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WO2024211234A1 - Conjugués anticorps-médicament et leurs utilisations - Google Patents

Conjugués anticorps-médicament et leurs utilisations Download PDF

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Publication number
WO2024211234A1
WO2024211234A1 PCT/US2024/022544 US2024022544W WO2024211234A1 WO 2024211234 A1 WO2024211234 A1 WO 2024211234A1 US 2024022544 W US2024022544 W US 2024022544W WO 2024211234 A1 WO2024211234 A1 WO 2024211234A1
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Prior art keywords
substituted
unsubstituted
substituent group
adc
pharmaceutically acceptable
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PCT/US2024/022544
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English (en)
Inventor
Yanwen Fu
Yufeng Hong
Ernest William Kovacs
Moli LIU
Hong Zhang
Lingna Li
Aaron SPRINGER
Original Assignee
Sorrento Therapeutics, Inc.
Vivasor, Inc.
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Publication of WO2024211234A1 publication Critical patent/WO2024211234A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • ADCs Antibody-Drug Conjugates
  • ADCs allow for the targeted delivery of a drug moiety to a tumor, and, in some embodiments intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).
  • ADCs are targeted chemotherapeutic molecules which combine properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing tumor cells (Teicher, B.A.
  • ADCs comprising an anti-CD25 antibody conjugated to camptothecin derivative toxins or duostatin derivative toxins through linker moieties.
  • the anti-CD25 antibody binds to CD25-expressing cancer cells and allows for selective uptake of the ADC into the cancer cells.
  • the ADCs provided herein selectively deliver an effective amount of the camptothecin derivative toxin or the duostatin derivative toxin to tumor tissue and reduce the non-specific toxicity associated with related ADCs.
  • the ADC compounds described herein include those with anticancer activity.
  • CD 25 is type I transmembrane protein expressed on the surface of both immune and non-immune cells. In addition to being overexpressed on certain cancer cells, CD25 is present on activated T-cells and B-cells, some thymocytes, and oligodendrocytes. CD25 is the alpha-chain of the heterotrimer IL-2 receptor.
  • CD25 On activated T-cells CD25 forms heterotrimers together with the beta- and gamma subunits (CD122 and CD132), thus forming a high-affinity receptor for IL- 2.
  • CD25 is highly expressed in many types of hematological malignancies, it is also highly expressed in activated circulating immune cells and regulatory T cells (Tregs).
  • CD25 (interleukin (IL)-2R ⁇ ) is part of the heterotrimeric IL-2 receptor that regulates normal immune function and is widely expressed on the surface of leukemias and lymphomas (Flynn M.J. et al., (2016) Mol Cancer Ther 15:2709–21; Zammarchi F.
  • Interleukin 2 plays an important role in the initiation and activation of T cellular responses and is involved in the development of CD4+CD25+ T regulatory cells (Tregs).
  • Tregs Regulatory T cells
  • TME immunosuppressive tumor microenvironment
  • Tregs and effector T cells appear to influence the outcome of immunotherapies, and poor prognosis in solid tumors is often associated with high tumor infiltration by Tregs and a low ratio of Teffs to Tregs (Arce V.F. et al., (2017) Immunity 46:577–86).
  • Teffs effector T cells
  • Various studies have analyzed the interaction between IL-2 and CD25 in mouse models. Blockade of IL-2 binding to CD25 by anti-CD25 antibodies such as PC61 (Moreau, J. L. et al., (1987) Eur. J.
  • CD25 is over-expressed in many types of cancers, including Hodgkin's and non-Hodgkin's Lymphoma, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, (FL), Mantle Cell lymphoma (MCL), chronic lymphatic lymphoma (CLL), Marginal Zone B-cell lymphoma (MZBL) and leukemias such as Hairy cell leukemia (HCL), Hairy cell leukemia variant (HCL-v), Acute Myeloid Leukemia (AML), and Acute Lymphoblastic Leukemia (ALL) such as Philadelphia chromosome-positive ALL (Ph+ALL) or
  • Solid tumors may lack expression of CD25, however, they may be tumors with high levels of infiltrating regulatory T-cells which may be treated with ADCs comprising an anti-CD25 antibody (Ménétrier-Caux C. et al., (2012) Targ Oncol 7:15-28; Arce V. et al., (2017) Immunity 46:1-10; Tanaka A. et al., (2017) Cell Res. 27(1):109-118).
  • the solid tumor that can be treated with ADCs comprising an anti- CD25 antibody may be pancreatic cancer, breast cancer, colorectal cancer, gastric and esophageal cancer, melanoma, non-small cell lung cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, and head and neck cancer.
  • Targeting CD25 not only blocks IL-2 from binding to the IL-2 receptor on the cancer cells, thus suppressing their growth, but also blocks the IL-2 from binding to the IL-2 receptor on the Tregs (where CD25 is also highly overexpressed), thus reducing their immunosuppressive activity.
  • Camptothecin is a cytotoxic quinoline alkaloid isolated from Camptotheca acuminta, a type of tree natively growing in China. CPT was discovered in the 1960s (Wall M.E. et al., 1966, J. Am. Chem. Soc. 88:3888-3890). The antitumor activity of Camptothecin depends on a highly specific inhibition of Topoisomerase-I (TOPO 1). The enzyme TOPO 1 cleaves one strand of double stranded DNA, partially unwinds the DNA, and then reanneals the strand to relieve tension.
  • TOPO 1 Topoisomerase-I
  • Camptothecin and its derivatives bind to the TOPO 1/DNA complex to prevent reannealing, which can cause cell death due to the accumulation of partially cleaved DNA (Hsiang Y. H., et al, 1985, J. Biol. Chem.260:14873-14878). [0013] The clinical application of camptothecin is limited due to its low solubility as well as serious side-effects (Joerger M. et al., 2015, Br. J. Clin. Pharmacol. 80:128-138; Joerger M. et al., 2015, Invest. New Drugs 33:472-479).
  • camptothecin derivatives have been developed to date, including topotecan (9-dimethyl amino-10-hydroxy camptothecin; TPT) and irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin; CPT-11)
  • topotecan (9-dimethyl amino-10-hydroxy camptothecin; TPT
  • irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin; CPT-11)
  • NPT topotecan
  • irinotecan 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin; CPT-11
  • the US Food and Drug Administration has approved these CPT derivatives for ovarian and colon cancer treatment (Vladu et al., 2000, Mol. Pharmacol.
  • camptothecin derivative is exatecan, which is a water soluble derivative of camptothecin (US patent Nos.10,195,288, 8,575,188). Unlike irinotecan currently used in clinical settings, an activation by an enzyme is unnecessary. Dxd is another useful camptothecin derivative.
  • camptothecin drugs are widely applied clinically, and the main indications are bone cancer, prostatic cancer, breast cancer, gastric cancer, pancreatic cancer, ovarian cancer, esophageal cancer, endometrial cancer and the like (Iqbal et al., 2014, Mol.
  • Dolastatins such as natural product Dolastatin 10, and its synthetic derivatives Monomehtyl Auristatin E (MMAE) and Monomethyl Auristatin F (MMAF) are products that show potent antineoplastic and tubulin inhibitory property. Because of their high toxicity, the direct use of Dolastatins as therapeutic agents has not been effective. Instead, they were conjugated to an antibody for targeted delivery to kill cancer cells.
  • ADCs antibody-drug conjugates
  • methods of preparing ADCs comprising an anti-CD25 antibody are methods for treating cancers, such as CD25-expressing cancers, using the ADCs disclosed herein.
  • the present disclosure provides an antibody drug conjugate (ADC), having an IgG antibody that binds to a CD25 target, conjugated at one or more cysteine sites of the IgG antibody.
  • the present disclosure provides an antibody drug conjugate (ADC), having an IgG antibody that binds to a CD25 target, conjugated at one or more lysine sites of the IgG antibody.
  • ADC antibody drug conjugate
  • the present disclosure provides an antibody drug conjugate (ADC), having a modified IgG antibody that binds to a CD25 target.
  • the present disclosure further provides a method for treating Hodgkin's and non-Hodgkin's Lymphoma and leukemias, comprising providing an effective amount of a CD25 ADC.
  • a method of treating a CD25-expressing cancer in a subject in need thereof including administering the ADC described herein (including in an aspect, embodiment, table, example, or claim), or a pharmaceutically acceptable salt thereof, to the subject.
  • an antibody drug conjugate (ADC) of formula (I) , formula ( II) , or formula (III) , or a pharmaceutically acceptable salt thereof said method including reacting an anti-CD25 antibody, or a modified antibody with a molecule of formula (P-I) , formula (P-II) , or formula (P-III) , or a pharmaceutically acceptable salt thereof, wherein B is a reactive moiety capable of forming a bond with an anti-CD25 antibody;
  • L 2 is a bond -C(O)- -NH- Amino Acid Unit, –(CH 2 CH 2 O) n –, –(CH 2 ) n –, –(4-aminobenzyloxycarbonyl)–, -O-, –(C(O)CH 2 CH 2 NH)–, –(C(O)N(R 2 )CH2CH2N(R 3 ))–, or any combination
  • FIG. 1 shows the chemical structures of linker-payload compounds that were linked to anti-CD25 antibody (MAA-V clone) to synthesize the ADCs used in the in vitro and in vivo efficacy studies.
  • FIG. 2 shows averaged results, of two replicates of CD25 receptor quantification experiments in various human cancer cell lines (SU-DHL-1, HDLM-2, Karpas 200T, and Daudi).
  • FIG.3A-D shows results of an in vitro efficacy study of anti-CD25 antibody (MAA-V clone) linked duostatin or camptothecin derivatives (ADCs) in: SU-DHL-1 (CD25 +) cells (FIG. 3A), HDLM-2 (CD25 +) cells (FIG. 3B), Karpas 299T (CD25 +) cells (FIG.3C), and Daudi (CD25-) cells (FIG. 3D).
  • FIG. 3A-D shows results of an in vitro efficacy study of anti-CD25 antibody (MAA-V clone) linked duostatin or camptothecin derivatives (ADCs) in: SU-DHL-1 (CD25 +) cells (FIG. 3A), HDLM-2 (CD25 +) cells (FIG. 3B), Karpas 299T (CD25 +) cells (FIG.3C), and Daudi (CD25-) cells (FIG. 3D).
  • FIG. 3A shows results
  • FIG. 4A-B shows results of an in vitro efficacy study of anti-CD25 antibody (MAA-V clone) linked camptothecin derivatives (ADCs) in: SU-DHL-1 (CD25 +) cells (FIG. 4A) and Daudi (CD25 -) cells (FIG.4B).
  • FIG. 5 shows results of an in vivo efficacy study in SU-DHL-1 xenograft in Nu/Nu nude mice of anti-CD25 antibody (MAA-V clone) linked camptothecin derivatives (ADCs), where the mice were treated once intravenously with 3 mg/kg of ADC.
  • FIG. 5 shows results of an in vivo efficacy study in SU-DHL-1 xenograft in Nu/Nu nude mice of anti-CD25 antibody (MAA-V clone) linked camptothecin derivatives (ADCs), where the mice were treated once intravenously with 3 mg/kg of ADC.
  • FIG. 7A-B shows results of an in vivo efficacy study in SU-DHL-1 xenograft in Nu/Nu nude mice of anti-CD25 antibody (MAA-V clone) linked duostatin or camptothecin derivatives (ADCs), where the mice were treated once intravenously with 3 mg/kg of ADC.
  • CD25-L078-118 ADC FIG.7A
  • CD25-L014-077 ADC FIG.7B
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone) [0035]
  • the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system.
  • “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art.
  • “about” or “approximately” can mean a range of up to 10% (i.e., ⁇ 10%) or more depending on the limitations of the measurement system.
  • about 5 mg can include any number between 4.5 mg and 5.5 mg.
  • the terms can mean up to an order of magnitude or up to 5-fold of a value.
  • Numerical ranges include the endpoints of the range. For example, “between 4.5 mg and 5.5 mg” includes 4.5 mg, 5.5 mg, and all values greater than 4.5 mg and less than 5.5 mg.
  • “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like. “Consisting essentially of or “consists essentially” likewise has the meaning ascribed in U.S.
  • polypeptide polypeptide
  • peptide and “protein” and other related terms used herein are used interchangeably to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • a "fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.
  • Polypeptides include mature molecules that have undergone cleavage. These terms encompass native and artificial proteins, protein fragments and polypeptide analogs (such as muteins variants chimeric proteins and fusion proteins) of a protein sequence as well as post-translationally, or otherwise covalently or non-covalently, modified proteins.
  • Two or more polypeptides e.g., 3 polypeptide chains
  • a polypeptide complex can be dimeric, trimeric, tetrameric, or higher order complexes depending on the number of polypeptide chains that form the complex.
  • cancer cancer
  • neoplasm and “tumor” are used interchangeably and, in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination.
  • the definition of a cancer cell includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor.
  • a “clinically detectable” tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation on physical examination, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • X-ray X-ray
  • Tumors may be a hematopoietic (or hematologic or hematological or blood-related) cancer, for example, cancers derived from blood cells or immune cells, which may be referred to as “liquid tumors.”
  • liquid tumors include leukemias such as chronic myelocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia; plasma cell malignancies such as multiple myeloma, MGUS and Waldenstrom's macroglobulinemia; lymphomas such as non-Hodgkin's lymphoma, Hodgkin's lymphoma; and the like.
  • leukemia refers broadly to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous) lymphoid (lymphogenous) or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood-leukemic or aleukemic (subleukemic).
  • Exemplary leukemias that may be treated with a compound or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia,
  • lymphoma refers to a group of cancers affecting hematopoietic and lymphoid tissues. It begins in lymphocytes, the blood cells that are found primarily in lymph nodes, spleen, thymus, and bone marrow. Two main types of lymphoma are non-Hodgkin lymphoma and Hodgkin’s disease. Hodgkin’s disease represents approximately 15% of all diagnosed lymphomas. This is a cancer associated with Reed-Sternberg malignant B lymphocytes. Non-Hodgkin’s lymphomas (NHL) can be classified based on the rate at which cancer grows and the type of cells involved.
  • B-cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, small lymphocytic lymphoma, Mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, extranodal (MALT) lymphoma, nodal (monocytoid B- cell) lymphoma, splenic lymphoma, diffuse large cell B-lymphoma, Burkitt’s lymphoma, lymphoblastic lymphoma, immunoblastic large cell lymphoma, or precursor B-lymphoblastic lymphoma.
  • Exemplary T-cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, cunateous T-cell lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma, mycosis fungoides, and precursor T-lymphoblastic lymphoma.
  • the term "cancer” refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g. humans), including leukemias, lymphomas, carcinomas and sarcomas.
  • the ADCs and methods provided herein are useful for treating CD25-expressing cancers.
  • the CD25-expressing cancer is a lymphoma or leukemia.
  • the CD25-expressing cancer is Adult T-cell leukemia/lymphoma (ATLL), non-Hodgkin’s lymphoma, including but not limited to, cutaneous T-cell lymphoma (CTCL), Hodgkin’s lymphoma, including but not limited to, Reed-Sternberg lymphoma, acute myeloid leukemia (AML), multiple myeloma, and non-small cell lung cancer (NSCLC).
  • ATLL Adult T-cell leukemia/lymphoma
  • CTCL cutaneous T-cell lymphoma
  • Hodgkin’s lymphoma including but not limited to, Reed-Sternberg lymphoma, acute myeloid leukemia (AML), multiple myeloma, and non-small cell lung cancer (NSCLC).
  • the CD25-expressing cancer is Hodgkin's and non-Hodgkin's Lymphoma, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, (FL), Mantle Cell lymphoma (MCL), chronic lymphatic lymphoma (CLL), Marginal Zone B-cell lymphoma (MZBL) and leukemias such as Hairy cell leukemia (HCL), Hairy cell leukemia variant (HCL-v), Acute Myeloid Leukemia (AML), and Acute Lymphoblastic Leukemia (ALL) such as Philadelphia chromosome-positive ALL (Ph+ALL) or Philadelphia chromosome- negative ALL (Ph ⁇ ALL) (Fielding A., (2010) Haematologica.95(1): 8-12).
  • DLBCL diffuse large B-cell lymphoma
  • FL Mantle Cell lymphoma
  • CLL chronic lymphatic lymphoma
  • MZBL Marginal Zone B-cell lymphoma
  • cancers which do not express CD25 are contemplated.
  • solid tumors with high levels of infiltrating T-cells such as infiltrating regulatory T-cells (Tregs) are contemplated.
  • solid tumors with high levels of infiltrating Tregs are contemplated.
  • solid tumors may be pancreatic cancer, breast cancer, colorectal cancer, gastric and esophageal cancer, melanoma, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, or head and neck cancer.
  • solid tumors may be sarcomas (including cancers that arise from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, vascular, hematopoietic or fibrous connective tissues), carcinomas (including tumors that arise from epithelial cells), mesothelioma, neuroblastoma, retinoblastoma, etc.
  • Cancers involving solid tumors include, without limitation, brain cancer, lung cancer, stomach cancer, duodenal cancer, esophageal cancer, breast cancer, rectal and colon cancer, kidney cancer, bladder cancer, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, mouth cancer, and sarcoma.
  • the CD25 protein is overexpressed in various human cancers and can be evaluated using a method generally carried out in the art, such as an immunohistochemical staining method (IHC) for evaluating the overexpression of the CD25 protein, or a fluorescence in situ hybridization method (FISH) for evaluating amplification of the CD25 gene.
  • IHC immunohistochemical staining method
  • FISH fluorescence in situ hybridization method
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • metalastasis can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part.
  • Metalastatic cancer is also called “Stage IV cancer.” Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer.
  • a second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor.
  • the metastatic tumor and its cells are presumed to be similar to those of the original tumor.
  • lung cancer metastasizes to the breast the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells.
  • the secondary tumor in the breast is referred to a metastatic lung cancer.
  • metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors.
  • non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors.
  • metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.
  • Exemplary cancers that may be treated with an ADC or method provided herein include lymphomas and leukemias.
  • cancers that may be treated with an ADC or method provided herein include Hodgkin's and non-Hodgkin's Lymphoma including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, (FL), Mantle Cell lymphoma (MCL), chronic lymphatic lymphoma (CLL), Marginal Zone B-cell lymphoma (MZBL) and leukemias such as Hairy cell leukemia (HCL), Hairy cell leukemia variant (HCL-v), Acute Myeloid Leukemia (AML), and Acute Lymphoblastic Leukemia (ALL) such as Philadelphia chromosome-positive ALL (Ph+ALL) or Philadelphia chromosome-negative ALL (Ph ⁇ ALL).
  • DLBCL diffuse large B-cell lymphoma
  • FL Mantle Cell lymphoma
  • CLL chronic lymphatic lymphoma
  • MZBL Marginal Zone B-cell lymphoma
  • HCL Hairy cell leukemia
  • HCL-v
  • the cancer is a metastatic cancer, refractory cancer, or recurrent cancer.
  • An "antibody” and “antibodies” and related terms used herein refers to an intact immunoglobulin or to an antigen binding portion thereof that binds specifically to an antigen. Antigen binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
  • Antibodies include recombinantly produced antibodies and antigen binding portions.
  • Antibodies include non-human, chimeric, humanized and fully human antibodies.
  • Antibodies include monospecific, multispecific (e.g., bispecific, trispecific and higher order specificities).
  • Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, heavy chain dimers. Antibodies include F(ab’)2 fragments, Fab’ fragments and Fab fragments. Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragment (scFv), camelized antibodies, affibodies, disulfide- linked Fvs (sdFv), anti-idiotypic antibodies (anti-Id), minibodies. Antibodies include monoclonal and polyclonal populations. Anti-CD25 antibodies are described herein.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes) each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • An "epitope" and related terms as used herein refers to a portion of an antigen that is bound by an antigen binding protein (e.g., by an antibody or an antigen binding portion thereof).
  • An epitope can comprise portions of two or more antigens that are bound by an antigen binding protein.
  • An epitope can comprise non-contiguous portions of an antigen or of two or more antigens (e.g., amino acid residues that are not contiguous in an antigen’s primary sequence but that, in the context of the antigen’s tertiary and quaternary structure, are near enough to each other to be bound by an antigen binding protein).
  • the variable regions, particularly the CDRs, of an antibody interact with the epitope.
  • Anti-CD25 antibodies, and antigen binding proteins thereof, that bind an epitope of a CD25 polypeptide are described herein.
  • An "antibody fragment”, “antibody portion”, “antigen-binding fragment of an antibody”, or “antigen-binding portion of an antibody” and other related terms used herein refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAb; diabodies; linear antibodies; single-chain antibody molecules (e.g.
  • Antigen binding portions of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer antigen binding properties to the antibody fragment.
  • Antigen-binding fragments of anti-CD25 antibodies are described herein.
  • An antigen binding protein can have, for example, the structure of an immunoglobulin.
  • an "immunoglobulin” refers to a tetrameric molecule. Each tetrameric molecule is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).
  • the variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two antigen binding sites.
  • an antigen binding protein can be a synthetic molecule having a structure that differs from a tetrameric immunoglobulin molecule but still binds a target antigen or binds two or more target antigens.
  • a synthetic antigen binding protein can comprise antibody fragments, 1-6 or more polypeptide chains, asymmetrical assemblies of polypeptides, or other synthetic molecules.
  • variable heavy chain refers to the variable region of an immunoglobulin heavy chain, including an Fv, scFv , dsFv or Fab
  • variable light chain refers to the variable region of an immunoglobulin light chain, including of an Fv, scFv , dsFv or Fab
  • variant region or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs).
  • FRs conserved framework regions
  • HVRs hypervariable regions
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J.
  • antibody functional fragments include, but are not limited to, complete antibody molecules, antibody fragments, such as Fv, single chain Fv (scFv), complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), Fab, F(ab)2' and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to target antigen (see, e.g., FUNDAMENTAL IMMUNOLOGY (Paul ed., 4th ed.2001).
  • antibody fragments can be obtained by a variety of methods, for example, digestion of an intact antibody with an enzyme, such as pepsin; or de novo synthesis.
  • Antibody fragments are often synthesized de novo either chemically or by using recombinant DNA methodology.
  • the term antibody includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., (1990) Nature 348:552).
  • antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies.
  • Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J. Immunol. 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al.(1993), PNAS. USA 90:6444, Gruber et al. (1994) J Immunol. 152:5368, Zhu et al. (1997) Protein Sci.6:781, Hu et al. (1996) Cancer Res.56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al.
  • antigen binding protein “antigen binding domain,” “antigen binding region,” or “antigen binding site” and related terms used herein refers to a protein comprising a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen.
  • antigen binding proteins include antibodies, antibody fragments (e.g., an antigen binding portion of an antibody), antibody derivatives, and antibody analogs.
  • the antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives.
  • Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to for example stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog.20:639-654.
  • PAMs peptide antibody mimetics
  • Antigen binding proteins that bind CD25 are described herein.
  • a dissociation constant (K D ) can be measured using a BIACORE surface plasmon resonance (SPR) assay.
  • SPR surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).
  • BIACORE surface plasmon resonance
  • antigen binding proteins of the invention may also be cross-reactive with other forms of CD25, for example primate CD25.
  • an antibody specifically binds to a target antigen if it binds to the antigen with a dissociation constant K D of 10 -5 M or less, or 10- 6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or less.
  • K D dissociation constant K D of 10 -5 M or less, or 10- 6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or less.
  • CD25 refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g.
  • CD25-expressing cancer refers to a cancer comprising cells that express CD25 on their surface. In embodiments, the term “CD25-expressing cancer” refers to a cancer comprising cells that internalize CD25 inside the cells.
  • anti- CD25 antibody and “an antibody that binds to CD25” refer to an antibody that is capable of binding CD25 with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting CD25
  • the extent of binding of an anti- CD25 antibody to an unrelated, non-CD25 protein is less than about 10% of the binding of the antibody to CD25 as measured, e.g., by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • an antibody that binds to CD25 has a dissociation constant (Kd) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, , ⁇ 5 nM , ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., 10 -8 M or less, e.g. from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M).
  • an anti- CD25 antibody binds to an epitope of CD25 that is conserved among CD25 from different species.
  • chimeric antibody refers to an antibody that contains one or more regions from a first antibody and one or more regions from one or more other antibodies.
  • one or more of the CDRs are derived from a human antibody.
  • all of the CDRs are derived from a human antibody.
  • the CDRs from more than one human antibody are mixed and matched in a chimeric antibody.
  • a chimeric antibody may comprise a CDR1 from the light chain of a first human antibody, a CDR2 and a CDR3 from the light chain of a second human antibody, and the CDRs from the heavy chain from a third antibody.
  • the CDRs originate from different species such as human and mouse, or human and rabbit, or human and goat.
  • the framework regions may be derived from one of the same antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody.
  • a portion of the heavy and/or light chain is identical with, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with, homologous to, or derived from an antibody (-ies) from another species or belonging to another antibody class or subclass.
  • Chimeric antibodies can be prepared from portions of any of the anti-CD25 antibodies described herein.
  • Effective functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
  • Fc C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • phagocytosis e.g., B cell receptor
  • B cell activation e.g., B cell receptor
  • the Fc region comprises at least a portion of the CH and CH3 regions, and may or may not include a portion of the hinge region. Two polypeptide chains each carrying a half Fc region can dimerize to form an Fc region.
  • An Fc region can bind Fc cell surface receptors and some proteins of the immune complement system.
  • An Fc region exhibits effector function, including any one or any combination of two or more activities including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), opsonization and/or cell binding.
  • An Fc region can bind an Fc receptor, including Fc ⁇ RI (e.g., CD64), Fc ⁇ RII (e.g, CD32) and/or Fc ⁇ RIII (e.g., CD16a).
  • Humanized antibody refers to an antibody having a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject.
  • certain amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody.
  • the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species.
  • one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the likely immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues either are not critical for immunospecific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to make humanized antibodies may be found in U.S. Pat. Nos.6,054,297, 5,886,152 and 5,877,293. [0070]
  • the term “human antibody” refers to antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences.
  • variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody).
  • human immunoglobulin sequences e.g., a fully human antibody.
  • These antibodies may be prepared in a variety of ways, examples of which are described below, including through recombinant methodologies or through immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes.
  • Fully human anti-CD25 antibodies and antigen binding proteins thereof are described herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • isolated means altered “by the hand of man” from its natural state, has been changed or removed from its original environment, or both.
  • nucleic acid or protein When the term “isolated” is applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis, high-performance liquid chromatography or mass spectrophotometry. A protein that is the predominant species present in a preparation is substantially purified.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, including but not limited to when such polynucleotide or polypeptide is introduced back into a cell, even if the cell is of the same species or type as that from which the polynucleotide or polypeptide was separated.
  • CDRs are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable domains of immunoglobulin heavy and light chains.
  • CDRs There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin.
  • CDRs may refer to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy and all light chain CDRs, if appropriate).
  • CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope.
  • CDRs of interest in this invention are derived from donor antibody variable heavy and light chain sequences, and include analogs of the naturally occurring CDRs, which analogs also share or retain the same antigen binding specificity and/or neutralizing ability as the donor antibody from which they were derived.
  • the CDR sequences of antibodies can be determined by the Kabat numbering system (Kabat et al; (Sequences of proteins of Immunological Interest NIH, 1987); alternatively they can be determined using the Chothia numbering system (Al-Lazikani et al., (1997) JMB 273, 927- 948), the contact definition method (MacCallum R M and Martin A C R and Thornton J M (1996), Journal of Molecular Biology, 262 (5), 732-745) or any other established method for numbering the residues in an antibody and determining CDRs known to the skilled in the art.
  • CDR sequences available to a skilled person include “AbM” (University of Bath) and “contact” (University College London) methods.
  • the minimum overlapping region using at least two of the Kabat, Chothia, AbM and contact methods can be determined to provide the “minimum binding unit”.
  • the minimum binding unit may be a sub- portion of a CDR.
  • “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
  • An “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • variant polypeptides and variants of polypeptides refers to a polypeptide comprising an amino acid sequence with one or more amino acid residues inserted into, deleted from and/or substituted into the amino acid sequence relative to a reference polypeptide sequence.
  • Polypeptide variants include fusion proteins.
  • a variant polynucleotide comprises a nucleotide sequence with one or more nucleotides inserted into, deleted from and/or substituted into the nucleotide sequence relative to another polynucleotide sequence.
  • Polynucleotide variants include fusion polynucleotides.
  • domain refers to a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
  • An “antibody single variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody variable domains.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Pb and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.
  • radioactive isotopes e.g.
  • a “chemotherapeutic agent” is a chemical compound useful in the treatment of a cancer.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan
  • calicheamicin especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-dox
  • an “antibody-drug conjugate” or “ADC” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
  • conjugated when referring to two moieties means the two moieties are bonded, wherein the bond or bonds connecting the two moieties may be covalent or non-covalent.
  • the two moieties are covalently bonded to each other (e.g. directly or through a covalently bonded intermediary).
  • the two moieties are non-covalently bonded (e.g.
  • An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non- human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. In certain embodiments, the subject is an adult, an adolescent, a child, or an infant.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Needleman and Wunsch, 1970, J. Mol.
  • the amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence for optimal alignment of the two sequences. Local alignment between two sequences only includes segments of each sequence that are deemed to be sufficiently similar according to a criterion that depends on the algorithm used to perform the alignment (e.g., EMBOSS Water).
  • identity refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region).
  • the percentage identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (Add. APL. Math. 2:482, 1981), by the global homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85: 2444, 1988), or by inspection.
  • GAP and BESTFIT can be employed to determine the optimal alignment of two sequences that have been identified for comparison. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used.
  • a comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between two polynucleotide sequences, may be accomplished using a mathematical algorithm. For example, the "percent identity" or “percent homology" of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters.
  • Expressions such as “comprises a sequence with at least X% identity to Y” with respect to a test sequence mean that, when aligned to sequence Y as described above, the test sequence comprises residues identical to at least X% of the residues of Y.
  • the amino acid sequence of a test antibody may be similar but not identical to any of the amino acid sequences of the polypeptides that make up the multi-specific antigen binding protein complexes described herein.
  • similarities between the test antibody and the polypeptides can be at least 95%, or at or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, to any of the polypeptides that make up the multi-specific antigen binding protein complexes described herein.
  • similar polypeptides can contain amino acid substitutions within a heavy and/or light chain.
  • the amino acid substitutions comprise one or more conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference in its entirety.
  • Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine tyrosine and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine.
  • Antibodies can be obtained from sources such as serum or plasma that contain immunoglobulins having varied antigenic specificity.
  • antibodies can be enriched for a particular antigenic specificity.
  • Such enriched preparations of antibodies usually are made of less than about 10% antibody having specific binding activity for the particular antigen. Subjecting these preparations to several rounds of affinity purification can increase the proportion of antibody having specific binding activity for the antigen.
  • Antibodies prepared in this manner are often referred to as "monospecific.” Monospecific antibody preparations can be made up of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% antibody having specific binding activity for the particular antigen.
  • Antibodies can be produced using recombinant nucleic acid technology as described below.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • expression vectors are referred to herein as “expression vectors.”
  • host cell “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • pharmaceutically acceptable salts is meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and the like.
  • inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic,
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19).
  • Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
  • the compounds of the present disclosure may exist as salts, such as with pharmaceutically acceptable acids.
  • the present disclosure includes such salts.
  • Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, proprionates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g. methyl iodide, ethyl iodide, and the like). These salts may be prepared by methods known to those skilled in the art.
  • the neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.
  • the present disclosure provides compounds, which are in a prodrug form.
  • Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present disclosure.
  • Prodrugs of the compounds described herein may be converted in vivo after administration.
  • prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment, such as, for example, when contacted with a suitable enzyme or chemical reagent.
  • Certain compounds of the present disclosure can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.
  • “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient.
  • Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like.
  • compositions can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • pharmaceutical excipients are useful in the present disclosure.
  • pharmaceutical formulation refers to a preparation which
  • administering refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • exemplary routes of administration for the formulations disclosed herein include intravenous intramuscular subcutaneous intraperitoneal spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the formulation is administered via a non-parenteral route, e.g., orally.
  • non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • An “effective amount” of an agent, e.g., a pharmaceutical formulation refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • the abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.
  • saccharide means carbohydrate (or sugar). In embodiments, the saccharide is a monosaccharide. In embodiments the saccharide is a polysaccharide The most basic unit of saccharide is a monomer of carbohydrate. The general formula is C n H 2n O n .
  • saccharide derivative means sugar molecules that have been modified with substituents other than hydroxyl groups. Examples include glycosylamines, sugar phosphates, and sugar esters.
  • a Charged Group means a chemical group bearing a positive or a negative charge, such as for example phosphate, phosphonate, sulfate, sulfonate, nitrate, carboxylate, carbonate, etc.
  • a Charged Group is at least 50% ionized in aqueous solution at least one pH in the range of 5-9.
  • a Charged Group is an anionic Charged Group.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals.
  • the alkyl may include a designated number of carbons (e.g., C1-C10 means one to ten carbons).
  • Alkyl is an uncyclized chain.
  • saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2- propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-).
  • An alkyl moiety may be an alkenyl moiety.
  • An alkyl moiety may be an alkynyl moiety.
  • An alkyl moiety may be fully saturated.
  • alkenyl may include more than one double bond and/or one or more triple bonds in addition to the one or more double bonds.
  • An alkynyl may include more than one triple bond and/or one or more double bonds in addition to the one or more triple bonds.
  • alkylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, -CH 2 CH 2 CH 2 CH 2 -.
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkenylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, or S), and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
  • heteroatom(s) e.g., O, N, S, Si, or P
  • the heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
  • Heteroalkyl is an uncyclized chain.
  • a heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • the term “heteroalkenyl,” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one double bond.
  • a heteroalkenyl may optionally include more than one double bond and/or one or more triple bonds in addition to the one or more double bonds.
  • heteroalkynyl by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one triple bond.
  • heteroalkynyl may optionally include more than one triple bond and/or one or more double bonds in addition to the one or more triple bonds.
  • heteroalkylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH 2 -CH 2 -S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O) 2 R'- represents both -C(O)2R'- and -R'C(O)2-.
  • heteroalkyl groups include those groups that are attached to the remainder of the molecule through a heteroatom, such as - C(O)R', -C(O)NR', -NR'R'', -OR', -SR', and/or -SO 2 R'.
  • heteroalkyl is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R'' or the like, it will be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity.
  • heteroalkyl should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R'' or the like.
  • cycloalkyl and heterocycloalkyl mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
  • cycloalkyl examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, 1-(1,2,5,6- tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1- piperazinyl, 2-piperazinyl, and the like.
  • the term “cycloalkyl” means a monocyclic, bicyclic, or a multicyclic cycloalkyl ring system.
  • monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated or unsaturated, but not aromatic.
  • cycloalkyl groups are fully saturated.
  • monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
  • Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fused bicyclic rings.
  • bridged monocyclic rings contain a monocyclic cycloalkyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CH2)w , where w is 1, 2, or 3).
  • bicyclic ring systems include, but are not limited to bicyclo[311]heptane bicyclo[221]heptane bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane.
  • fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
  • the bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring.
  • cycloalkyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • the fused bicyclic cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused bicyclic cycloalkyl is optionally substituted by one or two groups which are independently oxo or thia.
  • multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl.
  • multicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the base ring.
  • multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
  • a cycloalkyl is a cycloalkenyl.
  • the term “cycloalkenyl” is used in accordance with its plain ordinary meaning.
  • a cycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenyl ring system.
  • monocyclic cycloalkenyl ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups are unsaturated (i.e containing at least one annular carbon carbon double bond) but not aromatic.
  • monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl.
  • bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings.
  • bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CH2)w, where w is 1, 2, or 3).
  • alkylene bridge of between one and three additional carbon atoms
  • bicyclic cycloalkenyls include, but are not limited to, norbornenyl and bicyclo[2.2.2]oct 2 enyl.
  • fused bicyclic cycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
  • the bridged or fused bicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkenyl ring.
  • cycloalkenyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl.
  • multicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the base ring.
  • multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
  • a heterocycloalkyl is a heterocyclyl.
  • heterocyclyl as used herein, means a monocyclic, bicyclic, or multicyclic heterocycle.
  • the heterocyclyl monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic.
  • the 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S
  • the 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • the 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • the heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle.
  • heterocyclyl monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl
  • the heterocyclyl bicyclic heterocycle is a monocyclic heterocycle fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclic heteroaryl.
  • the heterocyclyl bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system.
  • bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, and octahydrobenzofuranyl.
  • heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia.
  • Multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl and a monocyclic or bicyclic heterocyclyl.
  • multicyclic heterocyclyl is attached to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring.
  • multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
  • multicyclic heterocyclyl groups include, but are not limited to 10H-phenothiazin-10-yl, 9,10- dihydroacridin-9-yl, 9,10-dihydroacridin-10-yl, 10H-phenoxazin-10-yl, 10,11-dihydro-5H- dibenzo[b,f]azepin-5-yl, 1,2,3,4-tetrahydropyrido[4,3-g]isoquinolin-2-yl, 12H- benzo[b]phenoxazin-12-yl, and dodecahydro-1H-carbazol-9-yl.
  • halo or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl.
  • halo(C 1 -C 4 )alkyl includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • acyl means, unless otherwise stated, -C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently.
  • a fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring.
  • heteroaryl refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized.
  • heteroaryl includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring).
  • a 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring.
  • a 6,6-fused ring heteroarylene refers to two rings fused together wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring.
  • a 6,5- fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring.
  • a heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom.
  • Non- limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1- naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4- imidazoly
  • Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.
  • a heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.
  • a fused ring heterocyloalkyl-aryl is an aryl fused to a heterocycloalkyl.
  • a fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl.
  • a fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl.
  • a fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl.
  • Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl- cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substitutents described herein.
  • Spirocyclic rings are two or more rings wherein adjacent rings are attached through a single atom.
  • the individual rings within spirocyclic rings may be identical or different.
  • Individual rings in spirocyclic rings may be substituted or unsubstituted and may have different substituents from other individual rings within a set of spirocyclic rings.
  • Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene and individual rings within a spirocyclic ring group may be any of the immediately previous list, including having all rings of one type (e.g.
  • heterocyclic spirocyclic rings means a spirocyclic rings wherein at least one ring is a heterocyclic ring and wherein each ring may be a different ring.
  • substituted spirocyclic rings means that at least one ring is substituted and each substituent may optionally be different.
  • alkylsulfonyl means a moiety having the formula -S(O 2 )-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' may have a specified number of carbons (e.g., “C1-C4 alkylsulfonyl”).
  • alkylarylene as an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker).
  • the alkylarylene group has the formula: or .
  • An alkylarylene moiety may be substituted (e.g. with a substituent group) on the alkylene moiety or the arylene linker (e.g.
  • alkylarylene is unsubstituted.
  • R, R', R'', R'', and R''' each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.
  • aryl e.g., aryl substituted with 1-3 halogens
  • substituted or unsubstituted heteroaryl substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.
  • each of the R groups is independently selected as are each R', R'', R''', and R''' group when more than one of these groups is present.
  • R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring.
  • -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like).
  • haloalkyl e.g., -CF3 and -CH2CF3
  • acyl e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like.
  • each of the R groups is independently selected as are each R', R'', R'', and R''' groups when more than one of these groups is present.
  • Substituents for rings e.g. cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene
  • substituents on the ring may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent).
  • the substituent may be attached to any of the ring atoms (obeying the rules of chemical valency) and in the case of fused rings or spirocyclic rings, a substituent depicted as associated with one member of the fused rings or spirocyclic rings (a floating substituent on a single ring), may be a substituent on any of the fused rings or spirocyclic rings (a floating substituent on multiple rings).
  • the multiple substituents may be on the same atom, same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent may optionally be different.
  • a point of attachment of a ring to the remainder of a molecule is not limited to a single atom (a floating substituent)
  • the attachment point may be any atom of the ring and in the case of a fused ring or spirocyclic ring, any atom of any of the fused rings or spirocyclic rings while obeying the rules of chemical valency.
  • a ring, fused rings, or spirocyclic rings contain one or more ring heteroatoms and the ring, fused rings, or spirocyclic rings are shown with one more floating substituents (including, but not limited to, points of attachment to the remainder of the molecule), the floating substituents may be bonded to the heteroatoms.
  • the ring heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen) in the structure or formula with the floating substituent, when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen, while obeying the rules of chemical valency.
  • Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups.
  • Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure.
  • the ring- forming substituents are attached to adjacent members of the base structure.
  • two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure.
  • the ring-forming substituents are attached to a single member of the base structure.
  • two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure.
  • the ring-forming substituents are attached to non-adjacent members of the base structure.
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR') p -U-, wherein T and U are independently - NR-, -O-, -CRR'-, or a single bond, and p is an integer of from 0 to 3.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently -CRR'-, -O-, -NR-, - S-, -S(O) -, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer of from 1 to 4.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR')s-X'- (C''R''R'')d-, where s and d are independently integers of from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or - S(O) 2 NR'-.
  • R, R', R'', and R''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • heteroatom or “ring heteroatom” are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
  • a “substituent group,” as used herein, means a group selected from the following moieties: (A) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO 4 H, -SO 2 NH 2 , ⁇ NHNH 2 , ⁇ ONH 2 , ⁇ NHC(O)NHNH 2 , -NHC(O)NH 2 , -NHSO 2 H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl 3 , -OCF 3 , -OCBr 3 , -OCI 3
  • a “size-limited substituent” or “ size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C 1 -C 20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and each substituted or unsubstituted heteroaryl is
  • a “lower substituent” or “ lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 3 -C 7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted phenyl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstitute
  • each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group.
  • each substituted or unsubstituted alkyl may be a substituted or unsubstituted C1-C20 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl
  • each substituted or unsubstituted aryl is a substituted or unsubstituted C 6 -C 10 aryl
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted or unsubstituted
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C20 alkylene
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene
  • each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C 3 -C 8 cycloalkylene
  • each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene
  • each substituted or unsubstituted arylene is a substituted or unsubstituted C 6 -C 10 arylene
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroarylene.
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted C 1 -C 8 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl
  • each substituted or unsubstituted aryl is a substituted or unsubstituted C 6 -C 10 aryl
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl.
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene
  • each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene
  • each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene
  • each substituted or unsubstituted arylene is a substituted or unsubstituted C 6 -C 10 arylene
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene.
  • the compound is a chemical species set forth in the Examples section, figures, or tables below.
  • a substituted or unsubstituted moiety e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroarylene) is unsubstituted (e.g., is an unsubstituted alkyl, unsubstituted cycloalkyl, substituted
  • a substituted or unsubstituted moiety e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroarylene) is substituted (e.g., is a substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alky
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene
  • is substituted with at least one substituent group wherein if the substituted moiety is substituted with a plurality of substituent groups, each substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of substituent groups, each substituent group is different.
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene
  • is substituted with at least one size-limited substituent group wherein if the substituted moiety is substituted with a plurality of size-limited substituent groups, each size-limited substituent group may optionally be different.
  • each size-limited substituent group is different.
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene
  • each lower substituent group is different.
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene
  • each substituent group, size-limited substituent group, and/or lower substituent group is different.
  • Certain compounds of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present disclosure.
  • the compounds of the present disclosure do not include those that are known in art to be too unstable to synthesize and/or isolate.
  • the present disclosure is meant to include compounds in racemic and optically pure forms.
  • Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
  • the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.
  • the term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.
  • tautomer refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.
  • Linker refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety.
  • linkers include a divalent radical.
  • linkers can comprise one or more amino acid residues.
  • the linker is a non-cleavable linker.
  • the linker is an enzyme- cleavable linker (e.g., Val-Cit or Val-Cit-PAB linker).
  • “Amino Acid Unit” has the formula , where R is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH 2 OH, —CH(OH)CH 3 , —CH 2 CH 2 SCH 3 , —CH 2 CONH 2 , —CH 2 COOH, —CH 2 CH 2 CONH 2 , —CH 2 CH 2 COOH, —(CH2)3NHC( ⁇ NH)NH2, —(CH2)3NH2, —(CH2)3NHCOCH3, —(CH2)3NHCHO, —(CH 2 ) 4 NHC( ⁇ NH)NH 2 , —(CH 2 ) 4 NH 2 , —(CH 2 ) 4 NHCOCH 3 , —(CH 2 ) 4 NHCHO, —(CH 2 ) 3 NHCONH 2 , —(CH 2 ) 4 NHCONH 2 , —CH 2 CH 2 CH
  • Amino Acid Unit includes not only naturally occurring amino acids but also minor amino acids, and non- naturally occurring amino acid analogs, such as citrulline, norleucine, selenomethionine, ⁇ - alanine, N-dimethyl lysine etc.
  • An amino acid unit may be referred to by its standard three-letter code for the amino acid (e.g., Ala, Cys, Asp, Glu, Val, Phe, Lys, etc.).
  • bioconjugate and “bioconjugate linker” refers to the resulting association between atoms or molecules of “bioconjugate reactive groups” or “bioconjugate reactive moieties”. The association can be direct or indirect.
  • a conjugate between a first bioconjugate reactive group e.g., –NH2, –C(O)OH, –N- hydroxysuccinimide, or –maleimide
  • a second bioconjugate reactive group e.g., thiol, sulfur-containing amino acid, amine, amine sidechain containing amino acid, or carboxylate
  • covalent bond or linker e.g. a first linker of second linker
  • indirect e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g.
  • bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e. the association of two bioconjugate reactive groups) including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon- heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).
  • bioconjugate chemistry i.e. the association of two bioconjugate reactive groups
  • nucleophilic substitutions e.g., reactions of amines and alcohols with acyl halides, active esters
  • electrophilic substitutions e.g., enamine reactions
  • additions to carbon-carbon and carbon- heteroatom multiple bonds e.g., Michael reaction, Diels-Alder addition.
  • the first bioconjugate reactive group e.g., maleimide moiety
  • the second bioconjugate reactive group e.g. a thiol
  • the first bioconjugate reactive group (e.g., haloacetyl moiety) is covalently attached to the second bioconjugate reactive group (e.g. a thiol).
  • the first bioconjugate reactive group (e.g., pyridyl moiety) is covalently attached to the second bioconjugate reactive group (e.g. a thiol).
  • the first bioconjugate reactive group (e.g., –N-hydroxysuccinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. an amine).
  • the first bioconjugate reactive group (e.g., fluorophenyl ester moiety) reacts with the second bioconjugate reactive group (e.g. an amine) to form a covalent bond.
  • the first bioconjugate reactive group (e.g., –sulfo–N-hydroxysuccinimide moiety) reacts with the second bioconjugate reactive group (e.g. an amine) to form a covalent bond.
  • bioconjugate reactive moieties used for bioconjugate chemistries herein include, for example: (a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.
  • haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;
  • dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido or maleimide groups;
  • aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;
  • sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides;
  • thiol groups which can be converted to disulf
  • bioconjugate reactive groups can be chosen such that they do not participate in, or interfere with, the chemical stability of the conjugate described herein. Alternatively, a reactive functional group can be protected from participating in the crosslinking reaction by the presence of a protecting group.
  • the bioconjugate comprises a molecular entity derived from the reaction of an unsaturated bond, such as a maleimide, and a thiol group.
  • an unsaturated bond such as a maleimide, and a thiol group.
  • “Analog,” or “analogue” is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound.
  • an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.
  • common organic and cell types abbreviations are defined as follows: Ac Acetyl ACN Acetonitrile Ala Alanine Asn Asparagine aq.
  • R 1 is H or –C 1 -C 8 alkyl
  • R 3 is H, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH 2 Br, -CH 2 F, -CH 2 I, -CN, -OR 3A , -NR 3A R 3B , -(CH 2 ) v OR 6 , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl;
  • R 4 is H, halogen, -OR 4A , -NR 4A R 4B , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl;
  • V is N, O, or C;
  • Z 1 is a substituted or unsubstituted
  • D wherein: R 1 is H or –C 1 -C 8 alkyl; R 3 is H, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 4 is H, halogen, or substituted or unsubstituted alkyl; V is N; and Z 2 is a substituted or unsubstituted arylene.
  • D’ is:
  • D is .
  • m is an integer from 1 to 8. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. [00160] In embodiments, n is an integer from 1 to 24. In embodiments, n is an integer from 1 to 4. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 3. In embodiments, n is 4. In embodiments, n is 5. In embodiments, n is 6. In embodiments, n is 7. In embodiments, n is 8.
  • n is 9. In embodiments, n is 10. In embodiments, n is 11. In embodiments, n is 12. In embodiments, n is 13. In embodiments, n is 14. In embodiments, n is 15. In embodiments, n is 16. In embodiments, n is 17. In embodiments, n is 18. In embodiments, n is 19. In embodiments, n is 20. In embodiments, n is 21. In embodiments, n is 22. In embodiments, n is 23. In embodiments, n is 24. [00161] In embodiments, the anti-CD25 antibody is a modified antibody. In embodiments, the modified antibody binds a transmembrane protein, e.g., an extracellular domain of a transmembrane protein.
  • the tr smembrane protein is a transmembrane receptor, such as a transmembrane receptor kinase.
  • the transmembrane receptor kinase is a transmembrane receptor tyrosine kinase.
  • the modified antibody binds a tyrosine kinase.
  • L 1 is a linker bound to the anti-CD25 antibody.
  • L 1 is a linker bound to one or two sulfur or nitrogen atoms on the anti-CD25 antibody.
  • L 1 is a linker bound to one sulfur atom on the anti-CD25 antibody.
  • L 1 is a linker bound to two sulfur atoms on the anti-CD25 antibody. In embodiments, L 1 is a linker bound to one nitrogen atom on the anti-CD25 antibody. In embodiments, L 1 is a linker bound to two nitrogen atoms on the anti-CD25 antibody. [00163] In embodiments, L 1 is a linker bound to a modified anti-CD25 antibody. [00164] In embodiments, L 1 is a linker bound to one cysteine molecule on the anti-CD25 antibody. In embodiments, L 1 is a linker bound to two cysteine molecules on the anti-CD25 antibody. In embodiments, L 1 is a linker bound to one lysine molecule on the anti-CD25 antibody.
  • L 1 is a linker bound to two lysine molecules on the anti-CD25 antibody. [00165] In embodiments, L 1 is a linker bound to a modified anti-CD25 antibody. [ [00167] In embodiments, . [ e . In embodiments, L is . In embodiments, L is . In e . [00169] the two CH2 moieties shown on the right side of the structure may each be bound to a different cysteine of the anti-CD25 antibody via a thiol group. , the two alkene carbons shown on the bottom of the structure may each be bound to a different cysteine of the anti-CD25 antibody via a thiol group.
  • L 1 is , the carbon may be bound to a cysteine of the anti-CD25 antibody via a thiol group.
  • L 2 is a bond, -C(O)-, -NH-, -Val-, -Phe-, -Lys-, -Gly-, -O- –(4-aminobenzyloxycarbonyl)–, –(C(O)N(R 2 )CH2CH2N(R 5 ))–, -Ser-, -Thr-, -Ala-, - ⁇ -Ala-, -citrulline- (Cit), –(CH 2 ) n –, –(CH 2 CH 2 O) n –, N-dimethyl lysine, or any combination thereof.
  • each R 2 and R 5 is independently H or substituted or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, each R 2 and R 5 is independently H. In embodiments, each R 2 and R 5 is independently substituted or unsubstituted alkyl. In embodiments, each R 2 and R 5 is independently substituted or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • each R 2 and R 5 is independently unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl). In embodiments, each R 2 and R 5 is independently substituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • each R 2 and R 5 is independently H or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl).
  • each R 2 and R 5 is independently substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl.
  • each R 2 and R 5 is independently substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl) In embodiments each R 2 and R 5 is independently unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl).
  • each R 2 and R 5 is independently substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • each R 2 and R 5 is independently methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, or hexyl.
  • each R 2 and R 5 is independently methyl.
  • each R 2 and R 5 is independently ethyl.
  • each R 2 and R 5 is independently propyl. In embodiments, each R 2 and R 5 is independently butyl.
  • L 2 is a bond, -C(O)-, -NH-, -Val-, -Phe-, -Lys-, -Gly-, –(4-aminobenzyloxycarbonyl)–, –(C(O)N(CH 3 )CH 2 CH 2 N(CH 3 ))–, -Ser-, -Thr-, -Ala-, - ⁇ -Ala-, -O-, -citrulline- (Cit), –(CH2)n–, –(CH2CH2O)n–, N-dimethyl lysine, or any combination thereof.
  • L 2 is -C(O)-, -NH-, -Val-, -Gly-, -Cit-, -Ala-, -O-, –(4- aminobenzyloxycarbonyl)–, –(CH2)n–, –(CH2CH2O)n–, –(C(O)N(CH3)CH2CH2N(CH3))–, N- dimethyl lysine, or any combination thereof.
  • L 2 is -C(O)-, -NH-, -Gly-, –(CH 2 ) n –, –(CH 2 CH 2 O) n –, or any combination thereof.
  • L 2 is -C(O)-, -NH-, -Val-, -Cit-, –(CH2CH2O)n–, –(4- aminobenzyloxycarbonyl)–, –(CH 2 ) n –, –(C(O)N(CH 3 )CH 2 CH 2 N(CH 3 ))–, N-dimethyl lysine, or any combination thereof.
  • L 2 is -C(O)-, -NH-, -Val-, –(4-aminobenzyloxycarbonyl)–, -Gly-,- citrulline- (-Cit-), –(CH2)n–, –(CH2CH2O)n–, N-dimethyl lysine, or any combination thereof.
  • L 2 is:
  • L is . In embodiments, L 2 is . In embodiments, L 2 is In embodiments, L 2 is . In embodiments, L 2 is . In embodiments, L 2 is . In embodiments, L 2 is H N O . In embodiments, L 2 is . In embodiments, L 2 is . In embodiments, L 2 is . In embodiments, L 2 is . In embodiments, L 2 is . In embodiments, L 2 is . In embodiments, L 2 is
  • L 2 is a bond. In embodiments, L 2 is -C(O)-. In embodiments, L 2 is -NH-. In embodiments, L 2 is -Val-. In embodiments, L 2 is -Phe-. In embodiments, L 2 is -Lys-. In embodiments, L 2 is –(4-aminobenzyloxycarbonyl)–. In embodiments, L 2 is –(CH 2 ) n –. In embodiments, L 2 is –(CH2CH2O)n–. In embodiments, L 2 is -Gly-. In embodiments, L 2 is -Ser-. In embodiments, L 2 is -Thr-.
  • L 2 is -Ala-. In embodiments, L 2 is - ⁇ -Ala-. In embodiments, L 2 is -Cit-. In embodiments, L 2 is -O-. In embodiments, L 2 is N-dimethyl lysine.
  • -L -L - is .
  • -L -L - is , where the two CH2 moieties shown on the left side of the structure may each be bound to a separate sulfur of the anti-CD25 antibody.
  • -L 1 -L 2 - is .
  • -L 1 -L 2 - is , where the two alkene carbons shown on the bottom of the structure may each be bound to a separate sulfur of the anti CD25 antibody.
  • -L 1 -L 2 - is In embodiments, -L 1 -L 2 - is , embodiments, -L 1 -L 2 - is .
  • -L 1 -L 2 - is . e o e s, - - - s . n embodiments, -L 1 -L 2 - i s .
  • -L-L- is .
  • -L 1 -L 2 - is embodiments, -L 1 -L 2 - is .
  • -L 1 -L 2 - is n embodiments, -L 1 -L 2 - is .
  • -L 1 -L 2 - is .
  • -L 1 -L 2 - is .
  • -L 1 -L 2 - is .
  • -L 1 -L 2 - is .
  • -L 1 -L 2 - is .
  • L 3 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene), substituted (e.g.
  • heteroarylene e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene, substituted (e.g.
  • heterocycloalkylene e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene
  • substituted e.g., with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted -OCH2-(heteroarylene e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene)
  • substituted e.g.
  • heterocycloalkyl e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl
  • substituted e.g., with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted -CH 2 NCH 2 -(heteroaryl e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl
  • L 3 is substituted with one or more substituent groups. In embodiments, L 3 is substituted with one or more size-limited substituent groups. In embodiments, L 3 is substituted with one or more lower substituent groups. [00186] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene), or substituted (e.g.
  • L 3 is substituted with one or more substituent groups. In embodiments, L 3 is substituted with one or more size-limited substituent groups. In embodiments, L 3 is substituted with one or more lower substituent groups.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • L 3 is substituted with one or more substituent groups
  • L 3 is substituted with one or more size-limited substituent groups.
  • L 3 is substituted with one or more lower substituent groups.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • L 3 is unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • heteroarylene e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene.
  • L 3 is unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) - OCH 2 -(heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl)).
  • L 3 is unsubstituted -OCH2-(heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene)).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -OCH2-(heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene)).
  • L 3 is unsubstituted -OCH 2 -(heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene)).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -CH2NCH2- (heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl)).
  • L 3 is unsubstituted -CH2NCH2- (heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl)).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -CH 2 NCH 2 -(heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl)).
  • heteroaryl e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl
  • L 3 is unsubstituted -CH2NCH2-(heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl)) [00189]
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 8 membered heterocycloalkylene.
  • L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) 3 to 8 membered heterocycloalkylene.
  • L 3 is unsubstituted 3 to 8 membered heterocycloalkylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(3 to 8 membered heterocycloalkyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -CH2NCH2-(3 to 8 membered heterocycloalkyl). In embodiments, L 3 is unsubstituted - CH2NCH2-(3 to 8 membered heterocycloalkyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) or unsubstituted -OCH2-(3 to 8 membered heterocycloalkylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -OCH2-(3 to 8 membered heterocycloalkylene). In embodiments, L 3 is unsubstituted -OCH 2 -(3 to 8 membered heterocycloalkylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 8 membered heterocycloalkylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 6 membered heterocycloalkylene.
  • L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) 3 to 6 membered heterocycloalkylene.
  • L 3 is unsubstituted 3 to 6 membered heterocycloalkylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) or unsubsituted -CH2NCH2-(3 to 6 membered heterocycloalkyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -CH 2 NCH 2 -(3 to 6 membered heterocycloalkyl). In embodiments, L 3 is unsubsituted - CH 2 NCH 2 -(3 to 6 membered heterocycloalkyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) or unsubsituted -OCH2-(3 to 6 membered heterocycloalkylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent grou or a lower substituent group) -OCH 2 -(3 to 6 membered heterocycloalkylene). In embodiments, L 3 is unsubsituted -OCH 2 -(3 to 6 membered heterocycloalkylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 6 membered heterocycloalkylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclobutylene, heterocyclopentylene or heterocyclohexylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclobutylene, heterocyclopentylene or heterocyclohexylene. In embodiments, L 3 is unsubstituted heterocyclobutylene, heterocyclopentylene or heterocyclohexylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(heterocyclobutyl, heterocyclopentyl, or heterocyclohexyl).
  • L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) -CH 2 NCH 2 -(heterocyclobutyl, heterocyclopentyl, or heterocyclohexyl). In embodiments, L 3 is unsubstituted -CH2NCH2- (heterocyclobutyl, heterocyclopentyl, or heterocyclohexyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(heterocyclobutylene, heterocyclopentylene, or heterocyclohexylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 -(heterocyclobutylene, heterocyclopentylene, or heterocyclohexylene). In embodiments, L 3 is unsubstituted -OCH2-(heterocyclobutylene, heterocyclopentylene, or heterocyclohexylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclobutylene, heterocyclopentylene or heterocyclohexylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclobutylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclobutylene.
  • L 3 is unsubstituted heterocyclobutylene.
  • L 3 is substituted (e g with a substituent group a size- limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 - (heterocyclobutyl).
  • L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) -CH2NCH2-(heterocyclobutyl).
  • L 3 is unsubstituted -CH 2 NCH 2 -(heterocyclobutyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(heterocyclobutylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 - (heterocyclobutylene). In embodiments, L 3 is unsubstituted -OCH 2 -(heterocyclobutylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclopentylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclopentylene. In embodiments, L 3 is unsubstituted heterocyclopentylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 - (heterocyclopentyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH2NCH2-(heterocyclopentyl). In embodiments, L 3 is unsubstituted -CH 2 NCH 2 -(heterocyclopentyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(heterocyclopentylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2- (heterocyclopentylene). In embodiments, L 3 is unsubstituted -OCH 2 -(heterocyclopentylene). [00197] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclohexylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclohexylene.
  • L 3 is unsubstituted heterocyclohexylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 - (heterocyclohexyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) -CH2NCH2-(heterocyclohexyl). In embodiments, L 3 is unsubstituted -CH2NCH2-(heterocyclohexyl).
  • L 3 is substituted (e.g., with a substituent group a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(heterocyclohexylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2- (heterocyclohexylene). In embodiments, L 3 is unsubstituted -OCH2-(heterocyclohexylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 10 membered heteroarylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 10 membered heteroarylene. In embodiments, L 3 is unsubstituted 5 to 10 membered heteroarylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(5 to 10 membered heteroaryl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH 2 NCH 2 -(5 to 10 membered heteroaryl). In embodiments, L 3 is unsubstituted -CH2NCH2-(5 to 10 membered heteroaryl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(5 to 10 membered heteroarylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2-(5 to 10 membered heteroarylene). In embodiments, L 3 is unsubstituted -OCH 2 -(5 to 10 membered heteroarylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 9 membered heteroarylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 9 membered heteroarylene. In embodiments, L 3 is unsubstituted 5 to 9 membered heteroarylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(5 to 9 membered heteroaryl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH2NCH2-(5 to 9 membered heteroaryl). In embodiments, L 3 is unsubstituted -CH2NCH2-(5 to 9 membered heteroaryl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(5 to 9 membered heteroarylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2-(5 to 9 membered heteroarylene).
  • L 3 is unsubstituted -OCH 2 -(5 to 9 membered heteroarylene) [00200] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 6 membered heteroarylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heteroarylene. In embodiments, L 3 is unsubstituted 5 to 6 membered heteroarylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(5 to 6 membered heteroaryl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH 2 NCH 2 -(5 to 6 membered heteroaryl). In embodiments, L 3 is unsubstituted -CH2NCH2-(5 to 6 membered heteroaryl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(5 to 6 membered heteroarylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2-(5 to 6 membered heteroarylene). In embodiments, L 3 is unsubstituted -OCH 2 -(5 to 6 membered heteroarylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene, thienylene, oxazolylene, or thiazolylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene, thienylene, oxazolylene, or thiazolylene.
  • L 3 is unsubstituted furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene, thienylene, oxazolylene, or thiazolylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH2NCH2- (furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl).
  • L 3 is unsubstituted -CH 2 NCH 2 -(furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene, thienylene, oxazolylene, or thiazolylene).
  • L 3 is substituted (eg with a substituent group a size-limited substituent group or a lower substituent group) -OCH 2 -(furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene, thienylene, oxazolylene, or thiazolylene).
  • L 3 is unsubstituted -OCH2-(furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene, thienylene, oxazolylene, or thiazolylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanylene.
  • L 3 is unsubstituted furanylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(furanyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) - CH2NCH2-(furanyl). In embodiments, L 3 is unsubstituted -CH2NCH2-(furanyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(furanylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 - (furanylene). In embodiments, L 3 is unsubstituted -OCH2-(furanylene). [00203] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyrrolylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyrrolylene. In embodiments, L 3 is unsubstituted pyrrolylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(pyrrolyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH 2 NCH 2 -(pyrrolyl). In embodiments, L 3 is unsubstituted -CH 2 NCH 2 -(pyrrolyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(pyrrolylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 -(pyrrolylene). In embodiments, L 3 is unsubstituted -OCH 2 -(pyrrolylene). [00204] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyridylene.
  • L 3 is substituted (e.g., with a substituent group a size-limited substituent group or a lower substituent group) pyridylene. In embodiments, L 3 is unsubstituted pyridylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(pyridyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) - CH 2 NCH 2 -(pyridyl).
  • L 3 is unsubstituted -CH 2 NCH 2 -(pyridyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(pyridylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 - (pyridylene). In embodiments, L 3 is unsubstituted -OCH2-(pyridylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyranylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyranylene. In embodiments, L 3 is unsubstituted pyranylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(pyranyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) - CH 2 NCH 2 -(pyranyl). In embodiments, L 3 is unsubstituted -CH 2 NCH 2 -(pyranyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(pyranylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 -(pyranylene).
  • L 3 is unsubstituted -OCH 2 -(pyranylene). [00206] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted imidazolylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) imidazolylene. In embodiments, L 3 is unsubstituted imidazolylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(imidazolyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH2NCH2-(imidazolyl). In embodiments, L 3 is unsubstituted -CH2NCH2-(imidazolyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(imidazolylene)
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2-(imidazolylene).
  • L 3 is unsubstituted -OCH2-(imidazolylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted thiazolylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) thiazolylene. In embodiments, L 3 is unsubstituted thiazolylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(thiazolyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH2NCH2-(thiazolyl).
  • L 3 is unsubstituted -CH2NCH2-(thiazolyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(thiazolylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 -(thiazolylene). In embodiments, L 3 is unsubstituted -OCH 2 -(thiazolylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted thienylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) thienylene. In embodiments, L 3 is unsubstituted thienylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(thienyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) - CH2NCH2-(thienyl). In embodiments, L 3 is unsubstituted -CH2NCH2-(thienyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(thienylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2- (thienylene).
  • L 3 is unsubstituted -OCH2-(thienylene). [00209] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted oxazolylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) oxazolylene. In embodiments, L 3 is unsubstituted oxazolylene.
  • L 3 is unsubstituted oxazolylene In embodiments L 3 is substituted (eg with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(oxazolyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH2NCH2-(oxazolyl). In embodiments, L 3 is unsubstituted -CH 2 NCH 2 -(oxazolyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(oxazolylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 -(oxazolylene). In embodiments, L 3 is unsubstituted -OCH 2 -(oxazolylene). [00210] In embodiments, R * is substituted (e.g.
  • R * is substituted with one or more substituent groups.
  • R * is substituted with one or more size-limited substituent groups. In embodiments, R * is substituted with one or more lower substituent groups. [00211] In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • heterocycloalkyl e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl.
  • R * is unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • R * is unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 8 membered heterocycloalkyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 3 to 8 membered heterocycloalkyl.
  • R * is unsubstituted 3 to 8 membered heterocycloalkyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 6 membered heterocycloalkyl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 3 to 6 membered heterocycloalkyl. In embodiments, R * is unsubstituted 3 to 6 membered heterocycloalkyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclobutyl, heterocyclopentyl or heterocyclohexyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclobutyl, heterocyclopentyl or heterocyclohexyl.
  • R * is unsubstituted heterocyclobutyl, heterocyclopentyl or heterocyclohexyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclobutyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclobutyl.
  • R * is unsubstituted heterocyclobutyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclopentyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclopentyl. In embodiments, R * is unsubstituted heterocyclopentyl. [00217] In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclohexyl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclohexyl. In embodiments, R * is unsubstituted heterocyclohexyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 10 membered heteroaryl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 10 membered heteroaryl. In embodiments, R * is unsubstituted 5 to 10 membered heteroaryl [00219] In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 9 membered heteroaryl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 9 membered heteroaryl. In embodiments, R * is unsubstituted 5 to 9 membered heteroaryl. [00220] In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 6 membered heteroaryl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heteroaryl.
  • R * is unsubstituted 5 to 6 membered heteroaryl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, or thiazolyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, or thiazolyl.
  • R * is unsubstituted furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, or thiazolyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanyl.
  • R * is unsubstituted furanyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyrrolyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyrrolyl.
  • R * is unsubstituted pyrrolyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyridyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyridyl. In embodiments, R * is unsubstituted pyridyl. [00225] In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyranyl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyranyl. In embodiments, R * is unsubstituted pyranyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted imidazolyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) imidazolyl.
  • R * is unsubstituted imidazolyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted thiazolyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) thiazolyl. In embodiments, R * is unsubstituted thiazolyl.
  • R 1 is H. In embodiments, R 1 is –C 1 -C 8 alkyl. [00229] In embodiments, R 1 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, or hexyl. In embodiments, R 1 is methyl. In embodiments, R 1 is ethyl. In embodiments, R 1 is propyl.
  • R 1 is isopropyl. In embodiments, R 1 is butyl. In embodiments, R 1 is isobutyl. In embodiments, R 1 is tert-butyl. In embodiments, R 1 is pentyl. In embodiments, R 1 is hexyl.
  • R 3 is H, halogen, -CCl 3 , -CBr 3 , -CF 3 , -CI 3 , -CHCl 2 , -CHBr 2 , -CHF 2 , -CHI 2 , -CH 2 Cl, -CH 2 Br, -CH 2 F, -CH 2 I, -CN, -OR 3A , -NR 3A R 3B , -(CH 2 ) v OR 6 , substituted or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), or substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • alkyl e.g., C1-C8 alkyl, C1-C6 alkyl, or C
  • R 3 is H, -OR 3A , -(CH2)vOR 6 , substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl), or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • alkyl e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl
  • substituted e.g., substituted with at least one substituent group, size-limited substituent group, or lower substitu
  • R 3 is a substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • R 3 is an unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C 1 -C 4 alkyl).
  • R 3 is a substituted (eg substituted with at least one substituent group, size-limited substituent group, or lower substituent group) heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 3 is an unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 3 is methyl, ethyl, propyl, butyl, –CH 2 OH, -CH2CH2OH, -CH2N3, -CH2CH2N3, -CH2OCH3, -CH2OCH2CH3, -CH2CH2OCH3, - , embodiments, R 3 is H, methyl, ethyl, propyl, butyl, –CH2OH, -CH2CH2OH, -CH2N3, -CH2CH2N3, -CH2OCH3, -CH2OCH2CH3, or -CH2CH2OCH3. In embodiments, R 3 is methyl, –CH2OH, or -CH2N3.
  • R 3 is methyl. In embodiments, R 3 is ethyl. In embodiments, R 3 is propyl. In embodiments, R 3 is butyl. In embodiments, R 3 is –CH2OH. In embodiments, R 3 is – CH2 CH2OH. In embodiments, R 3 is -CH2N3. In embodiments, R 3 is -CH2CH2N3. In embodiments, R 3 is -CH 2 OCH 3 . In embodiments, R 3 is -CH 2 OCH 2 CH 3 . In embodiments, R 3 is - CH 2 CH 2 OCH 3 . In embodiments, R 3 is -CH 2 CH 2 OCH 2 CH 3 . In embodiments, R 3 is -OH.
  • R 3 is H. In embodiments, R is . [00235] In embodiments, R 3 is methyl, – CH 2 OH, , or -CH2N3. In embodiments, R 3 is -CH2N3. [00236] In embodiments, v is an integer from 1 to 24. In embodiments, v is 1. In embodiments, v is 2. In embodiments, v is 3. In embodiments, v is 4. In embodiments, v is 5. In embodiments, v is 6. In embodiments, v is 7. In embodiments, v is 8. In embodiments, v is 9. In embodiments, v is 10. In embodiments, v is 11. In embodiments, v is 12. In embodiments, v is 13. In embodiments, v is 14.
  • v is 15. In embodiments, v is 16. In embodiments, v is 17. In embodiments, v is 18. In embodiments, v is 19. In embodiments, v is 20. In embodiments, v is 21. In embodiments, v is 22. In embodiments, v is 23. In embodiments, v is 24.
  • R 4 is H, halogen, -OR 4A , -NR 4A R 4B , substituted or unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl), or substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • alkyl e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl
  • heteroalkyl e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl.
  • R 4 is H, -OR 4A , substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • alkyl e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl
  • substituted e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group
  • unsubstituted heteroalkyl e
  • R 4 is H, or substituted or unsubstituted alkyl.
  • R 4 is H, or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl).
  • R 4 is a substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C1-C4 alkyl).
  • R 4 is an unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • R 4 is a substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 4 is an unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 4 is H, -OH, methyl, ethyl, propyl or butyl.
  • R 4 is H or -OH.
  • R 4 is H or methyl. In embodiments, R 4 is methyl. In embodiments, R 4 is ethyl. In embodiments, R 4 is propyl. In embodiments, R 4 is butyl. In embodiments, R 4 is H. In embodiments, R 4 is -OH. [00242] In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently H or substituted or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • alkyl e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl.
  • each R 3A , R 3B , R 4A , and R 4B is independently H or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • each R 3A , R 3B , R 4A , and R 4B is independently H.
  • each R 3A , R 3B , R 4A , and R 4B is independently substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C 1 -C 4 alkyl).
  • each R 3A , R 3B , R 4A , and R 4B is independently unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl).
  • each R 3A , R 3B , R 4A , and R 4B is independently H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, or pentyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently H. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently methyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently ethyl.
  • each R 3A , R 3B , R 4A , and R 4B is independently propyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently isopropyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently butyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently isobutyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently tert-butyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently pentyl.
  • R 6 is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -CO(CH2CH2O)wCH2CH2M, -CONH(CH2CH2O)wCH2CH2M, , a Charged Group, or a saccharide derivative, w is an integer from 1 to 24; M is -NH 2 , -OH, -COOH, or -OCH 3 ; R 10 is -OH, -OCH 3 or -COOH.
  • R 6 is H or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1- C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or
  • R 6 is H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • R s o . embodiments, R 6 is H.
  • w is an integer from 1 to 24. In embodiments, w is 1. In embodiments, w is 2. In embodiments, w is 3. In embodiments, w is 4. In embodiments, w is 5. In embodiments, w is 6.
  • w is 7. In embodiments, w is 8. In embodiments, w is 9. In embodiments, w is 10. In embodiments, w is 11. In embodiments, w is 12. In embodiments, w is 13. In embodiments, w is 14. In embodiments, w is 15. In embodiments, w is 16. In embodiments, w is 17. In embodiments, w is 18. In embodiments, w is 19. In embodiments, w is 20. In embodiments, w is 21. In embodiments, w is 22. In embodiments, w is 23. In embodiments, w is 24. [00250] In embodiments, M is -NH 2 , -OH, -COOH, or -OCH 3. In embodiments, M is -NH 2.
  • M is -OH. In embodiments, M is -COOH. In embodiments, M is -OCH3. [ . I e [00252]
  • R 6 is a saccharide derivative. In embodiments, R 6 is . [00253]
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkyl (e.g., C 3 -C 8 cycloalkyl, C 3 -C 6 cycloalkyl, or C 5 -C 6 cycloalkyl). In embodiments, Z 1 is a substituted (e.g.
  • cycloalkyl e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl.
  • Z 1 is an unsubstituted cycloalkyl (e.g., C 3 -C 8 cycloalkyl, C 3 -C 6 cycloalkyl, or C 5 -C 6 cycloalkyl).
  • Z 1 is a substituted (e.g.
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • Z 1 is an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted aryl (e.g., C 6 -C 10 aryl, C 10 aryl, or phenyl).
  • Z 1 is a substituted (e.g.
  • aryl e.g., C6-C10 aryl, C10 aryl, or phenyl
  • Z 1 is an unsubstituted aryl (e.g., C6-C10 aryl, C 10 aryl, or phenyl).
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • Z 1 is an unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • each Q is independently a halogen, methyl, ethyl, or propyl; and q is an integer from 1 to 5.
  • s .
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C 3 -C 6 cycloalkylene, or C 5 -C 6 cycloalkylene).
  • Z 2 is a substituted (e.g.
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted arylene (e.g., C 6 -C 10 arylene, C 10 arylene, or phenylene).
  • Z 2 is a substituted (e.g.
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted arylene (e.g., C 6 -C 10 arylene, C 10 arylene, or phenylene).
  • Z 2 is a substituted (e.g.
  • arylene e.g., C6-C10 arylene, C10 arylene, or phenylene
  • Z 2 is an unsubstituted arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene).
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • Z 2 is an unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • Z 2 is a substituted (e.g.
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 6 membered heteroarylene.
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heteroarylene.
  • Z 2 is an unsubstituted 5 to 6 membered heteroarylene.
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted phenylene.
  • Z 2 is a substituted (e.g.
  • Z 2 is an unsubstituted phenylene.
  • Z is an unsubstituted arylene.
  • Z is .
  • V is N. In embodiments, V is O. In embodiments, V is C.
  • -Z 2 -V- is or ; wherein each G is independently Cl, Br, I, F, -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -OCH 3 , -OCH 2 CH 3 , -OH, or -NH 2 ; and p is an integer from 0-4. [ e , - - - s .
  • an ADC of formula (IA) or formula (IIA): or (IA) (IIA) or a pharmaceutically acceptable salt thereof wherein: ring A is a substituted or unsubstituted heterocycloalkylene or a substituted or unsubstituted heteroarylene, connected to L 2 through a heteroatom Y; ring A’ is a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl, connected to D’ through a heteroatom Y; each Y is independently N, P, or S; and L 1 , L 2 , Ab, m, D, and D’ are each as defined herein including embodiments.
  • ring A is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene) or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • a substituted e.g. with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted heterocycloalkylene e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5
  • ring A is substituted with one or ore substituent groups In embodiments ring A is substituted with one or more size-limited substituent groups. In embodiments, ring A is substituted with one or more lower substituent groups. Ring A is connected to L 2 through a heteroatom Y. [00267] In embodiments, ring A’ is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl) or substituted (e.g.
  • ring A’ is substituted with one or more substituent groups.
  • ring A’ is substituted with one or more size-limited substituent groups.
  • ring A’ is substituted with one or more lower substituent groups.
  • Ring A' is connected to D’ through a heteroatom Y. In embodiments, each Y is N.
  • ring A is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 3 to 8 membered heterocycloalkylene, where ring A is connected to L 2 through a heteroatom Y.
  • ring A’ is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 3 to 8 membered heterocycloalkyl, where ring A' is connected to D’ through a heteroatom Y.
  • each Y is N.
  • ring A is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heterocycloalkylene, where ring A is connected to L 2 through a heteroatom Y.
  • ring A’ is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heterocycloalkyl, where ring A' is connected to D’ through a heteroatom Y.
  • each Y is N.
  • -NH- and -D are connected to different carbon atoms on ring W. In embodiments, -NH- and -D are connected to the same carbon atom on ring W.
  • ring W is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C 3 -C 6 cycloalkylene, or C 5 -C 6 cycloalkylene) or substituted (e.g.
  • ring W is substituted with one or more substituent groups.
  • ring W is substituted with one or more size-limited substituent groups.
  • ring W is substituted with one or more lower substituent groups.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C 3 -C 8 cycloalkylene.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C3-C8 cycloalkylene. In embodiments, ring W is an unsubstituted C 3 -C 8 cycloalkylene. [00280] In embodiments, ring W is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C3-C8 cycloalkylene.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclobutylene. In embodiments, ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclopentylene. In embodiments, ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclohexylene.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C 5 -C 6 arylene.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C5-C6 arylene.
  • ring W is an unsubstituted C5-C6 arylene.
  • ring W is a substituted with one or more (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) C5-C6 arylene.
  • ring C is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl) or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • a substituted e.g. with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted heterocycloalkyl e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5
  • ring C is substituted with one or more substituent groups. In embodiments, ring C is substituted with one or more size-limited substituent groups. In embodiments, ring C is substituted with one or more lower substituent groups. [00284] In embodiments, ring C is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl) or substituted (e.g.
  • ring C is substituted with one or more substituent groups.
  • ring C is substituted with one or more size-limited substituent groups.
  • ring C is substituted with one or more lower substituent groups.
  • ring C is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 9 membered heteroaryl.
  • ring C is an unsubstituted 5 to 9 membered heteroaryl.
  • ring C is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heteroaryl.
  • ring C is an unsubstituted 5 to 6 membered heteroaryl.
  • ring C is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 3 to 8 membered heterocycloalkyl.
  • ring C is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heterocycloalkyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl.
  • ring C is unsubstituted furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanyl. In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanyl. In embodiments, ring C is unsubstituted furanyl. [00290] In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyrrolyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyrrolyl. In embodiments, ring C is unsubstituted pyrrolyl. [00291] In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyridyl. In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyridyl. In embodiments, ring C is unsubstituted pyridyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyranyl
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyranyl.
  • ring C is unsubstituted pyranyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted imidazolyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) imidazolyl. In embodiments, ring C is unsubstituted imidazolyl. [00294] In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted thiazolyl. In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) thiazolyl. In embodiments, ring C is unsubstituted thiazolyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted thienyl. In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) thienyl. In embodiments, ring C is unsubstituted thienyl. [00296] In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted oxazolyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) oxazolyl. In embodiments, ring C is unsubstituted oxazolyl. [00297] In embodiments, ring C is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C 3 -C 6 cycloalkyl, or C 5 -C 6 cycloalkyl) or substituted (e.g.
  • ring C is substituted with one or more substituent groups.
  • ring C is substituted with one or more size-limited substituent groups.
  • ring C is substituted with one or more lower substituent groups.
  • ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C 3 -C 8 cycloalkyl.
  • ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C3-C8 cycloalkyl. In embodiments, ring C is an unsubstituted C 3 -C 8 cycloalkyl. In embodiments ring C is a substituted with one or more (eg with a substituent group, a size-limited substituent group or a lower substituent group) C 3 -C 8 cycloalkyl.
  • ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclobutyl. In embodiments, ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclopentyl. In embodiments, ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclohexyl.
  • ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C5-C6 aryl.
  • ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C5-C6 aryl.
  • ring C is an unsubstituted C5-C6 aryl.
  • ring C is a substituted with one or more (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) C 5 -C 6 aryl.
  • Z is N.
  • Z is O.
  • Z is S.
  • an ADC having the structure: ,
  • Drug loading is represented by m, the average number of drug moieties (i.e., D, D’, or D’’) per anti-CD25 antibody in an antibody drug conjugate (ADC) of formula (I), formula (II), or formula (III), and variations thereof. Drug loading may range from 1 to 20 drug moieties per antibody.
  • ADCs of formula (I), formula (II), or formula (III), and any embodiment, variation, or aspect thereof, include collections of antibodies conjugated with a range of drug moieties, from 1 to 20.
  • the average number of drug moieties per antibody in preparations of ADCs from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC.
  • the quantitative distribution of ADCs in terms of m may also be determined.
  • separation, purification, and characterization of homogeneous ADCs where m is a certain value from ADCs with other drug loadings may be achieved by means such as HIC or reverse phase HPLC or electrophoresis.
  • the average number of drug moieties (i.e., D, D’, or D’’) per anti-CD25 antibody may range from 1 to 8 drug moieties per antibody.
  • m may be limited by the number of attachment sites on the antibody.
  • an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.
  • the average drug loading for ADC ranges from 1 to about 8, or from about 3 to about 8.
  • L 1 is capable of forming a covalent bond with the thiol groups of the free cysteine(s) in the IgG antibody.
  • conjugation methods to derivatize a polypeptide with a payload can be accomplished by forming an amide bond with a lysine side chain. Due to the presence of large number of lysine side chain amines with similar reactivity, this conjugation strategy can produce very complex heterogeneous mixtures.
  • the compositions and methods provided herein provide conjugation through lysine, where, in some embodiments, enhanced selectivity of the lysine can result in a less heterogenous mixture.
  • the average drug loading for ADC ranges from 1 to about 20, from 1 to about 8, or from about 3 to about 8.
  • L 1 is capable of forming a covalent bond with the amine group(s) of the lysine(s) in the IgG antibody.
  • fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction.
  • antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug moiety; indeed, most cysteine thiol residues in antibodies exist as disulfide bridges.
  • an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarboxyethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups.
  • a reducing agent such as dithiothreitol (DTT) or tricarboxyethylphosphine (TCEP)
  • DTT dithiothreitol
  • TCEP tricarboxyethylphosphine
  • an antibody is subjected to reducing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.
  • the loading (drug/antibody ratio or “DAR”) of an ADC may be controlled in different ways, and for example, by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification. DAR can also be controlled by the reactivity of the groups reacting with the antibody or reactivity of the groups of the antibody.
  • the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody.
  • the average number of drugs per antibody may be calculated by using HIC, RP, UV, or LC-MS.
  • Individual ADC molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography (see, e.g., McDonagh et al (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al (2004) Clin.
  • a homogeneous ADC with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.
  • Anti-CD25 Antibodies i. Exemplary Antibodies and Antibody Sequences
  • the ADC comprises an antibody that binds to CD25.
  • CD25 has been reported to be upregulated, for example, in leukemias and lymphomas independent of baseline levels of CD25 expression.
  • the ADC compounds described herein comprise an anti-CD25 antibody.
  • the anti-CD25 antibody provided herein comprises a cysteine.
  • the anti-CD25 antibody is bound to a drug, via linker, through the sulfur of a cysteine residue.
  • the anti-CD25 antibody is bound to a drug, via linker, through the sulfur of two cysteine residues.
  • the anti-CD25 antibody provided herein comprises a lysine.
  • the anti-CD25 antibody is bound to a drug, via linker, through the amine of a lysine residue.
  • the anti-CD25 antibody is bound to a drug, via linker, through the amine of one or two lysine residues.
  • the ADC provided herein comprises an anti-CD25 antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a light chain complementarity determining region 1 (CDR1) a light chain CDR2 and a light chain CDR3, and the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3.
  • the light chain variable region comprises a light chain complementarity determining region 1 (CDR1) a light chain CDR2 and a light chain CDR3
  • the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3.
  • the ADC provided herein comprises an anti-CD25 antibody comprising at least one, two, three, four, five, or six CDRs selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising at least one CDR selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising at least two CDRs selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising at least three CDRs selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises anti-CD25 antibody comprising at least four CDRs selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising at least five CDRs selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising at least six CDRs selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising one CDR selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising two CDRs selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising three CDRs selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising four CDRs selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising five CDRs selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising six CDRs selected from (a) VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the anti-CD25 antibody comprises a VL CDR1 comprising the sequence of SEQ ID NO: 1, a VL CDR2 comprising the sequence of SEQ ID NO: 2, a VL CDR3 comprising the sequence of SEQ ID NO: 3, a VH CDR1 comprising the sequence of SEQ ID NO: 4, a VH CDR2 comprising the sequence of SEQ ID NO: 5, and a VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the anti-CD25 antibody comprises a VL CDR1 comprising the sequence of SEQ ID NO: 1.
  • the anti-CD25 antibody comprises a VL CDR2 comprising the sequence of SEQ ID NO: 2.
  • the anti-CD25 antibody comprises a VL CDR3 comprising the sequence of SEQ ID NO: 3. In embodiments, the anti-CD25 antibody comprises a VH CDR1 comprising the sequence of SEQ ID NO: 4. In embodiments, the anti-CD25 antibody comprises a VH CDR2 comprising the sequence of SEQ ID NO: 5. In embodiments, the anti-CD25 antibody comprises and a VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the ADC comprises an anti-CD25 antibody comprising the light chain CDR1 has the amino acid sequence of SEQ ID NO:1, the light chain CDR2 has the amino acid sequence of SEQ ID NO:2, the light chain CDR3 has the amino acid sequence of SEQ ID NO:3, the heavy chain CDR1 has the amino acid sequence of SEQ ID NO:4, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:5, and the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:6.
  • the anti-CD25 antibody comprises a VL having a sequence with at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7.
  • the anti-CD25 antibody comprises a VL having the sequence of SEQ ID NO: 7.
  • a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-CD25 antibody comprising that sequence retains the ability to bind to CD25.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 7.
  • a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 7.
  • the anti-CD25 antibody comprises the VL sequence of SEQ ID NO: 7, and includes post-translational modifications of that sequence. [00324] In embodiments, the anti-CD25 antibody comprises a VH having a sequence with at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8. In embodiments, the anti-CD25 antibody comprises a VH having the sequence of SEQ ID NO: 8.
  • a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-CD25 antibody comprising that sequence retains the ability to bind to CD25.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 8.
  • a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 8.
  • substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).
  • the anti-CD25 antibody comprises the VH sequence of SEQ ID NO: 8, and includes post-translational modifications of that sequence.
  • the anti-CD25 antibody is an IgG antibody.
  • the anti-CD25 antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.
  • the anti-CD25 antibody is an IgG1 or IgG4 antibody.
  • the anti-CD25 antibody is an IgG1 antibody.
  • an anti-CD25 antibody binds a human CD25.
  • the human CD25 has the amino acid sequence of SEQ ID NO: 16.
  • an anti-CD25 antibody is humanized.
  • an anti-CD25 antibody comprises CDRs as in any of the above embodiments, and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.
  • a humanized anti-CD25 antibody comprises (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6.
  • the anti-CD25 antibody is a monoclonal antibody, including a chimeric, humanized, or human antibody.
  • an anti-CD25 antibody is an antibody fragment, e.g., a Fv, Fab, Fab’, scFv, diabody, or F(ab’) 2 fragment.
  • the antibody is a substantially full-length antibody, e.g., an IgG1 antibody or other antibody class or isotype as defined herein.
  • an anti-CD25 antibody provided herein binds a human CD25 with an affinity of ⁇ 10 nM, or ⁇ 5 nM, or ⁇ 4 nM, or ⁇ 3 nM, or ⁇ 2 nM.
  • an anti- CD25 antibody binds a human CD25 with an affinity of ⁇ 0.0001 nM, or ⁇ 0.001 nM, or ⁇ 0.01 nM.
  • Standard assays known to the skilled artisan can be used to determine binding affinity. For example, whether an anti-CD25 antibody “binds with an affinity of” ⁇ 10 nM, or ⁇ 5 nM, or ⁇ 4 nM, or ⁇ 3 nM, or ⁇ 2 nM, can be determined using standard Scatchard analysis utilizing a non- linear curve fitting program (see, for example, Munson et al., Anal Biochem, 107: 220-239, 1980).
  • the anti-CD25 antibody provided herein has a dissociation constant (Kd) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM, and optionally is ⁇ 10 -13 M. (e.g. 10 -8 M or less, e.g. from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10- 13 M).
  • Kd dissociation constant
  • the antibody (e.g., anti-CD25 antibody) provided herein is an antibody fragment.
  • Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments, and other fragments described below.
  • Fab fragment antigen binding protein
  • Fab fragment antigen binding protein
  • Fab fragment antigen binding protein
  • Fab fragment antigen binding protein
  • Fab fragment antigen binding protein
  • Fab fragment antigen binding protein
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129- 134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein. iv.
  • the anti-CD25 antibody provided herein is a chimeric antibody.
  • Certain chimeric antibodies are described, e.g., in U.S. Patent No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
  • a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody.
  • Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a humanized antibody.
  • a non- human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci.
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol.151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front.
  • framework regions selected using the “best-fit” method see, e.g., Sims et al. J. Immunol.151:2296 (1993)
  • framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions see, e.g.,
  • the anti-CD25 antibody provided herein is a human antibody.
  • Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol.20:450-459 (2008).
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge.
  • Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes.
  • the endogenous immunoglobulin loci have generally been inactivated.
  • Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.
  • Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described.
  • Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain.
  • the anti-CD25 antibody provided herein is a multispecific antibody, e.g., a bispecific antibody.
  • Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In embodiments, one of the binding specificities is for CD25 and the other is for any other antigen.
  • bispecific antibodies may bind to two different epitopes of CD25. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express CD25. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.
  • Multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J.10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross- linking two or more antibodies or fragments (see, e.g., US Patent No.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. a) Substitution, Insertion, and Deletion Variants [00348] In embodiments, the anti-CD25 antibody provided herein has one or more amino acid substitutions.
  • Sites of interest for substitutional mutagenesis include the HVRs and FRs.
  • Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions.” More substantial changes are provided in Table 1 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Table 1. Exemplary Amino acid substitutions.
  • yr rp; e; r; er e Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity). [00350] Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity.
  • HVR HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol.207:179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al.
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (eg error prone PCR chain shuffling or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling.
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of HVR “hotspots” or SDRs.
  • each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085.
  • a residue or group of target residues e.g., charged residues such as arg, asp, his, lys, and glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g.
  • an anti-CD25 antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the antibody comprises an Fc region
  • the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region.
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody may be made in order to create antibody variants with certain improved properties.
  • antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al J Mol Biol 336:1239-1249 (2004); Yamane-Ohnuki et al Biotech. Bioeng.
  • Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys.249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol.
  • Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
  • Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • c) Fc Region Variants [00358]
  • one or more amino acid modifications may be introduced into the Fc region of an anti-CD25 antibody provided herein, thereby generating an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
  • a human Fc region sequence e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region
  • an amino acid modification e.g. a substitution
  • an antibody variant that possesses some but not all effector functions is contemplated, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fc ⁇ R binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 ® non- radioactive cytotoxicity assay (Promega, Madison, WI).
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652- 656 (1998).
  • C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056).
  • Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No.7,332,581).
  • Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No.
  • Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826).
  • Fc region residue 434 US Patent No. 7,371,826
  • a monoclonal antibody such as an anti-CD25 antibody, provided herein may be further modified (e.g., derivatized) to contain one or more additional non- proteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
  • Non- limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • copolymers of ethylene glycol/propylene glycol carboxymethylcellulose
  • dextran polyvinyl alcohol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc. ix.
  • Recombinant Methods and Compositions [00365] Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No.
  • exemplary host cells include eukaryotic cells, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • eukaryotic cells e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • lymphoid cell e.g., Y0, NS0, Sp20 cell.
  • An ADC of formula (I) may be prepared by several routes employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent (L 1 ) to form Ab-L 1 via a covalent bond, followed by reaction with a drug-linker molecule D-L 3 or D-L 3 -L 2 and (2) reaction of a nucleophilic group of a drug moiety D with a bivalent linker reagent (L 3 -L 2 -L 1 or L 3 -L 1 ) to form D-L 3 -L 1 or D-L 3 -L 2 -L 1 via a covalent bond,
  • An ADC of formula (II) may be prepared by several routes employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent (L 1 ) to form Ab-L 1 via a covalent bond, followed by reaction with a drug-linker molecule R * -D’ or R * -D’-L 2 and (2) reaction of a nucleophilic group of a drug- linker molecule R * -D’ with a bivalent linker reagent (L 2 -L 1 or L 1 ) to form R * -D’-L 1 or R * -D’-L 2 - L 1 via a covalent bond, followed by reaction with a nucleophilic group of an antibody or a reduced antibody.
  • An ADC of formula (III) may be prepared by several routes employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent (L 1 ) to form Ab-L 1 via a covalent bond, followed by reaction with a drug moiety D’’ or drug-linker molecule D’’-L 2 ; and (2) reaction of a nucleophilic group of a drug moiety D’’ with a bivalent linker reagent (L 2 and/or L 1 ) to form D’’-L 2 or D’’-L 2 -L 1 via a covalent bond, followed by reaction with a nucleophilic group of an antibody or a reduced antibody.
  • an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarboxyethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups.
  • DTT dithiothreitol
  • TCEP tricarboxyethylphosphine
  • the inter-chain cysteine residues can then be alkylated for example using maleimide.
  • the inter-chain cysteine residues can undergo bridging alkylation for example using bis sulfone linkers or propargyldibromomaleimide followed by Cu-click ligation.
  • the antibody can be conjugated through lysine amino acid.
  • Such conjugation can be a one-step conjugation or a two- step conjugation.
  • the one-step conjugation entails conjugation of the ⁇ -amino group of lysine residue to the drug-linker molecule (D-L 3 -L 2 -L 1 or D-L 3 -L 1 ) containing an amine-reactive group via amide bonds.
  • the one-step conjugation entails conjugation of the ⁇ -amino group of lysine residue to the drug-linker molecule (R * -D’-L 2 -L 1 or R * -D’-L 1 ) containing an amine-reactive group via amide bonds.
  • the one-step conjugation entails conjugation of the ⁇ -amino group of lysine residue to the drug-linker molecule (D’’-L 2 -L 1 or D’’-L 1 ) containing an amine-reactive group via amide bonds.
  • the amine-reactive group is an activated ester.
  • the antibody can be conjugated via a two-step conjugation.
  • the two-step conjugation entails a first step, where a bi-functional reagent containing both amine and thiol reactive functional groups is reacted with the lysine ⁇ -amino group(s).
  • the drug-linker molecule (D-L 3 -L 2 -L 1 , D-L 3 -L 1 , R * -D’-L 2 -L 1 , R * -D’-L 1 , D’’-L 2 -L 1 , or D’’-L 1 ) is conjugated to the thiol reactive group of the bifunctional reagent.
  • Several examples are provided by Jain et al., (2015), Pharm. Res., 32:3526- 3540.
  • the first step may involve the functionalization of the antibody with azide followed by a click chemistry reaction with an alkyne modified linker or drug-linker molecule (D-L 3 -L 2 -L 1 , D-L 3 -L 1 , R * -D’-L 2 -L 1 , R * -D’-L 1 , D’’-L 2 -L 1 , or D’’-L 1 ).
  • an alkyne modified linker or drug-linker molecule D-L 3 -L 2 -L 1 , D-L 3 -L 1 , R * -D’-L 2 -L 1 , R * -D’-L 1 , D’’-L 2 -L 1 , or D’’-L 1 ).
  • the first step may involve the functionalization of the antibody with an alkyne followed by a click chemistry reaction with an azide modified linker or drug-linker molecule (D-L 3 -L 2 -L 1 , D-L 3 -L 1 , R * -D’-L 2 -L 1 , R * -D’-L 1 , D’’-L 2 -L 1 , or D’’-L 1 ).
  • the first step may involve the functionalization of the antibody with an aldehyde followed by a click chemistry reaction with an alkoxyamine or hydrazine modified linker or drug-linker molecule (D-L 3 -L 2 -L 1 , D-L 3 -L 1 , R * -D’- L 2 -L 1 , R * -D’-L 1 , D’’-L 2 -L 1 , or D’’-L 1 ).
  • an alkoxyamine or hydrazine modified linker or drug-linker molecule D-L 3 -L 2 -L 1 , D-L 3 -L 1 , R * -D’- L 2 -L 1 , R * -D’-L 1 , D’’-L 2 -L 1 , or D’’-L 1 ).
  • the first step may involve the functionalization of the antibody with a tetrazine followed by a click chemistry reaction with a trans-cyclooctene or cyclopropene modified linker or drug-linker molecule (D-L 3 -L 2 -L 1 , D-L 3 - L 1 , R * -D’-L 2 -L 1 , R * -D’-L 1 , D’’-L 2 -L 1 , or D’’-L 1 ).
  • a trans-cyclooctene or cyclopropene modified linker or drug-linker molecule D-L 3 -L 2 -L 1 , D-L 3 - L 1 , R * -D’-L 2 -L 1 , R * -D’-L 1 , D’’-L 2 -L 1 , or D’’-L 1 ).
  • the first step may involve the functionalization of the antibody with a trans-cyclooctene or cyclopropene followed by a click chemistry reaction with a tetrazine modified linker or drug-linker molecule (D-L 3 -L 2 -L 1 , D-L 3 - L 1 , R * -D’-L 2 -L 1 , R * -D’-L 1 , D’’-L 2 -L 1 , or D’’-L 1 ).
  • a tetrazine modified linker or drug-linker molecule D-L 3 -L 2 -L 1 , D-L 3 - L 1 , R * -D’-L 2 -L 1 , R * -D’-L 1 , D’’-L 2 -L 1 , or D’’-L 1 .
  • an ADC of formula (I), formula (II), or formula (III) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-I), formula (P-II), or formula (P-III): , , (P-I) (P-II) (P-III) or a pharmaceutically acceptable salt thereof, wherein: B is a reactive moiety capable of forming a bond with the anti-CD25 antibody; L 2 is a bond, -C(O)-, -NH-, Amino Acid Unit, –(CH 2 CH 2 O) n –, –(CH 2 ) n –, -O-, –(4-aminobenzyloxycarbonyl)–,
  • R 1 is H or –C1-C8 alkyl
  • R 3 is H, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR 3A , -NR 3A R 3B , -(CH2)vOR 6 , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl;
  • R 4 is H, halogen, -OR 4A , -NR 4A R 4B , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl;
  • V is N, O, or C;
  • Z 1 is a substituted or unsubstit
  • the anti-CD25 antibody is modified with a reactive moiety such as an aldehyde, azide, alkyne, tetrazine, hydrazine, alkoxyamine, trans-cyclooctene or cyclopropene.
  • the anti-CD25 antibody is modified with an aldehyde.
  • the anti-CD25 antibody is modified with an azide.
  • the anti-CD25 antibody is modified with a tetrazine.
  • the anti-CD25 antibody is modified with an alkoxyamine.
  • the anti-CD25 antibody is modified with a hydrazine.
  • the anti-CD25 antibody is modified with a trans-cyclooctene. In embodiments, the anti-CD25 antibody is modified with a cyclopropene.
  • B is a reactive moiety capable of forming a bond with an anti-CD25 antibody.
  • Ab is a modified anti-CD25 antibody. [00372] In embodiments, Ab is modified with an aldehyde, azide, alkyne, tetrazine, hydrazine, alkoxyamine, trans-cyclooctene or cyclopropene. In embodiments, Ab is modified with an aldehyde. In embodiments, Ab is modified with an azide.
  • Ab is modified with a tetrazine. In embodiments, Ab is modified with an alkoxyamine. In embodiments, Ab is modified with a hydrazine. In embodiments, Ab is modified with a trans-cyclooctene. In embodiments, Ab is modified with a cyclopropene. In embodiments, a modified Ab is a modified anti-CD25 antibody.
  • n is an integer from 1 to 24. In embodiments, n is an integer from 1 to 4. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 3. In embodiments, n is 4. In embodiments, n is 5. In embodiments, n is 6. In embodiments, n is 7.
  • n is 8. In embodiments, n is 9. In embodiments, n is 10. In embodiments, n is 11. In embodiments, n is 12. In embodiments, n is 13. In embodiments, n is 14. In embodiments, n is 15. In embodiments, n is 16. In embodiments, n is 17. In embodiments, n is 18. In embodiments, n is 19. In embodiments, n is 20. In embodiments, n is 21. In embodiments, n is 22. In embodiments, n is 23. In embodiments, n is 24.
  • B is a reactive moiety capable of forming a bond with one or two thiol or amine groups of the anti-CD25 antibody, or with the modified anti-CD25 antibody.
  • the anti-CD25 antibody is modified with an azide, aldehyde, alkyne, tetrazine, hydrazine, alkoxyamine, trans-cyclooctene or cyclopropene.
  • B is an alkyne, azide, aldehyde, tetrazine, hydrazine, alkoxyamine, trans-cyclooctene, cyclopropene, activated ester, haloacetyl, cycloalkyne, maleimide, or bis- sulfone.
  • B is dibromomaleimide.
  • B is cyclooctyne.
  • the activated ester may be for example pentafluorophenyl ester, tetrafluorophenyl ester, trifluorophenyl ester, difluorophenyl ester, monofluorophenyl or ester, N- hydroxysuccinimide ester.
  • D’’ is wherein: R 1 is H or –C 1 -C 8 alkyl; R 3 is H, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 4 is H, halogen, or substituted or unsubstituted alkyl; V is N; and Z 2 is a substituted or unsubstituted arylene.
  • D’’ is: [ [ , [ [ , . , . embodiments, B is . In embodiments, B is . In embodiments, B is In embodiments, embodiments, B is . In embodiments, B is . In embodiments, B is .
  • B is . In embodiments, B . [ , , , , or . [00383] In embodiments, monoclonal antibodies, modified monoclonal antibodies, or anti- CD25 unmodified or modified antibodies (Ab) undergo conjugation reactions with the following reactive B moieties as follows:
  • L 2 is a cleavable or a non-cleavable linker as described in US Patents Nos. US 9,884,127, US 9,981,046, US 9,801,951, US 10,117,944, US 10,590,165, and US 10,590,165, and US Patent publications Nos. US 2017/0340750, and US 2018/0360985, all of which are incorporated herein in their entireties.
  • L 2 is a bond, -C(O)-, -NH-, -Val-, -Phe-, -Lys-, -Gly-, -O- –(4-aminobenzyloxycarbonyl)–, –(C(O)N(R 2 )CH2CH2N(R 5 ))–, -Ser-, -Thr-, -Ala-, - ⁇ -Ala-, -citrulline- (Cit), –(CH2)n–, –(CH2CH2O)n–, N-dimethyl lysine, or any combination thereof.
  • each R 2 and R 5 is independently H or substituted or unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl). In embodiments, each R 2 and R 5 is independently H. In embodiments, each R 2 and R 5 is independently substituted or unsubstituted alkyl. In embodiments, each R 2 and R 5 is independently substituted or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • each R 2 and R 5 is independently unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl). In embodiments, each R 2 and R 5 is independently substituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • each R 2 and R 5 is independently H or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • each R 2 and R 5 is independently substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl).
  • each R 2 and R 5 is independently unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). [00388] In embodiments, each R 2 and R 5 is independently methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, or hexyl. In embodiments, each R 2 and R 5 is independently methyl. In embodiments, each R 2 and R 5 is independently ethyl. In embodiments, each R 2 and R 5 is independently propyl.
  • each R 2 and R 5 is independently butyl. In embodiments, each R 2 and R 5 is independently isopropyl. In embodiments, each R 2 and R 5 is independently isobutyl. In embodiments, each R 2 and R 5 is independently tert-butyl. In embodiments, each R 2 and R 5 is independently isopentyl.
  • L 2 is a bond, -C(O)-, -NH-, -Val-, -Phe-, -Lys-, -Gly-, –(4-aminobenzyloxycarbonyl)–, –(C(O)N(CH3)CH2CH2N(CH3))–, -Ser-, -Thr-, -Ala-, - ⁇ -Ala-, -O-, -citrulline- (Cit), –(CH 2 ) n –, –(CH 2 CH 2 O) n –, N-dimethyl lysine, or any combination thereof.
  • L 2 is -C(O)-, -NH-, -Val-, -Gly-, -Cit-, -Ala-, -O-, –(4- aminobenzyloxycarbonyl)–, –(CH2)n–, –(CH2CH2O)n–, –(C(O)N(CH3)CH2CH2N(CH3))–, N- dimethyl lysine, or any combination thereof.
  • L 2 is -C(O)-, -NH-, -Gly-, –(CH 2 ) n –, –(CH 2 CH 2 O) n –, or any combination thereof.
  • L 2 is -C(O)-, -NH-, -Val-, -Cit-, –(CH2CH2O)n–, –(4- aminobenzyloxycarbonyl)–, –(CH 2 ) n –, –(C(O)N(CH 3 )CH 2 CH 2 N(CH 3 ))–, N-dimethyl lysine, or any combination thereof.
  • L 2 is -C(O)-, -NH-, -Val-, –(4-aminobenzyloxycarbonyl)–, -Gly-, -citrulline- (-Cit-), –(CH 2 ) n –, –(CH 2 CH 2 O) n –, N-dimethyl lysine, or any combination thereof.
  • L 2 is: , , o [ , o [ , . e .
  • L is .
  • L 2 is
  • L 2 is a bond. In embodiments, L 2 is -C(O)-. In embodiments, L 2 is -NH-. In embodiments, L 2 is -Val-. In embodiments, L 2 is -Phe-. In embodiments, L 2 is -Lys-. In embodiments, L 2 is –(4-aminobenzyloxycarbonyl)–. In embodiments, L 2 is –(CH2)n–. In embodiments, L 2 is –(CH 2 CH 2 O) n –. In embodiments, L 2 is -Gly-. In embodiments, L 2 is -Ser-. In embodiments, L 2 is -Thr-.
  • L 2 is -Ala-. In embodiments, L 2 is - ⁇ -Ala-. In embodiments, L 2 is -Cit-. In embodiments, L 2 is -O-. In embodiments, L 2 is N-dimethyl lysine. [00398] In embodiments, L 3 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene), substituted (e.g.
  • heteroarylene e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene, substituted (e.g.
  • heterocycloalkylene e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene
  • substituted e.g., with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted -OCH2-(heteroarylene e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene)
  • substituted e.g.
  • heterocycloalkyl e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl
  • substituted e.g., with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted -CH 2 NCH 2 -(heteroaryl e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl
  • L 3 is substituted with one or more substituent groups. In embodiments, L 3 is substituted with one or more size-limited substituent groups. In embodiments, L 3 is substituted with one or more lower substituent groups. [00399] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene), or substituted (e.g.
  • L 3 is substituted with one or more substituent groups. In embodiments, L 3 is substituted with one or more size-limited substituent groups. In embodiments, L 3 is substituted with one or more lower substituent groups.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • L 3 is substituted with one or more substituent groups.
  • L 3 is substituted with one or more size-limited substituent groups.
  • L 3 is substituted with one or more lower substituent groups.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • L 3 is unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • heteroarylene e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene.
  • L 3 is unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) - OCH 2 -(heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl)).
  • L 3 is unsubstituted -OCH2-(heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene)).
  • L 3 is substituted (e.g., with a substituent group a size-limited substituent group or a lower substituent group) -OCH 2 -(heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene)).
  • L 3 is unsubstituted -OCH2-(heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene)).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -CH 2 NCH 2 - (heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl)).
  • L 3 is unsubstituted -CH 2 NCH 2 - (heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl)).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -CH2NCH2-(heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl)).
  • L 3 is unsubstituted -CH2NCH2-(heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl)).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 8 membered heterocycloalkylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 3 to 8 membered heterocycloalkylene. In embodiments, L 3 is unsubstituted 3 to 8 membered heterocycloalkylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) or unsubstituted -CH2NCH2-(3 to 8 membered heterocycloalkyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -CH2NCH2-(3 to 8 membered heterocycloalkyl). In embodiments, L 3 is unsubstituted - CH2NCH2-(3 to 8 membered heterocycloalkyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) or unsubstituted -OCH2-(3 to 8 membered heterocycloalkylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -OCH2-(3 to 8 membered heterocycloalkylene). In embodiments, L 3 is unsubstituted -OCH 2 -(3 to 8 membered heterocycloalkylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 8 membered heterocycloalkylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 6 membered heterocycloalkylene.
  • L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) 3 to 6 membered heterocycloalkylene.
  • L 3 is unsubstituted 3 to 6 membered heterocycloalkylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) or unsubsituted -CH 2 NCH 2 -(3 to 6 membered heterocycloalkyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -CH2NCH2-(3 to 6 membered heterocycloalkyl). In embodiments, L 3 is unsubsituted - CH2NCH2-(3 to 6 membered heterocycloalkyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) or unsubsituted -OCH2-(3 to 6 membered heterocycloalkylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group, or a lower substituent group) -OCH2-(3 to 6 membered heterocycloalkylene). In embodiments, L 3 is unsubsituted -OCH 2 -(3 to 6 membered heterocycloalkylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 6 membered heterocycloalkylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclobutylene, heterocyclopentylene or heterocyclohexylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclobutylene, heterocyclopentylene or heterocyclohexylene. In embodiments, L 3 is unsubstituted heterocyclobutylene, heterocyclopentylene or heterocyclohexylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(heterocyclobutyl, heterocyclopentyl, or heterocyclohexyl).
  • L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) -CH 2 NCH 2 -(heterocyclobutyl, heterocyclopentyl, or heterocyclohexyl). In embodiments, L 3 is unsubstituted -CH2NCH2- (heterocyclobutyl, heterocyclopentyl, or heterocyclohexyl).
  • L 3 is substituted (e.g., with a substituent group a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(heterocyclobutylene, heterocyclopentylene, or heterocyclohexylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2-(heterocyclobutylene, heterocyclopentylene, or heterocyclohexylene). In embodiments, L 3 is unsubstituted -OCH 2 -(heterocyclobutylene, heterocyclopentylene, or heterocyclohexylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclobutylene, heterocyclopentylene or heterocyclohexylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclobutylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclobutylene.
  • L 3 is unsubstituted heterocyclobutylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 - (heterocyclobutyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) -CH2NCH2-(heterocyclobutyl). In embodiments, L 3 is unsubstituted -CH 2 NCH 2 -(heterocyclobutyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(heterocyclobutylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2- (heterocyclobutylene). In embodiments, L 3 is unsubstituted -OCH 2 -(heterocyclobutylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclopentylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclopentylene. In embodiments, L 3 is unsubstituted heterocyclopentylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 - (heterocyclopentyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH2NCH2-(heterocyclopentyl). In embodiments, L 3 is unsubstituted -CH2NCH2-(heterocyclopentyl). In embodiments, L 3 is substituted (e.g., with a substituent group a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(heterocyclopentylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2- (heterocyclopentylene). In embodiments, L 3 is unsubstituted -OCH2-(heterocyclopentylene). [00410] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclohexylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclohexylene.
  • L 3 is unsubstituted heterocyclohexylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2- (heterocyclohexyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) -CH 2 NCH 2 -(heterocyclohexyl). In embodiments, L 3 is unsubstituted -CH2NCH2-(heterocyclohexyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(heterocyclohexylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 - (heterocyclohexylene). In embodiments, L 3 is unsubstituted -OCH2-(heterocyclohexylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 10 membered heteroarylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 10 membered heteroarylene. In embodiments, L 3 is unsubstituted 5 to 10 membered heteroarylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(5 to 10 membered heteroaryl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH 2 NCH 2 -(5 to 10 membered heteroaryl). In embodiments, L 3 is unsubstituted -CH2NCH2-(5 to 10 membered heteroaryl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(5 to 10 membered heteroarylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2-(5 to 10 membered heteroarylene). In embodiments, L 3 is unsubstituted -OCH2-(5 to 10 membered heteroarylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 9 membered heteroarylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 9 membered heteroarylene. In embodiments, L 3 is unsubstituted 5 to 9 membered heteroarylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(5 to 9 membered heteroaryl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH 2 NCH 2 -(5 to 9 membered heteroaryl). In embodiments, L 3 is unsubstituted -CH2NCH2-(5 to 9 membered heteroaryl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(5 to 9 membered heteroarylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2-(5 to 9 membered heteroarylene). In embodiments, L 3 is unsubstituted -OCH 2 -(5 to 9 membered heteroarylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 6 membered heteroarylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heteroarylene. In embodiments, L 3 is unsubstituted 5 to 6 membered heteroarylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(5 to 6 membered heteroaryl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH2NCH2-(5 to 6 membered heteroaryl). In embodiments, L 3 is unsubstituted -CH2NCH2-(5 to 6 membered heteroaryl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(5 to 6 membered heteroarylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 -(5 to 6 membered heteroarylene). In embodiments, L 3 is unsubstituted -OCH 2 -(5 to 6 membered heteroarylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene thienylene oxazolylene or thiazolylene
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene, thienylene, oxazolylene, or thiazolylene.
  • L 3 is unsubstituted furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene, thienylene, oxazolylene, or thiazolylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH 2 NCH 2 - (furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl).
  • L 3 is unsubstituted -CH2NCH2-(furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene, thienylene, oxazolylene, or thiazolylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 -(furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene, thienylene, oxazolylene, or thiazolylene).
  • L 3 is unsubstituted -OCH 2 -(furanylene, pyrrolylene, pyridylene, pyranylene, imidazolylene, thienylene, oxazolylene, or thiazolylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanylene. In embodiments, L 3 is unsubstituted furanylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(furanyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) - CH2NCH2-(furanyl). In embodiments, L 3 is unsubstituted -CH2NCH2-(furanyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(furanylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2- (furanylene).
  • L 3 is unsubstituted -OCH2-(furanylene). [00416] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyrrolylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyrrolylene. In embodiments, L 3 is unsubstituted pyrrolylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(pyrrolyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH 2 NCH 2 -(pyrrolyl). In embodiments, L 3 is unsubstituted -CH 2 NCH 2 -(pyrrolyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(pyrrolylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2-(pyrrolylene). In embodiments, L 3 is unsubstituted -OCH2-(pyrrolylene). [00417] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyridylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyridylene. In embodiments, L 3 is unsubstituted pyridylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(pyridyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) - CH2NCH2-(pyridyl).
  • L 3 is unsubstituted -CH2NCH2-(pyridyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(pyridylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2- (pyridylene). In embodiments, L 3 is unsubstituted -OCH 2 -(pyridylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyranylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyranylene. In embodiments, L 3 is unsubstituted pyranylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(pyranyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) - CH 2 NCH 2 -(pyranyl). In embodiments, L 3 is unsubstituted -CH 2 NCH 2 -(pyranyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH2-(pyranylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 -(pyranylene).
  • L 3 is unsubstituted -OCH 2 -(pyranylene). [00419] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted imidazolylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) imidazolylene. In embodiments, L 3 is unsubstituted imidazolylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(imidazolyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH2NCH2-(imidazolyl). In embodiments, L 3 is unsubstituted -CH2NCH2-(imidazolyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(imidazolylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH 2 -(imidazolylene). In embodiments, L 3 is unsubstituted -OCH 2 -(imidazolylene).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted thiazolylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) thiazolylene. In embodiments, L 3 is unsubstituted thiazolylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH2NCH2-(thiazolyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -CH2NCH2-(thiazolyl). In embodiments, L 3 is unsubstituted -CH2NCH2-(thiazolyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(thiazolylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2-(thiazolylene).
  • L 3 is unsubstituted -OCH2-(thiazolylene). [00421] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted thienylene In embodiments L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) thienylene. In embodiments, L 3 is unsubstituted thienylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(thienyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) - CH2NCH2-(thienyl). In embodiments, L 3 is unsubstituted -CH2NCH2-(thienyl).
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(thienylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) -OCH2- (thienylene). In embodiments, L 3 is unsubstituted -OCH2-(thienylene). [00422] In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted oxazolylene.
  • L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) oxazolylene. In embodiments, L 3 is unsubstituted oxazolylene. In embodiments, L 3 is unsubstituted oxazolylene. In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -CH 2 NCH 2 -(oxazolyl).
  • L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) -CH 2 NCH 2 -(oxazolyl). In embodiments, L 3 is unsubstituted -CH2NCH2-(oxazolyl). In embodiments, L 3 is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted -OCH 2 -(oxazolylene). In embodiments, L 3 is substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) -OCH2-(oxazolylene).
  • L 3 is unsubstituted -OCH2-(oxazolylene).
  • R * is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl) or substituted (e.g.
  • R * is substituted with one or more substituent groups.
  • R * is substituted with one or more size-limited substituent groups.
  • R * is substituted with one or more lower substituent groups [00424]
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • R * is unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • R * is unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 8 membered heterocycloalkyl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 3 to 8 membered heterocycloalkyl. In embodiments, R * is unsubstituted 3 to 8 membered heterocycloalkyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 6 membered heterocycloalkyl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 3 to 6 membered heterocycloalkyl. In embodiments, R * is unsubstituted 3 to 6 membered heterocycloalkyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclobutyl, heterocyclopentyl or heterocyclohexyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclobutyl, heterocyclopentyl or heterocyclohexyl.
  • R * is unsubstituted heterocyclobutyl, heterocyclopentyl or heterocyclohexyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclobutyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclobutyl.
  • R * is unsubstituted heterocyclobutyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclopentyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclopentyl. In embodiments, R * is unsubstituted heterocyclopentyl. [00430] In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocyclohexyl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) heterocyclohexyl. In embodiments, R * is unsubstituted heterocyclohexyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 10 membered heteroaryl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 10 membered heteroaryl. In embodiments, R * is unsubstituted 5 to 10 membered heteroaryl. [00432] In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 9 membered heteroaryl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 9 membered heteroaryl. In embodiments, R * is unsubstituted 5 to 9 membered heteroaryl. [00433] In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 6 membered heteroaryl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heteroaryl.
  • R * is unsubstituted 5 to 6 membered heteroaryl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, or thiazolyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, or thiazolyl.
  • R * is unsubstituted furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, or thiazolyl [00435]
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanyl.
  • R * is unsubstituted furanyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyrrolyl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyrrolyl. In embodiments, R * is unsubstituted pyrrolyl. [00437] In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyridyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyridyl. In embodiments, R * is unsubstituted pyridyl. [00438] In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyranyl. In embodiments, R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyranyl. In embodiments, R * is unsubstituted pyranyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted imidazolyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) imidazolyl.
  • R * is unsubstituted imidazolyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted thiazolyl.
  • R * is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) thiazolyl. In embodiments, R * is unsubstituted thiazolyl.
  • R 1 is H. In embodiments, R 1 is –C1-C8 alkyl. [00442] In embodiments, R 1 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, or hexyl. In embodiments, R 1 is methyl. In embodiments, R 1 is ethyl. In embodiments, R 1 is propyl.
  • R 1 is isopropyl. In embodiments, R 1 is butyl. In embodiments, R 1 is isobutyl. In embodiments, R 1 is tert-butyl. In embodiments, R 1 is pentyl. In embodiments, R 1 is hexyl.
  • R 3 is H, halogen, -CCl 3 , -CBr 3 , -CF 3 , -CI 3 , -CHCl 2 , -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR 3A , -NR 3A R 3B , -(CH2)vOR 6 , substituted or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), or substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • alkyl e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl
  • R 3 is H, -OR 3A , -(CH2)vOR 6 , substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl), or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • alkyl e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl
  • heteroalkyl e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered hetero
  • R 3 is a substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C 1 -C 4 alkyl).
  • R 3 is an unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl).
  • R 3 is a substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 3 is an unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 3 is methyl, ethyl, propyl, butyl, –CH2OH, -CH 2 CH 2 OH, -CH 2 N 3 , -CH 2 CH 2 N 3 , -CH 2 OCH 3 , -CH 2 OCH 2 CH 3 , -CH 2 CH 2 OCH 3 , -CH 2 CH 2 OCH 2 CH 3, or .
  • R 3 is H, methyl, ethyl, propyl, butyl, –CH 2 OH, -CH 2 CH 2 OH, -CH 2 N 3 , -CH 2 CH 2 N 3 , -CH 2 OCH 3 , -CH 2 OCH 2 CH 3 , or -CH2CH2OCH3.
  • R 3 is methyl, –CH2OH, or -CH2N3.
  • R 3 is methyl.
  • R 3 is ethyl.
  • R 3 is propyl.
  • R 3 is butyl.
  • R 3 is –CH 2 OH.
  • R 3 is – CH2 CH2OH.
  • R 3 is -CH2N3. In embodiments, R 3 is -CH2CH2N3. In embodiments, R 3 is -CH2OCH3. In embodiments, R 3 is -CH2OCH2CH3. In embodiments, R 3 is - CH 2 CH 2 OCH 3 . In embodiments, R 3 is -CH 2 CH 2 OCH 2 CH 3 . In embodiments, R 3 is -OH. In embodiments, R 3 is H. In embodiments, [00448] In embodiments, R 3 is methyl, -CH 2 N 3 . In embodiments, R 3 is -CH 2 N 3 . [00449] In embodiments, v is an integer from 1 to 24. In embodiments, v is 1.
  • v is 2. In embodiments, v is 3. In embodiments, v is 4. In embodiments, v is 5. In embodiments, v is 6. In embodiments, v is 7. In embodiments, v is 8. In embodiments, v is 9. In embodiments, v is 10. In embodiments, v is 11. In embodiments, v is 12. In embodiments, v is 13. In embodiments, v is 14. In embodiments, v is 15. In embodiments, v is 16. In embodiments, v is 17. In embodiments, v is 18. In embodiments, v is 19. In embodiments, v is 20. In embodiments, v is 21. In embodiments, v is 22. In embodiments, v is 23. In embodiments, v is 24.
  • R 4 is H, halogen, -OR 4A , -NR 4A R 4B , substituted or unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl), or substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • alkyl e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl
  • heteroalkyl e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl.
  • R 4 is H, -OR 4A , substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • alkyl e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl
  • substituted e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group
  • unsubstituted heteroalkyl e
  • R 4 is H, or substituted or unsubstituted alkyl. In embodiments, R 4 is H, or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl).
  • R 4 is H, or substituted or unsubstituted alkyl.
  • R 4 is H, or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl).
  • R 4 is a substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • R 4 is an unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C 1 -C 4 alkyl).
  • R 4 is a substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 4 is an unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 4 is H, -OH, methyl, ethyl, propyl or butyl.
  • R 4 is H or -OH.
  • R 4 is H or methyl. In embodiments, R 4 is methyl. In embodiments, R 4 is ethyl. In embodiments, R 4 is propyl. In embodiments, R 4 is butyl. In embodiments, R 4 is H. In embodiments, R 4 is -OH. [00455] In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently H or substituted or unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl).
  • alkyl e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl.
  • each R 3A , R 3B , R 4A , and R 4B is independently H or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl).
  • each R 3A , R 3B , R 4A , and R 4B is independently H.
  • each R 3A , R 3B , R 4A , and R 4B is independently substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C 1 -C 4 alkyl).
  • each R 3A , R 3B , R 4A , and R 4B is independently unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).
  • each R 3A , R 3B , R 4A , and R 4B is independently H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, or pentyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently H. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently methyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently ethyl.
  • each R 3A , R 3B , R 4A , and R 4B is independently propyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently isopropyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently butyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently isobutyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently tert-butyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently pentyl.
  • R 6 is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -CO(CH 2 CH 2 O) w CH 2 CH 2 M, -CONH(CH 2 CH 2 O) w CH 2 CH 2 M, , a Charged Group, or a saccharide derivative, w is an integer from 1 to 24; M is -NH2, -OH, -COOH, or -OCH3; R 10 is -OH, -OCH3 or -COOH.
  • R 6 is H or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C 1 - C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C 3 -C 8 cycloalkyl, C 3 -C 6 cycloalkyl, or C 5 -C 6 cycloalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl
  • R 6 is H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • R 6 is H or .
  • R 6 is H.
  • w is an integer from 1 to 24. In embodiments, w is 1. In embodiments, w is 2. In embodiments, w is 3. In embodiments, w is 4. In embodiments, w is 5. In embodiments, w is 6.
  • w is 7. In embodiments, w is 8. In embodiments, w is 9. In embodiments, w is 10. In embodiments, w is 11. In embodiments, w is 12. In embodiments, w is 13. In embodiments, w is 14. In embodiments, w is 15. In embodiments, w is 16. In embodiments, w is 17. In embodiments, w is 18. In embodiments, w is 19. In embodiments, w is 20. In embodiments, w is 21. In embodiments, w is 22. In embodiments, w is 23. In embodiments, w is 24. [00463] In embodiments, M is -NH2, -OH, -COOH, or -OCH3. In embodiments, M is -NH2.
  • M is -OH. In embodiments, M is -COOH. In embodiments, M is -OCH 3. [ . I e m o men s, s . [00465] In embodiments, R 6 is a saccharide derivative. In embodiments, R 6 is . [00466] In embodiments, Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl).
  • cycloalkyl e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl.
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl).
  • Z 1 is an unsubstituted cycloalkyl (eg C3-C8 cycloalkyl C3-C6 cycloalkyl or C5-C6 cycloalkyl)
  • Z 1 is a substituted (e.g.
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • Z 1 is an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl).
  • Z 1 is a substituted (e.g.
  • aryl e.g., C6-C10 aryl, C10 aryl, or phenyl
  • Z 1 is an unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl).
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • Z 1 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • Z 1 is an unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • Z is or ; wherein each Q is independently a halogen, methyl, ethyl, or propyl; and q is an integer from 1 to 5.
  • Z 1 is .
  • Z 1 is , wherein Q and q are as described herein including in embodiments.
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene).
  • Z 2 is a substituted (e.g.
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted arylene (e.g., C 6 -C 10 arylene, C 10 arylene, or phenylene).
  • Z 2 is a substituted (e.g.
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene).
  • Z 2 is a substituted (e.g.
  • Z 2 is an unsubstituted arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene).
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • Z 2 is an unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • Z 2 is a substituted (e.g.
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 6 membered heteroarylene.
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heteroarylene.
  • Z 2 is an unsubstituted 5 to 6 membered heteroarylene.
  • Z 2 is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted phenylene.
  • Z 2 is a substituted (e.g.
  • Z 2 is an unsubstituted phenylene.
  • Z 2 is an unsubstituted arylene.
  • V is N.
  • V is O.
  • V is C.
  • - wherein each G is independently Cl, Br, I, F, -CH3, -CH2CH3, -CH2CH2CH3, -OCH3, -OCH2CH3, -OH, or -NH2; and p is an integer from 0-4.
  • an ADC of formula (IA) or formula (IIA) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-IA) or formula (P-IIA): (P-IA) (P-IIA) or a pharmaceutically acceptable salt thereof, wherein: ring A is a substituted or unsubstituted heterocycloalkylene or a substituted or unsubstituted heteroarylene, connected to L 2 through a heteroatom Y; ring A’ is a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl, connected to D’ through a heteroatom Y; Y is N, P, or S; and B, L 2 , D, and D’ are each as defined here
  • ring A is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene) or substituted (e.g.
  • ring A is substituted with one or more substituent groups.
  • ring A is substituted with one or more size-limited substituent groups.
  • ring A is substituted with one or more lower substituent groups.
  • Ring A is connected to L 2 through a heteroatom Y. In embodiments, Y is N. [00480] In embodiments, ring A’ is substituted (e.g.
  • ring A’ is substituted with one or more substituent groups.
  • ring A’ is substituted with one or more size-limited substituent groups. In embodiments, ring A’ is substituted with one or more lower substituent groups. Ring A' is connected to D’ through a heteroatom Y. In embodiments, Y is N. [00481] In embodiments, in formula (P-IA) or (P-IIA), ring A is a substituted with one or more (e.g., with a substitu t i li i b tit t l b tit t group) 3 to 8 membered heterocycloalkylene. In embodiments, ring A is connected to L 2 through a heteroatom Y. In embodiments, each Y is N.
  • ring A’ is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 3 to 8 membered heterocycloalkyl.
  • ring A' is connected to D’ through a heteroatom Y.
  • Y is N.
  • ring A is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heterocycloalkylene.
  • Ring A is connected to L 2 through a heteroatom Y.
  • each Y is N.
  • ring A’ is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heterocycloalkyl.
  • ring A' is connected to D’ through a heteroatom Y.
  • each Y is N.
  • an ADC of formula (IB) or formula (IIB) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-IB) or formula (P-IIB): or (P-IB) (P-IIB) or a pharmaceutically acceptable salt thereof, wherein: Y, D, D’, B, and L 2 are each as defined herein including embodiments.
  • an ADC of formula (IC) or formula (IIC) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-IC) or formula (P-IIC): or (P-IC) (P-IIC) or a pharmaceutically acceptable salt thereof; wherein D, D’, Y, B, and L 2 , are each as defined herein including embodiments.
  • an ADC of formula (ID) or formula (IID) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-ID) or formula (P-IID): (P-ID) (P-IID) or a pharmaceutically acceptable salt thereof; wherein D, D’, Y, B, and L 2 , are each as defined herein including embodiments.
  • an ADC of formula (ID1) or formula (IID1) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-ID1) or formula (P-IID1): (P-ID1) (P-IID1) or a pharmaceutically acceptable salt thereof; wherein D, D’, Y, B, and L 2 , are each as defined herein including embodiments.
  • an ADC of formula (IE) or formula (IIE) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-IE) or formula (P-IIE): or (P-IE) (P-IIE) or a pharmaceutically acceptable salt thereof; wherein D, D’, Y, B, and L 2 , are each as defined herein including embodiments.
  • an ADC of formula (IF) or formula (IIF) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-IF) or formula (P-IIF): or (P-IF) (P-IIF) or a pharmaceutically acceptable salt thereof; wherein D, D’, Y, B, and L 2 , are each as defined herein including embodiments.
  • an ADC of formula (IG) or formula (IH) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-IG) or formula (P-IH): (P-IG) (P-IH) or a pharmaceutically acceptable salt thereof, wherein: ring W is a substituted or unsubstituted cycloalkylene or a substituted or unsubstituted arylene; ring C is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and wherein D, B, and L 2 , are each as defined herein including embodiments.
  • -NH- and -D are connected to different carbon atoms on ring W. In embodiments, -NH- and -D are connected to the same carbon atom on ring W.
  • ring W is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene) or substituted (e.g.
  • ring W is substituted with one or more substituent groups.
  • ring W is substituted with one or more size-limited substituent groups.
  • ring W is substituted with one or more lower substituent groups.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C3-C8 cycloalkylene.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C3-C8 cycloalkylene.
  • ring W is an unsubstituted C3-C8 cycloalkylene.
  • ring W is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C 3 -C 8 cycloalkylene.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclobutylene.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclopentylene.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclohexylene.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C 5 -C 6 arylene.
  • ring W is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C 5 -C 6 arylene.
  • ring W is an unsubstituted C 5 -C 6 arylene. In embodiments, ring W is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C5-C6 arylene.
  • ring C is a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl) or substituted (e.g.
  • ring C is substituted with one or more substituent groups.
  • ring C is substituted with one or more size-limited substituent groups.
  • ring C is substituted with one or more lower substituent groups.
  • ring C is a substituted (e.g.
  • ring C is substituted with one or more substituent groups.
  • ring C is substituted with one or more size-limited substituent groups. In embodiments, ring C is substituted with one or more lower substituent groups. [00498] In embodiments, ring C is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 9 membered heteroaryl. In embodiments, ring C is an unsubstituted 5 to 9 membered heteroaryl. [00499] In embodiments, ring C is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heteroaryl.
  • ring C is an unsubstituted 5 to 6 membered heteroaryl.
  • ring C is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 3 to 8 membered heterocycloalkyl.
  • ring C is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heterocycloalkyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl.
  • ring C is unsubstituted furanyl, pyrrolyl, pyridyl, pyranyl, imidazolyl, thienyl, oxazolyl, or thiazolyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted furanyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) furanyl.
  • ring C is unsubstituted furanyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyrrolyl. In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyrrolyl. In embodiments, ring C is unsubstituted pyrrolyl. [00504] In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyridyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyridyl. In embodiments, ring C is unsubstituted pyridyl. [00505] In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted pyranyl. In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) pyranyl. In embodiments, ring C is unsubstituted pyranyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted imidazolyl. In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) imidazolyl. In embodiments, ring C is unsubstituted imidazolyl. [00507] In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted thiazolyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) thiazolyl. In embodiments, ring C is unsubstituted thiazolyl. [00508] In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted thienyl. In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) thienyl. In embodiments, ring C is unsubstituted thienyl.
  • ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted oxazolyl. In embodiments, ring C is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) oxazolyl. In embodiments, ring C is unsubstituted oxazolyl. [00510] In embodiments, ring C is a substituted (e.g.
  • ring C is substituted with one or more substituent groups.
  • ring C is substituted with one or more size-limited substituent groups. In embodiments, ring C is substituted with one or more lower substituent groups. [00511] In embodiments, ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C3-C8 cycloalkyl. In embodiments, ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C 3 -C 8 cycloalkyl. In embodiments, ring C is an unsubstituted C3-C8 cycloalkyl.
  • ring C is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C3-C8 cycloalkyl.
  • ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclobutyl.
  • ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclopentyl.
  • ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cyclohexyl.
  • ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C 5 -C 6 aryl.
  • ring C is a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C5-C6 aryl.
  • ring C is an unsubstituted C5-C6 aryl. In embodiments, ring C is a substituted with one or more (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) C5-C6 aryl.
  • an ADC of formula (IK) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-IK): (P-IK) or a pharmaceutically acceptable salt thereof, wherein: Z is S, N, or O; and wherein B, D, and L 2 , are each as defined herein including embodiments.
  • Z is N.
  • an ADC of formula (IL) or formula (IM) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-IL) or formula (P-IM): or (IL) (IM) or a pharmaceutically acceptable salt thereof; wherein D, Z, B, and L 2 , are each as defined herein including embodiments.
  • an ADC of formula (IN) or formula (IO) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-IN) or formula (P-IO): or a pharmaceutically acceptable salt thereof; wherein D, Z, B, and L 2 , are each as defined herein including embodiments.
  • an ADC of formula (IP) or formula (IQ) can be prepared by reacting an anti-CD25 antibody (Ab) with a molecule of formula (P-IP) or formula (P-IQ): or (IP) (IQ) or a pharmaceutically acceptable salt thereof; wherein D, Z, B, and L 2 , are each as defined herein including embodiments.
  • (P-I) is a molecule of formula: ,
  • (P-I) is a molecule of formula:
  • (P-II) is a molecule of formula: ,
  • (P-III) is a molecule of formula: ,
  • compositions [00524] In an aspect, provided herein is a pharmaceutical composition including an ADC as described herein, including embodiments, and a pharmaceutically acceptable carrier. In embodiments, the ADC as described herein is included in a therapeutically effective amount. [00525] In embodiments, the pharmaceutical composition is formulated as a tablet, a powder, a capsule, a pill, a cachet, or a lozenge as described herein. The pharmaceutical composition may be formulated as a tablet, capsule, pill, cachet, or lozenge for oral administration.
  • the pharmaceutical composition may be formulated for dissolution into a solution for administration by such techniques as, for example, intravenous administration.
  • the pharmaceutical composition may be formulated for oral administration, suppository administration, topical administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intranasal administration, subcutaneous administration, implantation, transdermal administration, or transmucosal administration as described herein.
  • the ADCs and pharmaceutical compositions thereof are particularly useful for parenteral administration, i.e., subcutaneously (s.c.), intrathecally, intraperitoneally, intramuscularly (i.m.) or intravenously (i.v.).
  • the ADCs and pharmaceutical compositions thereof are administered intravenously or subcutaneously.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
  • concentration of the antigen binding protein of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as about 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • the pharmaceutical composition may include optical isomers, diastereomers, enantiomers, isoforms, polymorphs, hydrates, solvates or products, or pharmaceutically acceptable salts of the compound described herein.
  • the compound described herein (including pharmaceutically acceptable salts thereof) included in the pharmaceutical composition may be covalently attached to a carrier moiety, as described above.
  • the compound described herein (including pharmaceutically acceptable salts thereof) included in the pharmaceutical composition is not covalently linked to a carrier moiety.
  • a combination of covalently and not covalently linked compound described herein may be in a pharmaceutical composition herein.
  • CD25 in solid tumors can have both tumor-promoting and tumor-suppressive effects, depending on the context.
  • CD25 When CD25 is overexpressed on the cancer cells it promotes the growth and proliferation of the cancer cells. Additionally, CD25 is often overexpressed on regulatory T cells (Tregs).
  • Tregs regulatory T cells
  • the role of CD25 in solid tumors may vary between different types of cancer.
  • an antibody drug conjugate comprising an IgG antibody, a conjugation linker moiety (L 1 ) that binds to the thiol of cysteine residues or to the amine of lysine residues of the IgG antibody, and to a drug moiety covalently bound to L 3 -L 2 -L 1 or L 2 -L 1 , or a drug moiety separately bound to both L 2 -L 1 and R * .
  • the IgG antibody binds to CD25.
  • an ADC provided herein is used in a method of inhibiting proliferation of a CD25-expressing cell, the method comprising exposing the cell to the ADC under conditions permissive for binding of the anti-CD25 antibody of the ADC on the surface of the cell, thereby inhibiting the proliferation of the cell.
  • the method is an in vitro or an in vivo method.
  • the cell is a Treg cell.
  • the cell is a T cell.
  • the cell is a NK cell.
  • Inhibition of cell proliferation in vitro may be assayed using the CellTiter-Glo TM Luminescent Cell Viability Assay, which is commercially available from Promega (Madison, WI). That assay determines the number of viable cells in culture based on quantitation of ATP present, which is an indication of metabolically active cells. See Crouch et al. (1993) J. Immunol. Meth. 160:81-88, US Pat. No. 6602677. The assay may be conducted in 96- or 384- well format, making it amenable to automated high-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs 6:398-404.
  • HTS high-throughput screening
  • the assay procedure involves adding a single reagent (CellTiter-Glo ® Reagent) directly to cultured cells. This results in cell lysis and generation of a luminescent signal produced by a luciferase reaction.
  • the luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in culture. Data can be recorded by luminometer or CCD camera imaging device.
  • the luminescence output is expressed as relative light units (RLU).
  • RLU relative light units
  • a method of treating a disease in a subject in need thereof including administering an effective amount of a pharmaceutical composition of the ADC as described herein.
  • the disease is cancer.
  • the cancer is associated with overexpression of CD25.
  • an ADC for use in a method of treating an individual having a CD25-expressing cancer the method comprising administering to the individual an effective amount of the ADC described herein.
  • an ADC for use in a method of treating an individual having a CD25-expressing cancer the method comprising administering the ADC described herein, to the individual in need thereof.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.
  • proliferative disease pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
  • proliferative conditions include, but are not limited to, benign, pre- malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumors (e.g. histiocytoma, glioma, astrocytoma, osteoma), cancers (e.g.
  • lung cancer small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carcinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), lymphomas, leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Cancers of interest include, but are not limited to, leukemias and lymphomas.
  • the cancers include, but are not limited to, Hodgkin's and non- Hodgkin's Lymphoma, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, (FL), Mantle Cell lymphoma (MCL), chronic lymphatic lymphoma (CLL), Marginal Zone B-cell lymphoma (MZBL) and leukemias such as Hairy cell leukemia (HCL), Hairy cell leukemia variant (HCL-v), Acute Myeloid Leukemia (AML), and Acute Lymphoblastic Leukemia (ALL) such as Philadelphia chromosome-positive ALL (Ph+ALL) or Philadelphia chromosome- negative ALL (Ph ⁇ ALL).
  • DLBCL diffuse large B-cell lymphoma
  • FL Mantle Cell lymphoma
  • CLL chronic lymphatic lymphoma
  • MZBL Marginal Zone B-cell lymphoma
  • leukemias such as Hairy cell leukemia (HCL), Hairy
  • the proliferative disease may be characterized by the presence of a neoplasm comprising both CD25+ and CD25 ⁇ cells.
  • the cancer is not associated with overexpression of CD25, in such embodiments, the cancer is a solid tumor. “Solid tumor” herein will be understood to include solid hematological cancers.
  • the cancer may be characterized by the presence of a neoplasm composed of CD25 ⁇ neoplastic cells, optionally wherein the CD25 ⁇ neoplastic cells are associated with CD25+ non-neoplastic cells such as CD25+ T-cells.
  • Solid tumors may be neoplasms, including non-hematological cancers, comprising or composed of CD25+ neoplastic cells.
  • Solid tumors may be neoplasms, including non- hematological cancers, infiltrated with CD25+ cells, such as CD25+ T-cells; such solid tumors may lack expression of CD25 (that is comprise or be composed of CD25 ⁇ neoplastic cells)
  • the solid tumor may be a tumor with high levels of infiltrating T-cells, such as infiltrating regulatory T-cells.
  • the solid tumor may be pancreatic cancer, breast cancer, colorectal cancer, gastric and esophageal cancer, leukemia and lymphoma, melanoma, non-small cell lung cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, and head and neck cancer.
  • the present disclosure provides for the use of an ADC in the manufacture or preparation of a medicament.
  • the medicament is for treatment of CD25-expressing cancer.
  • the medicament is for use in a method of treating CD25-expressing cancer, the method comprising administering to an individual having CD25-expressing cancer an effective amount of the medicament.
  • the medicament is for use in a method of treating CD25-expressing cancer, the method comprising administering the medicament to an individual having CD25-expressing cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.
  • the methods provided herein are for treating cancer in a mammal. In embodiments, the methods provided herein are for treating cancer in a human.
  • CD25 expressing cancers include, but are not limited to, Hodgkin's and non-Hodgkin's Lymphoma, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, (FL), Mantle Cell lymphoma (MCL), chronic lymphatic lymphoma (CLL), Marginal Zone B-cell lymphoma (MZBL) and leukemias such as Hairy cell leukemia (HCL), Hairy cell leukemia variant (HCL-v), Acute Myeloid Leukaemia (AML), and Acute Lymphoblastic Leukaemia (ALL) such as Philadelphia chromosome-positive ALL (Ph+ALL) or Philadelphia chromosome-negative ALL (Ph ⁇ ALL).
  • DLBCL diffuse large B-cell lymphoma
  • FL Mantle Cell lymphoma
  • CLL chronic lymphatic lymphoma
  • MZBL Marginal Zone B-cell lymphoma
  • HCL Hairy cell leukemia
  • examples of cancers to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer rectal cancer colorectal cancer endometrial or uterine carcinoma salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • the CD25-expressing cancer is metastatic.
  • the CD25-expressing cancer is relapsed cancer. In embodiments, the CD25-expressing cancer is recurrent cancer.
  • the cancer is lymphoma or leukemia. In embodiments, the cancer is lymphoma. In embodiments, the cancer is leukemia.
  • the lymphoma is Hodgkin’s or non-Hodgkin’s lymphoma. In embodiments, the lymphoma is Hodgkin’s lymphoma. In embodiments, the lymphoma is non- Hodgkin’s lymphoma.
  • the non-Hodgkin’s lymphoma is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, (FL), Mantle Cell lymphoma (MCL), chronic lymphatic lymphoma (CLL), or Marginal Zone B-cell lymphoma (MZBL).
  • the leukemia is Hairy cell leukemia (HCL), Hairy cell leukemia variant (HCL-v), Acute Myeloid leukemia (AML), or Acute Lymphoblastic leukemia (ALL) such as Philadelphia chromosome-positive ALL (Ph+ALL) or Philadelphia chromosome- negative ALL (Ph ⁇ ALL).
  • the ADCs disclosed herein can be used to treat CD25-expressing cancers that have not been previously treated with a therapeutic agent (i.e., as a first line treatment).
  • ADCs disclosed herein can be used to treat CD25-expressing cancers that are resistant to, refractory to and/or relapsed from treatment with another therapeutic agent (i.e., as a second line treatment).
  • ADCs disclosed herein can be used to treat CD25-expressing cancers that are resistant to, refractory to and/or relapsed from treatment with more than one other therapeutic agent (i.e., as a third line treatment or a fourth line treatment, etc.).
  • the ADCs described herein can be used either alone or in combination with other agents in a therapy.
  • an ADC as described herein may be co-administered with at least one additional therapeutic agent.
  • other therapeutic regimens may be combined with the administration of the ADC including, without limitation, radiation therapy and/or bone marrow and peripheral blood transplants, and/or a cytotoxic agent.
  • a cytotoxic agent is an agent or a combination of agents such as for example an immune- oncology drug.
  • immune-oncology drugs including but not limited to PD1 inhibitors, PD-L1 inhibitors, CLTL4 inhibitors, GITR agonists and OX40 agonists.
  • the cytotoxic agent may be, for example, a) a Bruton's Tyrosine Kinase inhibitor (BTKi), such as Ibrutinib (Imbruvica), Acalabrutinib/ACP-196, ONO/GS-4059, Spebrutinib/AVL-292/CC-292, HM71224 (Poseltinib) or BGB-3111 (Zanubrutinib); (b) a PD1 antagonist, such as pembrolizumab, nivolumab, MEDI0680, PDR001 (spartalizumab), Camrelizumab, AUNP12, Pidilizumab, Cemiplimab (REGN-2810), AMP-224, BGB-A317 (Tisleizumab), or BGB-108; (c) a PD-L1 antagonist, such as atezolizumab (Tecentriq), BMS- 936559
  • Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the ADC can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
  • the ADCs described herein can also be used in combination with radiation therapy.
  • Articles of Manufacture [00556] In a further aspect, provided herein is an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided.
  • the article of manufacture (a kit) comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the disorder and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an ADC as described herein.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an ADC as described herein; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as Ringer's solution or dextrose solution.
  • non-exemplified compounds according to the present disclosure can be successfully performed by modifications apparent to those skilled in the art, e.g., by utilizing other suitable reagents known in the art other than those described, or by making routing modifications of reaction conditions, reagents, and starting materials.
  • other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other compounds of the present disclosure.
  • Synthesis of compound 40 and related compounds was disclosed in US Patent Nos.10,590,165 and 9,981,046, which are incorporated herein in their entireties.
  • Example S6 Synthesis of Compound L078-059.
  • [00574] To a solution of compounds 16 (5mg, 38 ⁇ mol) and 17 (13mg, 38 ⁇ mol) in 5mL of 50% CH3CN/H2O added 0.5mL of Sat. NaHCO3 solution. The solution was stirred for 10 minutes. The mixture was purified by HPLC to give compound 18 (13mg).
  • [00575] To a solution of compounds 18 (13 mg, 38 ⁇ mol), 5 (Mesylate salt, 20mg, 38 ⁇ mol) and HATU (15mg, 38 ⁇ mol) in 2 mL of DMF was added DIEA (12mg, 95 ⁇ mol). The solution was stirred for 5 minutes followed by addition of 0.5 mL diethylamine.
  • Example S11 Synthesis of Compound L078-066LT.
  • compounds 27 14mg, 55 ⁇ mol
  • 5 (Mesylate salt, 30mg, 55 ⁇ mol) and HATU (21mg, 55 ⁇ mol) in 2mL of DMF was added DIEA (11mg, 83 ⁇ mol).
  • DIEA 11mg, 83 ⁇ mol
  • the solution was stirred for 10 minutes and purified by HPLC.
  • the resulting product (29mg) was treated with 50% TFA/DCM (2mL) for 30 minutes and purified by HPLC to give compound L078-066 as a TFA salt.
  • Example S18 Synthesis of Compound L078-091.
  • a solution of compounds 37 (HCl salt, 250mg, 1.48mmol), 38 (643mg, 2.95mmol) and NaOH (296mg, 7.4mmol) in MeOH (10mL)/H2O (1mL) was stirred for 5 hours.
  • the mixture was purified by HPLC to give compound 39 (305mg).
  • Example S21 Synthesis of Compound L078-093.
  • the residue was purified by HPLC to give compound L078-093 (51mg) as a pale-yellow powder. MS m/z 1353.0 (M+H).
  • Example S22 Synthesis of Compound L079-018.
  • Example S42 Synthesis of Compound L078-178.
  • a solution of compound L078-030 (TFA salt, 30 mg, 0.047 mmol), compound 89 (39 mg, 0.057 mmol), HOBT (5 mg) and DIEA (11 mg, 0.114 mmol) in DMF (3 mL) was stirred for 30 minutes followed by addition of 0.5 ml of diethylamine and then stirred for another 30 minutes. The mixture was purified by HPLC to give compound 125 (42 mg).
  • a solution of compound 130 (TFA salt, 21 mg, 0.031 mmol), compound 89 (21 mg, 0.031 mmol), HOBT (5 mg) and DIEA (10 mg) in DMF (3 mL) was stirred for 30 minutes followed by addition of 0.5 mL of diethylamine.
  • Example S45 Synthesis of Compound L081-034. [00661] A solution of compound 51 (500 mg, 0.839 mmol), HATU (319 mg, 0.839 mmol) and DIEA (108 mg, 0.839 mmol) in DMF (3 mL) was stirred for 1 minute. The solution was then added to a solution of compound 133 (CAS# 33527-91-2; 367 mg, 2.52 mmol) in DCM (20 mL) dropwise. DCM was evaporated under reduced pressure. The residue was purified by HPLC to give compound 134 (172 mg).
  • Compound 141 was dissolved in 50% CH3CN/H2O (5 mL). To the solution was added compound 74 (CAS# 663921-15-1; 78 mg, 0.294 mmol) and 10% NaHCO 3 (bring solution to pH 8.5). The solution was stirred for 30 minutes, concentrated, and purified to give compound 142 (157 mg). [00664] A solution of compound 142 (15 mg, 0.02 mmol), compound 80 (20 mg, 0.02 mmol), HATU (8 mg, 0.02 mmol) and DIEA (10 mg, 0.08 mmol) in DMF (3 mL) was stirred for 5 minutes. The mixture was purified by HPLC and concentrated to give compound 143 (17 mg).
  • Antibody-Drug Conjugates were prepared by conjugating a drug-linker compound, whose synthesis is provided above, with anti-CD25 antibody.
  • ADCs Antibody-Drug Conjugates
  • the procedure described for preparation of ADC-1 is used for conjugation of compounds comprising 2,3- bis(bromomethyl)quinoxaline to thiols of cysteine group(s).
  • the procedure described for preparation of ADC-2 is used for conjugation of compounds comprising maleimide to thiols of cysteine group(s).
  • Affinity purified anti-CD25 antibody was buffer exchanged into Conjugation Buffer (50 mM sodium phosphate buffer, pH 7.2, 5 mM EDTA) at a concentration of 1-20 mg/mL.
  • Conjugation Buffer 50 mM sodium phosphate buffer, pH 7.2, 5 mM EDTA
  • TCEP tricarboxyethylphosphine
  • ADC-conjugate ADC-1
  • ADC-2 Affinity purified anti-CD25 antibody was buffer exchanged into Conjugation Buffer in a manner identical to ADC-1 above. To a portion of this anti-CD25 antibody solution was added a freshly prepared 10 mM water solution of TCEP at 3-fold molar excess. The resulting mixture was incubated at room temperature 1-2 hours or 2-8°C overnight. The excess unreacted TCEP was then removed by several rounds of centrifugal ultrafiltration or TFF with fresh Conjugation Buffer.
  • ADC-2 Purification and analysis of the resulting ADC-conjugate (ADC-2) proceeded in a manner identical to that of ADC-1 above.
  • the resulting average DAR for was calculated based on comparative peak area integration of the HIC-HPLC chromatogram.
  • Confirmation of low percent ( ⁇ 5%) high molecular weight (HMW) aggregates for the resulting ADC-conjugate (ADC-2) was determined using analytical SEC-HPLC.
  • Preparation of ADC-3 [00672] To a solution of 0.5-50 mg/mL of anti-CD25 antibody in buffer at pH 6.0-9.0 with 0- 30% organic solvent, was added 0.1-10 eq of activated drug linker conjugate either portion wise or by continuous flow.
  • Example B1 CD25 Receptor Quantification in Various Cell Lines
  • Receptor Quantification Tumor cell cultures were harvested by non-enzymatic cell dissociation and plated in PBS + 2%FBS + 5 mM EDTA.
  • FIG. 2 shows a bar graph of CD25 receptor numbers in four human cancer cell lines, and a table summarizing the CD25 receptor quantification.
  • Example B2 In vitro Efficacy of Antibody-Drug Conjugates (ADCs).
  • ADCs Antibody-Drug Conjugates
  • 3A-3D (where Dxd is covalently bound to anti-CD25 MAA-V clone via maleimide-glycine-glycine-phenylalanine-glycine (GGFG) peptide linker; drug linker system of Daiichi used as positive control).
  • STI-1499-SET0218 was used as isotype control for anti-CD25 antibody, where Dxd is conjugated to an anti-SARS-COV-2 antibody.
  • Anti-CD25 antibody was conjugated to drug-linker compounds L014-077, L078-118, L078-119, L078-120, L078-121, or L078-044.
  • the anti-CD25 antibody comprised the light chain sequence of SEQ ID NO: 7 and the heavy chain sequence of SEQ ID NO: 8.
  • the resulting average drug-antibody-ratio (DAR) for CD25-L014-077 was 3.26.
  • the resulting average DAR for CD25-L078-044 was 3.72.
  • the resulting average DAR for CD25- SET0218 was 3.93.
  • the resulting average DAR for CD25-L078-118 was 3.83.
  • the resulting average DAR for CD25-L078-119 was 3.83.
  • the resulting average DAR for CD25-L078-120 was 3.65.
  • the resulting average DAR for CD25-L078-121 was 3.06.
  • CD25 ADCs The in vitro efficacies of CD25 ADCs were evaluated using the following human cancer cell lines: SU-DHL-1, HDLM-2, Karpas 299T (CD25+) and Daudi (CD25-), purchased from the American Type Culture Collection (ATCC; Manassas, VA) and the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Leibniz, Germany) and were routinely cultured in RPMI-1640 medium (Catalog #10041CV; Corning) supplemented with 10% fetal bovine serum (FBS; Catalog #MT35011CV; Corning) and 1X Penicillin-Streptomycin (Catalog #30-002-CI; Corning) and maintained at 37°C with 5% CO2 in a humidified environment.
  • ATCC American Type Culture Collection
  • VA Manassas, VA
  • DSMZ DSMZ, Leibniz, Germany
  • Tumor cells were harvested by detachment with cell stripper. Viable cell counts were made by Trypan blue exclusion using a Countess or Countess II automated cell counter.
  • Cell Viability Assay All cells were harvested and seeded into 384-well white wall clear bottom plates (Catalog #3765; Corning) at a density of 2500 cells/well in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1X Penicillin-Streptomycin (complete growth media). The outer wells of plates contained complete growth medium only and were used for background subtraction for the cell viability assay. Working solutions of test articles were prepared at 2X final concentrations with 5-fold serial dilutions in complete growth medium.
  • Cell treatment was performed in triplicates and maintained at 37°C for 120-hour assay. After treatment, cell viability was determined by CellTiter-Glo 20 assay (Catalog #G9243; Promega; Madison WI USA) based on the manufacturer’s instructions.
  • CellTiter Glo reagent reacts with ATP in metabolically active cells to give a luminescent readout that is directly proportional to the number of viable cells. Briefly, plates were removed from the incubator and equilibrated to room temperature before addition of CellTiter Glo reagent. Luminescence was measured using a Tecan Spark microplate reader (Tecan; Mannedorf, Switzerland).
  • FIGS. 3A-3D and 4A-4B and Tables 4A and 4B Cell viability for CD25-L014-077, CD25-L078-118, CD25-L078-119, CD25-L078- 120, CD25-L078-121, CD25-L078-044, CD25-L078-182, CD25-L081-036, CD25-L081-038, and controls (CD25-SET0218, STI-1499-SET0218, and Dxd toxin) are shown in FIGS. 3A-3D and 4A-4B and Tables 4A and 4B.
  • Linker-payload structures are shown in FIG.1.
  • STI-1499- SET0218 is isotype control for anti-CD25 antibody, where Dxd is conjugated to an anti-SARS- COV-2 antibody.
  • CD25-SET0218 is described above.
  • In vitro cytotoxic activities and targeting specificity of the ADCs described herein were evaluated against CD25-positive SU-DHL-1, CD25-positive HDLM-2, CD25-positive Karpas 299T and CD25-negative Daudi cancer cell lines using standard cell viability assays.
  • treatment with CD25-L014-077, CD25-L078-118, CD25-L078-119, CD25-L078-120, CD25-L078-121, CD25-L078-1044, CD25-L078-182, and CD25-SET0218 dose dependently reduced cell viability of SU-DHL-1 and did not show potent activity against Daudi cells.
  • CD25-SET0218 is not as cytotoxic as our ADCs CD25-L078-118, CD25-L078-182, or CD25-L014-077.
  • CD25-L078-182 is the most cytotoxic ADC with EC 50 of about 0.07 nM in SU-DHL-1 cell line (CD25 +) and EC50 of about 867 nM in Daudi cell line (CD25 -).
  • mice Upon receipt, groups of 7 mice were housed per cages in a controlled environment vivarium and allowed to acclimate for 72 hours prior to experimentation. Rodent chow and water were provided ad libitum. Animal health status was determined during the acclimation period. Each cage was identified by group number and study number, and mice were individually identified using ear tags. The study was conducted under approved IACUC protocols and were performed in the vivarium at Sorrento Therapeutics Inc (4955 Directors Places, San Diego, CA), which was managed by Explora BioLabs (San Diego, CA). [00687] Animals were observed twice weekly for general clinical conditions including tumor growth, viability, mortality, mobility, posture, body weight (BW) and other signs of distress.
  • BW body weight
  • Human anaplastic large cell lymphoma cell line SU-DHL-1 cells were cultured and expanded in RPMI 1640 medium (catalog # 10-041-CV, Corning, Corning, NY) supplemented with 10% FBS (catalog # FB-02, Omega Scientific, Tarzana, CA) at 37°C in a 5% CO2 humidified environment for a period of 2-3 weeks before harvesting for implantation.
  • mice of each group were treated with a single dose (3 mg/kg) of CD25-ADCs or the controls: PBS (vehicle) or anti-CD25 antibody (MAA-V clone), according to the regimens shown in Table 5A and 5B. All compounds were diluted in PBS to working concentrations which were calculated according to treatment regimens and an injection volume of 0.2 ml per mouse.
  • the body weight of all mice was measured twice a week for a period of 59 days after the single ADC dose, shown in FIG. 6 (this experiment corresponds to in vivo efficacy experiment the results of which are shown in FIG.5).
  • Table 5A Table 5A.
  • mice treated with CD25-L078-118, CD25-L078-119, or CD25-L078-120 show comparable tumor growth inhibition of 100% on day 24 after treatment.
  • Mice treated with CD25-L078-118 also show 100% tumor regression on day 24 after treatment, although after thirty days we start to see tumor regrowth.
  • Mice treated with CD25- L078-118, CD25-L078-119, or CD25-L078-120 had better efficacy than Daiichi’s ADC CD25- SET0218 (see Table 6).
  • the body weight of all mice was measured twice a week for a period of 59 days after the single ADC dose, shown in FIG. 6.
  • mice in the groups dosed with CD25-L078-044, CD25- L078-118, or CD25-L078-119 had a transient body weight reduction due to infection (C. bovis infection).
  • CD25-L078-119 died on day 2-3 after dosing. The rest of the mice recovered within one week after the infection, with their weight returning to normal.
  • mice treated with CD25-L014-077 not only show 100% tumor regression on day 12 after treatment, like the mice treated with CD25-L078-118, but the tumor does not regrow by day 19. In mice treated with CD25-L078-118, some mice show regrowth by day 19.
  • Table 6 shows percent tumor regression (TR%) and percent tumor inhibition (TGI%) for mice treated with ADCs (or controls) according to Table 5A (1 st experiment). [00702] Table 6. Tumor Growth inhibition rate (TGI%) and Tumor Regression Rate (TR%)

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Abstract

L'invention concerne entre autres, des conjugués anticorps-médicament (ADC) qui se lient à l'antigène CD25. Des compositions pharmaceutiques, et des méthodes de traitement du cancer à l'aide des ADC présentement décrits sont également divulguées.
PCT/US2024/022544 2023-04-05 2024-04-01 Conjugués anticorps-médicament et leurs utilisations WO2024211234A1 (fr)

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