1 cell lines, respectively, in a 96-hour BrdU assay.

[0026] [Figure 21] Illustrates the results of SDS-PAGE / Western blot analysis of the stability of ADC compounds T-D265C-30.2867 and T-D265C-30.0880, respectively, after 0, 4, and 10 days of incubation, respectively, in (A) human-, (B) mouse-, (C) cynomolgus monkey plasma, and (D) PBS (control).

[0027] [Figure 22] Illustrates the in vitro cytotoxic activity of ADC compound T-D265C-30.2867 against SBR-3 cells after 0, 4, and 10 days of incubation, respectively, in (A) human-, (B) mouse-, (C) cynomolgus monkey plasma, and (D) PBS (control).

[0028] [Diagram 23]Illustrates the in vitro cytotoxic activity of ADC compound T-D265C-30.2867 against NCI-N87 cells after 0, 4, and 10 days of incubation, respectively, in (A) human-, (B) mouse-, (C) cynomolgus monkey plasma, and (D) PBS (control).

[0029] [Figure 24] Illustrates the in vitro cytotoxic activity of ADC compound T-D265C-30.2867 against JIMT-1 cells after 0, 4, and 10 days of incubation, respectively, in (A) human-, (B) mouse-, (C) cynomolgus monkey plasma, and (D) PBS (control).

[0030] [Diagram 25] 1 illustrates the results of cytotoxic efficacy analysis of anti-Her2-ADC in an in vivo JIMT-1-cell xenograft tumor mouse model using ADC compounds T-D265C-30.2867, T-D265C-30.0880, and T-D265C-30.1699.

[0031] [Figure 26] 1 illustrates the results of cytotoxic efficacy analysis of anti-Her2-ADCs in an in vivo NCI-N87-cell xenograft tumor mouse model using ADC compounds T-D265C-30.2867, T-D265C-30.0880, and T-D265C-30.1699.

[0032] [Figure 27] FIG. 1 illustrates the in vitro cytotoxic activity of ADC compounds h3 / F11-D265C-Var16-30.2867 and h3 / F11-D265C-Var16-30.0880, respectively, against (A) LNCap, (B) 22RV1, and (C) PC3 cell lines in a 96-hour BrdU assay.

[0033] [Figure 28]1 illustrates the results of cytotoxic efficacy analysis of anti-PSMA-ADCs in an in vivo LNCap-cell xenograft tumor mouse model using ADC compounds h3 / F11-D265C-Var16-30.2867, h3 / F11-D265C-Var16-30.0880, and h3 / F11-D265C-Var16-30.2060.

[0034] [Figure 29] FIG. 1 illustrates the results of a tolerability study in monkeys using the anti-digoxigenin ADC compound DIG-D265C-30.2867 at different doses between 1 mg / kg and 20 mg / kg. The results of the evaluation of LDH, AST, and ALT parameters are shown.

[0035] [Diagram 30] Illustrates the results of a tolerability study in monkeys using DIG-D265C-30.2867. Pharmacokinetic data of the ADC in serum is shown.

[0036] [Diagram 31] 1 illustrates the results of a tolerability study in monkeys using DIG-D265C-30.2867. Pharmacokinetic data of amatoxin in serum is shown.

[0037] [Diagram 32] Illustrates the in vitro cytotoxic activity against JIMT-1 cells of ADC compounds comprising an anti-Her2 antibody with a D265C mutation (T-D265C) conjugated to structurally distinct amanitin derivatives (A, B).

[0038] [Diagram 33] Illustrates the in vitro cytotoxic activity against NCI-N87 cells of ADC compounds comprising an anti-Her2 antibody with a D265C mutation (T-D265C) conjugated to structurally distinct amanitin derivatives (A, B).

[0039] [Diagram 34]Illustrates the in vitro cytotoxic activity against SKBR-3 cells of ADC compounds comprising an anti-Her2 antibody with a D265C mutation (T-D265C) conjugated to structurally distinct amanitin derivatives (A, B).

[0040] [Diagram 35] Illustrates the in vitro cytotoxic activity against LNCap cells of ADC compounds comprising an anti-PSMA antibody with a D265C mutation (h3 / F11-D265C-Var16) conjugated to structurally distinct amanitin derivatives (A, B).

[0041] [Diagram 36] Illustrates the in vitro cytotoxic activity against 22RV1 cells of ADC compounds comprising an anti-PSMA antibody with a D265C mutation (h3 / F11-D265C-Var16) conjugated to structurally distinct amanitin derivatives (A, B).

[0042] [Figure 37] FIG. 1 illustrates the results of a cytotoxic efficacy analysis of various anti-PSMA-ADCs containing structurally distinct amanitin derivatives in an in vivo LNCap-cell xenograft tumor mouse model. Shown are the means ± SEM of all groups at ½ and ¼ of the maximum tolerated dose (MTD).

[0043] [Figure 38] Illustrates the results of cytotoxic efficacy analysis of various anti-PSMA-ADCs containing structurally distinct amanitin derivatives in an in vivo LNCap-cell xenograft tumor mouse model. Shown are the mean ± SEM of all groups at ½ the maximum tolerated dose (MTD).

[0044] [Figure 39] Illustrates the results of cytotoxic efficacy analysis of various anti-PSMA-ADCs containing structurally distinct amanitin derivatives in an in vivo LNCap-cell xenograft tumor mouse model. Shown are the means ± SEM of all groups at ¼ of the maximum tolerated dose (MTD).

[0045] [Diagram 40] 1 illustrates the results of a cytotoxicity potency analysis of various anti-PSMA-ADCs containing structurally different amanitin derivatives in an in vivo LNCap-cell xenograft tumor mouse model. Shown are the mean ± SEM of ADCs with linker attachment at amino acid 1 of amatoxin at ½ and ¼ of the maximum tolerated dose (MTD).

[0046] [Diagram 41] 1 illustrates the results of a cytotoxicity potency analysis of various anti-PSMA-ADCs containing structurally different amanitin derivatives in an in vivo LNCap-cell xenograft tumor mouse model. Shown are the mean ± SEM of ADCs with linker attachment at amino acid 4 of amatoxin at ½ and ¼ of the maximum tolerated dose (MTD).

[0047] [Diagram 42] Illustrates the results of cytotoxic efficacy analysis of various anti-PSMA-ADCs containing structurally different amanitin derivatives in an in vivo LNCap-cell xenograft tumor mouse model. Shown are the mean ± SEM of ADCs with cleavable linkers at ¼ of the maximum tolerated dose (MTD).

[0048] [Diagram 43] Illustrates the results of cytotoxic efficacy analysis of anti-Her2 ADCs containing the triple L234A / L235A / D265C mutation and the amatoxin-linker constructs HDP30.2060 and HDP30.2867, respectively (T-LALA-D265C-30.2060, T-LALA-D265C-30.2867, respectively) against Her2-positive NCI-N87 cells in a CDX mouse model.

[0049] [Diagram 44]Illustrates the results of cytotoxicity efficacy analysis of anti-PSMA ADCs containing the triple L234A / L235A / D265C mutation and the amatoxin-linker constructs HDP30.2060 and HDP30.2867 (h3 / F11-LALA-D265C-Var-30.2060, h3 / F11-LALA-D265C-Var-30.2867, respectively) against PSMA-positive LNCap cells in a CDX mouse model.

[0050] [Diagram 45] Illustrated are the results of a 96-hour BrdU assay to assess the in vitro cytotoxic potential of Her2-specific ADCs comprising antibodies with the L234A / L235A and / or D265C mutations, or a control mutation, against Her2-positive SKBR-3 cells (A, B, C, D).

[0051] [Figure 46] Illustrated are the results of a 96-hour CTG assay to assess the in vitro cytotoxic potential of Her2-specific ADCs comprising antibodies with the L234A / L235A and / or D265C mutations, or a control mutation, against Her2-negative THP-1 cells (A, B, C, D).

[0052] [Figure 47] Illustrated are the results of a 120-hour CTG assay to assess the in vitro cytotoxic potential of Her2-specific ADCs comprising antibodies with L234A / L235A and / or D265C mutations or control mutations against Her2-negative THP-1 cells (A, B, C, D). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] For clarity of this disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections below.

[0054] The present invention relates to a compound represented by formula (An) or (Bn) [ka] (wherein n is 2, 3, 4, 5, 6, 7, 8, or 9). or a derivative or analogue thereof.

[0055] In one embodiment, the present invention provides a compound of formula (A): [ka] Formula (A)(HDP 30.2867) or a derivative or analogue thereof, or an enantiomer or diastereomer thereof.

[0056] In a further embodiment, the present invention relates to a compound of formula (B) [ka] Formula (B) (HDP 30.0880) or a derivative or analogue thereof, or an enantiomer or diastereomer thereof.

[0057] The amatoxins or derivatives or analogues thereof can be used in the preparation of antibody drug conjugates (ADCs).

[0058] The ADCs according to the invention have been shown by the inventors to have particularly high plasma stability and tolerability and therefore an improved therapeutic window.

[0059] The present invention includes an antibody or antigen-binding fragment thereof conjugated to an amatoxin by a linker, the antibody or antigen-binding fragment having the formula (I): [ka] (In the formula: Q is S or a sulfoxide group; L is a non-cleavable linker; Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in an antibody or antigen-binding fragment thereof; and Ab is an antibody or an antigen-binding fragment thereof or a stereoisomer thereof.

[0060] The ADC has the formula (Ia): [ka] The structure may be:

[0061] The ADC has the formula (Ib): [ka] It may also have the structure:

[0062] In the ADC, L is a bond, -(C=O)-, a -C(O)NH- group, a -OC(O)NH- group, 1 ~C 6 Alkylene, C 1 ~C 6 Heteroalkylene, C 2 ~C 6 Alkenylene, C 2 ~C 6 Heteroalkenylene, C 2 ~C 6 Alkynylene, C 2 ~C 6 Heteroalkynylene, C 3 ~C 6 Cycloalkylene, heterocycloalkylene, arylene, heteroarylene, p is an integer of 1 to 6, -(CH 2 CH 2 O) p - group, or solubility enhancing group; Here, each C 1 ~C 6 Alkylene, C 1 ~C 6 Heteroalkylene, C 2 ~C6 Alkenylene, C 2 ~C 6 Heteroalkenylene, C 2 ~C 6 Alkynylene, C 2 ~C 6 Heteroalkynylene, C 3 ~C 6 The cycloalkylene, heterocycloalkylene, arylene, or heteroarylene can be optionally substituted by 1 to 5 substituents independently selected at each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro; Or each C 1 ~C 6 Alkylene, C 1 ~C 6 Heteroalkylene, C 2 ~C 6 Alkenylene, C 2 ~C 6 Heteroalkenylene, C 2 ~C 6 Alkynylene, C 2 ~C 6 Heteroalkynylene, C 3 ~C 6 The cycloalkylene, heterocycloalkylene, arylene, or heteroarylene may be optionally interrupted by one or more heteroatoms selected from O, S, and N.

[0063] The solubility enhancing group has the formula -O a -C(O)NH-SO 2 -NR 1 -, wherein: a is 0 or 1; and R 1 is hydrogen, C 1 ~C 24Alkyl group, C 3 ~C 24 Cycloalkyl groups, C 2 ~C 24 (Hetero)aryl groups, C 3 ~C 24 Alkyl(hetero)aryl groups, and C 3 ~C 24 (hetero)arylalkyl groups, each of which is selected from the group consisting of O, S, and NR 3 and optionally substituted or interrupted by one or more heteroatoms selected from, where R 3 is hydrogen and C 1 ~C 4 alkyl.

[0064] In the ADC, L is -(CH 2 ) n - units. Preferably, L is -(CH 2 ) n -It is.

[0065] In the ADC, Ab, Z, and L together represent Ab-ZL and have the following formula: [ka] where S is the sulfur atom of a cysteine ​​residue present in the antibody or antigen-binding fragment thereof.

[0066] In one embodiment, the invention comprises an antibody or antigen-binding fragment thereof conjugated to an amatoxin by a linker, the antibody or antigen-binding fragment having the formula (I): [ka] (In the formula: Q is S or a sulfoxide group; L is a cleavable linker; Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in an antibody or antigen-binding fragment thereof; and Ab is an antibody or an antigen-binding fragment thereof or a stereoisomer thereof.

[0067] The ADC has formula (Ia): [ka] The structure may be:

[0068] The ADC has formula (Ib): [ka] The structure may be:

[0069] In the ADC, L is a hydrazine, a disulfide, a thioether, an amino acid, a peptide of up to 10 amino acids, a p-aminobenzyl (PAB) group, a heterocyclic self-immolative group, C 1 ~C 6 Alkyl, C 1 ~C 6 Heteroalkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Heteroalkenyl, C 2 ~C 6 Alkynyl, C 2 ~C 6 Heteroalkynyl, C 3 ~C 6 Cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -(C=O)- group, -C(O)NH- group, -OC(O)NH- group, -(CH 2 CH 2 O) p -group, or one or more solubility enhancing groups; Here, each C 1 ~C 6 Alkyl, C 1 ~C 6 Heteroalkyl, C 2 ~C 6 Alkenyl, C 2~C 6 Heteroalkenyl, C 2 ~C 6 Alkynyl, C 2 ~C 6 Heteroalkynyl, C 3 ~C 6 The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can be optionally substituted by 1 to 5 substituents independently selected at each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro; Or each C 1 ~C 6 Alkyl, C 1 ~C 6 Heteroalkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Heteroalkenyl, C 2 ~C 6 Alkynyl, C 2 ~C 6 Heteroalkynyl, C 3 ~C 6 The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group may be optionally interrupted by one or more heteroatoms selected from O, S, and N.

[0070] The solubility enhancing group has the formula -O a -C(O)NH-SO 2 -NR 1 -, wherein: a is 0 or 1; and R 1 is hydrogen, C 1 ~C 24 Alkyl group, C 3 ~C 24 Cycloalkyl groups, C2 ~C 24 (Hetero)aryl groups, C 3 ~C 24 Alkyl(hetero)aryl groups, and C 3 ~C 24 (hetero)arylalkyl groups, each of which is selected from the group consisting of O, S, and NR 3 and optionally substituted or interrupted by one or more heteroatoms selected from, where R 3 is hydrogen and C 1 ~C 4 alkyl.

[0071] According to one embodiment of the invention, L comprises a peptide selected from the group consisting of Phe-Lys, Val-Lys, Phe-Ala, Phe-Cit, Val-Ala, Val-Cit, and Val-Arg. L may further comprise a PAB group.

[0072] In one embodiment, L has the formula: [ka] It is expressed by:

[0073] According to a preferred embodiment, the present invention comprises an antibody conjugated to an amatoxin, the antibody having the formula (II): [ka] or a stereoisomer thereof.

[0074] The ADC has the formula (IIa): [ka] The structure may be:

[0075] The ADC has the formula (IIb): [ka] The structure may be:

[0076] According to a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof specifically binds to an antigen expressed on the cell surface of a cancer cell, or a human stem cell, in particular a hematopoietic stem cell (HSC), or a T cell.

[0077] According to further preferred embodiments of the invention, the antibody or antigen-binding fragment thereof specifically binds to human Her2, PSMA, CD37, or CD123.

[0078] According to another preferred embodiment of the invention, the antibody or antigen-binding fragment thereof comprises an Fc region comprising at least one mutation selected from the group consisting of D265C, D265A, A118C, H435A, L234A, or L235A (according to the EU index).

[0079] According to another preferred embodiment of the invention, the antibody or antigen-binding fragment thereof specifically binds to PSMA and comprises a CDRH1 according to SEQ ID NO: 378, a CDRH2 according to SEQ ID NO: 379, a CDRH3 according to SEQ ID NO: 380, a CDRL1 according to SEQ ID NO: 381, a CDRL2 according to SEQ ID NO: 382, ​​and a CDRL3 according to SEQ ID NO: 383.

[0080] According to another preferred embodiment of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region according to SEQ ID NO:375 and a light chain variable region according to SEQ ID NO:377.

[0081] According to another preferred embodiment of the invention, the antibody comprises a heavy chain according to SEQ ID NO: 371, SEQ ID NO: 372, SEQ ID NO: 373, or SEQ ID NO: 374, and a light chain according to SEQ ID NO: 376, or an antigen-binding fragment thereof.

[0082] The present invention further relates to an antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof conjugated by a linker to an amatoxin or a derivative or analogue thereof, wherein the antibody or antigen-binding fragment thereof comprises an Fc region comprising at least two mutations consisting of L234A and L235A (according to the EU index).

[0083] According to a particularly preferred embodiment, said Fc region further comprises the mutation D265C (according to the EU index).

[0084] According to a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof specifically binds to an antigen expressed on the cell surface of a cancer cell, preferably a human cancer cell.

[0085] According to another preferred embodiment of the invention, the antibody or antigen-binding fragment thereof specifically binds to prostate-specific membrane antigen (PSMA), preferably human PSMA, or the Her2 antigen, CD37, or CD123.

[0086] A preferred embodiment relates to an antibody drug conjugate (ADC), wherein the antibody or antigen-binding fragment thereof specifically binds to PSMA and comprises a CDRH1 according to SEQ ID NO: 378, a CDRH2 according to SEQ ID NO: 379, a CDRH3 according to SEQ ID NO: 380, a CDRL1 according to SEQ ID NO: 381, a CDRL2 according to SEQ ID NO: 382, ​​and a CDRL3 according to SEQ ID NO: 383.

[0087] According to a preferred embodiment of the invention, the antibody specifically binds to PSMA or an antigen-binding fragment thereof and comprises a heavy chain variable region according to SEQ ID NO:375 and a light chain variable region according to SEQ ID NO:377.

[0088] According to another preferred embodiment of the invention, the antibody comprises a heavy chain according to SEQ ID NO: 372, SEQ ID NO: 373, or SEQ ID NO: 374, and a light chain according to SEQ ID NO: 376, or an antigen-binding fragment thereof.

[0089] In a further preferred embodiment, the antibody drug conjugate (ADC) comprises an antibody or antigen-binding fragment thereof conjugated to any compound selected from the group consisting of HDP30.2060, HDP30.2115, HDP30.2347, HDP30.1699, HDP30.2371, HDP30.0880, and HDP30.2867.

[0090] The present invention further relates to an antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds human PSMA and comprises a heavy chain having the amino acid sequence according to SEQ ID NO: 374 and a light chain having the amino acid sequence according to SEQ ID NO: 376, conjugated to the compound HDP30.2060. Most preferably, in said ADC, the compound HDP30.2060 is directly linked to the sulfur atom of cysteine ​​D265C (EU numbering) of said antibody.

[0091] The present invention further relates to an antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and that comprises a heavy chain having an amino acid sequence according to SEQ ID NO: 374 and a light chain having an amino acid sequence according to SEQ ID NO: 376, conjugated to the compound HDP30.2115. Most preferably, in said ADC, the compound HDP30.2115 is directly linked to the sulfur atom of cysteine ​​D265C of said antibody.

[0092] The present invention further relates to an antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and that comprises a heavy chain having an amino acid sequence according to SEQ ID NO: 374 and a light chain having an amino acid sequence according to SEQ ID NO: 376, conjugated to the compound HDP30.2347. Most preferably, in said ADC, the compound HDP30.2347 is directly linked to the sulfur atom of cysteine ​​D265C of said antibody.

[0093] The present invention further relates to an antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and comprises a heavy chain having an amino acid sequence according to SEQ ID NO: 374 and a light chain having an amino acid sequence according to SEQ ID NO: 376, conjugated to the compound HDP30.1699. Most preferably, in said ADC, the compound HDP30.1699 is directly linked to the sulfur atom of cysteine ​​D265C of said antibody.

[0094] The present invention further relates to an antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and comprises a heavy chain having an amino acid sequence according to SEQ ID NO: 374 and a light chain having an amino acid sequence according to SEQ ID NO: 376, conjugated to the compound HDP30.2371. Most preferably, in said ADC, the compound HDP30.2371 is directly linked to the sulfur atom of cysteine ​​D265C of said antibody.

[0095] The present invention further relates to an antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and comprises a heavy chain having an amino acid sequence according to SEQ ID NO: 374 and a light chain having an amino acid sequence according to SEQ ID NO: 376, conjugated to the compound HDP30.0880. Most preferably, in said ADC, the compound HDP30.0880 is directly linked to the sulfur atom of cysteine ​​D265C of said antibody.

[0096] The present invention further relates to an antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and comprises a heavy chain having an amino acid sequence according to SEQ ID NO: 374 and a light chain having an amino acid sequence according to SEQ ID NO: 376, conjugated to the compound HDP30.2867. Most preferably, in said ADC, the compound HDP30.2867 is directly linked to the sulfur atom of cysteine ​​D265C of said antibody.

[0097] In the antibody drug conjugates (ADCs) according to the invention, the drug to antibody ratio (DAR) is about 1, 2, 3, or 4, preferably the DAR is 2.

[0098] A further aspect of the invention relates to said ADC for use in treating cancer in a patient, particularly where the cancer is selected from the group consisting of breast cancer, pancreatic cancer, bile duct cancer, colorectal cancer, lung cancer, prostate cancer, ovarian cancer, prostate cancer, gastric cancer, renal cancer, malignant melanoma, blood system cancer, leukemia, and malignant lymphoma.

[0099] The invention further relates to the use of any of the above ADCs for the treatment of cancer in a patient, particularly where the cancer is selected from the group consisting of breast cancer, pancreatic cancer, bile duct cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, prostate cancer, gastric cancer, renal cancer, malignant melanoma, blood system cancer, leukemia, and malignant lymphoma.

[0100] According to another preferred embodiment of the invention, the antibody drug conjugate (ADC) comprises an antibody or an antigen-binding fragment thereof that specifically binds to an antigen expressed on the cell surface of a hematopoietic stem cell (HSC), preferably a human HSC.

[0101] A further embodiment of the invention relates to a method of depleting a population of cells in a human subject, comprising administering to the subject said ADC, wherein the ADC comprises an antibody, or antigen-binding fragment thereof, that specifically binds to an extracellular antigen expressed by cells in the population of cells.

[0102] A further embodiment of the invention relates to a method of pretreating a human subject for cell transplantation, comprising administering to the human subject said ADC such that endogenous stem cells or endogenous immune cells in the human subject are depleted, wherein the ADC specifically binds to an extracellular antigen expressed by the endogenous stem cells or endogenous immune cells.

[0103] Further embodiments of the invention relate to said methods further comprising administering to the human subject allogeneic stem cells or allogeneic immune cells.

[0104] Further embodiments of the invention relate to methods wherein the ADC specifically binds to an extracellular antigen expressed on an immune cell, and the subject has or is at risk of developing graft-versus-host disease (GVHD).

[0105] The invention further relates to a pharmaceutical composition comprising any of the above ADCs or combinations thereof and at least a pharma- ceutically acceptable carrier.

[0106] definition Unless otherwise stated, the following terms and phrases used herein shall have the following meanings:

[0107] The term "acyl" as used herein refers to -C(=O)R, where R is hydrogen (an "aldehyde"), C(=O) or -C(=O)R, as defined herein. 1 ~C 12 Alkyl, C 2 ~C 12 Alkenyl, C 2 ~C 12 Alkynyl, C 3 ~C 7 Carbocyclyl, C 6 ~C 20 aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl. Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, and acryloyl.

[0108] As used herein, the term "C 1 ~C 12 "Alkyl" refers to a straight or branched chain saturated hydrocarbon having 1 to 12 carbon atoms. Representative C 1 ~C 12 Alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl, and branched C 1 ~C 12 Alkyl includes, but is not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl. 1 ~C 12 The alkyl group can be unsubstituted or substituted.

[0109] As used herein, the term "alkenyl" refers to an alkyl group having at least one site of unsaturation, i.e., a carbon-carbon sp 2 C containing normal, secondary, or tertiary carbon atoms with double bonds 2 ~C 12 It refers to a hydrocarbon. Examples include, but are not limited to, ethylene or vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, etc. Alkenyl groups can be unsubstituted or substituted.

[0110] As used herein, "alkynyl" refers to a C alkyl group containing normal, secondary, or tertiary carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon sp triple bond. 2 ~C 12 Refers to a hydrocarbon. Examples include, but are not limited to, acetylenic and propargyl. Alkynyl groups can be unsubstituted or substituted.

[0111] As used herein, "aryl" refers to 6 ~C 20 It refers to a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. Aryl groups can be unsubstituted or substituted.

[0112] As used herein, "arylalkyl" refers to an alkyl group consisting of a carbon atom, typically a terminal or sp 3It refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a 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-naphthylethene-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like. An arylalkyl group contains 6 to 20 carbon atoms, e.g., the alkyl portion, including the alkanyl, alkenyl, or alkynyl group of the arylalkyl group, is 1 to 6 carbon atoms, and the aryl portion is 5 to 14 carbon atoms. An alkaryl group may be unsubstituted or substituted.

[0113] As used herein, "cycloalkyl" refers to a saturated carbocyclic radical that may be monocyclic or bicyclic. Cycloalkyl groups include rings having 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be unsubstituted or substituted.

[0114] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic radical that may be monocyclic or bicyclic. Cycloalkenyl groups include rings having 3 to 6 carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle. 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. Cycloalkenyl groups may be unsubstituted or substituted.

[0115] As used herein, a "heteroaralkyl" refers to an aryl group that is heteroaryl with a carbon atom, typically a terminal or sp 3It refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom is replaced by a heteroaryl radical. Exemplary heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like. Heteroarylalkyl groups contain 6-20 carbon atoms, e.g., the alkyl portion of the heteroarylalkyl group, including the alkanyl, alkenyl, or alkynyl group, is 1-6 carbon atoms, and the heteroaryl portion is 5-14 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S. The heteroaryl portion of the heteroarylalkyl group can be a monocyclic ring having 3-7 ring members (2-6 carbon atoms) or a bicyclic ring having 7-10 ring members (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S), e.g., bicyclo[4,5], [5,5], [5,6], or [6,6] systems.

[0116] As used herein, "heteroaryl" and "heterocycloalkyl" refer to an aromatic or non-aromatic ring system in which one or more ring atoms are heteroatoms, such as nitrogen, oxygen, and sulfur, respectively. The heteroaryl or heterocycloalkyl radical contains 2-20 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S. The heteroaryl or heterocycloalkyl may be a monocyclic ring having 3-7 ring members (2-6 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S) or a bicyclic ring having 7-10 ring members (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S), such as: bicyclo[4,5], [5,5], [5,6], or [6,6] systems. The heteroaryl and heterocycloalkyl may be unsubstituted or substituted.

[0117] Heteroaryl and heterocycloalkyl groups are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially 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), especially volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.

[0118] Examples of heteroaryl groups include, by way of example and not limitation, 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-quinolyl, 5H-quinolyl, 6H-quinolyl, 7H-quinolyl, 8H-quinolyl, 9H-quinolyl, 10H-quinolyl, 11H-quinolyl, 12H-quinolyl, 13H-quinolyl, 14H-quinolyl, 15H-quinolyl, 16H-quinolyl, 17H-quinolyl, 18H-quinolyl, 19 ...9H-quinolyl, 20H-quinolyl, 21H-quinolyl, 22H-quinolyl, 23H-quinolyl, 24H-quinolyl, 25H-quinolyl, 26H-quinolyl, 27H-quinolyl, 28H-quinolyl, 29H-quinolyl, 30H-quinolyl, 31H-quinolyl, 32H-quinolyl, 33H-quinolyl, 34H These include lysinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, benzotriazolyl, benzisoxazolyl, and isatinoyl.

[0119] Examples of heterocycloalkyl include, by way of example and not limitation, dihydroypyridyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinuclidinyl, and morpholinyl.

[0120] By way of example and not limitation, carbon bonded heteroaryl and heterocycloalkyl are bonded at the 2-, 3-, 4-, 5-, or 6-position of pyridine, the 3-, 4-, 5-, or 6-position of pyridazine, the 2-, 4-, 5-, or 6-position of pyrimidine, the 2-, 3-, 5-, or 6-position of pyrazine, the 2-, 3-, 4-, or 5-position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2-, 4-, or 5-position of oxazole, imidazole, or thiazole, the 3-, 4-, or 5-position of isoxazole, pyrazole, or isothiazole, the 2- or 3-position of aziridine, the 2-, 3-, or 4-position of azetidine, the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of quinoline, or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of isoquinoline. Even more typically, the carbon-linked heterocyclyl is 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.

[0121] By way of example and not limitation, nitrogen-linked heteroaryls and heterocycloalkyls are linked 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, 2-position of isoindole or isoindoline, 4-position of morpholine, and 9-position of carbazole or β-carboline. Even more typically, nitrogen-linked heterocyclyls include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0122] "Substituted," as used herein and as applied to any of the above alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, etc., means that one or more hydrogen atoms are each independently replaced by a substituent. Unless otherwise constrained by the definition of the particular substituent, the above chemical moieties, such as "alkyl," "alkylene," "heteroalkyl," "heteroalkylene," "alkenyl," "alkenylene," "heteroalkenyl," "heteroalkenylene," "alkynyl," "alkynylene," "heteroalkynyl," "heteroalkynylene," "cycloalkyl," "cycloalkylene," "heterocyclolalkyl," "heterocycloalkylene," "aryl," "arylene," "heteroaryl," and "heteroarylene" groups, may be optionally substituted. Exemplary substituents include -X, -R, -OH, -OR, -SH, -SR, NH 2 , -NHR, -N(R) 2 , -N + (R) 3 , -CX 3 , -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO 2 , -N 3 , -NC(=O)H, -NC(=O)R, -C(=O)H, -C(=O)R, -C(=O)NH2 , -C(=O)N(R) 2 , -SO 3 -, -SO 3 H, -S(=O) 2 R, -OS(=O) 2 OR, -S(=O) 2 NH 2 , -S(=O) 2 N(R) 2 , -S(=O)R, -OP(=O)(OH) 2 , -OP(=O)(OR) 2 , -P(=O)(OR) 2 , -PO 3 , -PO 3 H 2 , -C(=O)X, -C(=S)R, -CO 2 H, -CO 2 R, -CO 2 -, -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 at each occurrence from F, Cl, Br, and I; and each R is C 1 ~C 12 Alkyl, C 6 ~C 20 Aryl, C 3 ~C 14 is independently selected at each occurrence from heterocycloalkyl or heteroaryl, a protecting group, and a prodrug moiety. Whenever a group is described as "optionally substituted," that group can be substituted at each occurrence independently with one or more of the above substituents.

[0123] It should be understood that a particular radical naming convention can include monoradicals or diradicals, depending on the context. For example, if a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a diradical. For example, a substituent specified as an alkyl requiring two points of attachment includes -CH2 -, -CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 Other radical naming conventions explicitly indicate that the radical is a diradical, such as "alkylene," "alkenylene," "arylene," "heterocycloalkylene," etc.

[0124] Whenever a substituent is depicted as a diradical (i.e., having two points of attachment to the remainder of the molecule), it is to be understood that the substituent can be attached in any directional configuration, unless otherwise specified.

[0125] "Isomerism" means compounds that have identical molecular formulae but differ in the sequence of bonds of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers." Stereoisomers that are not mirror images of one another are termed "diastereoisomers" and stereoisomers that are non-superimposable mirror images of each other are termed "enantiomers" or sometimes "optical isomers."

[0126] A carbon atom bonded to four non-identical substituents is termed a "chiral center." "Chiral isomer" means a compound having at least one chiral center. Compounds with two or more chiral centers can exist as individual diastereomers or as a mixture of diastereomers, termed a "diastereomeric mixture." When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked according to the Cahn, Ingold, and Prelog ranking rules (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116). A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is called a "racemic mixture".

[0127] The compounds disclosed herein and in the claims may contain one or more asymmetric centers, and different diastereomers and / or enantiomers of each of the compounds may exist. Any description of a compound in the specification and claims is intended to include all enantiomers, diastereomers, and mixtures thereof, unless otherwise specified. Furthermore, any description of a compound in the specification and claims is intended to include both individual enantiomers, and any mixtures of enantiomers, whether racemic or otherwise, unless otherwise specified. When a compound structure is depicted as a specific enantiomer, it should be understood that the invention of the present application is not limited to that specific enantiomer. Thus, each enantiomer, optical isomer, and diastereomer of the structural formula of the present disclosure is contemplated herein. Although the structural formula of a compound in the present specification may conveniently represent a specific isomer in some cases, the present disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, and the like, with the understanding that not all isomers may have the same level of activity. A compound may exist in different tautomeric forms. Compounds according to the present disclosure are intended to include all tautomeric forms unless otherwise specified. When a compound structure is depicted as a particular tautomer, it should be understood that the invention of this application is not limited to that particular tautomer.

[0128] The compounds of any formula described herein include the compounds themselves, as well as their salts and solvates, if applicable. Salts can be formed, for example, between an anion and a positively charged group (e.g., amino) on the compounds of the present disclosure. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate). The term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Similarly, salts can be formed between a cation and a negatively charged group (e.g., carboxylate) on the compounds of the present disclosure. Suitable cations include ammonium cations such as sodium, potassium, magnesium, calcium, and tetramethylammonium ions. Some suitable substituted ammonium ions are ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and those derived from amino acids such as lysine and arginine. The compounds of the present disclosure also include salts containing quaternary nitrogen atoms.

[0129] Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0130] Furthermore, the compounds of the present disclosure, e.g., salts of the compounds, can exist in hydrated or non-hydrated (anhydrous) form, or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc. "Solvate" refers to a solvent addition form that contains a stoichiometric or non-stoichiometric amount of solvent. Some compounds have a tendency to incorporate a certain molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. When the solvent is water, the solvate formed is a hydrate. When the solvent is alcohol, the solvate formed is an alcoholate. A hydrate is a compound that is formed by dissolving one or more molecules of water with water at a molar ratio of 1 to 10,000,000. 2 It is formed by the combination of one molecule of a substance that retains its molecular state as O. Hydrates refer to, for example, monohydrates, dihydrates, trihydrates, etc.

[0131] Furthermore, crystalline polymorphism may exist for the compounds represented by the formulas disclosed herein or their salts. It is noted that any crystalline form, mixture of crystalline forms, or anhydrates or hydrates thereof are included within the scope of the present disclosure.

[0132] As used herein, the term "about" refers to a value that is within 10% above or below the stated value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

[0133] As used herein, the term "amatoxin" refers to a member of the amatoxin family of peptides produced by Amanita phalloides mushroom, or a variant or derivative thereof, such as a variant or derivative thereof capable of inhibiting RNA polymerase II activity. Suitable amatoxins and derivatives thereof are further described below. As described herein, amatoxins can be conjugated to an antibody or antigen-binding fragment thereof (thereby forming a conjugate (i.e., ADC)), for example, by a linker moiety (L). Exemplary methods of amatoxin conjugation and linkers useful for such processes are described below.

[0134] In the context of the present invention, the term "amatoxin" includes the fully cyclic peptide composed of 8 amino acids isolated from the genus Amanita and described in Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 5 (1978) 185-260), further fully chemical derivatives thereof; further fully and semi-synthetic analogues thereof; further fully synthetic analogues thereof made from building blocks according to the master structure of the natural compound (cyclic, 8 amino acids), further fully synthetic or semi-synthetic analogues containing non-hydroxylated amino acids instead of hydroxylated amino acids, further fully synthetic or semi-synthetic analogues in which the sulfoxide moiety is replaced by a sulfone, a thioether or by an atom different from sulfur, such as a carbon atom in the carbanalog of amanitin.

[0135] As used herein, a "derivative" of a compound refers to a species that has a similar chemical structure to the compound, but contains at least one chemical group not present in the compound and / or lacks at least one chemical group present in the compound. The compound to which the derivative is compared is known as the "parent" compound. Typically, a "derivative" can be produced from the parent compound by one or more chemical reaction steps.

[0136] As used herein, an "analog" of a compound is structurally related to, but not identical to, a compound and exhibits at least one activity of the compound. The compound to which the analog is compared is known as the "parent" compound. The above activities include, but are not limited to: binding activity to another compound; inhibitory activity, e.g., enzyme inhibitory activity; toxic effect; activating activity, e.g., enzyme activating activity. An analog is not required to exhibit such activity to the same extent as the parent compound. A compound is considered to be an analog within the context of this application if it exhibits a related activity to the extent of at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, and more preferably at least 50%) of the activity of the parent compound. Thus, "analogs of amatoxin," as used herein, refer to compounds that are structurally related to any one of -amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, and amanuric acid, and that exhibit at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, and more preferably at least 50%) inhibitory activity against mammalian RNA polymerase II compared to at least one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, and amanuric acid. "Analogs of amatoxin" suitable for use in the present invention may even exhibit higher inhibitory activity against mammalian RNA polymerase II than any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, or amanuric acid. Inhibitory activity is expressed as the concentration at which 50% inhibition occurs (IC 50 The inhibitory activity against mammalian RNA polymerase II can be indirectly determined by measuring the inhibitory activity against cell proliferation.

[0137] "Semi-synthetic analogues" refer to analogues obtained by chemical synthesis using compounds from natural sources (e.g., plant materials, bacterial cultures, fungal cultures, or cell cultures) as starting materials. Typically, the "semi-synthetic analogues" of the present invention are synthesized starting from compounds isolated from mushrooms of the Amanitaceae family. In contrast, "synthetic analogues" refer to analogues synthesized by so-called total synthesis from small (typically petrochemical) building blocks. Usually, this total synthesis is carried out without the aid of biological processes.

[0138] According to some embodiments of the invention, the amatoxin may be selected from the group consisting of α-amanitin, β-amanitin, amanin, amaninamide, and analogs, derivatives, and salts thereof.

[0139] Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins are those that have a functional group (e.g., a carboxyl group, an amino group, a hydroxyl group, a thiol, or a thiol capture group) that can react with a linker molecule or a target binding moiety, as defined below.

[0140] In the context of the present invention, the term "amanitin" refers in particular to a bicyclic structure based on an aspartic acid or asparagine residue at position 1, a proline residue, particularly a hydroxyproline residue, at position 2, an isoleucine, hydroxyisoleucine, or dihydroxyisoleucine residue at position 3, a tryptophan or hydroxytryptophan residue at position 4, a glycine residue at positions 5 and 7, an isoleucine residue at position 6, and a cysteine ​​residue at position 8, particularly a derivative of cysteine ​​that has been oxidized to a sulfoxide or sulfone derivative (see FIG. 1 for the numbering and representative examples of amanitin), and further includes all chemical derivatives thereof; further all and semi-synthetic analogues thereof; further all and semi-synthetic analogues thereof made from building blocks according to the master structure of the natural compound (cyclic, 8 amino acids), further all and semi-synthetic analogues comprising non-hydroxylated amino acids instead of hydroxylated amino acids, further all and semi-synthetic analogues, in each case any such derivative or analogue is functionally active by inhibiting mammalian RNA polymerase II.

[0141] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen, and includes monoclonal, genetically engineered and otherwise modified forms of antibodies, including, but not limited to, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi-, tri-, and tetra-specific antibodies, diabodies, triabodies, and tetrabodies), and antibody fragments such as, for example, Fab', F(ab') 2 Unless otherwise specified, the term "monoclonal antibody" (mAb) includes antigen-binding fragments of antibodies, including intact molecules as well as antibody fragments (e.g., Fab and F(ab')) that are capable of specifically binding to a target protein. 2 As used herein, Fab and F(ab') are intended to include both Fab and F(ab') fragments. 2 Fragment refers to an antibody fragment that lacks the Fc fragment of an intact antibody. Examples of these antibody fragments are described herein.

[0142] As used herein, the term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0143] The antibody of the present invention is generally isolated or recombinant. As used herein, "isolated" refers to a polypeptide, e.g., an antibody, that is separated and / or recovered from a cell or cell culture in which the polypeptide, e.g., the antibody, is expressed. Thus, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificity. For example, an isolated antibody that specifically binds to CD117 is substantially free of antibodies that specifically bind to antigens other than CD117.

[0144] The term "antigen-binding fragment" as used herein refers to a fragment of an antibody that retains the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Antibody fragments include, for example, Fab, F(ab') 2 Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) Fab fragments, VFv, diabodies, triabodies, affibodies, nanobodies, aptamers, or domain antibodies. L , V H , C L , and C H (ii) F(ab') 2 (iii) V fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; H and C H (iv) a single-arm V of an antibody; L and V H (v) an Fv fragment consisting of a V H and V L (vi) a dAb containing a domain; H (vii) a dAb fragment consisting of the V domain (see, e.g., Ward et al., Nature 341:544-546, 1989); H Or V L(viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more (e.g., 2, 3, 4, 5, or 6) isolated CDRs, which may optionally be linked by a synthetic linker. Additionally, the two domains of an Fv fragment, V L and V H are encoded by separate genes, but they have been L Area and V H The domains can be linked by a linker that allows them to be produced as a single protein chain that pairs to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Antibody fragments can be obtained using conventional techniques known to those of skill 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, by enzymatic or chemical cleavage of intact immunoglobulins, or, in certain cases, by chemical peptide synthesis procedures known in the art.

[0145] As used herein, the term "anti-CD117 antibody" or "antibody that binds to CD117" refers to an antibody that is capable of binding to CD117, e.g., human CD117, with sufficient affinity such that the antibody is useful in targeting CD117 as a diagnostic and / or therapeutic agent. The amino acid sequences of the two major isoforms of human CD117 are provided in SEQ ID NO: 145 (isoform 1) and SEQ ID NO: 146 (isoform 2). "Anti-CD117 ADC" refers to an ADC in which the antibody is an anti-CD117 antibody.

[0146] As used herein, the term "anti-CD45 antibody" or "antibody that binds CD45" refers to an antibody that is capable of binding to CD45, e.g., human CD117, with sufficient affinity such that the antibody is useful in targeting CD45 as a diagnostic and / or therapeutic agent. An "anti-CD45 ADC" refers to an antibody where the antibody is an anti-CD45 antibody.

[0147] As used herein, the term "anti-CD137 antibody" or "antibody that binds CD137" refers to an antibody that is capable of binding to CD137, e.g., human CD137, with sufficient affinity such that the antibody is useful in targeting CD137 as a diagnostic and / or therapeutic agent. An "anti-CD137 ADC" refers to an ADC in which the antibody is an anti-CD137 antibody.

[0148] As used herein, the term "anti-CD2 antibody" or "antibody that binds CD2" refers to an antibody that is capable of binding to CD2, e.g., human CD2, with sufficient affinity such that the antibody is useful in targeting CD2 as a diagnostic and / or therapeutic agent. "Anti-CD2 ADC" refers to an ADC in which the antibody is an anti-CD2 antibody.

[0149] As used herein, the term "anti-CD5 antibody" or "antibody that binds CD5" refers to an antibody that is capable of binding to CD5, e.g., human CD5, with sufficient affinity such that the antibody is useful in targeting CD5 as a diagnostic and / or therapeutic agent. "Anti-CD5 ADC" refers to an ADC in which the antibody is an anti-CD5 antibody.

[0150] As used herein, the term "bispecific antibody" refers to an antibody, e.g., monoclonal, often human or humanized, that is capable of binding to at least two different antigens. For example, one of the binding specificities may be directed against a hematopoietic stem cell surface antigen, CD117 (e.g., GNNK+CD117), and the other may specifically bind to a different hematopoietic stem cell surface antigen or another cell surface protein, such as a receptor or receptor subunit involved in a signaling pathway that enhances cell growth, among others.

[0151] As used herein, the term "complementarity 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 framework regions (FR). The amino acid positions that border the hypervariable regions of an antibody may vary depending on the context and the various definitions known in the art. Some positions within the variable domain may be considered hybrid hypervariable positions, in that these positions may be considered to be within the hypervariable region by one set of criteria, while being considered to be outside the hypervariable region by a different set of criteria. One or more of these positions may also be found in an extended hypervariable region. The antibodies described herein may contain modifications at these hybrid hypervariable positions. Naturally occurring heavy and light chain variable domains each contain four framework regions that are primarily in a β-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in each chain are held together in close proximity to 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, immunoglobulin amino acid residue numbering is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified (any antibody numbering scheme may be utilized, including but not limited to IMGT and Chothia).

[0152] As used herein, the terms "precondition" and "preconditioning" refer to a process by which a patient prepares to receive a transplant, e.g., a transplant comprising hematopoietic stem cells. Such procedures promote the engraftment of the hematopoietic stem cell transplant (e.g., as inferred from a sustained increase in the amount of viable hematopoietic stem cells in a blood sample isolated from the patient following a conditioning procedure and subsequent hematopoietic stem cell transplant). According to the methods described herein, a patient can be pretreated for hematopoietic stem cell transplantation therapy by administration to the patient of an ADC capable of binding to an antigen expressed by hematopoietic stem cells, such as CD117 (e.g., GNNK+CD117). Administration of an ADC capable of binding to an HSC antigen to a patient in need of hematopoietic stem cell transplantation therapy can promote the engraftment of the hematopoietic stem cell graft, e.g., by selectively depleting endogenous hematopoietic stem cells, thereby creating a void that is filled by exogenous hematopoietic stem cell transplantation.

[0153] As used herein, the term "conjugate" refers to a compound formed by chemically linking a reactive functional group of one molecule, such as an antibody or an antigen-binding fragment thereof, with an appropriately reactive functional group of another molecule, such as a cytotoxin as described herein. The conjugate may include a linker between the two molecules that are linked together. Examples of linkers that may be used to form the conjugate include peptide-containing linkers, such as those that contain naturally occurring or non-naturally occurring amino acids, such as D-amino acids. Linkers can be prepared using a variety of strategies described herein and known in the art. Depending on the reactive moieties 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). Of note, the term "conjugate" (when referring to a compound) is also referred to interchangeably herein as "drug conjugate," "antibody drug conjugate," or "ADC."

[0154] As used herein, the term "coupling reaction" refers to a chemical reaction in which two or more substituents suitable for reaction with each other react to form a chemical moiety that (e.g., covalently) links the molecular fragments attached to each substituent. Coupling reactions include those in which a reactive substituent attached to a fragment that is a cytotoxin, such as a cytotoxin known in the art or described herein, reacts with a suitably reactive substituent attached to a fragment that is an antibody or antigen-binding fragment thereof, such as an antibody, antigen-binding fragment thereof, or a specific anti-CD117 antibody that binds to CD117 (e.g., GNNK+CD117), known in the art or described herein. Examples of suitably reactive substituents include nucleophile / electrophile pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, among others), diene / dienophile pairs (e.g., azide / alkyne pairs, among others). Coupling reactions include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine condensation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huesgen cycloaddition, among others), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reactivity modes known in the art or described herein.

[0155] As used herein, "competitive repopulating unit (CRU)" refers to a unit of measure of long-term engrafting stem cells that can be detected following in vivo transplantation.

[0156] As used herein, the term "donor" refers to a human or animal from which one or more cells are isolated prior to administration of the cells or their progeny to a recipient. The one or more cells can be, for example, a population of hematopoietic stem cells.

[0157] As used herein, the term "diabody" refers to a bivalent antibody comprising two polypeptide chains, each of which is a V H Domain and V LV linked by a linker that is too short to allow intramolecular association of the domains (e.g., a linker consisting of five amino acids) H and V L This refers to a bivalent antibody that contains a V domain. This configuration forces each domain to pair with a complementary domain on another polypeptide chain to form a homodimeric structure. Thus, the term "triabody" refers to a trivalent antibody that contains three peptide chains, each of which is a V domain in the same peptide chain. H Domain and V L A single V linked by a linker that is too short (e.g., a linker consisting of 1-2 amino acids) to allow intramolecular association of the domains. H Domain and one V L This refers to a trivalent antibody that contains a V domain. To fold into its native structure, a peptide constructed in this way typically has adjacent V domains of peptide chains. H and V L They trimerize so that the domains are in spatial proximity to each other (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993).

[0158] As used herein, "drug antibody ratio" or "DAR" refers to the number of drugs, e.g., amatoxins, attached to an antibody in a conjugate. The DAR of an ADC can range from 1 to 8, although higher loadings are possible depending on the number of attachment sites on the antibody. In certain embodiments, the conjugate has a DAR of 1, 2, 3, 4, 5, 6, 7, or 8.

[0159] As used herein, the term "endogenous" describes a substance, such as a molecule, cell, tissue, or organ, that is naturally found in a particular organism, such as a human patient (e.g., a hematopoietic stem cell or cell of the hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen presenting cell, macrophage, dendritic cell, natural killer cell, T lymphocyte, or B lymphocyte, etc.).

[0160] As used herein, the term "engraftment potential" is used to refer to the ability of hematopoietic stem and progenitor cells to repopulate tissues, whether such cells are naturally circulating or provided by transplantation. The term encompasses all events surrounding and leading up to engraftment, including tissue establishment and colonization of cells in the tissue of interest. Engraftment efficiency or rate can be assessed or quantified using any clinically acceptable parameter as known to those of skill in the art, and may include, for example, estimation of competitive repopulating units (CRU); incorporation or expression of markers in the tissue where the stem cells have settled, colonized, or engrafted; or evaluation of disease progression in the subject, survival of hematopoietic stem and progenitor cells, or survival of the recipient. Engraftment can also be determined by measuring the number of white blood cells in peripheral blood during the post-transplant period. Engraftment can also be estimated by measuring bone marrow cell recovery by donor cells in a bone marrow aspirate sample.

[0161] As used herein, the term "exogenous" describes a substance, such as a molecule, cell, tissue, or organ that is not naturally found in a particular organism, such as a human patient (e.g., a hematopoietic stem cell or cell of the hematopoietic lineage, e.g., megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen presenting cell, macrophage, dendritic cell, natural killer cell, T lymphocyte, or B lymphocyte, etc.). Exogenous substances include those provided to an organism from an external source, or to a culture extracted therefrom.

[0162] The terms "Fc", "Fc region" and "Fc domain" as used herein refer to the portion of an IgG antibody that is related to the crystallizable fragment obtained by papain digestion of the IgG molecule. The Fc region comprises the C-terminal halves of the two heavy chains of an IgG molecule, which are linked by disulfide bonds. It does not have any antigen binding activity, but contains carbohydrate moieties and binding sites for complement and Fc receptors, including the FcRn receptor. The Fc region comprises the second constant domain CH2 (e.g., residues at EU positions 231-340 of IgG1) and the third constant domain CH3 (e.g., residues at EU positions 341-447 of human IgG1). As used herein, the Fc region comprises the "lower hinge region" (e.g., residues at EU positions 233-239 of IgG1). Fc refers to this region in isolation as well as in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at several positions in the Fc domain, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, and therefore slight differences may exist between the sequences presented herein 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 human IgG1, IgG2, IgG3, and IgG4 heavy chains can be found in several sequence databases, such as the Uniprot database (www.uniprot.org), under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG1_HUMAN), respectively. An example of a "WT" Fc region is provided in SEQ ID NO: 122 (providing the heavy chain constant region including the Fc region).

[0163] The term "modified Fc region" or "variant Fc region" as used herein refers to an IgG Fc domain that contains one or more amino acid substitutions, deletions, insertions or alterations introduced anywhere within the Fc region.

[0164] The terms "full-length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody in its substantially intact form and not an antibody fragment as defined herein. In one embodiment, the ADCs described herein include intact antibodies. Thus, for an IgG antibody, an intact antibody includes two heavy chains, each including a variable region, a constant region, and an Fc region, and two light chains, each including a variable region and a constant region. More specifically, an intact IgG includes two light chains, each including a light chain variable region (VL) and a light chain constant region (CL), and two heavy chains, each including a heavy chain variable region (VH) and three heavy chain constant regions (CH1, CH2, and CH3). CH2 and CH3 represent the Fc region of the heavy chain.

[0165] As used herein, the term "framework region" or "FW region" comprises the amino acid residues adjacent to the CDRs of an antibody or antigen-binding fragment thereof. FW region residues may be present, for example, in human antibodies, humanized antibodies, monoclonal antibodies, antibody fragments, Fab fragments, single-chain antibody fragments, scFv fragments, antibody domains, and bispecific antibodies, among others.

[0166] As used herein, the term "hematopoietic stem cell" ("HSC") refers to an immature blood cell that has the capacity to self-renew and differentiate into mature blood cells of various lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), platelets (e.g., megakaryoblasts, megakaryocytes that produce platelets, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Such cells express the CD34 + It may contain CD34 cells. +The cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are believed to comprise a subpopulation of cells with stemness as defined above, while in mice, HSCs are CD34-. Furthermore, HSCs are also referred to as long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). LT-HSCs and ST-HSCs are differentiated based on functional potential and cell surface marker expression. 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). 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, and IL7ra), whereas ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL7ra). Furthermore, ST-HSCs are less quiescent and more proliferative than LT-HSCs under homeostatic conditions. However, LT-HSCs have higher self-renewal potential (i.e., they survive throughout adulthood and can be continuously transplanted into subsequent recipients), while ST-HSCs have limited self-renewal (i.e., they only survive for a limited period of time and do not have continuous transplantation potential). Any of these HSCs can be used in the methods described herein. ST-HSCs are particularly useful because they are highly proliferative and therefore can generate differentiated progeny more quickly.

[0167] As used herein, the term "hematopoietic stem cell functional potential" refers to functional properties of hematopoietic stem cells including: 1) pluripotency (referring to the ability to differentiate into a number of different blood lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, megakaryocytes that produce platelets, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells); 2) self-renewal (referring to the ability of hematopoietic stem cells to give rise to daughter cells with equal potential to the mother cell, and further, this ability can occur repeatedly over the life of an individual without exhaustion); and 3) the ability of hematopoietic stem cells or their progeny to be reintroduced into a transplant recipient and rapidly colonize the hematopoietic stem cell niche and re-establish productive, sustained hematopoiesis.

[0168] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or during gene rearrangement, or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Human antibodies can 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 naturally occurring human antibodies. For example, an Fv may include a linker peptide, such as from 2 to about 8 glycine or other amino acid residues, connecting the variable region of the heavy chain to the variable region of the light chain. Such linker peptides are believed to be of human origin. Human antibodies can 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 can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes (see, e.g., WO 1998 / 24893; WO 1992 / 01047; WO 1996 / 34096; WO 1996 / 33735; U.S. Pat. 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).

[0169] A "humanized" antibody refers to an antibody that contains minimal sequence derived from a non-human immunoglobulin. Thus, a "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequence derived from a non-human antibody. All or substantially all of the FW region may be of human immunoglobulin sequence. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin consensus sequence. Methods for antibody humanization are known in the art and are described, for example, in Riechmann et al., Nature 332:323-7, 1988; U.S. Patent No. 5,530,101; U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; and U.S. Patent No. 6,180,370.

[0170] As used herein, the term "antibody-like protein" refers to a protein that has been engineered (e.g., by mutagenesis of Ig loops) to specifically bind to a target molecule. Typically, such antibody-like proteins contain at least one variable peptide loop connected to both ends of a protein scaffold. This double structural constraint greatly increases the binding affinity of antibody-like proteins to a level comparable to that of antibodies. The length of the variable peptide loop typically consists of 10-20 amino acids. The scaffold protein can be any protein with good solubility. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include, but are not limited to, affibodies, anticalins, and engineered ankyrin repeat proteins (Binz et al., 2005). Antibody-like proteins can be derived from large libraries of mutants, for example by panning from large phage display libraries, and can be isolated similar to regular antibodies. Antibody-like binding proteins can also be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins.

[0171] As used herein, the term "Fab" refers to an IgG fragment containing the antigen-binding region, said fragment consisting of one constant domain and one variable domain from each heavy and light chain of the antibody.

[0172] As used herein, the term "F(ab) 2 " relates to an IgG fragment that consists of two Fab fragments linked together by a disulfide bond.

[0173] As used herein, the term "scFv" refers to a single chain fragment that is a fusion of the variable regions of the heavy and light chains of an immunoglobulin linked by a short linker that usually contains serine (S) and / or glycine (G) residues. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of a linker peptide.

[0174] According to one aspect of the invention, the invention relates to said conjugate or pharmaceutical composition as described above for its use in the treatment of cancer.

[0175] According to one aspect of the invention, the invention relates to said conjugate or pharmaceutical composition as described for its use in the treatment of B-lymphocyte associated malignancies or B-cell mediated autoimmune diseases, especially for its use in the treatment of non-Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, granulomatosis with polyangiitis, and microscopic polyangiitis and pemphigus vulgaris.

[0176] As used herein, patients "in need of" hematopoietic stem cell transplantation include patients who exhibit defects or deficiencies in one or more blood cell types, as well as patients with stem cell disease, autoimmune disease, cancer, or other pathologies described herein. Hematopoietic stem cells generally 1) exhibit pluripotency, such that they can differentiate into a number of different blood lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), platelets (e.g., megakaryoblasts, megakaryocytes that produce platelets, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells), 2) exhibit self-renewal, such that they can give rise to daughter cells with potential equal to that of the parent cell, and 3) exhibit the ability to colonize the hematopoietic stem cell niche and establish productive and sustained hematopoiesis upon reintroduction into the transplant recipient. Thus, hematopoietic stem cells can be administered to a patient with a deficiency or deficiency in one or more cell types of the hematopoietic lineage to reconstitute the defective or deficient cell population in vivo. For example, the patient can be afflicted with cancer, and the deficiency can occur due to administration of a chemotherapeutic agent or other pharmaceutical agent that selectively or non-selectively depletes the cancer cell population. Additionally or alternatively, the patient can be afflicted with a hemoglobulinopathy (e.g., non-malignant hemoglobinopathy), such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome. The subject can be afflicted with adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome. The subject can have or be afflicted with an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune disease. Additionally or alternatively, the subject may have or be suffering from a malignant tumor, such as neuroblastoma or a hematological cancer. For example, the subject may have leukemia, lymphoma, or myeloma. In some embodiments, the subject has acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.In some embodiments, the subject has a myelodysplastic syndrome. In some embodiments, the subject has an autoimmune disease, such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, type 1 diabetes, or another autoimmune condition described herein. In some embodiments, the subject is in need of chimeric antigen receptor T cell (CART) therapy. In some embodiments, the subject has or is otherwise afflicted with a metabolic storage disease. Subjects may be those suffering from glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipid storage diseases, metachromatic leukodystrophy, or any other disease or disorder that may benefit from the treatments and therapies disclosed herein, including, but not limited to, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic scleroderma, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and “Bone Marrow Transplantation for Non-Malignant Disease,” ASH Education A patient may suffer from a metabolic disease or may otherwise be afflicted with a metabolic disease selected from the group consisting of other diseases or disorders, including those described in J. Med. Physiol. 1:319-338 (2000), the disclosure of which is incorporated herein by reference in its entirety as it relates to diseases that can be treated by administration of hematopoietic stem cell transplantation therapy. Additionally or alternatively, a patient "in need" of hematopoietic stem cell transplantation may be one who suffers from or does not suffer from one of the above diseases, but who nevertheless exhibits reduced levels (e.g., compared to those of an otherwise healthy subject) 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.One of skill in the art can readily determine whether the levels of one or more of the above cell types, or other blood cell types, are decreased relative to an otherwise healthy subject, for example, by flow cytometry and fluorescence-activated cell sorting (FACS), among other procedures known in the art.

[0177] As used herein, a "neutral antibody" refers to an antibody or antigen-binding fragment thereof that is not capable of significantly neutralizing, blocking, inhibiting, abrogating, reducing, or interfering with the activity of a specified or defined target (e.g., CD117), including receptor-to-ligand binding or enzyme-to-substrate interaction. In one embodiment, a neutral anti-CD117 antibody or fragment thereof is an anti-CD117 antibody that does not substantially inhibit SCF-dependent cell proliferation and does not cross-block SCF binding to CD117. An example of a neutral antibody is Ab67 (or an antibody having the binding region of Ab67). In contrast, an "antagonist" anti-CD117 antibody is capable of inhibiting SCF-dependent proliferation and cross-blocking SCF binding to CD117. An example of an antagonist antibody is Ab55 (or an antibody having the binding region of Ab55).

[0178] As used herein, the term "recipient" refers to a patient receiving a transplant, such as a transplant that includes a population of hematopoietic stem cells. The transplanted cells administered to the recipient can be, for example, autologous, syngeneic, or allogeneic cells.

[0179] 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 skin), pancreatic juice, chorionic villus samples, and cells).

[0180] As used herein, the term "scFv" refers to a single chain Fv antibody in which the variable domains of the heavy and light chains from an antibody are combined to form one chain. An scFv fragment contains the variable region of an antibody light chain (V L) (e.g., CDR-L1, CDR-L2, and / or CDR-L3) and the variable region of the antibody heavy chain (V H ) (e.g., CDR-H1, CDR-H2, and / or CDR-H3). L Area and V H The linker linking the regions can be a peptide linker composed of proteinogenic amino acids. Alternative linkers can be used to increase the resistance of the scFv fragment to proteolysis (e.g., linkers containing D-amino acids) to increase the solubility of the scFv fragment (e.g., hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeated glycine and serine residues), improve the biophysical stability of the molecule (e.g., linkers containing cysteine ​​residues that form intra- or intermolecular disulfide bonds), or reduce the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites). It will also be understood by those skilled in the art that the variable regions of the scFv molecules described herein can be modified to vary in amino acid sequence from the original antibody molecule from which they are derived. For example, nucleotide or amino acid substitutions resulting in conservative substitutions or changes in amino acid residues (e.g., in CDR and / or framework residues) can be made to preserve or increase the ability of the scFv to bind to the antigen recognized by the corresponding antibody.

[0181] As used herein, the term "specific binding" or "specifically binds" refers to the ability of an antibody to recognize and bind to a particular protein structure (epitope) rather than proteins in general. If an antibody is specific for epitope "A", then the presence of a molecule containing epitope A (or free unlabeled A) in a reaction involving labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody. By way of example, an antibody "specifically binds" to a target if, when labeled, it can be competed away from its target by the corresponding unlabeled antibody. In one embodiment, an antibody "specifically binds" to a target with at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less (less than 10 -12 A number less than 10, for example -13 (meaning D In one embodiment, the antibody specifically binds to a target, e.g., CD117, CD45, CD2, CD5, CD137, CD134, or CD252, when the antibody has the following structure: -7 The dissociation constant (K D In one embodiment, K D (M) is determined according to standard biolayer interferometry (BLI). off (1 / s) is determined according to standard biolayer interferometry (BLI). However, it is understood that an antibody may be capable of specifically binding to two or more antigens that are related in sequence. For example, in one embodiment, an antibody may specifically bind to both human and non-human (e.g., mouse or non-human primate) orthologues of CD117, CD45, CD2, CD5, CD137, CD134, or CD252.

[0182] As used herein, the terms "subject" and "patient" refer to an organism, such as a human, receiving treatment for a particular disease or condition described herein. For example, a patient, such as a human patient, may receive treatment prior to hematopoietic stem cell transplantation therapy to promote engraftment of exogenous hematopoietic stem cells.

[0183] As used herein, the phrase "substantially removed from the blood" refers to a time point after administration of a therapeutic agent to a patient when the concentration of the therapeutic agent, e.g., ADCs containing amatoxins, in a blood sample isolated from a patient is such that the therapeutic agent is not detectable by conventional means (e.g., the therapeutic agent is not detectable above the noise threshold of the device or assay used to detect the therapeutic agent). Antibodies or antibody fragments can be detected using a variety of techniques known in the art, such as ELISA-based detection assays known in the art or described herein. Additional assays that can be used to detect antibodies or antibody fragments include immunoprecipitation techniques and immunoblot assays, among others known in the art.

[0184] As used herein, the phrase "stem cell disease" refers broadly to any disease, disorder, or condition that can be treated or cured by pretreating a target tissue of a subject and / or by removing endogenous stem cell populations in the target tissue (e.g., removing endogenous hematopoietic stem or progenitor cell populations from the bone marrow tissue of a subject) and / or by engrafting or transplanting stem cells into the target tissue of a subject. For example, type I diabetes has been shown to be curable by hematopoietic stem cell transplantation and may benefit from pretreatment according to the compositions and methods described herein. Additional diseases 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 Schwachman-Diamond syndrome. Additional diseases that can be treated using the patient conditioning and / or hematopoietic stem cell transplantation methods described herein include inherited blood diseases (e.g., sickle cell anemia) and autoimmune diseases such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease. Additional diseases that can be treated using the conditioning and / or transplantation methods described herein include malignancies such as neuroblastoma or blood system cancers such as leukemia, lymphoma, and myeloma. For example, the cancer can be acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Additional diseases that can be treated using the conditioning and / or transplantation methods described herein include myelodysplastic syndromes. In some embodiments, the subject has or is otherwise afflicted with a metabolic storage disease.For example, a subject may have a disease or disorder, such as glycogen storage disease, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipid storage diseases, metachromatic leukodystrophy, or any other disease or disorder that may benefit from the treatments and therapies disclosed herein, including, but not limited to, severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic scleroderma, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and “Bone Marrow Transplantation for Non-Malignant Disease,” ASH Education The patient may be suffering from or otherwise suffering from a metabolic disease selected from the group consisting of other diseases or disorders including those described in U.S. Pat. No. 6,319,232, US ...

[0185] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, including electroporation, lipofection, calcium phosphate precipitation, and DEAE-dextran transfection.

[0186] As used herein, the term "treat" or "treatment" refers to reducing the severity and / or frequency of disease symptoms, eliminating disease symptoms and / or the underlying etiology of said symptoms, reducing the frequency or likelihood of disease symptoms and / or their underlying etiology, and ameliorating or repairing damage caused directly or indirectly by a 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 therapy as described herein. Additional beneficial outcomes include increasing the cell number or relative concentration of hematopoietic stem cells in a patient in need of hematopoietic stem cell transplantation following conditioning therapy and subsequent administration of an exogenous hematopoietic stem cell graft to the patient. Beneficial results of the therapies described herein may also include an increase in the cell number or relative concentration of one or more 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, after conditioning therapy and subsequent hematopoietic stem cell transplantation therapy. Additional beneficial results may also include a decrease in the amount of disease-causing cell populations, such as populations of cancer cells (e.g., CD117+ leukemia cells) or autoimmune cells (e.g., CD117+ autoimmune lymphocytes, such as CD117+ T cells expressing T cell receptors that cross-react with autoantigens). Insofar as the methods of the present invention are directed to preventing disease, it is understood that the term "prevent" does not require that the pathology be completely halted. Rather, as used herein, the term preventing refers to the ability of one of skill in the art to identify a population susceptible to a disease such that administration of a compound of the invention can occur prior to the onset of the disease. The term does not mean that the condition is completely avoided.

[0187] As used herein, the terms "variant" and "derivative" are used interchangeably and refer to natural, synthetic, and semi-synthetic analogs of the compounds, peptides, proteins, or other substances described herein. Variants or derivatives of the compounds, peptides, proteins, or other substances described herein may retain or enhance the biological activity of the original substance.

[0188] As used herein, the term "vector" includes nucleic acid vectors such as plasmids, DNA vectors, plasmids, RNA vectors, viruses, or other suitable replicons. The expression vectors described herein may include polynucleotide sequences and additional sequence elements used, for example, for protein expression and / or integration of these polynucleotide sequences into the genome of mammalian cells. Particular vectors that can be used for the expression of the antibodies and antibody fragments of the invention include plasmids that include control sequences such as promoter and enhancer regions that direct gene transcription. Other useful vectors for the expression of antibodies and antibody fragments include polynucleotide sequences that increase the rate of translation of these genes or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements may include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. The expression vectors described herein may also include polynucleotides that encode markers for the selection of cells that contain such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.

[0189] Antibody-Drug Conjugates (ADCs) The antibodies and antigen-binding fragments thereof described herein may be conjugated (linked) to a cytotoxic molecule (i.e., a cytotoxin) to form an antibody-drug conjugate (ADC). As used herein, the terms "cytotoxin," "cytotoxic moiety," and "drug" are used interchangeably.

[0190] In particular, the ADCs disclosed herein comprise an antibody (including an antigen-binding fragment thereof) conjugated to an amatoxin, such as an amatoxin described in formula (V), where the cytotoxic moiety has a cytotoxic or cytostatic effect when not conjugated to the antibody. In various embodiments, the cytotoxic moiety exhibits reduced or no cytotoxicity when bound in the complex, but resumes cytotoxicity after cleavage from the linker. In various embodiments, the cytotoxic moiety maintains cytotoxicity without cleavage from the linker. In some embodiments, a cytotoxic molecule is conjugated to a cell-internalizing antibody or antigen-binding fragment thereof disclosed herein such that following cellular uptake of the antibody or fragment thereof, the cytotoxin has access to its intracellular target, e.g., to mediate the death of hematopoietic cells. Thus, the ADCs of the present disclosure have the general formula: Ab-(ZL-Cy) n where the antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently attached) via a chemical moiety (Z) to a linker (L) leading to a cytotoxic moiety (Cy).

[0191] Thus, an antibody or antigen-binding fragment thereof may be conjugated to several drug moieties as indicated by the integer n, where n represents the average number of cytotoxins per antibody and may range, for example, from about 1 to about 20. Any number of cytotoxins may be conjugated to the antibody, for example, 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in the preparation of ADCs from the conjugation reaction may be characterized by conventional means, such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the ADCs, represented by n, may also be determined. In some cases, separation, purification, and characterization of a homogenous ADC with a particular value of n from ADCs with other drug loadings may be achieved by means such as reverse-phase HPLC or electrophoresis.

[0192] In some antibody-drug conjugates, n may be limited by the number of attachment sites on the antibody. For example, if the attachment is a cysteine ​​thiol, the antibody may have only one or a few cysteine ​​thiol groups, or only one or a few sufficiently reactive thiol groups through which the linker may be attached. Generally, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be linked to drug moieties; primarily, cysteine ​​thiol residues in antibodies exist as disulfide bridges. In certain embodiments, antibodies may be reduced under partial or total reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiol groups. In certain embodiments, higher drug loading, e.g., n greater than 5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates.

[0193] In certain embodiments, less than the theoretical maximum drug moiety is conjugated to the antibody during the conjugation reaction. The antibody may contain lysine residues that do not react with the drug-linker intermediate or linker reagent, for example, as described below. Only the most reactive lysine groups can react with amine-reactive linker reagents. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups, such as lysine or cysteine.

[0194] The loading (drug / antibody ratio) of ADCs can be controlled in a variety of ways, for example, (i) by limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) by limiting the conjugation reaction time or temperature, (iii) partial or limited reduction conditions of cysteine ​​thiol modifications, (iv) by recombinant techniques, manipulating the amino acid sequence of the antibody such that the number and position of cysteine ​​residues are adjusted to control the number and / or position of linker-drug connections.

[0195] cytotoxin The cytotoxin of the antibody drug conjugates described herein is an amatoxin or a derivative thereof. Amatoxins are potent and selective inhibitors of RNA polymerase II, thereby also inhibiting transcription and protein biosynthesis in affected cells. As used herein, the term "amatoxin" refers to a member of the amatoxin family of peptides produced by Amanita phalloides mushroom, or a variant or derivative thereof, such as a variant or derivative thereof capable of inhibiting RNA polymerase II activity. Amatoxins are fixed bicyclic octapeptides with the basic sequence Ile-Trp-Gly-Ile-Gly-Cys-Asn (or Asp)-Pro, bridged by a connection between the Cys sulfur and the 2-position of the Trp indole ring to form tryptathionine. Depending on the particular amatoxin, specific amino acid substitutions are changed by post-translational modification (i.e., Pro to Hyp; Ile to DHIle; and Trp to 5-OH Trp).

[0196] Amatoxins can be isolated from various mushroom species (e.g., Amanita phalloides, Galerina marginata, Lepiota brunneo-incarnata) or prepared semi-synthetically or synthetically. Different mushroom species contain varying amounts of different amatoxin family members. A member of this family, α-amanitin, is known to be an extremely potent inhibitor of eukaryotic RNA polymerase II, and to a lesser extent RNA polymerase III, thereby inhibiting transcription and protein biosynthesis. Wieland, Int. J. Pept. Protein Res. 1983, 22(3):257-276.

[0197] The structures of various naturally occurring amatoxins are represented in formula (III) and attached Table 1, and are disclosed, for example, in Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459, which is incorporated herein by reference in its entirety. [ka]

[0198] [Table 1]

[0199] The antibody or antigen-binding fragment thereof that recognizes and binds to an antigen expressed on the cell surface of human stem cells or T cells can be conjugated to an amatoxin such as α-amanitin or a derivative thereof, as described, for example, in U.S. Pat. Nos. 9,233,173 and 9,399,681 and U.S. Patent Application Publication Nos. 2016 / 0089450, 2016 / 0002298, 2015 / 0218220, and 2014 / 0294865, the disclosures of which are incorporated herein by reference, as they relate to amatoxins such as α-amanitin and covalent linkers that can be used for covalent conjugation. Exemplary methods of amatoxin conjugation and linkers useful for such processes are described herein. Exemplary linker-containing amatoxins useful for conjugation to antibodies or antigen-binding fragments according to the compositions and methods are also described herein.

[0200] As used herein, the term "amatoxin derivative" or "amanitin derivative" refers to an amatoxin that has been chemically modified at one or more positions relative to a naturally occurring amatoxin, such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, amanuric acid, or proamanitin. In each case, the derivative may be obtained by chemical modification of a naturally occurring compound ("semi-synthetic") or may be obtained from a completely synthetic source. Synthetic routes to various amatoxin derivatives are disclosed, for example, in U.S. Pat. No. 9,676,702 and Perrin et al., J. Am. Chem. Soc. 2018, 140, p. 6513-6517, each of which is incorporated herein by reference in its entirety for synthetic methods of preparing and derivatizing amatoxins.

[0201] In some embodiments, the amatoxin or derivative thereof has the formula (V): [ka] or its enantiomer or diastereomer.

[0202] In some embodiments, Q is S. In some embodiments, Q is a sulfoxide group.

[0203] In one embodiment, the amatoxin or derivative thereof has the formula (Va): [ka] It is expressed by:

[0204] In some embodiments, Q is S. In some embodiments, Q is a sulfoxide group.

[0205] In this particular embodiment, the matoxin or derivative thereof has the formula (Vb): [ka] It is expressed by:

[0206] In some embodiments, Q is S. In some embodiments, Q is a sulfoxide group.

[0207] Additional amatoxins that may be used for conjugation to antibodies or antigen-binding fragments thereof in accordance with the compositions and methods described herein are described, for example, in WO 2016 / 142049; WO 2016 / 071856; WO 2017 / 149077; WO 2018 / 115466; and WO 2017 / 046658, the disclosures of which are incorporated by reference herein in their entireties.

[0208] Linker The term "linker" as used herein means a bivalent chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody or fragment thereof (Ab) to an amatoxin described herein, e.g., an amatoxin of formula (IV), (IVa), (IVb), (V), (Va), or (Vb), to form an antibody drug conjugate (ADC).

[0209] Covalent attachment of an antibody to an amatoxin requires that the linker has two reactive functional groups, i.e., bivalency in the sense of reactivity. Bivalent linker reagents useful for connecting two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups, are known and methods have been described along with their resulting conjugates (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, p. 234-242).

[0210] Thus, the linker has two reactive ends, one for conjugation to an antibody and the other for conjugation to an amatoxin. The antibody conjugation reactive end (reactive moiety, defined herein as Z') of the linker is typically a chemical moiety capable of conjugation to an antibody, for example, via a cysteine ​​thiol or lysine amine group on the antibody, typically a thiol-reactive group such as a Michael acceptor (such as in a maleimide), a leaving group such as a chloro, bromo, iodo, or R-sulfanyl group, or an amine-reactive group such as a carboxyl group. Conjugation of the linker to an antibody is described more fully below.

[0211] The amatoxin conjugation reactive end of the linker is typically a chemical moiety capable of conjugation to an amatoxin by formation of a bond with a reactive substituent in the amatoxin molecule. Non-limiting examples include formation of an amide bond, e.g., with a basic amine or carboxyl group on the amatoxin, respectively, with a carboxyl or basic amine group on the linker, or formation of an ether, e.g., by alkylation of an OH group on the amatoxin with a leaving group on the linker.

[0212] When the term "linker" is used in describing a conjugated form of a linker, one or both of the reactive ends are absent (such as reactive moiety Z' converted to chemical moiety Z as described below) or incomplete (such as only a carbonyl of a carboxylic acid) due to the formation of bonds between the linker and / or the amatoxin, and between the linker and / or the antibody or antigen-binding fragment thereof. Such conjugation reactions are further described below.

[0213] A variety of linkers can be used to conjugate the described antibodies, antigen-binding fragments, and ligands to cytotoxic molecules. In general, linkers suitable for the present disclosure can be substantially stable in circulation, but allow for release of the amatoxin into or near the target cell. In some embodiments, certain linkers suitable for the present disclosure can be classified as "cleavable" or "non-cleavable". In general, cleavable linkers contain one or more functional groups that are cleaved in response to a physiological environment. For example, cleavable linkers can contain an enzyme substrate (e.g., valine-alanine) that degrades in the presence of an intracellular enzyme (e.g., cathepsin B), an acid-cleavable group (e.g., hydrozone) that degrades in the acidic environment of a cellular compartment, or a reducing group (e.g., disulfide) that degrades in an intracellular reducing environment. In contrast, non-cleavable linkers are generally released from the ADC during degradation (e.g., lysosomal degradation) of the antibody portion of the ADC inside the target cell.

[0214] Non-cleavable linkers Suitable non-cleavable linkers for use herein include a bond, -(C=O)-, C 1 ~C 6 Alkylene, C 1 ~C 6 Heteroalkylene, C 2 ~C 6 Alkenylene, C 2 ~C 6 Heteroalkenylene, C 2 ~C 6 Alkynylene, C 2 ~C 6 Heteroalkynylene, C 3 ~C 6 The alkyl group may further include one or more groups selected from cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which may be optionally substituted and / or may contain one or more heteroatoms (e.g., S, N, or O) in place of one or more carbon atoms. Non-limiting examples of such groups include (CH 2 ) p , (C=O)(CH 2 ) p , and polyethylene glycol (PEG; (CH2 CH 2 O) p ) units, where p is an integer from 1 to 6, which is independently selected in each occurrence.

[0215] In some embodiments, the linker L is a bond, -(C=O)-, a -C(O)NH- group, a -OC(O)NH- group, 1 ~C 6 Alkylene, C 1 ~C 6 Heteroalkylene, C 2 ~C 6 Alkenylene, C 2 ~C 6 Heteroalkenylene, C 2 ~C 6 Alkynylene, C 2 ~C 6 Heteroalkynylene, C 3 ~C 6 Cycloalkylene, heterocycloalkylene, arylene, heteroarylene, p is an integer of 1 to 6, -(CH 2 CH 2 O) p -group, or one or more solubility enhancing groups; Here, each C 1 ~C 6 Alkylene, C 1 ~C 6 Heteroalkylene, C 2 ~C 6 Alkenylene, C 2 ~C 6 Heteroalkenylene, C 2 ~C 6 Alkynylene, C 2 ~C 6 Heteroalkynylene, C 3 ~C 6The cycloalkylene, heterocycloalkylene, arylene, or heteroarylene can be optionally substituted by 1 to 5 substituents independently selected at each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro; In some embodiments, each C 1 ~C 6 Alkylene, C 1 ~C 6 Heteroalkylene, C 2 ~C 6 Alkenylene, C 2 ~C 6 Heteroalkenylene, C 2 ~C 6 Alkynylene, C 2 ~C 6 Heteroalkynylene, C 3 ~C 6 The cycloalkylene, heterocycloalkylene, arylene, or heteroarylene may be optionally interrupted by one or more heteroatoms selected from O, S, and N.

[0216] In some embodiments, each C 1 ~C 6 Alkylene, C 1 ~C 6 Heteroalkylene, C 2 ~C 6 Alkenylene, C 2 ~C 6 Heteroalkenylene, C 2 ~C 6 Alkynylene, C 2 ~C 6 Heteroalkynylene, C 3 ~C 6The cycloalkylene, heterocycloalkylene, arylene, or heteroarylene can be optionally interrupted by one or more heteroatoms selected from O, S, and N, and can be optionally substituted by 1 to 5 substituents independently selected at each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0217] In some embodiments, the linker L has the formula -O a -C(O)NH-SO 2 -N(R 1 )-, wherein a is 0 or 1; R 1 is hydrogen, C 1 ~C 24 Alkyl group, C 3 ~C 24 Cycloalkyl groups, C 2 ~C 24 (Hetero)aryl groups, C 3 ~C 24 Alkyl(hetero)aryl groups, and C 3 ~C 24 (hetero)arylalkyl groups, each of which is selected from the group consisting of O, S, and NR 3 and optionally substituted or interrupted by one or more heteroatoms selected from, where R 3 is hydrogen and C 1 ~C 4 Such solubility-enhancing groups are described, for example, in U.S. Pat. No. 9,636,421 and U.S. Patent Application Publication No. 2017 / 0298145, the disclosures of each of which are incorporated herein by reference in their entireties.

[0218] In some embodiments, the formula -O a -C(O)NH-SO 2 -N(R 1 The solubility enhancing group of - is C 1 ~C 6 Alkylene or -(CH 2 CH 2 O) p Non-limiting examples of such solubility-enhancing groups include those depicted in Table 2 above.

[0219] [Table 2]

[0220] In some embodiments, the non-cleavable linker is -(CH 2 ) n - units, where n is an integer from 2 to 12, for example, from 2 to 6. In some embodiments, the non-cleavable linker is -(CH 2 ) n -, where n is 1, 2, 3, 4, 5, or 6. In some embodiments, the non-cleavable linker comprises the formula: [ka] n is 6, represented by -(CH 2 ) n -It is.

[0221] Cleavable Linkers In some embodiments, the linker conjugating the antibody or antigen-binding fragment thereof to the amatoxin is cleavable under intracellular conditions such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. The cleavable linker is designed to exploit the difference in the local environment, e.g., the extracellular and intracellular environments, including, for example, pH, reduction potential, or enzyme concentration, to trigger the release of the amatoxin in the target cell. In general, the cleavable linker is relatively stable in circulation, but is particularly susceptible to cleavage in the intracellular environment by one or more mechanisms, including, but not limited to, the activity of proteases, peptidases, and glucuronidases. The cleavable linker used herein is substantially stable in circulating plasma and / or outside the target cell, and can be cleaved at some effective rate inside or in the immediate vicinity of the target cell.

[0222] Suitable cleavable linkers include those that can 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, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference as it relates to linkers suitable for covalent conjugation). Suitable cleavable linkers can include chemical moieties such as, for example, hydrazines, disulfides, thioethers, or dipeptides.

[0223] Linkers that are hydrolyzable under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, and the like (see, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661, the disclosures of each of which are incorporated by reference in their entireties as they relate to linkers suitable for covalent conjugation. Such linkers are relatively stable under neutral pH conditions, such as in blood, and are unstable below pH 5.5 or 5.0, which is approximately the pH of the lysosome.

[0224] Linkers that are cleavable under reducing conditions include, for example, disulfides. A variety of disulfide linkers are known in the art, such as SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), which can be formed using SPDB and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987)). See also US Pat. No. 4,880,935, the disclosures of each of which relate to linkers suitable for covalent conjugation and are incorporated herein by reference in their entireties.

[0225] A linker susceptible to enzymatic hydrolysis may be, for example, a peptide-containing linker that is cleaved by intracellular peptidases or protease enzymes, including but not limited to lysosomal or endosomal proteases. One advantage of using intracellular proteolytic release of a therapeutic agent is that the agent is typically attenuated when conjugated, and the serum stability of the conjugate is typically high. In some embodiments, the peptidyl linker is at least two amino acids long, or at least three amino acids long. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Examples of suitable peptides include those that include amino acids such as valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine. The amino acid residues that make up the amino acid linker component include those that occur naturally, as well as minor amino acids and non-naturally occurring amino acid analogs such as citrulline. Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). In some embodiments, the linker comprises a dipeptide such as Val-Cit, Ala-Val, or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit. Linkers comprising dipeptides such as Val-Cit or Phe-Lys are disclosed, for example, in U.S. Pat. No. 6,214,345, the disclosure of which relates to linkers suitable for covalent conjugation and is incorporated herein by reference in its entirety. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit.

[0226] Suitable linkers for conjugating the antibodies, antigen-binding fragments described herein to cytotoxic molecules include those capable of releasing amatoxins by a 1,6-elimination process. Chemical moieties capable of this elimination process include p-aminobenzyl (PAB) groups, 6-maleimidohexanoic acid, pH-sensitive carbonates, and other reagents described in Jain et al., Pharm. Res. 32:3526-3540, 2015, the disclosure of which is incorporated herein by reference in its entirety as it relates to suitable linkers for covalent conjugation.

[0227] In some embodiments, the linker comprises a "self-immolative" group such as PAB or PABC (para-aminobenzyloxycarbonyl) as described above, e.g., in Carl et al., J. Med. Chem. (1981) 24:479-480; Chakravarty et al (1983) J. Med. Chem. 26:638-644; U.S. Pat. No. 6,214,345; U.S. Pat. Appl. Pub. No. 20030130189; U.S. Pat. Appl. Pub. No. 20030096743; U.S. Pat. No. 6,759,509; U.S. Pat. Appl. Pub. No. 20040052793; U.S. Pat. No. 6,218,519; U.S. Pat. No. 6,835,807; U.S. Pat. No. 6,268,488; U.S. Patent Application Publication No. 20040018194; WO 98 / 13059; U.S. Patent Application Publication No. 20040052793; U.S. Patent No. 6,677,435; U.S. Patent No. 5,621,002; U.S. Patent Application Publication No. 20040121940; WO 2004 / 032828. Other such chemical moieties ("self-immolative linkers") that are capable of this process include methylene carbamates and heteroaryl groups such as aminothiazoles, aminoimidazoles, aminopyrimidines, and the like. Linkers containing such heterocyclic self-immolative groups are disclosed, for example, in US Patent Publication Nos. 20160303254 and 20150079114, as well as U.S. Patent Publication Nos. 7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237; U.S. Patent Publication No. 2005 / 0256030; de Groot et al. (2001) J. Org. Chem. 66:8815-8830; and U.S. Patent No. 7,223,837. In some embodiments, a dipeptide is used in combination with a self-immolative linker.

[0228] In some embodiments, the linker L is a hydrazine, a disulfide, a thioether, an amino acid, a peptide of up to 10 amino acids, a p-aminobenzyl (PAB) group, a heterocyclic self-immolative group, C 1 ~C6 Alkyl, C 1 ~C 6 Heteroalkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Heteroalkenyl, C 2 ~C 6 Alkynyl, C 2 ~C 6 Heteroalkynyl, C 3 ~C 6 Cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -(C=O)- group, -C(O)NH- group, -OC(O)NH- group, -(CH 2 CH 2 O) p -group, or one or more solubility enhancing groups; Here, each C 1 ~C 6 Alkyl, C 1 ~C 6 Heteroalkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Heteroalkenyl, C 2 ~C 6 Alkynyl, C 2 ~C 6 Heteroalkynyl, C 3 ~C 6 A cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can be optionally substituted with from 1 to 5 substituents independently selected at each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0229] In some embodiments, each C 1 ~C 6 Alkyl, C1 ~C 6 Heteroalkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Heteroalkenyl, C 2 ~C 6 Alkynyl, C 2 ~C 6 Heteroalkynyl, C 3 ~C 6 The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups may be optionally interrupted by one or more heteroatoms selected from O, S, and N.

[0230] In some embodiments, each C 1 ~C 6 Alkyl, C 1 ~C 6 Heteroalkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Heteroalkenyl, C 2 ~C 6 Alkynyl, C 2 ~C 6 Heteroalkynyl, C 3 ~C 6 A cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can be optionally interrupted by one or more heteroatoms selected from O, S, and N, and can be optionally substituted by one to five substituents independently selected at each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0231] One of skill in the art will appreciate that one or more of the listed groups may be a divalent (diterminal) species, e.g., C 1 ~C 6It will be appreciated that the radical may be present in the form of an alkylene, etc.

[0232] In some embodiments, the linker L has the formula -O a -C(O)NH-SO 2 -N(R 1 )-, wherein a is 0 or 1; R 1 is hydrogen, C 1 ~C 24 Alkyl group, C 3 ~C 24 Cycloalkyl groups, C 2 ~C 24 (Hetero)aryl groups, C 3 ~C 24 Alkyl(hetero)aryl groups, and C 3 ~C 24 (hetero)arylalkyl groups, each of which is selected from the group consisting of O, S, and NR 3 and optionally substituted or interrupted by one or more heteroatoms selected from, where R 3 is hydrogen and C 1 ~C 4 Such solubility-enhancing groups are described, for example, in U.S. Pat. No. 9,636,421 and U.S. Patent Application Publication No. 2017 / 0298145, the disclosures of each of which are incorporated herein by reference in their entireties.

[0233] In some embodiments, the formula -O a -C(O)NH-SO 2 -N(R 1 The solubility enhancing group of - is C 1 ~C 6 Alkylene or -(CH 2 CH 2 O) p - group. Non-limiting examples of such solubility-enhancing groups include those depicted in Table 2 above.

[0234] In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In one embodiment, the p-aminobenzyl group is disposed between the cytotoxic agent and the protease cleavage site in the linker. In one embodiment, the p-aminobenzyl group is part of a p-aminobenzyloxycarbonyl unit. In one embodiment, the p-aminobenzyl group is part of a p-aminobenzylamide unit.

[0235] In some embodiments, the linker comprises a dipeptide selected from the group consisting of Phe-Lys, Val-Lys, Phe-Ala, Phe-Cit, Val-Ala, Val-Cit, and Val-Arg. In some embodiments, the linker comprises one or more of PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0236] In some embodiments, the linker is a peptide, an oligosaccharide, -(CH 2 ) p -, -(CH 2 CH 2 O) p -, PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0237] In some embodiments, the linker is -(C=O)(CH 2 ) p -Includes units.

[0238] In some embodiments, the linker has the formula: [ka] The PAB-Ala-Val-propionyl derivative is represented by the formula:

[0239] In some embodiments, the linker has the formula: [ka] The compound includes PAB-Cit-Val-propionyl, represented by the formula:

[0240] Such PAB-dipeptide-propionyl linkers are disclosed, for example, in WO 2017 / 149077, which is incorporated by reference in its entirety. Additionally, the cytotoxins disclosed in WO 2017 / 149077 are incorporated by reference herein.

[0241] It will be appreciated by those skilled in the art that any one or more of the chemical groups, moieties, and features disclosed herein can be combined in numerous ways to form linkers useful for conjugating the antibodies and amatoxins disclosed herein. Additional linkers useful in conjunction with the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0242] Linker-Amatoxin and Linker-Antibody Conjugation In certain embodiments, the linker is reacted with an amatoxin or derivative thereof according to any of formulas (V), (Va), or (Vb) under appropriate conditions to form a linker-amatoxin conjugate. In certain embodiments, a reactive group is used on the amatoxin or linker to form a covalent bond.

[0243] The amatoxin-linker conjugate is then reacted under appropriate conditions with an antibody, derivatized antibody, or antigen-binding fragment thereof to form an ADC. Alternatively, the linker can be reacted first with an antibody, derivatized antibody, or antigen-binding fragment thereof to form a linker-antibody conjugate, which is then reacted with an amatoxin to form an ADC. Such conjugation reactions are now more fully described.

[0244] Several different reactions are available for covalent attachment of linkers or amatoxin-linker conjugates to antibodies or antigen-binding fragments thereof. Suitable attachment points on antibody molecules include, but are not limited to, the amine groups of lysines, the free carboxylic acid groups of glutamic and aspartic acids, the sulfhydryl groups of cysteine, and various moieties of aromatic amino acids. For example, non-specific covalent attachment can be performed using a carbodiimide reaction to link a carboxy (or amino) group on the linker to an amino (or carboxy) group on the antibody moiety. Additionally, bifunctional agents such as dialdehydes or imidoesters can also be used to link an amino group on the linker to an amino group on the antibody moiety. Also available for attachment of an amatoxin to the antibody moiety is the Schiff base reaction. This method involves periodate oxidation of glycol or hydroxy groups on either the antibody or the linker, thereby forming an aldehyde, which is then reacted with the linker or antibody, respectively. Covalent bond formation occurs through the formation of a Schiff base between the aldehyde and the amino group. Isothiocyanates can also be used as coupling agents to covalently attach amatoxins or antibody moieties to linkers. Other techniques are known to those of skill in the art and are within the scope of this disclosure.

[0245] Linkers useful for conjugation to the antibodies or antigen-binding fragments described herein include, but are not limited to, linkers comprising a chemical moiety Z formed by a coupling reaction between an antibody and a reactive chemical moiety on the linker (referred to herein as a reactive substituent, Z'), as depicted in Table 3 below. The wavy lines indicate the points of attachment to the antibody or antigen-binding fragment and the cytotoxic molecule, respectively.

[0246] [Table 3] TIFF2025060760000025.tif202156TIFF2025060760000026.tif222156TIFF2025060760000027.tif87156

[0247] One of skill in the art will recognize that the linker and the reactive substituent Z' attached to the reactive substituent on the antibody or antigen-binding fragment thereof will participate in a covalent coupling reaction to produce the chemical moiety Z, and will appreciate that the reactive substituent Z'. Thus, antibody-drug conjugates that are useful in conjunction with the methods described herein can be formed by reacting an antibody or antigen-binding fragment thereof with a linker or amatoxin-linker conjugate, as described herein, which contains a reactive substituent Z' suitable for reacting with a reactive substituent on the antibody or antigen-binding fragment thereof to form the chemical moiety Z.

[0248] As depicted in Table 3, examples of suitably reactive substituents Z' on the linker and reactive substituents on the antibody or antigen-binding fragment thereof include nucleophile / electrophile pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, among others), diene / dienophile pairs (e.g., azide / alkyne pairs, or diene / α,β-unsaturated carbonyl pairs, among others). Coupling reactions between reactive substituents to form the chemical moiety Z include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine or hydroxylamine condensation, hydrazine formation, amidation, esterification, disulfide formation, cycloadditions (e.g., [4+2] Diels-Alder cycloadditions, [3+2] Huesgen cycloadditions, among others), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reactivity modes known in the art or described herein. In some embodiments, the reactive substituent Z' is an electrophilic functional group suitable for reaction with a nucleophilic functional group on an antibody or antigen-binding fragment thereof.

[0249] Reactive substituents that may be present in the antibodies or antigen-binding fragments thereof disclosed herein include, but are not limited to, nucleophilic groups such as (i) N-terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups when the antibody is glycosylated. Reactive substituents that may be present in the antibodies or antigen-binding fragments thereof disclosed herein include, but are not limited to, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine ​​residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids. In some embodiments, reactive substituents present in the antibodies or antigen-binding fragments thereof disclosed herein include amine or thiol moieties. Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine ​​bridge would thus theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by converting amines to thiols by reaction of lysines with 2-iminothiolane (Traut's Reagent). Reactive thiol groups can be introduced into antibodies (or fragments thereof) by introducing one, two, three, four, or more cysteine ​​residues (e.g., preparation of mutant antibodies containing one or more non-natural cysteine ​​amino acid residues). U.S. Pat. No. 7,521,541 teaches engineering antibodies by the introduction of reactive cysteine ​​amino acids.

[0250] In some embodiments, the reactive substituent Z' attached to the linker is a nucleophilic group that is reactive with an electrophilic group present on an antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. Nucleophilic groups (eg, a) heteroatom can react with an electrophilic group on an antibody to form a covalent bond to the antibody. Useful nucleophilic groups include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide.

[0251] In some embodiments, the chemical moiety Z is the product of a reaction between a reactive nucleophilic substituent present in an antibody or antigen-binding fragment thereof, such as an amine or thiol moiety, and a reactive electrophilic substituent Z' connected to a linker. For example, Z' can be a Michael acceptor (e.g., a maleimide), an activated ester, an electron-deficient carbonyl compound, or an aldehyde, among others.

[0252] Some representative, non-limiting examples of reactive substituents Z′ and the resulting chemical moieties Z are provided in Table 4.

[0253] [Table 4]

[0254] For example, suitable linkers for the synthesis of linker-antibody conjugates and ADCs include, but are not limited to, reactive substituents Z', such as maleimide or haloalkyl groups attached to the linker. These can be attached to the linker by reagents such as succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among others described in Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation.

[0255] In some embodiments, the reactive substituent Z' connected to the linker L is a maleimide, an azide, or an alkyne. An example of a maleimide-containing linker is the non-cleavable maleimidocaproyl-based linker, which is particularly useful for the conjugation of microtubule disrupting agents such as auristatins. Such linkers are described by Doronina et al., Bioconjugate Chem. 17:14-24, 2006, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation.

[0256] In some embodiments, the reactive substituent Z' is -(C=O)- or -NH(C=O)- such that the linker can be attached to an antibody or antigen-binding fragment thereof through an amide or urea moiety, respectively, resulting from reaction of the -(C=O)- or -NH(C=O)- group with an amino group on the antibody or antigen-binding fragment thereof.

[0257] In some embodiments, the reactive substituent Z' is an N-maleimidyl group, a halogenated N-alkylamide group, a sulfonyloxy N-alkylamide group, a carbonate group, a sulfonyl halide group, a thiol group or derivatives thereof, an alkynyl group containing an internal carbon-carbon triple bond, a (hetero)cycloalkynyl group, a bicyclo[6.1.0]non-4-yn-9-yl group, an alkenyl group containing an internal carbon-carbon double bond, a cycloalkenyl group, a tetrazinyl group, an azide group, a phosphine group, a nitrile oxide group, a nitrone group, a nitrile imine group, a diazo group, a ketone group, an (O-alkyl)hydroxylamino group, a hydrazine group, a halogenated N-maleimidyl group, a 1,1-bis(sulfonylmethyl)methylcarbonyl group or elimination derivatives thereof, a carbonyl halide group, or an allenamide group, each of which can be optionally substituted. In some embodiments, the reactive substiuent comprises a cycloalkene group, a cycloalkyne group, or an optionally substituted (hetero)cycloalkynyl group.

[0258] In some embodiments, the chemical moiety Z is selected from Table 3 or Table 4. In some embodiments, the chemical moiety Z is [ka] where S is a sulfur atom representing a reactive substituent present in an antibody or antigen-binding fragment thereof that specifically binds to an antigen expressed on the cell surface of human stem cells or T cells (e.g., from an -SH group of a cysteine ​​residue).

[0259] In some embodiments, the linker-reactive substituents, together as L-Z′, prior to conjugation with the antibody or antigen-binding fragment thereof, have the structure: [ka] where the wavy line indicates the point of attachment to the substituent on the amatoxin. This linker-reactive substituent L-Z' may alternatively be referred to as N-β-maleimidopropyl-Val-Ala-para-aminobenzyl (BMP-Val-Ala-PAB). The wavy line at the end of the linker indicates the point of attachment to the amatoxin. In some embodiments, the linker L and chemical moiety Z, together after conjugation to an antibody, LZ-Ab, have the structure: [ka] where S is a sulfur atom representing a reactive substituent present in an antibody or antigen-binding fragment thereof that specifically binds to an antigen expressed on the cell surface of human stem cells or T cells (e.g., from an -SH group of a cysteine ​​residue). The wavy line at the end of the linker indicates the point of attachment to the amatoxin.

[0260] In some embodiments, the linker-reactive substituents, together as L-Z′, prior to conjugation with the antibody or antigen-binding fragment thereof, have the structure: [ka] This linker-reactive substituent, which may alternatively be referred to as 1-n-hexyl-maleimide, is a non-cleavable linker. The wavy line at the end of the linker indicates the point of attachment to the amatoxin.

[0261] In some embodiments, the linker L and chemical moiety Z, together after conjugation to the antibody, form LZ-Ab, with the structure: [ka] where S is a sulfur atom representing a reactive substituent present in an antibody or antigen-binding fragment thereof that specifically binds to an antigen expressed on the cell surface of a cell, e.g., a tumor cell or a human stem cell or T cell (e.g., from an -SH group of a cysteine ​​residue). The wavy line at the end of the linker indicates the point of attachment to the amatoxin. One of skill in the art will recognize that in this embodiment, the linker-reactive substituent structure L-Z' includes a maleimide as group Z' prior to conjugation with the antibody or antigen-binding fragment thereof. Among those useful in conjunction with the compositions and methods described herein, the above linker moieties and amatoxin-linker conjugates are described, for example, in U.S. Patent Publication No. 2015 / 0218220 and WO 2017 / 149077, the disclosures of each of which are incorporated herein by reference in their entireties.

[0262] In one embodiment, the cytotoxin of the ADCs disclosed herein is an amatoxin or derivative thereof represented by any of Formula (V), (Va), or (Vb). One of skill in the art will recognize that such amatoxins offer numerous possibilities for attachment points to a linker.

[0263] In some embodiments, the amatoxin has the structure of formula (V) and the linker is connected to the OH group of the hydroxyltryptophan residue by an ether bond. In such embodiments, the ADC has the structure of formula (I): [ka] (In the formula: each as disclosed herein Q is S; L is a linker; Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the antibody or antigen-binding fragment thereof; and Ab is antibody) or a stereoisomer thereof.

[0264] In some embodiments, the amatoxin has the structure of formula (Va) and the linker is connected to the OH group of the hydroxyltryptophan residue by an ether bond. In such embodiments, the ADC has the structure of formula (Ia): [ka] It can be represented by:

[0265] In some embodiments, the amatoxin has the structure of formula (Vb) and the linker is connected to the OH group of the hydroxyltryptophan residue by an ether bond. In such embodiments, the ADC has the structure of formula (Ib): [ka] It can be represented by:

[0266] In some embodiments, the linker L of an ADC of Formula (I), (Ia), or (Ib) is a non-cleavable linker. In some embodiments, the non-cleavable linker L is a bond, -(C=O)-, a -C(O)NH- group, a -OC(O)NH- group, a -C(O)NH- group, 1 ~C 6 Alkylene, C 1 ~C 6 Heteroalkylene, C 2 ~C 6 Alkenylene, C 2 ~C 6 Heteroalkenylene, C 2 ~C 6 Alkynylene, C 2 ~C 6 Heteroalkynylene, C 3 ~C 6 Cycloalkylene, heterocycloalkylene, arylene, heteroarylene, p is an integer of 1 to 6, -(CH 2 CH 2 O) p -group, or one or more solubility enhancing groups; Here, each C 1 ~C 6 Alkylene, C 1 ~C 6 Heteroalkylene, C2 ~C 6 Alkenylene, C 2 ~C 6 Heteroalkenylene, C 2 ~C 6 Alkynylene, C 2 ~C 6 Heteroalkynylene, C 3 ~C 6 the cycloalkylene, heterocycloalkylene, arylene, or heteroarylene can be optionally substituted by 1 to 5 substituents independently selected at each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro; or Each C 1 ~C 6 Alkylene, C 1 ~C 6 Heteroalkylene, C 2 ~C 6 Alkenylene, C 2 ~C 6 Heteroalkenylene, C 2 ~C 6 Alkynylene, C 2 ~C 6 Heteroalkynylene, C 3 ~C 6 The cycloalkylene, heterocycloalkylene, arylene, or heteroarylene may be optionally interrupted by one or more heteroatoms selected from O, S, and N.

[0267] In some embodiments, the non-cleavable linker L has the formula -O a -C(O)NH-SO 2 -NR 1 - a solubility enhancing group of the formula: a is 0 or 1; and R 1is hydrogen, C 1 ~C 24 Alkyl group, C 3 ~C 24 Cycloalkyl groups, C 2 ~C 24 (Hetero)aryl groups, C 3 ~C 24 Alkyl(hetero)aryl groups, and C 3 ~C 24 (hetero)arylalkyl groups, each of which is selected from the group consisting of O, S, and NR 3 and optionally substituted or interrupted by one or more heteroatoms selected from 3 is hydrogen and C 1 ~C 4 alkyl).

[0268] In some embodiments, the non-cleavable linker L is —(CH 2 ) n In some embodiments, the non-cleavable linker L comprises a -(CH 2 ) n -It is.

[0269] In some embodiments, Ab, Z, and the non-cleavable linker L, taken together as Ab-ZL, have the formula: [ka] (wherein S is a sulfur atom representing a reactive substituent present in an antibody or antigen-binding fragment thereof that specifically binds to an antigen expressed on a cell, e.g., the surface of a human stem cell or T cell (e.g., derived from an -SH group of a cysteine ​​residue). The wavy line at the end of the linker indicates the point of attachment to the amatoxin.

[0270] In some embodiments, the ADC according to formula (I) has formula (II): [ka] It is represented by its stereoisomer or a pharma- ceutically acceptable salt thereof.

[0271] In some embodiments, the ADC according to formula (II) has formula (IIa): [ka] It is represented by its stereoisomer or a pharma- ceutically acceptable salt thereof.

[0272] In some embodiments, the ADC according to formula (II) has formula (IIb): [ka] It is represented by its stereoisomer or a pharma- ceutically acceptable salt thereof.

[0273] Surprisingly, according to the present disclosure, it has been found that ADCs comprising an amatoxin and a non-cleavable linker conjugating the amatoxin to the antibody portion of the ADC have improved tolerability compared to ADCs comprising an amatoxin and a cleavable linker. For example, in some embodiments, the improved tolerability can be an increased therapeutic index. In some embodiments, the improved tolerability can be a lower or no increase in one or more blood liver enzyme levels (e.g., AST, ALT, ADH, or total bilirubin) at a particular dose of an ADC comprising a non-cleavable linker compared to an ADC comprising a cleavable linker.

[0274] In some embodiments, the linker L of an ADC of Formula (I), (Ia), or (Ib) is a cleavable linker. In some embodiments, the cleavable linker L is a hydrazine, a disulfide, a thioether, an amino acid, a peptide of up to 10 amino acids, a p-aminobenzyl (PAB) group, a heterocyclic self-immolative group, a C 1 ~C 6 Alkyl, C 1 ~C 6 Heteroalkyl, C 2 ~C 6 Alkenyl, C 2 ~C6 Heteroalkenyl, C 2 ~C 6 Alkynyl, C 2 ~C 6 Heteroalkynyl, C 3 ~C 6 Cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -(C=O)- group, -C(O)NH- group, -OC(O)NH- group, -(CH 2 CH 2 O) p -group, or one or more solubility enhancing groups; Here, each C 1 ~C 6 Alkyl, C 1 ~C 6 Heteroalkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Heteroalkenyl, C 2 ~C 6 Alkynyl, C 2 ~C 6 Heteroalkynyl, C 3 ~C 6 The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can be optionally substituted by 1 to 5 substituents independently selected at each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro; Or each C 1 ~C 6 Alkyl, C 1 ~C 6 Heteroalkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Heteroalkenyl, C 2 ~C 6Alkynyl, C 2 ~C 6 Heteroalkynyl, C 3 ~C 6 The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups may be optionally interrupted by one or more heteroatoms selected from O, S, and N.

[0275] In some embodiments, the cleavable linker L has the formula -O a -C(O)NH-SO 2 -NR 1 - a solubility enhancing group of the formula: a is 0 or 1; and R 1 is hydrogen, C 1 ~C 24 Alkyl group, C 3 ~C 24 Cycloalkyl groups, C 2 ~C 24 (Hetero)aryl groups, C 3 ~C 24 Alkyl(hetero)aryl groups, and C 3 ~C 24 (hetero)arylalkyl groups, each of which is selected from the group consisting of O, S, and NR 3 wherein R 3 is hydrogen and C 1 ~C 4 alkyl).

[0276] In some embodiments, the cleavable linker L comprises a peptide selected from the group consisting of Phe-Lys, Val-Lys, Phe-Ala, Phe-Cit, Val-Ala, Val-Cit, and Val-Arg.

[0277] In some embodiments, the cleavable linker L further comprises a PAB group.

[0278] In some embodiments, the cleavable linker L has the formula: [ka] It is expressed by:

[0279] Preparation of antibody-drug conjugates ADCs of the formula Ab-(ZL-Cy) disclosed herein, such as ADCs of any of formula (I), (Ia), or (Ib), (II), (IIa), or (IIb): n In the ADCs, an antibody or antigen-binding fragment thereof (Ab) is conjugated to one or more cytotoxic drug moieties (Cy; e.g., amatoxins), e.g., about 1 to about 20 cytotoxic moieties per antibody, via a linker, L, and a chemical moiety, Z, each as disclosed herein. In some embodiments, n is 1. In some embodiments, n is about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 3 to about 5. In some embodiments, n is about 1, about 2, about 3, or about 4.

[0280] The ADCs of the disclosure can be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those of skill in the art, including: (1) reaction of a reactive substituent of an antibody or antigen-binding fragment thereof with a bivalent linker reagent to form Ab-ZL, as described previously herein, followed by reaction with a cytotoxic moiety, Cy; or (2) reaction of a reactive substituent of a cytotoxic moiety with a bivalent linker reagent to form Cy-L-Z', as described previously herein, followed by reaction with a reactive substituent of an antibody or antigen-binding fragment thereof, as described previously herein, to form a compound of the formula Ab-(ZL-Cy). n Formation of the ADC. Additional methods for preparing the ADC are described herein.

[0281] In one embodiment, an antibody or antigen-binding fragment thereof may bear one or more carbohydrate groups, which may be chemically modified to bear one or more sulfhydryl groups, which are then conjugated via the sulfur atoms of the sulfhydryl groups to form an ADC, as described herein above.

[0282] In another embodiment, an antibody may have one or more carbohydrate groups that may be oxidized to provide aldehyde (-CHO) groups (see, e.g., Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). ADCs are then formed by conjugation via the corresponding aldehyde as previously described herein. Other protocols for the modification of proteins for the attachment or association of cytotoxins are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), which is incorporated herein by reference.

[0283] Methods for conjugation of linker-drug moieties to cell-targeting proteins, such as antibodies, immunoglobulins or fragments thereof, can be found, for example, in U.S. Pat. No. 5,208,020; U.S. Pat. No. 6,441,163; WO 2005037992; WO 2005081711; and WO 2006 / 034488, all of which are expressly incorporated by reference in their entirety.

[0284] Alternatively, a fusion protein comprising the antibody and cytotoxic agent can be produced, for example, by recombinant techniques or peptide synthesis. The length of DNA can include regions encoding the two parts of the conjugate, either contiguous to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0285] In some embodiments, the ADC of Formula (IIa) has the structure: [ka] The amatoxin-linker complex Cy-L-Z′ can be prepared by conjugation of a thiol group on the antibody to an amatoxin-linker complex Cy-L-Z′ represented by:

[0286] This amatoxin-linker conjugate can be prepared according to Schemes 1-3 starting from commercially available 6-hydroxytryptophan (1). Compound 1 can be reacted with tert-butyloxycarbonyl anhydride ((BOC) 2 Hexahydropyrroloindole 3 can be produced as a mixture of cis and trans isomers upon irradiation of protected hydroxytryptophan 2 in the presence of oxygen and a sensitizer (Rose Bengal). The preparation of compound 3 (as well as a procedure for the preparation of Va from compound 2) is provided in WO 2019 / 030173, the disclosure of which is incorporated herein by reference in its entirety. Commercially available 9-fluorenylmethoxycarbonyl (FMOC) protected 4-hydroxyproline (4) can be alkylated with allyl bromide and subsequently attached to a tetrahydropyranyl (THP) polystyrene resin under mildly acidic conditions (pyridinium paratoluenesulfonate; PPTS) to give resin-bound allyl ester 5, which can be deprotected with palladium tetrakistriphenylphosphine palladium and dimethylbarbituric acid to give intermediate 6. The protected amino acid 7 can be prepared according to the methods reported in WO 2014 / 009025, the disclosure of which is incorporated herein in its entirety. The protected amine 7 can then be coupled to the resin-bound hydroxyproline 6 to give the peptide 8. Scheme 1 [ka]

[0287] Peptide 8 can then undergo multiple coupling and deprotection reactions to provide monocyclic intermediate 9 (Scheme 2). Amino acids FmocAsn(Trt)OH, FmocCys(OTrt)OH, FmocGlyOH, FmocIleOH, FmocGlyOH, and hexahydropyrroloindole 3 can be used in successive solid-phase coupling reactions to yield intermediate 9. Each amino acid can be coupled using PyBOP / HOBT in dichloromethane and dimethylformamide (DMF) in the presence of diisopropylethylamine (DIEA). Deprotection can be performed with 20% piperidine in DMF. Scheme 2 [ka]

[0288] After the final coupling reaction, intermediate 9 can be cleaved from the solid support resin with trifluoroacetic acid in the presence of triisopropylsilane to give peptide 10. Treatment of 10 with diphenylphosphoryl azide (DPPA) and DIEA induced macrocyclization, and deprotection with ammoniacal methanol gave the amatoxin derivative 11 (i.e., formula Va, where Q=S).

[0289] Maleimidohexyl amatoxin conjugate 14 can be prepared from compound 11 according to Scheme 3. Diels-Alder adduct 12 can be prepared from maleimide and 2,5-dimethylfuran and then alkylated with 1,6-dibromohexane to give protected linker 13. Compound 11 can be alkylated with compound 13 in dimethylsulfoxide (DMSO) in the presence of sodium hydroxide, followed by heating to 100° C. in DMSO to give amatoxin-linker conjugate 14. Procedures for preparing compounds 12 and 13, as well as the O-alkylation of related amatoxins (α-amanitin), have been previously reported in U.S. Patent Application Publication No. 2018 / 0043033, the disclosure of which is incorporated herein by reference in its entirety. Scheme 3 [ka]

[0290] antibody The ADC compositions and methods disclosed herein include agents that facilitate the selective delivery of such ADCs to a population of cells in a target tissue (e.g., cancer or tumor cells, or hematopoietic stem cells in the bone marrow stem cell niche). The cell targeting specificity of the ADC is determined by an antigen-binding protein, such as an antibody or antigen-binding portion thereof.

[0291] In one embodiment, the present invention relates to human CD45, CD49d (VLA-4), CD49f (VLA-6), CD51, CD84, CD90, CD117, CD133, CD134, CD184 (CXCR4), HLA-DR, CD11a, CD18, CD34, CD41 / 61, CD43, CD58, CD71, CD97, CD162, CD166, CD205 and CD361, CD13, CD33, CD34, CD44, CD4, CD59, CD84 / CD150, CD90 / Thy 1. The present invention encompasses ADCs comprising antibodies and antigen-binding fragments thereof that specifically bind to CD93, CD105 / endoglin, CD123 / IL-3R, CD126 / IL-6R, CD133, CD135 / Flt3 receptor, CD166 / ALCAM, prominin 2, erythropoietin R, endothelial cell-selective adhesion molecule, CD244, Tie1, Tie2, MPL, G-CSFR, CSF3R, IL-1R, gp130, leukemia inhibitory factor receptor, oncostatin M receptor, embodin, and IL-18R.Other examples of antigens that can be bound by the ADCs disclosed herein include CD7, CDw12, CD13, CD15, CD19, CD21, CD22, CD29, CD30, CD33, CD34, CD36, CD38, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD45RA, CD45RB, CD45RC, CD45RO, CD48, CD 49b, CD49d, CD49e, CD49f, CD50, CD53, CD55, CD64a, CD68, CD71, CD72, CD73, CD81, CD82, CD85A, CD85K, CD90, CD99, CD104, CD105, CD109, CD110, CD111, CD112, CD114, CD115, CD123, CD124, CD126, CD127, CD13 0, CD131, CD133, CD135, CD138, CD151, CD157, CD162, CD164, CD168, CD172a, CD173, CD174, CD175, CD17 5s, CD176, CD183, CD191, CD200, CD201, CD205, CD217, CD220, CD221, CD222, CD223, CD224, CD225, CD226 , CD227, CD228, CD229, CD230, CD235a, CD235b, CD236, CD236R, CD238, CD240, CD242, CD243, CD277, CD292, CDw293, CD295, CD298, CD309, CD318, CD324, CD325, CD338, CD344, CD349 or CD350. Other examples of antigens that can be bound by the ADCs disclosed herein include, but are not limited to, CD11a, CD18, CD37, CD47, CD52, CD58, CD62L, CD69, CD74, CD97, CD103, CD132, CD156a, CD179a, CD79b, CD184, CD232, CD244, CD252, CD302, CD305, CD317, and CD361.

[0292] In certain embodiments, the antibodies or antigen-binding fragments thereof in the ADCs described herein have particular dissociation rates that are particularly advantageous when used as part of a conjugate. For example, an anti-CD117 antibody, in certain embodiments, has a dissociation rate of 1×10 as measured by biolayer interferometry (BLI). -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 In some embodiments, the antibody or antigen-binding fragment thereof has an off rate constant (Koff) for human CD117 and / or rhesus monkey CD117 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, or about 1 nM or less as measured by a Biolayer Interferometry (BLI) assay. D and binds to cell surface antigens (e.g., human CD117 and / or rhesus CD117).

[0293] Anti-CD117 antibody In one embodiment, the present invention encompasses ADCs comprising antibodies and antigen-binding fragments thereof that specifically bind to CD117, such as GNNK+CD117. Such ADCs can be used, for example, as therapeutic agents for (i) treating cancers and autoimmune diseases characterized by CD117+ cells, and (ii) promoting engraftment of transplanted hematopoietic stem cells in patients in need of transplantation therapy. These therapeutic activities can be triggered, for example, by the binding of isolated anti-CD117 antibodies, antigen-binding fragments thereof, that bind to CD117 (e.g., GNNK+CD117) expressed on the surface of cells, such as cancer cells, autoimmune cells, or hematopoietic stem cells, and the subsequent induction of cell death. Depletion of endogenous hematopoietic stem cells provides a niche in which transplanted hematopoietic stem cells can colonize and subsequently establish productive hematopoiesis. In this way, transplanted hematopoietic stem cells can successfully engraft in patients, such as human patients suffering from stem cell diseases described herein.

[0294] Antibodies and antigen-binding fragments capable of binding human CD117 (also known as c-Kit, mRNA NCBI Reference Sequence: NM_000222.2, protein NCBI Reference Sequence: NP_000213.1), including those capable of binding GNNK+CD117, may be used in conjunction with the compositions and methods described herein to condition patients for hematopoietic stem cell transplantation therapy. Polymorphisms affecting the coding region or extracellular domain of CD117 in a significant percentage of the population are currently unknown in non-oncology indications. There are at least four identified isoforms of CD117, and additional isoforms may be expressed in tumor cells. Two of the CD117 isoforms are located in the intracellular domain of the protein, and two are in the outer membrane-proximal region. The two extracellular isoforms, GNNK+ and GNNK-, differ in the presence (GNNK+) or absence (GNNK-) of four amino acid sequences. 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. The GNNK+ isoform can be used as an immunogen to generate antibodies capable of binding to CD117, since antibodies raised against this isoform would include GNNK+ and GNNK- proteins. The amino acid sequences of human CD117 isoforms 1 and 2 are set forth in SEQ ID NOs: 145 and 146, respectively. In certain embodiments, the anti-human CD117 (hCD117) antibodies disclosed herein are capable of binding to both isoform 1 and isoform 2 of human CD117.

[0295] As described below, a yeast library screen of human antibodies was performed to identify novel anti-CD117 antibodies and fragments thereof with diagnostic and therapeutic applications. Antibody 54 (Ab54), antibody 55 (Ab55), antibody 56 (Ab56), antibody 57 (Ab57), antibody 58 (Ab58), antibody 61 (Ab61), antibody 66 (Ab66), antibody 67 (Ab67), antibody 68 (Ab68), and antibody 69 (Ab69) were human antibodies identified in this screen. These antibodies cross-react with human CD117 and rhesus CD117. Furthermore, these antibodies disclosed herein are capable of binding to both isoforms of human CD117, namely isoform 1 (SEQ ID NO: 145) and isoform 2 (SEQ ID NO: 146).

[0296] The amino acid sequences of the various binding regions of anti-CD117 antibodies, including Ab54, Ab55, Ab56, Ab57, Ab58, Ab61, Ab66, Ab67, Ab68, and Ab69, are set forth in the sequence listing below. Included in the present invention are human anti-CD117 antibodies comprising the CDRs set forth in the sequence listing below, as well as ADCs comprising human anti-CD117 antibodies comprising the variable regions set forth in the sequence listing below.

[0297] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 55. The heavy chain variable region (VH) amino acid sequence of antibody 55 (i.e., Ab55) is set forth in SEQ ID NO:19 (see sequence listing). The VH CDR domain amino acid sequence of antibody 55 is set forth in SEQ ID NO:21 (VH CDR1); SEQ ID NO:22 (VH CDR2), and SEQ ID NO:23 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 55 is set forth in SEQ ID NO:20 (see sequence listing). The VL CDR domain amino acid sequence of antibody 55 is set forth in SEQ ID NO:24 (VL CDR1); SEQ ID NO:25 (VL CDR2), and SEQ ID NO:26 (VL CDR3). The heavy chain constant region of antibody 55 is set forth in SEQ ID NO:122. The light chain constant region of antibody 55 is set forth in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 21, 22, and 23, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 24, 25, and 26. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 20, and a heavy chain variable region set forth in SEQ ID NO: 19.

[0298] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 54. The heavy chain variable region (VH) amino acid sequence of antibody 54 (i.e., Ab54) is set forth in SEQ ID NO:29 (see sequence listing). The VH CDR domain amino acid sequence of antibody 54 is set forth in SEQ ID NO:31 (VH CDR1); SEQ ID NO:32 (VH CDR2), and SEQ ID NO:33 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 54 is set forth in SEQ ID NO:30 (see sequence listing). The VL CDR domain amino acid sequence of antibody 54 is set forth in SEQ ID NO:34 (VL CDR1); SEQ ID NO:35 (VL CDR2), and SEQ ID NO:36 (VL CDR3). The heavy chain constant region of antibody 54 is set forth in SEQ ID NO:122. The light chain constant region of antibody 54 is set forth in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 31, 32, and 33, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 34, 35, and 36. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 30, and a heavy chain variable region set forth in SEQ ID NO: 29.

[0299] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 56. The heavy chain variable region (VH) amino acid sequence of antibody 56 (i.e., Ab56) is set forth in SEQ ID NO:39 (see sequence listing). The VH CDR domain amino acid sequence of antibody 56 is set forth in SEQ ID NO:41 (VH CDR1); SEQ ID NO:42 (VH CDR2), and SEQ ID NO:43 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 56 is set forth in SEQ ID NO:40 (see sequence listing). The VL CDR domain amino acid sequence of antibody 56 is set forth in SEQ ID NO:44 (VL CDR1); SEQ ID NO:45 (VL CDR2), and SEQ ID NO:46 (VL CDR3). The heavy chain constant region of antibody 56 is set forth in SEQ ID NO:122. The light chain constant region of antibody 56 is set forth in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 41, 42, and 43, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 44, 45, and 46. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 40, and a heavy chain variable region set forth in SEQ ID NO: 39.

[0300] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to antibody 57. The heavy chain variable region (VH) amino acid sequence of antibody 57 (i.e., Ab57) is set forth in SEQ ID NO:49 (see sequence listing). The VH CDR domain amino acid sequence of antibody 57 is set forth in SEQ ID NO:51 (VH CDR1); SEQ ID NO:52 (VH CDR2), and SEQ ID NO:53 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 57 is set forth in SEQ ID NO:50 (see sequence listing). The VL CDR domain amino acid sequence of antibody 57 is set forth in SEQ ID NO:54 (VL CDR1); SEQ ID NO:55 (VL CDR2), and SEQ ID NO:56 (VL CDR3). The heavy chain constant region of antibody 57 is set forth in SEQ ID NO:122. The light chain constant region of antibody 57 is set forth in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 51, 52, and 53, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 54, 55, and 56. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 50, and a heavy chain variable region set forth in SEQ ID NO: 49.

[0301] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 58. The heavy chain variable region (VH) amino acid sequence of antibody 58 (i.e., Ab58) is set forth in SEQ ID NO:59 (see sequence listing). The VH CDR domain amino acid sequence of antibody 58 is set forth in SEQ ID NO:61 (VH CDR1); SEQ ID NO:62 (VH CDR2), and SEQ ID NO:63 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 58 is set forth in SEQ ID NO:60 (see sequence listing). The VL CDR domain amino acid sequence of antibody 58 is set forth in SEQ ID NO:64 (VL CDR1); SEQ ID NO:65 (VL CDR2), and SEQ ID NO:66 (VL CDR3). The heavy chain constant region of antibody 58 is set forth in SEQ ID NO:122. The light chain constant region of antibody 58 is set forth in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 61, 62, and 63, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 64, 65, and 66. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 60, and a heavy chain variable region set forth in SEQ ID NO: 59.

[0302] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 61. The heavy chain variable region (VH) amino acid sequence of antibody 61 (i.e., Ab61) is set forth in SEQ ID NO:69 (see sequence listing). The VH CDR domain amino acid sequence of antibody 61 is set forth in SEQ ID NO:71 (VH CDR1); SEQ ID NO:72 (VH CDR2), and SEQ ID NO:73 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 61 is set forth in SEQ ID NO:70 (see sequence listing). The VL CDR domain amino acid sequence of antibody 61 is set forth in SEQ ID NO:74 (VL CDR1); SEQ ID NO:75 (VL CDR2), and SEQ ID NO:76 (VL CDR3). The heavy chain constant region of antibody 61 is set forth in SEQ ID NO:122. The light chain constant region of antibody 61 is set forth in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 71, 72, and 73, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 74, 75, and 76. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 70, and a heavy chain variable region set forth in SEQ ID NO: 69.

[0303] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 66. The heavy chain variable region (VH) amino acid sequence of antibody 66 (i.e., Ab66) is set forth in SEQ ID NO:79 (see sequence listing). The VH CDR domain amino acid sequence of antibody 66 is set forth in SEQ ID NO:81 (VH CDR1); SEQ ID NO:82 (VH CDR2), and SEQ ID NO:83 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 66 is set forth in SEQ ID NO:80 (see sequence listing). The VL CDR domain amino acid sequence of antibody 66 is set forth in SEQ ID NO:84 (VL CDR1); SEQ ID NO:85 (VL CDR2), and SEQ ID NO:86 (VL CDR3). The heavy chain constant region of antibody 66 is set forth in SEQ ID NO:122. The light chain constant region of antibody 66 is set forth in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 81, 82, and 83, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 84, 85, and 86. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 80, and a heavy chain variable region set forth in SEQ ID NO: 79.

[0304] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 67. The heavy chain variable region (VH) amino acid sequence of antibody 67 is set forth in SEQ ID NO:9 (see Table 2). The VH CDR domain amino acid sequence of antibody 67 is set forth in SEQ ID NO:11 (VH CDR1); SEQ ID NO:12 (VH CDR2), and SEQ ID NO:13 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 67 is set forth in SEQ ID NO:10 (see Table 2). The VL CDR domain amino acid sequence of antibody 67 is set forth in SEQ ID NO:14 (VL CDR1); SEQ ID NO:15 (VL CDR2), and SEQ ID NO:16 (VL CDR3). The full-length heavy chain (HC) of antibody 67 is set forth in SEQ ID NO:110, and the full-length heavy chain constant region of antibody 67 is set forth in SEQ ID NO:122. The light chain (LC) of antibody 67 is set forth in SEQ ID NO: 109. The light chain constant region of antibody 67 is set forth in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 11, 12, and 13, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 14, 15, and 16. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable heavy chain comprising the amino acid residues set forth in SEQ ID NO: 9, and a heavy chain variable region set forth in SEQ ID NO: 10. In further embodiments, an anti-CD117 antibody comprises a heavy chain comprising SEQ ID NO: 110, and a light chain comprising SEQ ID NO: 109.

[0305] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 68. The heavy chain variable region (VH) amino acid sequence of antibody 68 (i.e., Ab68) is set forth in SEQ ID NO:89 (see sequence listing). The VH CDR domain amino acid sequence of antibody 68 is set forth in SEQ ID NO:91 (VH CDR1); SEQ ID NO:92 (VH CDR2), and SEQ ID NO:93 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 68 is set forth in SEQ ID NO:90 (see sequence listing). The VL CDR domain amino acid sequence of antibody 68 is set forth in SEQ ID NO:94 (VL CDR1); SEQ ID NO:95 (VL CDR2), and SEQ ID NO:96 (VL CDR3). The heavy chain constant region of antibody 68 is set forth in SEQ ID NO:122. The light chain constant region of antibody 68 is set forth in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 91, 92, and 93, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 94, 95, and 96. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 90, and a heavy chain variable region set forth in SEQ ID NO: 89.

[0306] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 69. The heavy chain variable region (VH) amino acid sequence of antibody 69 (i.e., Ab69) is set forth in SEQ ID NO:99 (see sequence listing). The VH CDR domain amino acid sequence of antibody 69 is set forth in SEQ ID NO:101 (VH CDR1); SEQ ID NO:102 (VH CDR2), and SEQ ID NO:103 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 69 is set forth in SEQ ID NO:100 (see sequence listing). The VL CDR domain amino acid sequence of antibody 69 is set forth in SEQ ID NO:104 (VL CDR1); SEQ ID NO:105 (VL CDR2), and SEQ ID NO:106 (VL CDR3). The heavy chain constant region of antibody 69 is set forth in SEQ ID NO:122. The light chain constant region of antibody 69 is set forth in SEQ ID NO: 121. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 101, 102, and 103, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 104, 105, and 106. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 100, and a heavy chain variable region set forth in SEQ ID NO: 99.

[0307] Additionally, the amino acid sequences of the various binding regions of the anti-CD117 antibodies Ab77, Ab79, Ab81, Ab85, Ab86, Ab87, Ab88, and Ab89 are set forth in the sequence listing provided below. Anti-CD117 antibodies having these sequences can also be used in the ADCs described herein.

[0308] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 77. The heavy chain variable region (VH) amino acid sequence of antibody 77 (i.e., Ab77) is set forth in SEQ ID NO:147 (see sequence listing). The VH CDR domain amino acid sequence of antibody 77 is set forth in SEQ ID NO:263 (VH CDR1); SEQ ID NO:2 (VH CDR2), and SEQ ID NO:3 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 77 is set forth in SEQ ID NO:231 (see sequence listing). The VL CDR domain amino acid sequence of antibody 77 is set forth in SEQ ID NO:264 (VL CDR1); SEQ ID NO:265 (VL CDR2), and SEQ ID NO:266 (VL CDR3). The heavy chain constant region of antibody 77 is set forth in SEQ ID NO:269. The light chain constant region of antibody 77 is set forth in SEQ ID NO: 283. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 263, 2, and 3, and a light chain variable region CDR set forth in SEQ ID NOs: 264, 265, and 266. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 231, and a heavy chain variable region set forth in SEQ ID NO: 147.

[0309] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 79. The heavy chain variable region (VH) amino acid sequence of antibody 79 (i.e., Ab79) is set forth in SEQ ID NO:147 (see sequence listing). The VH CDR domain amino acid sequence of antibody 79 is set forth in SEQ ID NO:263 (VH CDR1); SEQ ID NO:2 (VH CDR2), and SEQ ID NO:3 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 79 is set forth in SEQ ID NO:233 (see sequence listing). The VL CDR domain amino acid sequence of antibody 79 is set forth in SEQ ID NO:267 (VL CDR1); SEQ ID NO:265 (VL CDR2), and SEQ ID NO:266 (VL CDR3). The heavy chain constant region of antibody 79 is set forth in SEQ ID NO:269. The light chain constant region of antibody 79 is set forth in SEQ ID NO: 283. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 263, 2, and 3, and a light chain variable region CDR set forth in SEQ ID NOs: 267, 265, and 266. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 233, and a heavy chain variable region set forth in SEQ ID NO: 147.

[0310] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 81. The heavy chain variable region (VH) amino acid sequence of antibody 81 (i.e., Ab81) is set forth in SEQ ID NO:147 (see sequence listing). The VH CDR domain amino acid sequence of antibody 81 is set forth in SEQ ID NO:263 (VH CDR1); SEQ ID NO:2 (VH CDR2), and SEQ ID NO:3 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 81 is set forth in SEQ ID NO:235 (see sequence listing). The VL CDR domain amino acid sequence of antibody 81 is set forth in SEQ ID NO:264 (VL CDR1); SEQ ID NO:268 (VL CDR2), and SEQ ID NO:266 (VL CDR3). The heavy chain constant region of antibody 81 is set forth in SEQ ID NO:269. The light chain constant region of antibody 81 is set forth in SEQ ID NO: 283. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 263, 2, and 3, and a light chain variable region CDR set forth in SEQ ID NOs: 264, 268, and 266. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 235, and a heavy chain variable region set forth in SEQ ID NO: 147.

[0311] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 85. The heavy chain variable region (VH) amino acid sequence of antibody 85 (i.e., Ab86) is set forth in SEQ ID NO:243 (see sequence listing). The VH CDR domain amino acid sequence of antibody 85 is set forth in SEQ ID NO:245 (VH CDR1); SEQ ID NO:246 (VH CDR2), and SEQ ID NO:247 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 85 is set forth in SEQ ID NO:242 (see sequence listing). The VL CDR domain amino acid sequence of antibody 85 is set forth in SEQ ID NO:248 (VL CDR1); SEQ ID NO:249 (VL CDR2), and SEQ ID NO:250 (VL CDR3). The heavy chain constant region of antibody 85 is set forth in SEQ ID NO:269. The light chain constant region of antibody 85 is set forth in SEQ ID NO: 283. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 245, 246, and 247, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 248, 249, and 250. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 244, and a heavy chain variable region set forth in SEQ ID NO: 243.

[0312] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 86. The heavy chain variable region (VH) amino acid sequence of antibody 86 (i.e., Ab86) is set forth in SEQ ID NO:251 (see sequence listing). The VH CDR domain amino acid sequence of antibody 86 is set forth in SEQ ID NO:245 (VH CDR1); SEQ ID NO:253 (VH CDR2), and SEQ ID NO:3 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 86 is set forth in SEQ ID NO:252 (see sequence listing). The VL CDR domain amino acid sequence of antibody 86 is set forth in SEQ ID NO:254 (VL CDR1); SEQ ID NO:249 (VL CDR2), and SEQ ID NO:255 (VL CDR3). The heavy chain constant region of antibody 86 is set forth in SEQ ID NO:269. The light chain constant region of antibody 86 is set forth in SEQ ID NO: 283. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 245, 253, and 3, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 254, 249, and 255. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 252, and a heavy chain variable region set forth in SEQ ID NO: 251.

[0313] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 87. The heavy chain variable region (VH) amino acid sequence of antibody 87 (i.e., Ab87) is set forth in SEQ ID NO:243 (see sequence listing). The VH CDR domain amino acid sequence of antibody 87 is set forth in SEQ ID NO:245 (VH CDR1); SEQ ID NO:246 (VH CDR2), and SEQ ID NO:247 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 87 is set forth in SEQ ID NO:256 (see sequence listing). The VL CDR domain amino acid sequence of antibody 87 is set forth in SEQ ID NO:257 (VL CDR1); SEQ ID NO:5 (VL CDR2), and SEQ ID NO:255 (VL CDR3). The heavy chain constant region of antibody 87 is set forth in SEQ ID NO:269. The light chain constant region of antibody 87 is set forth in SEQ ID NO: 283. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 245, 246, and 247, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 257, 5, and 255. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 256, and a heavy chain variable region set forth in SEQ ID NO: 243.

[0314] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 88. The heavy chain variable region (VH) amino acid sequence of antibody 88 (i.e., Ab88) is set forth in SEQ ID NO:258 (see sequence listing). The VH CDR domain amino acid sequence of antibody 88 is set forth in SEQ ID NO:245 (VH CDR1); SEQ ID NO:259 (VH CDR2), and SEQ ID NO:3 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 88 is set forth in SEQ ID NO:256 (see sequence listing). The VL CDR domain amino acid sequence of antibody 88 is set forth in SEQ ID NO:257 (VL CDR1); SEQ ID NO:5 (VL CDR2), and SEQ ID NO:255 (VL CDR3). The heavy chain constant region of antibody 88 is set forth in SEQ ID NO:269. The light chain constant region of antibody 88 is set forth in SEQ ID NO: 283. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 245, 259, and 3, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 257, 5, and 255. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 256, and a heavy chain variable region set forth in SEQ ID NO: 258.

[0315] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 89. The heavy chain variable region (VH) amino acid sequence of antibody 89 (i.e., Ab89) is set forth in SEQ ID NO:260 (see sequence listing). The VH CDR domain amino acid sequence of antibody 89 is set forth in SEQ ID NO:245 (VH CDR1); SEQ ID NO:2 (VH CDR2), and SEQ ID NO:3 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 89 is set forth in SEQ ID NO:252 (see sequence listing). The VL CDR domain amino acid sequence of antibody 89 is set forth in SEQ ID NO:254 (VL CDR1); SEQ ID NO:249 (VL CDR2), and SEQ ID NO:255 (VL CDR3). The heavy chain constant region of antibody 89 is set forth in SEQ ID NO:269. The light chain constant region of antibody 89 is set forth in SEQ ID NO: 283. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs:245, 2, and 3, and a light chain variable region CDR set forth in SEQ ID NOs:254, 249, and 255. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO:252, and a heavy chain variable region set forth in SEQ ID NO:260.

[0316] In one embodiment, the invention provides an ADC comprising an anti-CD117 antibody or antigen-binding fragment thereof comprising a binding region, e.g., CDRs, variable region, corresponding to that of antibody 249. The heavy chain variable region (VH) amino acid sequence of antibody 249 (i.e., Ab249) is set forth in SEQ ID NO:238 (see sequence listing). The VH CDR domain amino acid sequence of antibody 249 is set forth in SEQ ID NO:286 (VH CDR1); SEQ ID NO:2 (VH CDR2), and SEQ ID NO:287 (VH CDR3). The light chain variable region (VL) amino acid sequence of antibody 249 is set forth in SEQ ID NO:242 (see sequence listing). The VL CDR domain amino acid sequence of antibody 249 is set forth in SEQ ID NO:288 (VL CDR1); SEQ ID NO:249 (VL CDR2), and SEQ ID NO:289 (VL CDR3). The heavy chain constant region of antibody 249 is set forth in SEQ ID NO:269. The light chain constant region of antibody 249 is set forth in SEQ ID NO: 283. Thus, in certain embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a set of variable heavy chain CDRs (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 286, 2, and 287, and a set of light chain variable region CDRs set forth in SEQ ID NOs: 288, 249, and 289. In other embodiments, an anti-CD117 antibody, or antigen binding portion thereof, comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 242, and a heavy chain variable region set forth in SEQ ID NO: 238.

[0317] Further included in the present disclosure are anti-CD117 antibody drug conjugates (ADCs) comprising the binding regions (heavy and light chain CDRs or variable regions) set forth in SEQ ID NOs: 147-168. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 148. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 149. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 150. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 151. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 152. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 153. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 154. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 155. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 156. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:157.In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 158. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 159. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 160. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 161. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 162. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 163. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 164 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 165. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 166 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 167. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 168 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 169. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 170 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 171. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:172 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:173.In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 174 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 175. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 176 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 177. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 178 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 179. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 180 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 181. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 172 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 182. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 183 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 184. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 185 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 186. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 187 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 188. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 189 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 190. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 191 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 192. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:193 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:194.In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 195 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 196. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 197 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 198. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 199 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 200. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 201 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 190. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 202 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 203. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:204 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:205. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:206 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:207. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:208 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:209. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:210 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:211. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:212 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:213. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:214 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:215.In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:216 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:217. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:218 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:219. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:220 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:221. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:222 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:223. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:224 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:225. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:226 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:227. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:228. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:229. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:230. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:231. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:232.In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 233. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 234 ... In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 235. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 236.

[0318] In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, of the ADCs described herein comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 237. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 243 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 244. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 251 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 252. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 243 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 256. In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 258 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 256. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:260 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:252. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:238 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:239. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:239. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:147 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:240. In one embodiment, an anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:238 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:241.In one embodiment, the anti-CD117 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO:238 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO:242.

[0319] Some of the anti-CD117 antibodies described herein are neutral antibodies in that the antibodies do not substantially inhibit CD117 activity on cells expressing CD117.Neutral antibodies can be identified, for example, using in vitro stem cell factor (SCF)-dependent cell proliferation assay.In SCF-dependent cell proliferation assay, neutral CD117 antibodies will not kill CD34+ cells that are dependent on SCF to divide, because the neutral antibodies do not block SCF from binding to CD117, such as by inhibiting CD117 activity.

[0320] Neutral antibodies can be used for diagnostic purposes given their ability to specifically bind human CD117, but when conjugated with a cytotoxin such as those described herein, they are also effective for killing cells expressing CD117. Typically, the antibodies used in the conjugates have agonistic or antagonistic activity unique to the antibody. However, approaches unique to the conjugates are described herein, particularly in situations where the conjugates are used as conditioning agents prior to stem cell transplantation. While antagonist antibodies combined with cytotoxins alone or as conjugates can be effective given the killing capacity of the antibody alone in addition to the cytotoxin, pretreatment with conjugates containing neutral anti-CD117 antibodies presents an alternative strategy where the activity of the antibody is secondary to the effect of the cytotoxin, but the internalization and affinity properties of the antibody, such as dissociation rate, are important for effective delivery of the cytotoxin.

[0321] Examples of neutral anti-CD117 antibodies include Ab58, Ab61, Ab66, Ab67, Ab68, and Ab69. Comparison of the amino acid sequences of the CDRs of the neutral anti-CD117 antibody CDRs reveals consensus sequences between the two groups of identified neutral antibodies. Ab58 and Ab61 share the same light chain CDRs and HC CDR3, with slight variations in HC CDR1 and HC CDR2. The consensus sequences of HC CDR1 and CDR2 are set forth in SEQ ID NOs: 133 and 134. Ab66, Ab67, Ab68, and Ab69 are also neutral antibodies. Ab66, Ab67, Ab68, and Ab69 share the same light chain CDRs and the same HC CDR3, but these antibodies have variations within their HC CDR1 and HC CDR2 regions. The consensus sequences of these antibodies in the HC CDR1 and HC CDR2 regions are given in SEQ ID NOs:139 and 140, respectively.

[0322] Antagonist antibodies are also provided herein, including Ab54, Ab55, Ab56, and Ab57. Although Ab54, Ab55, Ab56, and Ab57 share the same light chain CDRs and the same HC CDR3, these antibodies have variations within their HC CDR1 and HC CDR2 regions. The consensus sequences of these antibodies in the HC CDR1 and HC CDR2 regions are provided in SEQ ID NOs: 127 and 128, respectively.

[0323] In one embodiment, the anti-CD117 antibody or antigen-binding fragment thereof comprises a variable region having an amino acid sequence at least 95%, 96%, 97% or 99% identical to a SEQ ID NO: disclosed herein. Alternatively, the anti-CD117 antibody or antigen-binding fragment thereof comprises a CDR comprising a SEQ ID NO: disclosed herein, together with a variable region framework region described herein having an amino acid sequence at least 95%, 96%, 97% or 99% identical to a SEQ ID NO: disclosed herein.

[0324] The anti-CD117 antibodies described herein may be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multiple chain or single chain antibodies, and / or binding fragments that specifically bind to human CD117, 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 be of or 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-CD117 antibody is an IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0325] Antibodies for use in conjunction with the methods described herein include variants of the above-mentioned antibodies, such as antibody fragments that contain or lack an Fc domain, as well as 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 equivalent regions thereof of the antibodies or antibody fragments described herein (e.g., 10 Exemplary antigen-binding fragments of the above antibodies include dual variable immunoglobulin domains, single chain Fv molecules (scFv), diabodies, triabodies, nanobodies, antibody-like protein scaffolds, Fv fragments, Fab fragments, F(ab') ... 2 molecule, and tandem di-scFv.

[0326] In one embodiment, anti-CD117 antibodies are provided that contain one or more radiolabeled amino acids. Radiolabeled anti-CD117 antibodies can be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molecules is another possible feature). Non-limiting examples of polypeptide labels 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., in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (USRE35,500), 5,648,471, and 5,697,902. For example, radioisotopes can be conjugated by the chloramine T method.

[0327] Anti-CD45 antibody In one embodiment, the invention encompasses ADCs comprising antibodies and antigen-binding fragments thereof that specifically bind to a CD45 polypeptide, e.g., a human CD45 polypeptide, and uses thereof. In an exemplary embodiment, the antibody or antigen-binding fragment thereof that specifically binds to a CD45 polypeptide comprises a heavy chain variable region and a light chain variable region.

[0328] CD45 is a hematopoietic cell-specific transmembrane protein tyrosine phosphatase essential for T- and B-cell antigen receptor-mediated signal transduction. CD45 contains a large extracellular domain and a phosphatase-containing cytosolic domain. CD45 can act as both a positive and negative regulator, depending on the nature of the stimulus and the cell type involved. Although there are many possible permutations in the CD45 gene, only six isoforms have been identified so far in humans. The isoforms are RA, RO, RB, RAB, RBC, and RABC (Hermiston et al. 2003 "CD45: a critical regulator of signaling thresholds in immune cells." Annu Rev Immunol. 2:107-137). CD45RA is expressed on naive T cells, and CD45RO is expressed on activated and memory T cells, some B cell subsets, activated monocytes / macrophages, and granulocytes. CD45RB is expressed on peripheral B cells, naive T cells, thymocytes, and weakly on macrophages and dendritic cells.

[0329] In certain embodiments, the anti-CD45 antibody is selected from apamitamab (also known as 90Y-BC8, Iomab-B, BC8; e.g., as described in U.S. Patent Publication No. 20170326259, WO 2017155937, 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 WO2003048327, WO2016016442, US20170226209, US20160152733, U.S. Pat. No. 9,701,756; US20110076270, or U.S. Pat. No. 7,825,222, each of which is incorporated by reference.

[0330] Anti-CD137 antibody The present invention encompasses ADCs comprising antibodies and antigen-binding fragments thereof that specifically bind to a CD137 polypeptide, e.g., a human CD137 polypeptide, and uses thereof. In an exemplary embodiment, the antibody or antigen-binding fragment thereof that specifically binds to a CD137 polypeptide comprises a heavy chain variable region and a light chain variable region.

[0331] T cells have been shown to express CD137 because this antigen is a costimulatory molecule of the transmembrane TNF receptor superfamily, is expressed on a variety of hematopoietic cells, and promotes T cell activation and regulates T cell proliferation and survival (see, e.g., Cannons et al., J. Immunol. 167:1313-1324, 2001, the disclosure of which is incorporated herein by reference as it relates to expression of CD137 by T cells). CD137 has alternatively been named tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-1BB, or induced by lymphocyte activation (ILA).

[0332] In certain embodiments, the anti-CD137 antibody is ADG106 (e.g., as described in U.S. Patent Publication No. 20190055314, WO 2019037711, WO 2019036855); AGEN2373 (e.g., as described in WO 2018191502, U.S. Patent Publication No. 20180344870); ATOR-1017 (e.g., as described in WO 2018091740, U.S. Patent Publication No. 20180118841), PE0166 (e.g., as described in Song et al. AACR 2019, Abstract 2397 / 21), urelumab (also known as BMS-663513; see, e.g., International Publication No. WO 2004010947, International Publication No. WO 2005035584, U.S. Patent Application Publication No. 20090068192, U.S. Patent No. 7659384, U.S. Patent No. 8475790, U.S. Patent No. 8137667, U.S. Patent Application Publication No. 20100183621 , U.S. Pat. No. 8,716,452; U.S. Pat. App. Pub. No. 20120141494; U.S. Pat. No. 9,382,328; U.S. Pat. App. Pub. No. 20140193422; WO 2016029073; U.S. Pat. App. Pub. No. 20160368998; WO 2017181034; U.S. Pat. App. Pub. No. 20190062445; Chin et al. Nature communications. 9.1(2018):4679.; Segal et al. Clinical Cancer Research. 23.8(2017):1929-1936; and utomilumab (also known as PF-05082566, MOR-7480.See, e.g., WO 2012032433, U.S. Patent Application Publication No. 20120237498, U.S. Patent Application Publication No. 20140178368, WO 2012145183, WO 2015119923, WO 2015179236, U.S. Patent Application Publication No. 20160152722, U.S. Patent Application Publication No. 20190031765, WO 2017130076, Chin et al. Nature communications. 9.1(2018):4679.; Segal et al. Clinical Cancer Research. 24.8(2018):1816-1823; Fisher et al. Cancer Immunology, Immunotherapy. 61.10(2012):1721-1733), each of which is incorporated by reference.

[0333] Other anti-CD137 antibodies are described, for example, in WO 2018134787, WO 2019020774, WO 2017077085, U.S. Patent Application Publication No. 20180327504, U.S. Patent Application Publication No. 20190099488, U.S. Patent Application Publication No. 2019006045, U.S. Patent Application Publication No. 20190015508, WO 2019014328, U.S. Patent Application Publication No. 2019020775 ... Publication No. 20190071510, International Publication No. 2018127787, U.S. Patent Application Publication No. 20180258177, U.S. Patent No. 10174122, International Publication No. 2016110584, International Publication No. 2018017761, International Publication No. 2018098370, U.S. Patent Application Publication No. 20130149301, International Publication No. 2019027754, International Publication No. 20181 No. 56740, U.S. Patent Application Publication No. 20160244528, WO 2016134358, U.S. Patent No. 10233251, U.S. Patent Application Publication No. 20170226215, U.S. Patent Application Publication No. 20160083474, WO 2017049452, U.S. Patent Application Publication No. 20180282422, WO 2015188047, WO 2010132 389, U.S. Patent Application Publication No. 20120076722, U.S. Patent Application Publication No. 20110177104, WO 2011031063, U.S. Patent Application Publication No. 20080305113, U.S. Patent Application Publication No. 20080008716, U.S. Patent No. 7829088, U.S. Patent Application Publication No. 20090041763, WO 2006126835, Soederstroemet al. Circulation J. 81.12(2017):1945-1952; Makkouk,et al. Annals of Oncology 28.2(2016):415-420; Martinez-Forero et al. J.of Immunology.190.12(2013):6694-6706; Dubrot et al. Cancer immunology, immunotherapy. 59.8(2010):1223-1233; each of which is incorporated by reference.

[0334] In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to an anti-CD137 antibody of the present invention, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an anti-CD137 antibody of the present invention. In a particular embodiment, the antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain of an anti-CD137 antibody of the present invention or a variant thereof, wherein the variant (i) differs from the anti-CD137 antibody in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from the anti-CD137 antibody in at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from the anti-CD137 antibody in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions; (i) differs from the anti-CD137 antibody in deletions and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the anti-CD137 antibody, where in any of (i)-(iv), the amino acid substitutions may be conservative or non-conservative amino acid substitutions; and the modified heavy chain variable region may have increased biological activity relative to the heavy chain variable region of the anti-CD137 antibody, while retaining the CD137-binding specificity of the antibody.

[0335] In one embodiment, the anti-CD137 antibody that may be used in the methods and compositions (including ADCs) described herein is the murine anti-CD137 antibody BBK2 (Thermo Fisher; MS621PABX) or an anti-CD137 antibody that comprises an 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 murine monoclonal antibody (IgG1, κ) that binds to the extracellular domain of the human 4-1BB recombinant protein (4-1BB is also known as CD137). In certain embodiments, the methods and compositions of the disclosure include an anti-CD137 antibody that comprises the binding region (e.g., CDR) of the BBK2 antibody. In another embodiment, the methods and compositions of the disclosure include an antibody that competitively inhibits the BBK2 antibody from binding to its epitope on CD137. In certain embodiments, the anti-CD137 antibody is a humanized BBK2 or a chimeric BBK2.

[0336] In one embodiment, the methods and compositions described herein include a chimeric anti-CD137 (ch-BBK2) antibody comprising the variable heavy and light chain regions of BBK2. In a particular embodiment, the chimeric BBK2 antibody is an IgG1 antibody comprising a human constant region. The heavy chain amino acid sequence of ch-BBK2 is set forth in SEQ ID NO:290, and the light chain amino acid sequence of ch-BBK2 is set forth in SEQ ID NO:291. The CDR regions (CDR1, CDR2, and CDR3) of each of the heavy and light chain sequences are set forth below in bold. The CDR regions of BBK2 may be defined according to Kabat numbering. The CDRs defined by Kabat numbering are set forth below for each of the heavy and light chain sequences (set forth below in bold). The variable regions of BBK2 are in italics. [ka] [ka]

[0337] Thus, in one embodiment, the VH CDR amino acid sequence of the anti-CD137 antibody BBK2 (including ch-BBK2) is SYWIN (VH CDR1; SEQ ID NO: 292); NIYPSDSYTNYNQKFKD (VH CDR2; SEQ ID NO: 293) and NGVEGYPHYYAMEY (VH CDR3; SEQ ID NO: 294) and the VL CDR amino acid sequence of the anti-CD137 antibody BBK2 (including ch-BBK2) is RASQDLSNHLY (VL CDR1; SEQ ID NO: 295); YTSRLHS (VL CDR2; SEQ ID NO: 296) and QQGYTLPYT (VL CDR3; SEQ ID NO: 297).

[0338] The heavy chain variable region of BBK2 is set forth in SEQ ID NO:298: [ka] The light chain variable region of BBK2 is set forth in SEQ ID NO: 299: [ka] Anti-CD137 antibodies (including anti-CD137 ADCs) can comprise the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs:298 and 299, respectively.

[0339] In one embodiment, 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 comprising the amino acid sequence of SEQ ID NO:292, a CDR2 comprising the amino acid sequence of SEQ ID NO:293, and a CDR3 comprising the amino acid sequence of SEQ ID NO:294; and a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:295, a CDR2 comprising the amino acid sequence of SEQ ID NO:296, and a CDR3 comprising the amino acid sequence of SEQ ID NO:297.

[0340] In one embodiment, 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 comprising the amino acid sequence of SEQ ID NO:292, a CDR2 comprising the amino acid sequence of SEQ ID NO:293, and a CDR3 comprising the amino acid sequence of SEQ ID NO:294; and a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:295, a CDR2 comprising the amino acid sequence of SEQ ID NO:296, and a CDR3 comprising the amino acid sequence of SEQ ID NO:297.

[0341] Thus, BBK2, humanized BBK2, or chimeric BBK2 antibodies can be used in the anti-CD137 ADCs and methods described herein. Each of these antibodies can be conjugated to any of the cytotoxins described below using methods known in the art and described herein.

[0342] Anti-CD5 antibody In certain embodiments, the compositions and methods described herein include ADCs comprising an antibody or fragment thereof that specifically binds to human CD5. Human CD5 is also referred to as LEU1 or T1. Human CD5 is a type I transmembrane glycoprotein found on the surface of thymocytes, T lymphocytes, and a subset of B lymphocytes. Two isoforms of human CD5 have been identified. Isoform 1 contains 438 amino acids and is described in Jones.et al.(1988) Nature 323(6086),346-349 and in (NCBI Reference Sequence: NP_001333385.1): [ka]

[0343] T cells have been shown to express CD5, a cell adhesion molecule that is involved in both the proliferation response of activated T cells and T cell helper functions. It has also been shown to function as a receptor, delivering costimulatory signals to T cells by interacting with CD72, a cell surface protein found only on B cells. Antibodies or antigen-binding fragments thereof that bind CD5 can suppress T cell activation and T cell-mediated immune responses to hematopoietic stem cell grafts, for example, by inhibiting the interaction between CD5 and CD72. Antibodies and antigen-binding fragments thereof that bind CD5 can also be used to directly kill CD5+ T cells, for example, by conjugating the antibody or antigen-binding fragment thereof to a cytotoxin (such as those described herein or known in the art) or by using an unconjugated antibody or antigen-binding fragment thereof that can recruit complement proteins to T cells.

[0344] Furthermore, a subset of activated B cells has been shown to express CD5, an expression pattern that is particularly prevalent among autoreactive B cells (Werner-Favre et al., European Journal of Immunology 19:1209-1231 (1989), the disclosure of which is incorporated herein by reference in its entirety). CD5 has also been shown to be expressed by a subset of NK cells; in particular, among patients with multiple myeloma, which have been shown to harbor a population of low density CD5+ (CD5LOW+) NK cells, this surface antigen being involved in NK cell activation (Ishiyama et al., Anticancer Research 14:725-730 (1994), the disclosure of which is incorporated herein by reference in its entirety). Thus, antibodies or antigen-binding fragments thereof that specifically bind to CD5 can be used to attenuate the activation of B cells and NK cells. Antibodies or antigen-binding fragments thereof that bind CD5 can also be used to directly kill CD5+ B cells and NK cells, for example, by conjugating the antibody or antigen-binding fragment thereof to a cytotoxin (such as a cytotoxin described herein or known in the art), or by using an unconjugated antibody or antigen-binding fragment thereof capable of recruiting complement proteins to the B cell or NK cell.

[0345] The present invention encompasses ADCs comprising antibodies and antigen-binding fragments thereof that specifically bind to a CD5 polypeptide, e.g., a human CD5 polypeptide, and uses thereof. In an exemplary embodiment, the antibody or antigen-binding fragment thereof that specifically binds to a CD5 polypeptide comprises a heavy chain variable region and a light chain variable region.

[0346] In one embodiment, the ADC comprises an antibody comprising a heavy chain variable region comprising one or more complementarity determining regions (CDRs). In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:341. In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO:342. In one embodiment, the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO:343. In one embodiment, the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO:341, SEQ ID NO:342, and SEQ ID NO:343. In one embodiment, the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO:341, SEQ ID NO:342, and SEQ ID NO:343. In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO:341, a VH CDR2 comprising SEQ ID NO:342, and a VH CDR3 comprising SEQ ID NO:343.

[0347] In one embodiment, the ADC comprises an antibody comprising a light chain variable region comprising one or more complementarity determining regions (CDRs). In one embodiment, the light chain variable region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO:344. In one embodiment, the light chain variable region comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO:345. In one embodiment, the light chain variable region comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO:346. In one embodiment, the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO:344, SEQ ID NO:345, and SEQ ID NO:346. In one embodiment, the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO:344, SEQ ID NO:345, and SEQ ID NO:346. In one embodiment, the light chain variable region comprises a VL CDR1 comprising SEQ ID NO:344, a VL CDR2 comprising SEQ ID NO:345, and a VL CDR3 comprising SEQ ID NO:346.

[0348] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 comprising SEQ ID NO:341, a VH CDR2 comprising SEQ ID NO:342, and a VH CDR3 comprising SEQ ID NO:343, and a light chain variable region comprising a VL CDR1 comprising SEQ ID NO:344, a VL CDR2 comprising SEQ ID NO:345, and a VL CDR3 comprising SEQ ID NO:346.

[0349] In certain embodiments, one or more of the CDRs (i.e., one or more heavy chain CDRs having SEQ ID NOs: 341-343 and / or one or more light chain CDRs having SEQ ID NOs: 344-346) may contain conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD5 specificity of the antibody (i.e., specificity similar to that of an antibody or antigen-binding fragment thereof comprising the heavy chain CDRs of SEQ ID NOs: 341-343 and the light chain CDRs of SEQ ID NOs: 344-346).

[0350] In certain embodiments, the anti-CD5 antibody or antigen-binding fragment thereof is murine antibody 5D7, or a humanized version thereof. Murine antibody 5D7 binds to human CD5 and is described in US Patent Application Publication No. 20008 / 0245027, the contents of which are incorporated by reference with respect to the antibody sequences disclosed herein. SEQ ID NOs:353-358, as set forth in the Sequence Summary Table, correspond to the CDRs of murine anti-CD5 antibody 5D7. A humanized version of anti-CD5 antibody 5D7 is set forth in SEQ ID NO:359 (humanized heavy chain variable region) and SEQ ID NO:360 (humanized light chain variable region). In one embodiment, the ADCs and uses thereof described herein include antibodies comprising the CDRs set forth in SEQ ID NOs:353-358. In one embodiment, the ADCs and uses thereof described herein include antibodies comprising the heavy and light chain variable regions set forth in SEQ ID NOs:359 and 360, respectively.

[0351] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 359. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 359, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 359. In a particular embodiment, the antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO: 359 or a variant of SEQ ID NO: 359, wherein the variant (i) differs from SEQ ID NO: 359 in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 359 in at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 359 in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions. and / or (iv) an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:359, where in any of (i)-(iv), the amino acid substitutions may be conservative or non-conservative amino acid substitutions; and the modified heavy chain variable region may have enhanced biological activity compared to the heavy chain variable region of SEQ ID NO:359 while retaining the CD5 binding specificity of the antibody, i.e., having similar binding specificity to an antibody comprising SEQ ID NO:359 or an antigen-binding fragment thereof.

[0352] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 360. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 360, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 360. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain comprising SEQ ID NO: 360 or a variant of SEQ ID NO: 360, wherein the variant (i) differs from SEQ ID NO: 360 in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 360 in at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 360 in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions. 360, and / or (iv) an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:360, where in any of (i)-(iv) the amino acid substitutions can be conservative or non-conservative amino acid substitutions; and the modified light chain variable region can have enhanced biological activity compared to the light chain variable region of SEQ ID NO:360 while retaining the CD5 binding specificity of the antibody, i.e., having similar binding specificity to an antibody comprising SEQ ID NO:360, or an antigen-binding fragment thereof.

[0353] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO:359, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:359, and a light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO:360, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:360. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:359 and a light chain variable region comprising SEQ ID NO:360.

[0354] In another embodiment, the anti-CD5 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:353. In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO:354. In one embodiment, the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO:355. In one embodiment, the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO:353, SEQ ID NO:354, and SEQ ID NO:355. In one embodiment, the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO:353, SEQ ID NO:354, and SEQ ID NO:355. In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO:353, a VH CDR2 comprising SEQ ID NO:354, and a VH CDR3 comprising SEQ ID NO:355.

[0355] In one embodiment, the light chain variable region comprises one or more complementarity determining regions (CDRs). In one embodiment, the light chain variable region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO:356. In one embodiment, the light chain variable region comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO:357. In one embodiment, the light chain variable region comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO:358. In one embodiment, the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO:356, SEQ ID NO:357, and SEQ ID NO:358. In one embodiment, the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO:356, SEQ ID NO:357, and SEQ ID NO:358. In one embodiment, the light chain variable region comprises a VL CDR1 comprising SEQ ID NO:356, a VL CDR2 comprising SEQ ID NO:357, and a VL CDR3 comprising SEQ ID NO:358.

[0356] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 comprising SEQ ID NO:353, a VH CDR2 comprising SEQ ID NO:354, and a VH CDR3 comprising SEQ ID NO:355, and a light chain variable region comprising a VL CDR1 comprising SEQ ID NO:356, a VL CDR2 comprising SEQ ID NO:357, and a VL CDR3 comprising SEQ ID NO:358.

[0357] In certain embodiments, one or more of the CDRs (i.e., one or more heavy chain CDRs having SEQ ID NOs: 353-355, and / or one or more light chain CDRs having SEQ ID NOs: 356-358) may have conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD5 specificity of the antibody (i.e., specificity similar to that of an antibody or antigen-binding fragment thereof comprising the heavy chain CDRs of SEQ ID NOs: 353-355 and the light chain CDRs of SEQ ID NOs: 356-358).

[0358] Antibodies and antigen-binding fragments thereof capable of binding to the CD5 antigen can be identified using techniques known in the art and described herein, such as immunization methods, computational modeling techniques, and in vitro selection methods, such as the phage display and cell-based display platforms described below.

[0359] Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those having one or both of the following variable regions, or an amino acid sequence having at least 85% sequence identity thereto (e.g., an amino acid sequence having 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity thereto): V having the following amino acid sequence L [ka] and V having the following amino acid sequence H [ka]

[0360] The above V L and V H Antibodies and antigen-binding fragments thereof comprising the sequences are described, for example, in U.S. Pat. No. 5,869,619, the disclosure of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof, such as he1 antibodies. In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises the sequences of SEQ ID NO:325 and SEQ ID NO:326. L and V H In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises the V chain of SEQ ID NO:325 and SEQ ID NO:326. L and V H In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises the CDRs contained in the V chain of SEQ ID NO: 325 and SEQ ID NO: 326. L and V H CDRs included in the V L and V H The remainder of the sequence is SEQ ID NO:325 and SEQ ID NO:326. L and V H having at least 85% sequence identity to the sequence (eg, 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity).

[0361] In some embodiments, the anti-CD5 antibody, or antigen-binding fragment thereof, comprises the following CDRs: CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO:327); CDR-H2 having the amino acid sequence NTHTGE (SEQ ID NO: 328); CDR-H3 having the amino acid sequence RGYDWYFDV (SEQ ID NO: 329); CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO:330); CDR-L2 having the amino acid sequence RANRLVD (SEQ ID NO: 331); and CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO:332).

[0362] Additional anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those having one or both of the following variable regions, or an amino acid sequence having at least 85% sequence identity thereto (e.g., an amino acid sequence having 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity thereto): V having the following amino acid sequence L [ka] V having the following amino acid sequence H [ka]

[0363] Above V L and V H Antibodies and antigen-binding fragments thereof comprising the sequences are described, for example, in U.S. Pat. No. 5,869,619, the disclosure of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof, such as he3 antibodies. In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises the sequences of SEQ ID NO:327 and SEQ ID NO:328. L and V H V of an antibody containing chains L and V H In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises the CDRs contained in the V chain of SEQ ID NO: 327 and SEQ ID NO: 328. L and V H CDRs included in the V L and V H The remainder of the sequence is SEQ ID NO:327 and SEQ ID NO:328. L and V H having at least 85% sequence identity to the sequence (eg, 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity).

[0364] In some embodiments, the anti-CD5 antibody, or antigen-binding fragment thereof, comprises the following CDRs: CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 335); CDR-H2 having the amino acid sequence NTHYGE (SEQ ID NO: 336); CDR-H3 having the amino acid sequence RRGYDWYFDV (SEQ ID NO: 337); CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO:338); CDR-L2 having the amino acid sequence RANRLES (SEQ ID NO: 339); and CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO:340).

[0365] Antibodies and antigen-binding fragments thereof comprising the above CDR sequences are described, for example, in U.S. Pat. No. 5,869,619, the disclosure of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0366] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, the anti-CD5 antibodies described in U.S. Pat. Nos. 5,821,123; 5,766,886; 5,770,196; 7,153,932; 5,621,083; 6,649,742; 6,146,631; 5,756,699; 5,744,580; 6,376,217; 5,837,491; and 6,146,850, the disclosures of each of which are incorporated herein by reference as they relate to anti-CD5 antibodies and antigen-binding fragments thereof.

[0367] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, those produced by the hybridoma cell line deposited as ATCC CRL 8000 (anti-CD5 murine antibody OKT1). Such antibodies are described in U.S. Pat. Nos. 4,515,894; 4,657,760; and 4,363,799, the disclosures of each of which are incorporated herein by reference as they relate to anti-CD5 antibodies and antigen-binding fragments thereof.

[0368] Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those having one or more or all of the following CDRs: CDR-H1 having the amino acid sequence GYSITSGYY (SEQ ID NO:341); CDR-H2 having the amino acid sequence ISYSGFT (SEQ ID NO:342); CDR-H3 having the amino acid sequence AGDRTGSWFAY (SEQ ID NO:343); CDR-L1 having the amino acid sequence QDISNY (SEQ ID NO:344); CDR-L2 having the amino acid sequence ATS (SEQ ID NO: 345); and CDR-L3 having the amino acid sequence LQYASYPFT (SEQ ID NO:346).

[0369] Antibodies and antigen-binding fragments thereof comprising the above CDR sequences are described, for example, in U.S. Pat. No. 8,679,500, the disclosure of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0370] Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those having one or more or all of the following CDRs: CDR-H1 having the amino acid sequence GYIFTNYG (SEQ ID NO: 347); CDR-H2 having the amino acid sequence INTYNGEP (SEQ ID NO: 348); CDR-H3 having the amino acid sequence ARGDYYGYEDY (SEQ ID NO:349); CDR-L1 having the amino acid sequence QGISNY (SEQ ID NO:350); CDR-L2 having the amino acid sequence YTS (SEQ ID NO: 351); and CDR-L3 having the amino acid sequence QQYSKLPWT (SEQ ID NO:352).

[0371] Antibodies and antigen-binding fragments thereof comprising the above CDR sequences are described, for example, in US Pat. No. 8,679,500.

[0372] Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those having one or more or all of the following CDRs: CDR-H1 having the amino acid sequence FSLSTSGMG (SEQ ID NO: 353); CDR-H2 having the amino acid sequence WWDDD (SEQ ID NO: 354); CDR-H3 having the amino acid sequence RRATGTGFDY (SEQ ID NO:355); CDR-L1 having the amino acid sequence QDVGTA (SEQ ID NO:356); CDR-L2 having the amino acid sequence WTSTRHT (SEQ ID NO:357); and CDR-L3 having the amino acid sequence YNSYNT (SEQ ID NO: 358).

[0373] Antibodies and antigen-binding fragments thereof comprising the above CDR sequences are described, for example, in U.S. Patent Application Publication No. 2008 / 0254027, the disclosure of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0374] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those described in WO 1992 / 014491, such as, for example, the anti-CD5 antibody produced by the hybridoma cell line deposited at the Institut Pasteur under No. 1-1025 on Jan. 10, 1991. The disclosure of WO 1992 / 014491 is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0375] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, the anti-CD5 antibodies described in U.S. Pat. Nos. 6,010,902 and 7,192,736, U.S. Patent Application Publication Nos. 2011 / 0250203 and 2017 / 0129128, as well as WO 2016 / 172606; WO 1994 / 023747; and WO 1996 / 041608; the disclosures of each of which are incorporated by reference herein as they relate to anti-CD5 antibodies and antigen-binding fragments thereof.

[0376] In some embodiments, anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those that contain a combination of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions set forth in the table below.

[0377] [Table 5] TIFF2025060760000056.tif208156

[0378] Antibodies and antigen-binding fragments thereof comprising the above CDR sequences of the above table are described, for example, in U.S. Patent Application Publication No. 2011 / 0250203, the disclosure of which is incorporated by reference herein as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0379] Anti-CD2 antibody The compositions and methods described herein, in certain embodiments, encompass an antibody or fragment thereof that specifically binds to human CD2. Human CD2 is also referred to as T cell surface antigen T11 / Leu-5, T11, CD2 antigen (p50), and sheep erythrocyte 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): [ka]

[0380] The second isoform of CD2 is 377 amino acids and is identified herein as NCBI reference sequence: NP_001315538.1.

[0381] T cells and NK cells have been shown to express CD2, which is a cell adhesion molecule and a specific marker for such lymphocytes. For example, CD2 interacts with other adhesion molecules, such as lymphocyte function-associated antigen-3 (LFA-3 / CD58), to enhance T cell activation. Antibodies and antigen-binding fragments thereof capable of binding to CD2 can suppress T cell activation and T cell-mediated immune responses to hematopoietic stem cell grafts, for example, by inhibiting the interaction of CD2 with LFA-3. Antibodies and antigen-binding fragments thereof that bind to this cell surface antigen can be identified using techniques known in the art and described herein, including immunization methods, computational modeling techniques, and in vitro selection methods, such as the phage display and cell-based display platforms described below.

[0382] In certain embodiments, the invention encompasses ADCs and uses thereof that comprise an antibody or antigen-binding fragment thereof that specifically binds to a CD2 polypeptide, e.g., a human CD2 polypeptide.

[0383] In one embodiment, the ADC comprises an anti-CD2 antibody comprising a heavy chain variable region comprising one or more complementarity determining regions (CDRs). In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:300. In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO:301. In one embodiment, the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO:302. In one embodiment, the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO:300, SEQ ID NO:301, and SEQ ID NO:302. In one embodiment, the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO:300, SEQ ID NO:301, and SEQ ID NO:302. In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO:300, a VH CDR2 comprising SEQ ID NO:301, and a VH CDR3 comprising SEQ ID NO:302.

[0384] In one embodiment, the ADC comprises an anti-CD2 antibody comprising a light chain variable region comprising one or more complementarity determining regions (CDRs). In one embodiment, the light chain variable region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO:303. In one embodiment, the light chain variable region comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO:304. In one embodiment, the light chain variable region comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO:305. In one embodiment, the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO:303, SEQ ID NO:304, and SEQ ID NO:305. In one embodiment, the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO:303, SEQ ID NO:304, and SEQ ID NO:305. In one embodiment, the light chain variable region comprises a VL CDR1 comprising SEQ ID NO:303, a VL CDR2 comprising SEQ ID NO:304, and a VL CDR3 comprising SEQ ID NO:305.

[0385] In an exemplary embodiment, the anti-CD2 antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising a VH CDR1 comprising SEQ ID NO:300, a VH CDR2 comprising SEQ ID NO:301, and a VH CDR3 comprising SEQ ID NO:302, and a light chain variable region comprising a VL CDR1 comprising SEQ ID NO:303, a VL CDR2 comprising SEQ ID NO:304, and a VL CDR3 comprising SEQ ID NO:305.

[0386] In certain embodiments, one or more of the CDRs (i.e., one or more heavy chain CDRs having SEQ ID NOs: 300-302, and / or one or more light chain CDRs having SEQ ID NOs: 303-305) may have conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD2 specificity of the antibody (i.e., specificity similar to that of an antibody or antigen-binding fragment thereof comprising the heavy chain CDRs of SEQ ID NOs: 300-302 and the light chain CDRs of SEQ ID NOs: 303-305).

[0387] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 306. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 306, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 306. In a particular embodiment, the antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO: 306 or a variant of SEQ ID NO: 306, wherein the variant (i) differs from SEQ ID NO: 306 in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 306 in at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 306 in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions. 306, and / or (iv) an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:306, where in any of (i)-(iv), the amino acid substitutions may be conservative or non-conservative amino acid substitutions; and the modified heavy chain variable region may have increased biological activity compared to the heavy chain variable region of SEQ ID NO:306 while retaining the CD2 binding specificity of the antibody, i.e., having similar binding specificity to an antibody or antigen-binding fragment thereof comprising SEQ ID NO:306. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region that differs from the amino acid sequence set forth in SEQ ID NO:306 by 1, 2, 3, or 4 amino acids. For example, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region that differs from the amino acid sequence set forth in SEQ ID NO:306 by 1, 2, 3, or 4 of positions 12, 13, 28, and / or 48. In one embodiment, the heavy chain variable region differs from the amino acid sequence set forth in SEQ ID NO: 306 at positions 12, 13, 28, and 48. In one embodiment, the heavy chain variable region contains one, two, three, or four of the following substitutions relative to the sequence set forth in SEQ ID NO: 306: K12Q; K13R; T28I; and M48V.In one embodiment, the heavy chain variable region contains the substitutions K12Q; K13R; T28I; and M48V relative to SEQ ID NO:306.

[0388] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 307. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 307, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 307. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain comprising SEQ ID NO: 307 or a variant of SEQ ID NO: 307, wherein the variant (i) differs from SEQ ID NO: 307 in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 307 in at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 307 in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions. 7, and / or (iv) an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:307, where in any of (i)-(iv) the amino acid substitutions can be conservative or non-conservative amino acid substitutions; and the modified light chain variable region retains the CD2 binding specificity of the antibody, i.e., has similar binding specificity to an antibody comprising SEQ ID NO:307, or an antigen-binding fragment thereof, while having enhanced biological activity compared to the light chain variable region of SEQ ID NO:307.

[0389] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO:306, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO:306, and a light chain variable region comprising an amino acid sequence having at least about 95% identity to SEQ ID NO:307, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO:307. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:306 and a light chain variable region comprising SEQ ID NO:307. In one embodiment, the antibody is an Ab1 antibody comprising a heavy chain variable region comprising SEQ ID NO:306 and a light chain variable region comprising SEQ ID NO:307.

[0390] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 308. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 308, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO: 308. In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO:308, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO:308, and a light chain variable region comprising an amino acid sequence having at least about 95% identity to SEQ ID NO:309, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO:309. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:308 and a light chain variable region comprising SEQ ID NO:309. In one embodiment, the antibody is an Ab1a antibody comprising a heavy chain variable region comprising SEQ ID NO:308 and a light chain variable region comprising SEQ ID NO:309.

[0391] In one embodiment, the heavy chain variable region comprises one or more complementarity determining regions (CDRs). In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:3131. In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO:314. In one embodiment, the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO:315. In one embodiment, the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO:313, SEQ ID NO:314, and SEQ ID NO:315. In one embodiment, the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO:313, SEQ ID NO:314, and SEQ ID NO:315. In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO:313, a VH CDR2 comprising SEQ ID NO:314, and a VH CDR3 comprising SEQ ID NO:315.

[0392] In one embodiment, the heavy chain variable region comprises one or more complementarity determining regions (CDRs). In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:313. In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO:314. In one embodiment, the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO:316. In one embodiment, the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO:313, SEQ ID NO:314, and SEQ ID NO:316. In one embodiment, the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO:313, SEQ ID NO:314, and SEQ ID NO:316. In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO:313, a VH CDR2 comprising SEQ ID NO:314, and a VH CDR3 comprising SEQ ID NO:316.

[0393] In one embodiment, the light chain variable region comprises one or more complementarity determining regions (CDRs). In one embodiment, the light chain variable region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO:317. In one embodiment, the light chain variable region comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO:318. In one embodiment, the light chain variable region comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO:319. In one embodiment, the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO:317, SEQ ID NO:318, and SEQ ID NO:319. In one embodiment, the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO:317, SEQ ID NO:318, and SEQ ID NO:319. In one embodiment, the light chain variable region comprises a VL CDR1 comprising SEQ ID NO:317, a VL CDR2 comprising SEQ ID NO:318, and a VL CDR3 comprising SEQ ID NO:319.

[0394] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 comprising SEQ ID NO:313, a VH CDR2 comprising SEQ ID NO:314, and a VH CDR3 comprising SEQ ID NO:315, and a light chain variable region comprising a VL CDR1 comprising SEQ ID NO:317, a VL CDR2 comprising SEQ ID NO:318, and a VL CDR3 comprising SEQ ID NO:319.

[0395] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 comprising SEQ ID NO:313, a VH CDR2 comprising SEQ ID NO:314, and a VH CDR3 comprising SEQ ID NO:316, and a light chain variable region comprising a VL CDR1 comprising SEQ ID NO:317, a VL CDR2 comprising SEQ ID NO:318, and a VL CDR3 comprising SEQ ID NO:319.

[0396] In certain embodiments, one or more of the CDRs (i.e., one or more heavy chain CDRs having SEQ ID NOs: 313-316, and / or one or more light chain CDRs having SEQ ID NOs: 317-318) may contain conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD2 specificity of the antibody (i.e., comprising a heavy chain CDR of SEQ ID NO: 313-315 and a light chain CDR of SEQ ID NO: 18-20; or a specificity similar to that of an antibody or antigen-binding fragment thereof comprising a heavy chain CDR of SEQ ID NO: 313, 314, 316 and a light chain CDR of SEQ ID NO: 317-319).

[0397] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 320. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least about 95% identity to SEQ ID NO: 320, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO: 320. In certain embodiments, the antibody comprises a modified heavy chain (HC) variable region comprising a HC variable domain comprising SEQ ID NO:320 or a variant of SEQ ID NO:320, where the variant (i) differs from SEQ ID NO:320 in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO:320 in at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO:320 in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions. and / or (iv) an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO:320, where in any of (i)-(iv) the amino acid substitutions may be conservative or non-conservative amino acid substitutions; and the modified heavy chain variable region may have enhanced biological activity compared to the heavy chain variable region of SEQ ID NO:320 while retaining the CD2 binding specificity of the antibody, i.e., having similar binding specificity to an antibody comprising SEQ ID NO:320 or an antigen-binding fragment thereof.

[0398] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 321. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least about 95% identity to SEQ ID NO: 321, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO: 321. In certain embodiments, the antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO:320 or a variant of SEQ ID NO:321, where the variant (i) differs from SEQ ID NO:321 in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO:321 in at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO:321 in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions. and / or (iv) an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO:321, where in any of (i)-(iv) the amino acid substitutions may be conservative or non-conservative amino acid substitutions; and the modified heavy chain variable region may have enhanced biological activity compared to the heavy chain variable region of SEQ ID NO:321 while retaining the CD2 binding specificity of the antibody, i.e., having similar binding specificity to an antibody comprising SEQ ID NO:321 or an antigen-binding fragment thereof.

[0399] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 322. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least about 95% identity to SEQ ID NO: 322, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO: 322. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain comprising SEQ ID NO:322 or a variant of SEQ ID NO:322, where the variant (i) differs from SEQ ID NO:322 in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO:322 in at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO:322 in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions. and / or (iv) comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO:322, where in any of (i)-(iv) the amino acid substitutions may be conservative or non-conservative amino acid substitutions; and the modified light chain variable region retains the CD2 binding specificity of the antibody, i.e., has similar binding specificity to an antibody comprising SEQ ID NO:322, or an antigen-binding fragment thereof, while having enhanced biological activity compared to the light chain variable region of SEQ ID NO:322.

[0400] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO:320, e.g., at least about 95%, about 96%, about 97%, about 98%, or about 99%, or 100% identity to SEQ ID NO:320, and a light chain variable region comprising an amino acid sequence having at least about 95% identity to SEQ ID NO:322, e.g., at least about 95%, about 96%, about 97%, about 98%, or about 99%, or 100% identity to SEQ ID NO:322. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:320 and a light chain variable region comprising SEQ ID NO:322.

[0401] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least about 95% identity to SEQ ID NO:321, e.g., at least about 95%, about 96%, about 97%, about 98%, or about 99%, or 100% identity to SEQ ID NO:321, and a light chain variable region comprising an amino acid sequence having at least about 95% identity to SEQ ID NO:322, e.g., at least about 95%, about 96%, about 97%, about 98%, or about 99%, or 100% identity to SEQ ID NO:322. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:321 and a light chain variable region comprising SEQ ID NO:322.

[0402] Anti-CD2 antibodies that can 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:300); b. CDR-H2 having the amino acid sequence RIDPEDGSIDYVEKFKK (SEQ ID NO:301); c. CDR-H3 having the amino acid sequence GKFNYRFAY (SEQ ID NO:302); d. CDR-L1 having the amino acid sequence RSSQSLLHSSGNTYLN (SEQ ID NO:303); e. CDR-L2 having the amino acid sequence LVSKLES (SEQ ID NO:304); and f. CDR-L3 having the amino acid sequence MQFTHYPYT (SEQ ID NO:305).

[0403] Antibodies and antigen-binding fragments thereof comprising the above CDR sequences are described, for example, in U.S. Pat. No. 6,849,258, the disclosure of which is incorporated herein by reference as it relates to anti-CD2 antibodies and antigen-binding fragments thereof.

[0404] Antibodies and fragments thereof disclosed in U.S. Pat. Nos. 5,730,979; 5,817,311; 5,951,983; and 7,592,006, such as LO-CD2a, BTI-322, and antibodies produced by the hybridoma cell line deposited under ATCC Deposit No. HB 11423 (e.g., antibodies or antigen-binding fragments thereof that contain one or more or all of the CDR sequences of antibody LO-CD2a isolated from the hybridoma cell line deposited under ATCC Deposit No. HB 11423), can be used in conjunction with the compositions and methods disclosed herein. Exemplary antibodies that can be used in conjunction with the compositions and methods described herein include humanized antibodies that contain one or more or all of the CDR sequences of an antibody isolated from the hybridoma cell line deposited under ATCC Deposit No. HB 11423, such as MEDI-507. MEDI-507 is a humanized anti-CD2 monoclonal antibody comprising the CDR-H and CDR-L sequences (a) to (f) above, and is described in Branco et al., Transplantation 68:1588-1596 (1999). MEDI-507 is further described in WO 99 / 03502A1 and WO 1994 / 020619A1; U.S. Patent No. 7,592,006, U.S. Patent No. 6,849,258, U.S. Patent No. 5,951,983, U.S. Patent No. 5,817,311, and U.S. Patent No. 5,730,979; and U.S. Patent Application Publication No. 2011 / 0280868, U.S. Patent Application Publication No. 2004 / 0265315, and U.S. Patent Application Publication No. 2011 / 0091453, the disclosures of each of which are incorporated herein by reference as they relate to anti-CD2 antibodies and antigen-binding fragments thereof, such as the anti-CD2 antibody MEDI-507. In one embodiment, the anti-CD2 antibody is siplizumab or an antigen-binding fragment thereof.

[0405] Other anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, those described in U.S. Patent Nos. 6,541,611 and 7,250,167, the disclosures of which are incorporated herein by reference as they relate to anti-CD2 antibodies and antigen-binding fragments thereof, such as anti-CD2 antibody LO-CD2b and the antibody produced by the hybridoma cell line deposited under ATCC Deposit No. PTA-802. Exemplary antibodies that can be used in conjunction with the compositions and methods described herein include humanized antibodies that include one or more or all of the CDR sequences of the antibody isolated from the hybridoma cell line deposited under ATCC Deposit No. PTA-802.

[0406] Other anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, those described in U.S. Patent Nos. 5,795,572 and 5,807,734, each of which is incorporated by reference herein as the disclosures thereof relate to anti-CD2 antibodies and antigen-binding fragments thereof, such as the anti-CD2 antibody produced by the hybridoma cell line deposited under ATCC Deposit No. HB 69277. For example, anti-CD2 antibodies and antigen-binding fragments thereof that can be used in conjunction with the compositions and methods described herein include those that comprise a hinge region having the amino acid sequence of EPKSSDKTHTSPPSP (SEQ ID NO:316), such as an scFv fragment that comprises a hinge region having the amino acid sequence of EPKSSDKTHTSPPSP (SEQ ID NO:316). Incorporation of a hinge region having the amino acid sequence of SEQ ID NO:316 can be advantageous as this hinge motif is mutated relative to the wild-type hinge region sequence to remove potentially reactive cysteine ​​residues that may promote undesired oxidative dimerization of single-chain antibody fragments such as scFv fragments.

[0407] Other anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include those described in U.S. Patent No. 6,764,688, such as anti-CD2 antibody TS2 / 18 and the antibody produced by the hybridoma cell line deposited under ATCC Deposit No. HB-195. The disclosure of U.S. Patent No. 6,764,688 is incorporated herein by reference as it relates to anti-CD2 antibodies and antigen-binding fragments thereof.

[0408] Other anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, the anti-CD2 antibodies described in U.S. Pat. Nos. 6,162,432, 6,558,662, 7,408,039, 7,332,157, 7,638,121, 7,939,062, and 7,115,259, U.S. Patent Application Publication Nos. 2006 / 0084107, 2014 / 0369974, 2002 / 0051784, and 2013 / 0183322, the disclosures of each of which are incorporated herein by reference as they relate to anti-CD2 antibodies and antigen-binding fragments thereof, and WO 1992 / 016563.

[0409] Anti-Her2 antibody Antibodies specific for the Her2 antigen are known to those skilled in the art, for example trastuzumab.

[0410] Anti-PSMA antibody Antibodies specific for prostate-specific membrane antigen (PSMA) included in the ADC according to the present invention are disclosed in WO 2020 / 025564 A1, the disclosure of which is incorporated herein by reference in its entirety.

[0411] Fc mutations The antibodies or binding fragments described herein may also include modifications and / or mutations that may alter the properties of the antibody and / or fragment, such as those that increase half-life, increase or decrease ADCC, as known in the art.

[0412] In one embodiment, the antibody or binding fragment thereof has a variant Fc region, the variant Fc region comprising at least one amino acid modification relative to a wild-type Fc region such that the molecule has an altered affinity for FcγR. Certain amino acid positions within the Fc region are known to directly contact FcγR based on crystallographic studies, 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). Thus, the antibodies described herein (e.g., anti-CD117, CD45, CD137, CD2, CD5, CD262, or CD134) may comprise a variant Fc region comprising a modification of at least one residue that directly contacts FcγR based on structural and crystallographic analyses. In one embodiment, the Fc region of the antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991), expressly incorporated herein by reference. "EU index as in Kabat" refers to the numbering of human IgG1 EU antibodies. EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety). In one embodiment, the Fc region comprises a D265A mutation. In one embodiment, the Fc region comprises a D265C mutation. In some embodiments, the Fc region of the antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index as in Kabat. In one embodiment, the Fc region comprises a L234A mutation. In some embodiments, the Fc region of the antibody (or 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 a L235A mutation.In yet another embodiment, the Fc region comprises the L234A and L235A mutations.In a further embodiment, the Fc region of an antibody of an ADC described herein comprises the D265C, L234A, and L235A mutations.

[0413] In certain embodiments, the variant IgG Fc domain comprises one or more amino acid substitutions that reduce or eliminate binding affinity to FcγR and / or C1q compared to a wild-type Fc domain that does not comprise one or more 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, antibodies comprising modified Fc regions (e.g., comprising L234A, L235A, and D265C mutations) have substantially reduced or abolished effector functions.

[0414] Affinity for the Fc region can be measured using a variety of techniques known in the art, including, for example, but not limited to, equilibration methods (e.g., enzyme-linked immunoabsorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)), or by surface plasmon resonance assays or other mechanism kinetic-based assays (e.g., BIACORE™ analysis or Octet™ analysis (forteBIO)), and other methods such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). These and other methods may utilize labels on one or more of the components being investigated and / or utilize a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. One example of a competitive binding assay is a radioimmunoassay, which involves incubation of the antibody of interest with a labeled antigen in the presence of increasing amounts of unlabeled antigen and detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest to a particular antigen and the binding off-rate can be determined from the data by Scatchard plot analysis. Competition with a second antibody can 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.

[0415] Antibodies can be used, for example, (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. (2009) Eur J Immunol 29:2819-25), including at 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 T250Q, M252Y, 1253A, S254T, T256E, P2571, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R mutations.

[0416] Thus, in one embodiment, the Fc region comprises a mutation that results in a reduced half-life. An antibody with a short half-life may be advantageous when the antibody is expected to function as a temporary therapy, for example in certain cases of the pretreatment step described herein, where the antibody is administered followed by the administration of HSCs. Ideally, the antibody would be substantially removed before delivery of the HSCs, which generally express the antigen targeted by the ADCs described herein, such as CD117, but which, unlike endogenous stem cells, is not the target of the ADC. In one embodiment, the Fc region comprises a mutation at position 435 (EU index according to Kabat). In one embodiment, the mutation is an H435A mutation.

[0417] In one embodiment, the antibodies described herein have a half-life of 24 hours or less, a half-life of 22 hours or less, a half-life of 20 hours or less, a half-life of 18 hours or less, a half-life of 16 hours or less, a half-life of 14 hours or less, a half-life of 13 hours or less, 12 hours or less, or a half-life of 11 hours or less. In one embodiment, the half-life of the antibody is between 11 hours and 24 hours; between 12 hours and 22 hours; between 10 hours and 20 hours; between 8 hours and 18 hours; or between 14 hours and 24 hours.

[0418] In some embodiments, the Fc region comprises two or more mutations that confer a reduced half-life and substantially reduce or completely abolish the effector function of the antibody. In some embodiments, the Fc region comprises a mutation that results in a reduced half-life and a mutation in at least one residue that can directly contact FcγR (e.g., based on structural and crystallographic analysis). In one embodiment, the Fc region comprises a H435A mutation, a L234A mutation, and a L235A mutation. In one embodiment, the Fc region comprises a H435A mutation and a D265C mutation. In one embodiment, the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, and a D265C mutation.

[0419] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin (e.g., an amatoxin) via a cysteine ​​residue in the Fc domain of the antibody or antigen-binding fragment thereof. In some embodiments, the cysteine ​​residue is introduced by 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-CD117 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 a D265C mutation. In one embodiment, the Fc region comprises a D265C and H435A mutations. In one embodiment, the Fc region comprises a D265C, L234A, and L235A mutations. In one embodiment, the Fc region comprises a D265C, L234A, L235A, and H435A mutations.

[0420] In some embodiments of these aspects, the cysteine ​​residue naturally occurs in the Fc domain of the antibody or antigen-binding fragment thereof. For example, the Fc domain can be an IgG Fc domain, such as a human IgG1 Fc domain, and the cysteine ​​residue can be selected from the group consisting of Cys261, Csy321, Cys367, and Cys425.

[0421] For example, in one embodiment, the Fc region of antibody 67 has been modified to include a D265C mutation (e.g., SEQ ID NO:111). In another embodiment, the Fc region of antibody 67 has been modified to include a D265C, L234A, and L235A mutation (e.g., SEQ ID NO:112). In yet another embodiment, the Fc region of antibody 67 has been modified to include a D265C and H435A mutation (e.g., SEQ ID NO:113). In a further embodiment, the Fc region of antibody 67 has been modified to include a D265C, L234A, L235A, and H435A mutation (e.g., SEQ ID NO:114).

[0422] With respect to antibody 55, in one embodiment, the Fc region of antibody 55 has been modified to include a D265C mutation (e.g., SEQ ID NO:117). In another embodiment, the Fc region of antibody 55 has been modified to include a D265C, L234A, and L235A mutation (e.g., SEQ ID NO:118). In yet another embodiment, the Fc region of antibody 55 has been modified to include a D265C and H435A mutation (e.g., SEQ ID NO:119). In a further embodiment, the Fc region of antibody 55 has been modified to include a D265C, L234A, L235A, and H435A mutation (e.g., SEQ ID NO:120).

[0423] The Fc region of any one of antibody 54, antibody 55, antibody 56, antibody 57, antibody 58, antibody 61, antibody 66, antibody 67, antibody 68, or antibody 69 may be modified to include a D265C mutation (e.g., as in SEQ ID NO:123); D265C, L234A, and L235A mutations (e.g., as in SEQ ID NO:124); D265C and H435A mutations (e.g., as in SEQ ID NO:125); or D265C, L234A, L235A, and H435A mutations (e.g., as in SEQ ID NO:126).

[0424] The variant Fc domains described herein are defined according to the amino acid modifications that compose them. For all amino acid substitutions discussed herein for the Fc region, the numbering is always according to the EU index. Thus, for example, D265C is an Fc variant in which an aspartic acid (D) at EU position 265 is substituted with a cysteine ​​(C) relative to the parent Fc domain. Similarly, for example, D265C / L234A / L235A defines a variant Fc variant with substitutions at EU positions 265 (D to C), 234 (L to A), and 235 (L to A) relative to the parent Fc domain. A variant may also be designated according to its final amino acid composition at the mutated EU amino acid positions. For example, a L234A / L235A variant may be referred to as LALA. It is noted that the order in which the substitutions are provided may be arbitrary.

[0425] In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable region having an amino acid sequence at least 95%, 96%, 97% or 99% identical to a SEQ ID NO: disclosed herein. Alternatively, the antibody or antigen-binding fragment thereof comprises a CDR comprising a SEQ ID NO: disclosed herein, together with a framework region of a variable region described herein having an amino acid sequence at least 95%, 96%, 97% or 99% identical to a SEQ ID NO: disclosed herein.

[0426] In one embodiment, the 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 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 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.

[0427] How to Identify Antibodies Provided herein are novel ADCs that can be used, for example, in conditioning regimens for stem cell transplantation. In light of the disclosure herein, other antibodies that can be used in the ADCs and methods of the invention can be identified.

[0428] Methods for high throughput screening of antibody or antibody fragment libraries for molecules capable of binding to cell surface antigens (e.g., CD117, CD45, CD2, CD5, CD134, CD252, CD137) can be used to identify affinity matured antibodies useful for treating cancer, autoimmune diseases, and pre-treating patients (e.g., human patients) in need of hematopoietic stem cell therapy as described herein. Such methods include in vitro display techniques known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display, among others. The use of phage display to isolate ligands that bind biologically important 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, the disclosures of which are incorporated herein by reference as they relate to in vitro display techniques. Randomized combinatorial peptide libraries have been constructed to select polypeptides that bind to cell surface antigens, as described in Kay, Perspect. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, 1996, the disclosures of which are incorporated herein by reference as they relate to the discovery of antigen-binding molecules. Proteins, such as multimeric proteins, have been successfully phage displayed as functional molecules (see, e.g., EP 0349578; EP 4527839; and EP 0589877, each of which is incorporated by reference herein as it relates to the use of in vitro display techniques for the discovery of antigen-binding molecules, and Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992).Additionally, functional antibody fragments such as Fab and scFv fragments have been expressed in in vitro display formats (see, e.g., McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, the disclosures of each of which are incorporated herein by reference as they relate to in vitro display platforms for the discovery of antigen-binding molecules). These techniques can be used, among others, to identify and improve the affinity of antibodies that bind, for example, CD117, CD45, CD2, CD5, CD134, CD252, CD137 (e.g., GNNK+CD117), which can then be used to deplete endogenous hematopoietic stem cells in patients (e.g., human patients) in need of hematopoietic stem cell transplantation therapy.

[0429] In addition to in vitro display techniques, computational modeling techniques can be used to design and identify in silico antibodies and antibody fragments that bind to cell surface antigens (e.g., CD117, CD45, CD2, CD5, CD134, CD252, CD137). For example, using computational modeling techniques, one of skill in the art can screen libraries of antibodies and antibody fragments in silico for molecules capable of binding to a specific epitope, such as an extracellular epitope, of the antigen. Antibodies and antigen-binding fragments thereof identified by these computational techniques can be used in conjunction with the therapeutic methods described herein, such as the cancer and autoimmune disease therapeutic methods described herein and the patient pretreatment procedures described herein.

[0430] Additional techniques can be used to identify antibodies and antigen-binding fragments thereof that bind to cell surface antigens (e.g., CD117) on the surface of cells (e.g., cancer cells, autoimmune cells, or hematopoietic stem cells) and are internalized by the cells, e.g., by receptor-mediated endocytosis. For example, the in vitro display techniques described above can be adapted to screen for antibodies and antigen-binding fragments thereof that bind to cell surface antigens (e.g., CD117) on the surface of cancer cells, autoimmune cells, or hematopoietic stem cells and are subsequently internalized. Phage display represents one such technique that can be used in conjunction with this screening paradigm. To identify antibodies and fragments thereof that bind to cell surface antigens (e.g., CD117) and are subsequently internalized by cancer cells, autoimmune cells, or hematopoietic stem cells, one of skill in the art can adapt, for example, 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, mutagenesis techniques known i...

Claims

1. Formula (An) or Formula (Bn) 【Chemistry 1】 wherein n is 2, 3, 4, 5, 6, 7, 8, or 9. or a derivative or analog thereof.

2. Formula (A) 【Chemistry 2】 Formula (A) (HDP 30.2867) or a derivative or analog thereof, or an enantiomer or diastereomer thereof.

3. Formula (B) 【Chemistry 3】 Formula (B) (HDP 30.0880) or a derivative or analog thereof, or an enantiomer or diastereomer thereof.

4. 4. An amatoxin or a derivative or analogue thereof according to any one of claims 1 to 3 for use in the preparation of an antibody-drug conjugate (ADC).

5. The compound comprises an antibody or antigen-binding fragment thereof conjugated to an amatoxin by a linker, and has the formula (I): 【Chemistry 4】 (In the formula: Q is S or a sulfoxide group; L is a non-cleavable linker; Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the antibody or antigen-binding fragment thereof; and Ab is the antibody or antigen-binding fragment thereof. or a stereoisomer thereof.

6. Formula (Ia): 【Chemistry 5】 6. The ADC of claim 5 having the structure:

7. Formula (Ib): 【Chemistry 6】 6. The ADC of claim 5 having the structure:

8. L is a bond, -(C=O)-, -C(O)NH- group, -OC(O)NH- group, C １ ~C ６ Alkylene, C １ ~C ６ Heteroalkylene, C ２ ~C ６ Alkenylene, C ２ ~C ６ Heteroalkenylene, C ２ ~C ６ Alkynylene, C ２ ~C ６ Heteroalkynylene, C ３ ~C ６ Cycloalkylene, heterocycloalkylene, arylene, heteroarylene, p is an integer of 1 to 6 -(CH ２ CH ２ O) ｐ -group, or one or more solubility enhancing groups; Here, each C １ ~C ６ Alkylene, C １ ~C ６ Heteroalkylene, C ２ ~C ６ Alkenylene, C ２ ~C ６ Heteroalkenylene, C ２ ~C ６ Alkynylene, C ２ ~C ６ Heteroalkynylene, C ３ ~C ６ the cycloalkylene, heterocycloalkylene, arylene, or heteroarylene can be optionally substituted by 1 to 5 substituents independently selected at each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro; Or, each C １ ~C ６ Alkylene, C １ ~C ６ Heteroalkylene, C ２ ~C ６ Alkenylene, C ２ ~C ６ Heteroalkenylene, C ２ ~C ６ Alkynylene, C ２ ~C ６ Heteroalkynylene, C ３ ~C ６ The ADC of any one of claims 5 to 7, wherein the cycloalkylene, heterocycloalkylene, arylene, or heteroarylene can be optionally interrupted by one or more heteroatoms selected from O, S, and N.

9. The solubility enhancing group is of the formula -O ａ -C(O)NH-SO ２ -NR １ - has During the ceremony: a is 0 or 1; and R １ is hydrogen, C １ ~C ２４ Alkyl group, C ３ ~C ２４ Cycloalkyl group, C ２ ~C ２４ (Hetero)aryl groups, C ３ ~C ２４ Alkyl(hetero)aryl groups, and C ３ ~C ２４ (hetero)arylalkyl groups, each of which is selected from the group consisting of O, S, and NR ３ and optionally substituted or interrupted by one or more heteroatoms selected from, where R ３ is hydrogen and C １ ~C ４ 9. The ADC of claim 8, wherein each of the groups is independently selected from the group consisting of alkyl.

10. L is -(CH ２ ) ｎ The ADC of any one of claims 5 to 9, comprising a - unit.

11. L is -(CH ２ ) ｎ The ADC of claim 10,

12. Ab, Z, and L taken together as Ab-Z-L have the formula: 【Chemistry 7】 (wherein S is the sulfur atom of a cysteine ​​residue present in the antibody or antigen-binding fragment thereof).

13. The compound comprises an antibody or antigen-binding fragment thereof conjugated to an amatoxin by a linker, and has the formula (I): 【Chemistry 8】 (In the formula: Q is S or a sulfoxide group; L is a cleavable linker; Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the antibody or antigen-binding fragment thereof; and Ab is the antibody or antigen-binding fragment thereof. or a stereoisomer thereof.

14. Formula (Ia): 【Chemistry 9】 14. The ADC of claim 13 having the structure:

15. Formula (Ib): 【Chemistry 10】 14. The ADC of claim 13 having the structure:

16. L is a hydrazine, a disulfide, a thioether, an amino acid, a peptide of up to 10 amino acids, a p-aminobenzyl (PAB) group, a heterocyclic self-immolative group, C １ ~C ６ Alkyl, C １ ~C ６ Heteroalkyl, C ２ ~C ６ Alkenyl, C ２ ~C ６ Heteroalkenyl, C ２ ~C ６ Alkynyl, C ２ ~C ６ Heteroalkynyl, C ３ ~C ６ Cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -(C=O)- group, -C(O)NH- group, -OC(O)NH- group, -(CH ２ CH ２ O) ｐ -group, or one or more solubility enhancing groups; Here, each C １ ~C ６ Alkyl, C １ ~C ６ Heteroalkyl, C ２ ~C ６ Alkenyl, C ２ ~C ６ Heteroalkenyl, C ２ ~C ６ Alkynyl, C ２ ~C ６ Heteroalkynyl, C ３ ~C ６ cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups can be optionally substituted by 1 to 5 substituents independently selected at each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro; Or each C １ ~C ６ Alkyl, C １ ~C ６ Heteroalkyl, C ２ ~C ６ Alkenyl, C ２ ~C ６ Heteroalkenyl, C ２ ~C ６ Alkynyl, C ２ ~C ６ Heteroalkynyl, C ３ ~C ６ 16. The ADC of any one of claims 13 to 15, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can be optionally interrupted by one or more heteroatoms selected from O, S, and N.

17. The solubility enhancing group is of the formula -O ａ -C(O)NH-SO ２ -NR １ - has During the ceremony: a is 0 or 1; and R １ is hydrogen, C １ ~C ２４ Alkyl group, C ３ ~C ２４ Cycloalkyl group, C ２ ~C ２４ (Hetero)aryl groups, C ３ ~C ２４ Alkyl(hetero)aryl groups, and C ３ ~C ２４ (hetero)arylalkyl groups, each of which is selected from the group consisting of O, S, and NR ３ and optionally substituted or interrupted by one or more heteroatoms selected from, where R ３ is hydrogen and C １ ~C ４ 17. The ADC of claim 16, wherein each of said groups is independently selected from the group consisting of alkyl.

18. 18. The ADC of any one of claims 13-17, wherein L comprises a peptide selected from the group consisting of Phe-Lys, Val-Lys, Phe-Ala, Phe-Cit, Val-Ala, Val-Cit, and Val-Arg.

19. 20. The ADC of claim 18, further comprising a PAB group.

20. L is of the formula: 【Chemistry 11】 20. The ADC of claim 19, wherein the ADC is represented by:

21. The antibody conjugated to an amatoxin has the formula (II): 【Chemistry 12】 or a stereoisomer thereof.

22. Formula (IIa): 【Chemistry 13】 22. The ADC of claim 21 having the structure:

23. Formula (IIb): 【Chemistry 14】 22. The ADC of claim 21 having the structure:

24. 24. The ADC of any one of claims 5 to 23, wherein the antibody or antigen-binding fragment thereof specifically binds to an antigen expressed on the cell surface of a cancer cell, or a human stem cell, in particular a hematopoietic stem cell (HSC), or a T cell.

25. The ADC of any one of claims 5 to 24, wherein the antibody or antigen-binding fragment thereof specifically binds to human Her2, PSMA, CD37, or CD123.

26. 26. The ADC of any one of claims 5 to 25, wherein the antibody or antigen-binding fragment thereof comprises an Fc region comprising at least one mutation selected from the group consisting of D265C, D265A, A118C, L234A, or L235A (according to the EU index).

27. The ADC of any one of claims 5 to 23, wherein the antibody or antigen-binding fragment thereof specifically binds to PSMA and comprises a CDRH1 according to SEQ ID NO: 378, a CDRH2 according to SEQ ID NO: 379, a CDRH3 according to SEQ ID NO: 380, a CDRL1 according to SEQ ID NO: 381, a CDRL2 according to SEQ ID NO: 382, ​​and a CDRL3 according to SEQ ID NO:

383.

28. 28. The ADC of claim 27, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region according to SEQ ID NO: 375 and a light chain variable region according to SEQ ID NO:

377.

29. 29. The ADC of claim 28, wherein the antibody comprises a heavy chain according to SEQ ID NO: 371, SEQ ID NO: 372, SEQ ID NO: 373, or SEQ ID NO: 374 and a light chain according to SEQ ID NO: 376, or an antigen-binding fragment thereof.

30. 1. An antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof conjugated to an amatoxin or a derivative or analogue thereof by a linker, wherein the antibody or antigen-binding fragment thereof comprises an Fc region comprising at least two mutations consisting of L234A and L235A (according to the EU index).

31. 31. The antibody-drug conjugate (ADC) of claim 30, wherein the Fc region further comprises the mutation D265C (according to the EU index).

32. 32. The antibody-drug conjugate (ADC) of claim 30 or 31, wherein the antibody or antigen-binding fragment thereof specifically binds to an antigen expressed on the cell surface of a cancer cell, preferably a human cancer cell.

33. 33. The antibody-drug conjugate (ADC) of claim 32, wherein the antibody or antigen-binding fragment thereof specifically binds to prostate-specific membrane antigen (PSMA), Her2 antigen, CD37, or CD123.

34. The antibody drug conjugate (ADC) of any one of claims 30 to 32, wherein the antibody or antigen-binding fragment thereof specifically binds to PSMA and comprises a CDRH1 according to SEQ ID NO: 378, a CDRH2 according to SEQ ID NO: 379, a CDRH3 according to SEQ ID NO: 380, a CDRL1 according to SEQ ID NO: 381, a CDRL2 according to SEQ ID NO: 382, ​​and a CDRL3 according to SEQ ID NO:

383.

35. The antibody-drug conjugate (ADC) of claim 34, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region according to SEQ ID NO: 375 and a light chain variable region according to SEQ ID NO:

377.

36. 36. The antibody drug conjugate (ADC) of claim 35, wherein the antibody comprises a heavy chain according to SEQ ID NO: 372, SEQ ID NO: 373, or SEQ ID NO: 374 and a light chain according to SEQ ID NO: 376, or an antigen-binding fragment thereof.

37. The antibody-drug conjugate (ADC) of any one of claims 30 to 36, wherein the antibody or antigen-binding fragment thereof is conjugated to any compound selected from the group consisting of HDP30.2060, HDP30.2115, HDP30.2347, HDP30.1699, HDP30.2371, HDP30.0880, and HDP30.2867.

38. An antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and comprises a heavy chain having an amino acid sequence according to SEQ ID NO:374 and a light chain having an amino acid sequence according to SEQ ID NO:376, conjugated to the compound HDP30.2060.

39. An antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and comprises a heavy chain having an amino acid sequence according to SEQ ID NO:374 and a light chain having an amino acid sequence according to SEQ ID NO:376, conjugated to the compound HDP30.2115.

40. An antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and comprises a heavy chain having an amino acid sequence according to SEQ ID NO:374 and a light chain having an amino acid sequence according to SEQ ID NO:376, conjugated to the compound HDP30.2347.

41. An antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and comprises a heavy chain having an amino acid sequence according to SEQ ID NO:374 and a light chain having an amino acid sequence according to SEQ ID NO:376, conjugated to the compound HDP30.1699.

42. An antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and comprises a heavy chain having an amino acid sequence according to SEQ ID NO:374 and a light chain having an amino acid sequence according to SEQ ID NO:376, conjugated to the compound HDP30.2371.

43. An antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and comprises a heavy chain having an amino acid sequence according to SEQ ID NO:374 and a light chain having an amino acid sequence according to SEQ ID NO:376, conjugated to the compound HDP30.0880.

44. An antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human PSMA and comprises a heavy chain having an amino acid sequence according to SEQ ID NO:374 and a light chain having an amino acid sequence according to SEQ ID NO:376, conjugated to the compound HDP30.2867.

45. 45. The antibody drug conjugate (ADC) of any one of claims 37 to 44, having a drug to antibody ratio (DAR) of about 1, 2, 3, or 4, preferably a DAR of 2.

46. 46. ​​The ADC of any one of claims 5 to 45 for use in treating cancer in a patient, particularly selected from the group consisting of breast cancer, pancreatic cancer, bile duct cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, prostate cancer, gastric cancer, renal cancer, malignant melanoma, blood system cancer, leukemia, and malignant lymphoma.

47. 32. The antibody-drug conjugate (ADC) of claim 30 or 31, wherein the antibody or antigen-binding fragment thereof specifically binds to an antigen expressed on the cell surface of a hematopoietic stem cell (HSC), preferably a human HSC.

48. 16. A method of depleting a population of cells in a human subject, comprising administering to the subject an ADC of any one of claims 5 to 24 and 47, wherein the ADC comprises an antibody or antigen-binding fragment thereof that specifically binds to an extracellular antigen expressed by cells in the population of cells.

49. A method of pretreating a human subject for cell transplantation, comprising administering to the human subject an ADC according to any one of claims 5 to 23 and 47, such that endogenous stem cells or endogenous immune cells in the human subject are depleted, wherein the ADC specifically binds to an extracellular antigen expressed by the endogenous stem cells or endogenous immune cells.

50. 50. The method of claim 48 or 49, further comprising administering to the human subject allogeneic stem cells or allogeneic immune cells.

51. 51. The method of any one of claims 48-50, wherein the ADC specifically binds to an extracellular antigen expressed on an immune cell, and the subject has graft-versus-host disease (GVHD) or is at risk of developing graft-versus-host disease.

52. 48. A pharmaceutical composition comprising the ADC of any one of claims 5 to 47 and at least a pharma- ceutically acceptable carrier.

53. 46. ​​Use of the ADC of any one of claims 5 to 45 for the treatment of cancer in a patient, particularly selected from the group consisting of breast cancer, pancreatic cancer, bile duct cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, prostate cancer, gastric cancer, renal cancer, malignant melanoma, blood system cancer, leukemia, and malignant lymphoma.

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