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WO2020089815A1 - Antibody conjugates comprising sting agonist - Google Patents

Antibody conjugates comprising sting agonist Download PDF

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Publication number
WO2020089815A1
WO2020089815A1 PCT/IB2019/059323 IB2019059323W WO2020089815A1 WO 2020089815 A1 WO2020089815 A1 WO 2020089815A1 IB 2019059323 W IB2019059323 W IB 2019059323W WO 2020089815 A1 WO2020089815 A1 WO 2020089815A1
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Prior art keywords
independently selected
formula
substituted
alkyl
crc
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PCT/IB2019/059323
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French (fr)
Inventor
Xueshi Hao
Porino VA
Yongqin Wan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis AG
Chinook Therapeutics Inc
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Novartis AG
Aduro Biotech Inc
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Publication of WO2020089815A1 publication Critical patent/WO2020089815A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • 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
    • 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
    • A61K47/6851—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 determinant of a tumour cell
    • A61K47/6855—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 determinant of a tumour cell the tumour determinant being from breast cancer cell
    • 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
    • A61K47/6851—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 determinant of a tumour cell
    • A61K47/6863—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 determinant of a tumour cell the tumour determinant being from stomach or intestines cancer cell
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents

Definitions

  • the invention provides antibody conjugates, also known as immunconjugates, comprising agonists of STING (stimulator of interferon genes) receptor, and the use of such conjugates for the treatment of cancer.
  • Innate immunity is a rapid nonspecific immune response that fights against
  • environmental insults including, but not limited to, pathogens such as bacteria or viruses.
  • Adaptive immunity is a slower but more specific immune response, which confers long-lasting or protective immunity to the host and involves differentiation and activation of naive T lymphocytes into CD4+ T helper ceils and/or CD8+ cytotoxic T cells, to promote cellular and humoral immunity.
  • Antigen presentation cells of the innate immune system such as dendritic ceils or macrophages, serve as a critical link between the innate and adaptive immune systems by phagocytosing and processing the foreign antigens and presenting them on the cell surface to the T cells, thereby activating T cell response.
  • STING (stimulator of interferon genes) is an endoplasmic reticulum adaptor that facilitates innate immune signaling (Ishikawa and Barber, Nature 2008, 455(7213):674 ⁇ 678). It was reported that STING comprises four putative transmembrane regions (Ouyang et. a!., immunity (2012) 36, 1073), predominantly resides in the endoplasmic reticulum and is able to activate NF-kB, STAT6, and IRF3 transcription pathways to induce expression of type I interferon (e.g , !FN-Q and IFN-b) and exert a potent anti-viral state following expression
  • type I interferon e.g , !FN-Q and IFN-b
  • the invention provides immunoconjugates comprising antibodies conjugated with STING agonists, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof and combinations thereof, which are useful for the treatment of diseases, in particular, cancer.
  • the invention further provides methods of treating, preventing, or ameliorating cancer comprising administering to a subject in need thereof an effective amount of an immunoconjugate of the invention.
  • immunoconjugate and“antibody conjugate” are used interchangeably herein.
  • the invention also provides compounds comprising STING agonists and a linker which are useful to conjugate to an antibody and thereby make the immunostimmuiatory conjugates (or immune Stimulator Antibody Conjugates (ISACs)) of the invention.
  • Various embodiments of the invention are described herein.
  • this application discloses an immunoconjugate comprising an antibody (Ab), or a functional fragment thereof, coupled to an agonist of Stimulator of Interferon Genes (STING) receptor (D) via a linker (L), wherein the linker optionally comprises one or more cleavage elements.
  • Ab antibody
  • STING Stimulator of Interferon Genes
  • the immunoconjugate comprises Formula (I):
  • Ab is an antibody or a functional fragment thereof
  • L is a linker comprising one or more cleavage elements
  • D is a drug moiety that has agonist activity against STING receptor
  • n is an integer from 1 to 8.
  • n is an integer from 1 to 20.
  • the immunoconjugate comprises Formula (I):
  • Ab is an antibody or a functional fragment thereof
  • L is a linker
  • D is a drug moiety that binds to STING receptor
  • n is an integer from 1 to 8.
  • n is an integer from 1 to 20:
  • the immunconjugate comprises Formula (I):
  • Ab is an antibody or a functional fragment thereof
  • L is a linker
  • D is a drug moiety that binds to STING receptor
  • n is an integer from 1 to 8.
  • n is an integer from 1 to 20;
  • the immunoconjugate delivers D, or a cleavage product thereof, to a cell targeted by the Ab, and wherein D, or the cleavage product thereof, has STING agonist activity.
  • the immunoconjugate comprises Formula (I):
  • Ab is an antibody or a functional fragment thereof
  • L is a linker comprising one or more cleavage elements
  • D is a drug moiety that binds to STING receptor
  • n is an integer from 1 to 8.
  • n is an integer from 1 to 20;
  • the immunoconjugate releases D, or a cleavage product thereof, in a cell targeted by the Ab, and wherein D, or the cleavage product thereof, has STING agonist activity.
  • the immunoconjugate comprises Formula (I):
  • Ab is an antibody or a functional fragment thereof
  • L is a linker comprising one or more cleavage elements
  • D is a drug moiety that has agonist activity against STING receptor
  • n is an integer from 1 to 8.
  • n is an integer from 1 to 20:
  • the immunoconjugate releases D, or a cleavage product thereof, in a cell targeted by the Ab, and wherein D, or the cleavage product thereof, has STING agonist activity in the cell.
  • the present application discloses an immunoconjugate for delivery of a STING receptor agonist to a ceil, the immunoconjugate comprising Formula (I):
  • Ab is an antibody or a functional fragment thereof
  • L is a linker comprising one or more cleavage elements
  • D is a drug moiety that binds to STING receptor
  • n is an integer from 1 to 8.
  • n is an integer from 1 to 20;
  • the immunoconjugate specifically binds to an antigen expressed on the ceil surface and is internalized into the cell, and wherein D, or a cleavage product thereof, is cleaved from L and has STING agonist activity as determined by one or more STING agonist assays selected from: an interferon stimulation assay, a hSTING wt assay, a THP1 ⁇ Dua! assay, a TANK binding kinase 1 (TBK1) assay, or an interferon-y-inducible protein 10 (IP-10) secretion assay.
  • STING agonist assays selected from: an interferon stimulation assay, a hSTING wt assay, a THP1 ⁇ Dua! assay, a TANK binding kinase 1 (TBK1) assay, or an interferon-y-inducible protein 10 (IP-10) secretion assay.
  • D or the cleavage product thereof, has STING agonist activity if it. binds to STING and is able to stimulate production of one or more STING-dependent cytokines in a STING-expressing cell at least 1 .1 -fold, 1.2-fold, 1.3-fold, 1 .4-fold, 1.5-fold, 1.6- fold, 1 7-fold, 1.8-fold, 1.9-fold, 2-fold or greater than an untreated STING-expressing cell.
  • the STING-dependent cytokine is selected from interferon, type 1 interferon, IFN-a, !FN-b, type 3 interferon, IRNl, IP10, TNF, !L-6, CXCL9, CCL4, CXCL1 1 ,
  • D or the cleavage product thereof, has STING agonist activity if it binds to STING and is able to stimulate phosphorylation of TBK1 in a STING- expressing cell at least 1.1-fold, 1.2-fold, 1.3-fo!d, 1.4-fold , 1 .5-fold, 1 .8-fold, 1.7-fold, 1 .8-foid, 1 .9-foid, 2-foid or greater than an untreated STING-expressing cell.
  • D has STING agonist activity if it binds to STING and is able to stimulate expression of a iuciferase reporter gene controlled by interferon (IFN)-stimu!ated response elements in a STING-expressing cell at an EC 5 o of 20 micromolar (mM), 15 mM, 10 m M, 9 mM, 8 mM, 7 m M, 6 m M, 5 m M, 4 m M, 3 m M, 2 m M, 1 mM, or less.
  • IFN interferon
  • D or the cleavage product thereof, has STING agonist activity if it binds to STING and is able to stimulate expression of a iuciferase reporter gene controlled by interferon (IFN)-stimuiated response elements in a STING-expressing cell to a level equal to or greater than the level of stimulation of 50 mM of 2’3’-cGAIVSP.
  • IFN interferon
  • the STING-expressing cell is THP1 -Dual cell
  • the Iuciferase reporter gene is the IRF-Lucia reporter gene in THP1 -Dual cell
  • the STING agonist activity is determined by the THP1 ⁇ Duai assay described herein
  • the iuciferase reporter gene is the 5xlSRE-m!FNb ⁇ GL4 reporter gene and the STING-expressing ceil is a ceil expressing wild-type human STING protein, and optionally the STING agonist activity is determined by the hSTING wt assay described in Table 3A.
  • the immunoconjugate stimulates IP-10 secretion from a STING- expressing cell targeted by the Ab at an EC 5 o of 5 nanomoiar (nM) or less in an IP-10 secretion assay.
  • the immunoconjugate is parentera!ly administered.
  • the immunoconjugate comprises an Ab that specifically binds a target antigen.
  • the target antigen is a tumor antigen.
  • the Ab is human or humanized. In other embodiments, the Ab is a monoclonal antibody.
  • the Ab comprises a modified Fc region in one embodiment, the Ab comprises cysteine at one or more of the following positions, which are numbered according to EU numbering:
  • L is attached to the Ab via conjugation to one or more modified cysteine residues in the Ab.
  • L is conjugated to the Ab via modified cysteine residues at positions 152 and 375 of the heavy chain of the Ab, wherein the positions are determined according to EU numbering.
  • L is conjugated via a maieimide linkage to the cysteine.
  • D is a dinucleotide in some cases, D is a cyclic dinucieotide (CDN).
  • CDN cyclic dinucieotide
  • D is a compound selected from any one of the compounds of Table 1 and Table 2.
  • D is a compound selected from
  • the present application discloses immunconjugates wherein L is a cleavable linker comprising one or more cleavage elements.
  • L comprises two or more cleavage elements, and each cleavage element is independently selected from a self-immoiative spacer and a group that is susceptible to cleavage.
  • the cleavage is selected from acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphodiesterase- induced cleavage, phosphatase-induced cleavage, protease- induced cleavage, lipase-induced cleavage, or disulfide bond cleavage.
  • the Linker-Drug Moiety (- (L-(D) m )), wherein m is 1 , has a structure selected from:
  • Lc is a linker component and each Lc is independently selected from a linker component as disclosed herein;
  • D is the compound selected from any one of embodiments 1 to 186;
  • each cleavage element (C E ) is independently selected from a self-immolative spacer and a group that is susceptible to cleavage selected from acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage.
  • the Linker (L) ot the Linker- Drug Moiety (-(L-(D) )), wherein m is 1 has a structure selected from:
  • Le is a linker component and each Lc is independently selected from a linker component as disclosed herein;
  • each cleavage element (C £ ) is independently selected from a self-immoiatlve spacer and a group that is susceptible to cleavage selected from acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage.
  • L comprises a structural component selected from: ;
  • the immunoconjugate is selected from the following:
  • each Gi is independently selected from
  • XB is C, and each Z2 is N;
  • Y 5 is -CH 2 -,-NH-,-0-or-S;
  • Ys is -CH 2 -,-NH-,-0-or-S;
  • Y- / is O or S
  • Ys is O or S;
  • Yg is -GH 2 -, -NH-, -0- or -S;
  • Yio is -CH 2 -, -NH-, -O- or -S;
  • q 1 , 2 or 3:
  • each R 1 is independently a partially saturated or aromatic monocyclic heterocyclyl or
  • each R 1a is independently a partially saturated or aromatic monocyclic heterocyclyl or
  • each R 1b is independently a partially saturated or aromatic monocyclic heterocyclyl or
  • R ,b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH 2 , -NHNH 2 , -NHOH, F, Cl, Br, CrC 6 a!kyi and a GrGsalkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 , or wherein when Ab is attached to R 1b then R 1b is substituted with -NHLiR 115 , -NHNHLiR 115 , -NHOL1R 115 - or - attached to the R 115 moiety;
  • each R 2 is independently selected from H and Ci-C 6 alkyl
  • each R 6 is independently selected from H, Ci-C 6 alkyl and C i-C 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 7 is independently selected from H, CrC 6 alky! and CrC 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 8 is independently selected from H, CrC 6 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 9 is independently selected from H, CrC 6 alkyl and CrC 6 aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 2a is independently selected from H and CrC 6 aikyl
  • each R 4a is independently selected from H, Ci-C 5 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 6a is independently selected from H, C -C 3 alkyi and CrCgalkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 7a is independently selected from H, C -C 3 alkyi and CrCgalkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R Sa is independently selected from H, Ci-C 3 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 9a is independently selected from H, Ci-C 6 alkyi and CrC 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ; eac yl, Ci
  • each R 11 is independently selected from H and CrC 6 aikyl
  • each R 12 is independently selected from H and CrC 6 aikyl
  • R 3 and R 6 are connected to form -Q-G i-C 6 aikylene, such that when R 3 and R s are connected, the O is bound at the R 3 position
  • R 3a and R 6a are connected to form -0-Ci-C 6 alkylene, such that when R 3a and R Sa are connected, the O is bound at the R 3a position;
  • R 2 and R 3 are connected to form -0-CrC 6 alkyiene, such that when R 2 and R 3 are connected, the O is bound at the R 3 position;
  • R 2a and R 3a are connected to form -0 ⁇ CrC 6 alkylene, such that when R 2a and R 3a are connected, the O is bound at the R 3a position;
  • R 4 and R 3 are connected to form -O-CrCeaikylene, such that when R 4 and R 3 are connected, the O is bound at the R 3 position;
  • R 4a and R 3a are connected to form -0-CrG s alkylene, such that when R 4a and R 3a are connected, the O is bound at the R 3a position;
  • R 5 and R 6 are connected to form -O-CrCeaikylene, such that when R 5 and R 6 are connected, the O is bound at the R 5 position;
  • R 5a and R 6a are connected to form -0-Ci-G 6 alkylene, such that when R 5a and R Sa are connected, the O is bound at the R 5a position;
  • R 5 and R 7 are connected to form -O-CrCeaikylene, such that when R 5 and R 7 are connected, the O is bound at the R 5 position; optionally R 5a and R 73 , are connected to form -0-C r C B alkyiene, such that when R 53 and R 73 are connected, the O is bound at the R 53 position:
  • Li is a linker
  • R 1 indicates the point of attachment to Ab
  • R 13 is H or methyi
  • R 14 is H, -CHs or phenyl
  • R 110 is independently selected from H, Gi-C 6 aikyl, F, Cl, and -OH;
  • R 111 is independently selected from H, Gi-C 6 alkyi, F, Cl, -NH 2 , -OCHs, -OCH 2 CH 3 ,
  • each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 and 18;
  • Ab is an antibody or fragment thereof
  • y is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the immunconjugates comprise a structure selected from:
  • the immunoeonjugate has in vivo anti-tumor activity.
  • the present application also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising an i munconjugate as disclosed herein and a pharmaceutically acceptable excipient.
  • the present application also discloses an immunoeonjugate as disclosed herein for use in combination with one or more additional therapeutic agents.
  • the additional therapeutic agent is selected from the group consisting of an inhibitor of a co- inhibitory molecule, an activator of a co-stimulatory molecule, a cytokine, an agent that reduces cytokine release syndrome (CRS), a chemotherapy, a targeted anti-cancer therapy, an oncolytic
  • the additional therapeutic agent is an inhibitor of a co-inhibitory molecule, an activator of a co-stimulatory molecule, or a cytokine, wherein:
  • the co-inhibitory molecule is selected from Programmed death-1 (PD-1), Programmed death- ligand 1 (PD-L1), Lymphocyte activation gene-3 (LAG-3), or T-celi immunoglobulin domain and mucin domain 3 (TIM-3),
  • PD-1 Programmed death-1
  • PD-L1 Programmed death- ligand 1
  • LAG-3 Lymphocyte activation gene-3
  • TIM-3 T-celi immunoglobulin domain and mucin domain 3
  • the co-stimulatory molecule is Glucocorticoid-induced TNFR-related protein (G!TR), and (ill) the cytokine is IL-15 complexed with a soluble form of IL-15 receptor alpha (IL-15Ra).
  • G!TR Glucocorticoid-induced TNFR-related protein
  • IL-15Ra IL-15 receptor alpha
  • immunconjugate a pharmaceutical composition thereof, or a composition comprising an immunoconjugate in combination with one or more additional therapeutic agents, as disclosed herein.
  • the present application also discloses use of an immunconjugate, a pharmaceutical composition thereof, or a composition comprising an immunoconjugate in combination with one or more additional therapeutic agents, as disclosed herein for treatment of a cancer in a subject in need thereof.
  • this application discloses an immunconjugate, a pharmaceutical composition thereof, or a composition comprising an immunoconjugate in combination with one or more additional therapeutic agents, as disclosed herein for use in the treatment of cancer.
  • an immunconjugate in yet another embodiment, disclosed herein is the use an immunconjugate, a pharmaceutical composition thereof, or a composition comprising an immunoconjugate in combination with one or more additional therapeutic agents, as disclosed herein in the manufacture of a medicament for use in the treatment of cancer.
  • the cancer is selected from sarcomas, adenocarcinomas, blastemas, carcinomas, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, lymphoid cancer, colon cancer, renal cancer, urothelial cancer, prostate cancer, cancer of the pharynx, rectal cancer, renal cell carcinoma, cancer of the small intestine, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, colorectal cancer, cancer of the anal region, cancer of the peritoneum, stomach or gastric cancer, esophageal cancer, salivary gland carcinoma, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, penile carcinoma, glioblastoma,
  • neuroblastoma cervical cancer , Hodgkin lymphoma, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small iniesiine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias inciuding acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CMS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, neuroendocrine tumors (inciud
  • myelogenous leukemia AML
  • acute lymphoid leukemia ALL
  • chronic myelogenous leukemia CML
  • chronic lymphoid leukemia CLL
  • myelodysplastic syndromes B-cell acute lymphoid leukemia (“BALL”)
  • T-cell acute lymphoid leukemia TALL
  • B-cell acute lymphoid leukemia B cell prolymphocytic leukemia
  • blast ic plasmacytoid dendritic ceil neoplasm Burkitt's lymphoma
  • diffuse large B ceil lymphoma Follicular lymphoma
  • Hairy cell leukemia small ceil- or a large cell-follicular lymphoma
  • malignant lymphopro!iferative conditions MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, piasmablastic lymphoma
  • the immunoconjugate is administered to the subject.
  • the present application also discloses an Immunconjugate, a pharmaceutical composition thereof, or a composition comprising an immunoconjugate in combination with one or more additional therapeutic agents, as disclosed herein for use as a medicament.
  • this application discloses a compound having a structure selected from Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or Formula (F) or stereoisomers or pharmaceutically acceptable salts thereof,
  • each Gi is independently selected from where the * of Gi indicates the point of attachment to ⁇ CR 8 R 9 ⁇ ;
  • XB is C, and each Z 2 is N ;
  • X D is C, and each Z 4 is N;
  • Ys is -CH 2 -, -NH-, -G- or -S;
  • Y 6 is -CH 2 -, -NH-, -G- or -S;
  • Y 7 is O or S
  • Y 8 is O or S
  • Y 9 is -CHr, -NH-, -O- or -S;
  • Y 10 is -CH 2 -, -NH-, -O- or -S;
  • q is 1 , 2 or 3;
  • each R 2 is independently selected from H and Ci-C 6 alkyi
  • each R 3 is independently selected from -NHL1R 15 , -NHNHL1R 15 , -NHOL1R 15 , -
  • NHN CR 12 (CH 2 ) r .LiR 15 , H, -OH, -SH, F, Cl, Br, I, NHOH, -NHNH 2 , NH 2 and a C r C 6 alkyi substituted with a NHOH, NHNH 2 or NH 2 ; each R 4 is independently selected from H, CrC 6 alkyl and Ci-C 6 aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 6 is independently selected from H, Ci-C 6 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, !, OH, CN, and N 3 ;
  • each R 7 is independently selected from H, Ci-C 6 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 8 is selected from H, Ci-C 3 alkyl and CrC 6 aikyl substituted by 1 , 2 or 3 substituents
  • each R 9 is selected from H, Ci-C 3 alkyl and CrC 6 aikyl substituted by 1 , 2 or 3 substituents
  • R 2a is selected from H and Ci-C 6 alkyl
  • R 4a is selected from H, Ci-C 3 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents
  • R 6a is selected from H, Ci-C 6 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents
  • R 78 is selected from H, Ci-C 3 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents
  • R 8a is selected from H, Ci-C 3 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents
  • R 9a is selected from H, Ci-Csalkyl and Ci-C 6 alkyl substituted by 1 , 2 or 3 substituents
  • each R 10 is independently selected from the group consisting of H, Ci-Csalkyl, C r
  • R 11 is selected from H and Ci-C 6 alkyl
  • R 12 is selected from H and CrC 6 alkyi
  • R 3 and R 6 are connected to form -0-Ci-C 6 alkylene, such that when R 3 and R 6 are connected, the O is bound at the R 3 position
  • R 38 and R 6a are connected to form -0-Ci-C 3 aikylene, such that when R 3a and R 6a are connected, the O is bound at the R 3a position;
  • R 2 and R 3 are connected to form -G-Ci-C 6 aikylene, such that when R 2 and R 3 are connected, the O is bound at the R 3 position;
  • R 2a and R 3a are connected to form -0-C r C B alkyiene, such that when R 2a and R 3a are connected, the O is bound at the R 3a position;
  • R 4 and R 3 are connected to form -Q-CrC 6 aikyiene, such that when R 4 and R 3 are connected, the O is bound at the R 3 position;
  • R 4a and R 3a are connected to form -0-Gi-C 6 alkylene, such that when R 4a and R 38 are connected, the O is bound at the R 3a position;
  • R 5 and R 6 are connected to form -0-C i-C 6 aikylene, such that when R 5 and R s are connected, the O is bound at the R 5 position;
  • R 5a and R 6a are connected to form -0-Ci-C 3 alkylene, such that when R 38 and R 68 are connected, the O is bound at the R 5a position;
  • R 5 and R 7 are connected to form ⁇ 0-Ci ⁇ C 6 aikylene, such that when R 5 and R 7 are connected, the O is bound at the R 5 position;
  • R 5a and R 7a are connected to form -0-Ci-C 6 alkylene, such that when R £,a and R 7a are connected, the O is bound at the R 5a position;
  • R 17 is 2-pyridyl or 4-pyridyl
  • each R 11 is independently selected from H and CrCealkyi
  • each R 12 is independently selected from H and CrC 6 alkyl
  • each m is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10;
  • each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 and 18;
  • each R 110 is independently selected from H, Ci-C 6 aikyl, F, Cl, and ⁇ -GH;
  • each R 111 is independently selected from H, CrCealkyi, F, Cl, -NH2, -OCH3, -QCH2CH3, -
  • R 1 , R 1a or R 1b Is substituted with -NHL R 15 , -NHNHL R 15 , - least one of R 3 , R 5 , R 33 or R 53 is -NHL1R 15 , - 2 ) n LiR 15 .
  • the compound is selected from:
  • the compound is selected from:
  • this application discloses a compound having a structure selected from Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or Formula (F) or stereoisomers or pharmaceutically acceptable salts thereof, wherein:
  • XB is C, and each Z 2 is N;
  • Y 6 is -CH2-, -NH-, -O- or -S;
  • Y 7 is O or S
  • Y 8 is O or S
  • Y 9 is -CH 2 -, -NH-, -O- or -S;
  • Y10 is -CH 2 -, -NH-, -O- or -S;
  • q is 1 , 2 or 3;
  • R 1 is a partially saturated or aromatic monocyclic heterocyclyi or partially saturated or aromatic fused bicyclic heterocyclyi containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R 1 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH 2 , -NHNH 2 , -NHOH, F, Cl, Br, Ci-C 6 aikyi and a Ci-Csalkyi substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N 3 ;
  • R 18 is a partially saturated or aromatic monocyclic heterocyclyi or partially saturated or aromatic fused bicyclic heterocyclyi containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R 18 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH 2 , -NHNH 2 , -NHOH, F, Cl, Br, Ci-C 6 alkyl and a Ci-C 3 alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N 3 ;
  • R 1b is a partially saturated or aromatic monocyclic heterocyclyi or partially saturated or aromatic fused bicyclic heterocyclyi containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R 1b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH 2 , -NHNH 2 , -NHOH, F, Cl, Br, CrC 6 alkyl and Ci-Cealkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 2 is independently selected from H and Ci-C 6 alkyl
  • each R 3 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 and a Ci-C 6 alkyi substituted with a NHOH, NHNH 2 or NH 2 ;
  • each R 4 is independently selected from H, CrC 6 alkyl and Ci-C 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 5 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 and a C Cealkyi substituted with a NHOH, NHNH 2 or NH 2 ;
  • each R 6 Is independently selected from H, CrCgalkyl and CrC 6 aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 7 is independently selected from H, CrC 6 alkyl and Ci-C 6 aiky! substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 8 is independently selected from H, CrC 6 aikyi and C r C B alky! substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 9 is independently selected from H, Gi-C 6 aikyl and CrC 6 alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Gi, Br, I, OH, CN, and N 3 ;
  • R 28 is independently selected from H and Ci-C 6 alkyl
  • R 38 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 and a C Csa!ky! substituted with a NHOH, NHNH 2 or NH 2 ;
  • R 4a is independently selected from H, CrC 6 alkyl and CrC 6 alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 5a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 and a C r Cealkyi substituted with a NHOH, NHNH 2 or NH 2 ;
  • R 6a is independently selected from H, Ci-C 6 alkyl and Ci-C 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 7a is independently selected from H, Ci-C 6 alkyl and Ci-C 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 8a is independently selected from H, CrC 6 alky! and CrC 6 alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 98 is independently selected from H, CrC 6 alky! and CrC 6 alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N 3 ;
  • each R 1C is independently selected from the group consisting yl, Cr
  • R 11 is Independently selected from H and C r C B alkyi;
  • R 12 is independently selected from H and C r C B alkyi
  • R 3 and R 6 are connected to form -0-CrC B alkylene, such that when R 3 and R 6 are connected, the O is bound at the R 3 position
  • R 3a and R 6a are connected to form -G-Ci-C 6 a!kyiene, such that when R 3a and R Sa are connected, the O is bound at the R 3a position;
  • R 2 and R 3 are connected to form -0-Ci-C 6 alkylene, such that when R 2 and R 3 are connected, the O is bound at the R 3 position; optionally R 2a and R 3a , are connected to form -OCr-C B alkyiene, such that when R 2a and R 3a are connected, the O is bound at the R 3a position:
  • R 4 and R 3 are connected to form -Q-Ci-C 6 aiky!ene, such that when R 4 and R 3 are connected, the O is bound at the R 3 position;
  • R 4a and R 3a are connected to form -0-CrC 6 aikylene, such that when R 4a and R 3a are connected, the O is bound at the R 3a position:
  • R 5 and R 6 are connected to form -Q-G i-C 6 aikylene, such that when R 5 and R s are connected, the O is bound at the R 5 position;
  • R 5a and R 6a are connected to form -0-Ci-C 6 alkylene, such that when R 58 and R 6a are connected, the O is bound at the R 5a position;
  • R 5 and R 7 are connected to form -0-CrC 6 alkylene, such that when R 5 and R 7 are connected, the O is bound at the R 5 position,
  • R £,a and R 7a are connected to form -O-GrCsalkylene, such that when R 5a and R 7a are connected, the O is bound at the R 5a position.
  • the compound is selected from any one of the compounds of
  • FIG. 1 is a line graph showing the anti-HER2 mAb1 -C4 conjugate inhibits N87 gastric tumor growth in mice.
  • FIG. 2 is a line graph showing the anti-HER2 mAb1 -C4 conjugate is well tolerated in the N87 gastric tumor xenograft mice.
  • alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation.
  • C r Csaikyi refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms, and which is attached to the rest of the molecuie by a single bond.
  • Non-iimiting examples of "Ci-C 6 aiky! groups include methyi, ethyi, 1 -methyiethyi , n-propyi, isopropyl, n- butyi, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl and hexyl.
  • alkenyl refers to a straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one double bond.
  • C2-C 6 alkenyr refers to a straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one double bond, having from two to six carbon atoms, which is attached to the rest of the molecuie by a single bond.
  • C2-C 3 alkenyi groups include ethenyl, prop-1 -enyi, but-1-enyl, pent-1 -enyl, pent-4-enyl and penta-1 ,4-dienyl.
  • alkynyl refers to a straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one triple bond
  • C2-C 6 alkynyi refers to a straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one triple bond, having from two to six carbon atoms, and which is attached to the rest of the molecuie by a single bond.
  • C2-C B alkynyr groups include ethynyl, prop-1 -ynyl, but-1-ynyi, pent-1-ynyl, pent-4-ynyl and penta-1 ,4-diynyl.
  • alkylene refers to a bivalent straight or branched hydrocarbon chain radical.
  • Ci-C 6 alkylene refers to a bivalent straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms.
  • alkenyiene refers to a bivalent straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one double bond.
  • C2-C 3 aikenylene refers to a bivalent straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one double bond, and having from two to six carbon atoms.
  • alkynylene refers to a bivalent straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one triple bond.
  • C2-C 3 alkynyiene refers to a bivalent straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one triple bond and having from two to six carbon atoms.
  • Ci-C 5 alkoxyalkyiene refers to a radical of the formula -Ra-Q- Ra, where each Ra is independently a Creaikyi radical as defined above.
  • the oxygen atom may be bonded to any carbon atom in either alkyl radical.
  • Examples of Ci. 5 alkoxy include, but are not limited to, methoxy-methyl, methoxy-ethyl, ethoxy-ethyl, 1 -ethoxy-propyl and 2-methoxy-butyl.
  • cycloalkyl refers to a saturated, monocyclic, fused bicyciic, fused tricyclic or bridged polycyclic ring system.
  • Non-limiting examples ef fused bicyciie or bridged polycyclic ring systems include bieyc!o[1 1 1 ]pentane, bicyclo[2.1 1]hexane, bicyclo[2.2.1]heptane, bieycio[3.1.Ijheptane, bicy o[3.2.1 joctane, bicycio[2.2 2]octane and adamantanyl.
  • Non-limiting examples monocyclic C 3 -C 8 eycioaikyl groups include cyclopropyl, cyciobutyi, cyciopentyl and cyclohexyl groups.
  • heteroalkyi refers to an "alkyl” moiety wherein at least one of the carbon atoms has been replaced with a heteroatom such as O S, or N.
  • heterocycloalkyl or“C 4 -C 8 heterocycloalkyl as used herein refers to a monocyclic ring structure having 3 to 6 ring members, wherein one to two of the ring members are independently selected from N, NH, NR 16 , O or ⁇ S ⁇ , wherein R 16 is Ci-C 6 alkyl.
  • Non-limiting examples of 3-6 membered heterocycloalkyl groups include aziridin-1 -yi, aziridin-2-yl, aziridin-3-yl, azetadinyl, azetadin-1 -yl, azetadin-2-yl, azetadin-3-yl, oxetanyl, oxetan-2-yl, oxetan-3-yl, oxetan-4-yi, thietanyl, thietan-2-yl, thietan-3-yi, thietan-4-yl, pyrrolidinyl, pyrrolidin-1 -yl, pyrro!idin-2-yl, pyrro!idin-3-yl, pyrrolidin-4-yl, pyrro!idin-5-yl, tetrahydrofuranyl, tetrahydrofuran-2-
  • heterocyclyl includes partially saturated or aromatic monocyclic or fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S. in a preferred embodiment, the heteroatoms are nitrogen.
  • substituents include oxo, halo, Ci. 6 alkyl, Ci- 6 alkoxy, amino, Ci- 5 alkyiamino, di-Ci- 5 alkyiamino.
  • the heterocyclic group can be attached at a heteroatom or a carbon atom.
  • the system can be fully aromatic (i.e. both rings are aromatic).
  • the heterocyclyl can be referred to as heteroaryl.
  • aromatic bicyclic heteroaryl include 9-10 membered fused bicyclic heteroaryl having 2-5 heteroatoms, preferably nitrogen atoms.
  • Non-limiting examples are: pyrrolo[2,3-bjpyrldlnyl, pyrroio[3,2-c]pyridinyL pyrrolo[3,2-c]pyridinyl, pyrrolo[3,2-b]pyridinyl, imidazo[4,5-b]pyridinyl, imidazo[4,5-c]pyridinyl, pyrazolo[4,3-d]pyridinyl, pyrazo!Q[4,3-c]pyridinyi, pyrazolo[3,4- cjpyridiny!, pyrazolo[3,4-d]pyridinyl, pyrazolo[3,4-b]pyridinyl, imidazo[1 ,2-a]pyridinyl,
  • bicyciic heierocyclyl ring systems include heierocyclyl ring systems wherein one of the fused rings is aromatic but the other is non-aromatic.
  • the heierocyclyl is said to be partially saturated.
  • partially saturated bicyciic system are for example dihydropurinones such as 2-amino- 1 ,9-dihydro-6H-purin-9-yl-6-one and 1 ,9-
  • Heterocyclyi also includes a 5- or 6- membered ring aromatic heterocyciyl having 2 to 3 heteroatom (preferably nitrogen) (also referred to as 5- to 6-membered heteroaryl).
  • monocyclic heteroaryl are: imidazolyi, pyrazolyl, thiazolyl, isothiazoiyi, 1 , 2, 3-oxadiazolyi, 1 ,2,4- oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazolyi, 1 ,2,4-th iadiazolyl, 1 ,2,5- thiadiazoiyl, 1 ,3,4-thiadiazo!yi, isothiazol-3-yl, isothiazol-4-yl, isothiazo!-5-yl, oxazol-2-yi, oxazo!- 4-yl, Qxazol-5-yi,
  • Heterocyclyi also includes 6-membered monocyclic partially saturated ring having 1-3 heteroatoms (preferably nitrogen).
  • Examples of partially saturated monocyclic heterocyclyi are pyrimidine-one and pyrimidine-dione, specifically pyrimidin-2(1 H)-one and pyrimidin-1 -yl-2,4(1 /-/, 3H)-dione.
  • Heterocyclyi can exist in various tautomeric forms.
  • a heterocyclyi moiety when substituted with an oxo group next to a nitrogen atom, the invention also pertains to its hydroxy tautomeric form.
  • 2-amino- 1 ,9-dihydro-6H-purin-6-one can tautomerize into 2-amino-9H-purin-6-ol.
  • the tautomerization is represented as foliow:
  • tautomer is used to designate 2 molecules with the same molecular formula but different connectivity, which can interconvert in a rapid equilibrium.
  • tautomers are phosporothioic acid which can exist in an equilibrium as shown below.
  • phosphoric acid exists as 2 tautomeric forms which interconvert in an equilibrium.
  • Drug moiety refers to a compound which binds to Stimulator of interferon Genes (STING) receptor and which comprises one or more functional groups each of which is capable of forming a covalent bond with a linker.
  • functional groups include, but are not limited to, primary amines, secondary amines, hydroxyls, thiols, aikenes, aikynes and azides in certain embodiments, such functional groups include reactive groups of Table 4 provided herein.
  • “sugar moiety” or“sugar moieties”, as used herein, refers to the following ring structures of the compounds of the invention ,
  • a wavy line indicates the point of attachment of the partial structure to the rest of the molecule.
  • HER2 refers to a transmembrane tyrosine kinase receptor of the epidermal growth factor (EGF) receptor family.
  • EGF epidermal growth factor
  • HER2 comprises an extracellular binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain.
  • HER2 does not have a ligand binding domain of its own and therefore cannot bind growth factors.
  • HER2 binds tightly to other ligand-bound EGF receptor family members such as HER1 or HER3, to form a heterodimer, stabilizing ligand binding and enhancing kinase-mediated activation of downstream signalling pathways.
  • the human HER2/NEU gene is mapped to chromosomal location 17q 12, and the genomic sequence of HER2/NEU gene can be found in GenBank at NG_007503.1. in human, there are five HER2 isoforms: A, B, C, D, and E: the term“HER2” is used herein to refer collectively to all HER2 isoforms.
  • a human HER2 protein also encompasses proteins that have over its full length at least about 70%, 71 %, 72%, 73%, 74%, 75%, 78%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 88%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with HER2 isoforms: A, B, C, D, and E, wherein such proteins still have at least one of the functions of HER2
  • the mRNA and protein sequences for human HER2 isoform A, the longest isoform, are:
  • ERBB2 erfo-fo2 receptor tyrosine kinase 2
  • transcript variant 1 mR!MA [HM__004448.3]
  • Receptor tyrosine-protein kinase erbB-2 isoform a precursor [Homo sapiens] EMP_ 004439,2]
  • HER2 isoform B NMJJ01005862.2 (mRNA)--> NPJJ01005862.1 (protein);
  • HER2 isoform C NMJ3G1289936 1 (mRNA)— > NPJJ01276865.1 (protein):
  • HER2 isoform D NM_001289937.1 (mRNA)— > NP__G01276866.1 (protein):
  • HER2 isoform E NMJJ01289938.1 (mRNA)— NPJ3Q1276887.1 (protein).
  • antibody refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources.
  • a naturally occurring“antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region
  • VH a heavy chain constant region
  • the heavy chain constant region is comprised of three domains, CH1 , CH2 and CHS.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl- terminus in the following order: FR1 , GDR1 , FR2, GDR2, FRS, CDRS, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that Interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the Immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1 q) of the classical complement system.
  • An antibody can be a monoclonal antibody, human antibody, humanized antibody, camelised antibody, or chimeric antibody.
  • the antibodies can be of any isotype (e.g., igG, IgE, IgM, IgD, IgA and IgY), class (e.g., igG1 , lgG2, lgG3, igG4, lgA1 and !gA2) or subclass.
  • isotype e.g., igG, IgE, IgM, IgD, IgA and IgY
  • class e.g., igG1 , lgG2, lgG3, igG4, lgA1 and !gA2
  • subclass e.g., igG1, , lgG2, lgG3, igG4, lgA1 and !gA2
  • antibody fragment or“antigen-binding fragment” or“functional fragment” refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), came!id VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • An antigen binding fragment can also be incorporated into single domain antibodies, naxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v ⁇ NAR and bis-scFv (see, e.g., Holiinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Patent No.: 6,703,199, which describes fibronectin polypeptide minibodies).
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker e.g., a short flexible polypeptide linker
  • an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-ierminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-iinker-VL.
  • CDR complementarity determining region
  • HCDR1 , HCDR2, and HCDR3 three CDRs in each heavy chain variable region
  • LCDR1 , LCDR2, and LCDR3 three CDRs in each light chain variable region
  • the precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991),“Sequences of Proteins of Immunological Interest,” 5th Ed.
  • the CDRs correspond to the amino acid residues that are defined as part of the Kabat CDR, together with the amino acid residues that are defined as part of the Chothia CDR.
  • the CDRs defined according to the“Chothia” number scheme are also sometimes referred to as“hypervariabie loops.”
  • VH heavy chain variable domain
  • HCDR1 e.g., insertion(s) after position 35
  • HGDR2 HGDR2
  • HCDR3 CDR amino acid residues in the light chain variable domain
  • the CDR amino acids in the VH are numbered 26-32 (HCDR1) (e.g., insertion(s) after position 31), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1) (e.g., inseriion(s) after position 30), 50-52 (LCDR2), and 91-95 (LCDR3)
  • HCDR1 e.g., insertion(s) after position 31
  • HCDR2 e.g., insertion(s) after position 31
  • HCDR2 e.g., insertion(s) after position 31
  • HCDR2 e.g., insertion(s) after position 31
  • HCDR2 e.g., insertion(s) after position 31
  • HCDR2 e.g., insertion(s) after position 31
  • HCDR2 e.g., insertion(s) after position 31
  • HCDR2 e.g
  • the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDRS) (numbering according to“Kabat”).
  • CDR1 the CDR amino acid residues in the VH
  • CDR2 CDR amino acid residues in the VL
  • CDRS 89-97
  • the CDR regions of an antibody can be determined using the program IMGT/DomainGap Align.
  • epitope includes any protein determinant capable of specific binding to an immunoglobulin or otherwise interacting with a molecule.
  • Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope may be“linear” or “conformational.” Conformational and linear epitopes are distinguished in that the binding to the former but not. the latter is lost in the presence of denaturing solvents.
  • phrases“monoclonal antibody” or“monoclonal antibody composition” as used herein refers to polypeptides, including antibodies, bispeeific antibodies, etc., that have substantially identical amino acid sequence or are derived from the same genetic source. This term also includes preparations of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • human antibody includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences, e.g., human ger iine sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik, et ai (2000.
  • immunoglobulin variable domains e.g., CDRs
  • CDRs immunoglobulin variable domains
  • weii known numbering schemes e.g , the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia
  • ImMunoGenTics (IMGT) numbering (see, e.g., Sequences of Proteins of Immunological interest, U.S. Department of Health and Human Services (1991), eds. Kabat et al.; AI Lazikani et al., (1997) J. Mol. Bio. 273:927 948); Kabat et al., (1991) Sequences of Proteins of immunological Interest, 5th edit., N!H Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol.
  • the human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germiine of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • recombinant human antibody includes ail human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DMA sequences.
  • recombinant means such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences in certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, whiie derived from and related to human germiine VH and VL sequences, may not naturally exist within the human antibody germiine repertoire in vivo.
  • Fc region refers to a polypeptide comprising the CHS, CH2 and at least a portion of the hinge region of a constant domain of an antibody.
  • an Fc region may include a CH4 domain, present in some antibody classes.
  • An Fc region may comprise the entire hinge region of a constant domain of an antibody.
  • the invention comprises an Fc region and a CH1 region of an antibody.
  • the invention comprises an Fc region CH3 region of an antibody in another embodiment, the invention comprises an Fc region, a CH1 region and a Ckappa/lambda region from the constant domain of an antibody in one embodiment, a binding molecule of the invention comprises a constant region, e.g., a heavy chain constant region in one embodiment, such a constant region is modified compared to a wild-type constant region.
  • the polypeptides of the invention disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1 , CH2 or CHS) and/or to the light chain constant region domain (CL).
  • Example modifications include additions, deletions or substitutions of one or more amino acids in one or more domains. Such changes may be included to optimize effector function, half-life, etc.
  • binding specificity refers to the ability of an individual antibody combining site to react with one antigenic determinant and not with a different antigenic determinant.
  • the combining site of the antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. Binding affinity of an antibody is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody it is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody.
  • affinity refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody“arm” interacts through weak non-covending forces with antigen at numerous sites: the more interactions, the stronger the affinity.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site- directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoieucine, proline, phenylalanine, methionine
  • beta- branched side chains e.g., threonine, valine, isoieucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • one or more amino acid residues within an antibody can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested using the functional assays described herein.
  • the term“homologous” or“identity” refers to the subunit sequence identity between two polymeric molecules, e.g , between two nucleic acid molecules, such as, two DMA molecules or two RNA molecules, or between two polypeptide molecules. Wien a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 1 Q), are matched or homologous, the two sequences are 90% homologous.
  • Percentage of“sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage can be calculated by determining the number of positions at which the identical 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 resuit by 100 to yield the percentage of sequence identity.
  • the output is the percent identity of the subject sequence with respect to the query sequence.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm in a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.corn), using either a Biossum 62 matrix or a PAM25Q matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.corn), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a Biossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the aigorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11 -17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See www.nGbi.nim.nih.gov
  • cancer and“cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cancer include, but are not limited to, solid tumors and hematological cancers, including carcinoma, lymphoma, biastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovia!
  • 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 cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, neuroblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract 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, testicular cancer, esophageal cancer, tumors of the biliary tract, as well as head and neck cancer. Additional cancer indications are disclosed herein.
  • tumor antigen or“cancer associated antigen” interchangeably refer to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of a cancer ceil, either entirely or as a fragment (e.g., MHC/peptide), and which is useful for the preferential targeting of a pharmacological agent to the cancer ceil.
  • a tumor antigen is a marker expressed by both normal ceils and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells.
  • a tumor antigen is a ceil surface molecule that is
  • a tumor antigen is a ceil surface molecule that is inappropriately synthesized in the cancer ceil, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal ceil.
  • a tumor antigen will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment (e.g., MHC/peptide), and not synthesized or expressed on the surface of a normal cell.
  • MHC Major histocompatibility complex
  • TCRs T ceil receptors
  • tumor-supporting antigen or“cancer-supporting antigen” interchangeably refer to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of a ceil that is, itself, not cancerous, but supports the cancer cells, e.g , by promoting their growth or survival e.g., resistance to immune cells.
  • the tumor-supporting antigen itself need not play a role in supporting the tumor cells so long as the antigen is present on a cell that supports cancer ceils.
  • A“HER2-positive cancer” or“HER2-expressing cancer” is a cancer comprising ceils that have HER2 protein present at their ceil surface. Many methods are known in the art for detecting or determining the presence of HER2 on a cancer ceil. For example, in some embodiments, the presence of HER2 on the cell surface may be determined by
  • IHC immunohistochemistry
  • flow cytometry Western blotting
  • immunofluorescent assay immunofluorescent assay
  • radioimmunoassay RIA
  • enzyme-linked immunosorbent assay ELISA
  • homogeneous time resolved fluorescence HTRF
  • PET positron emission tomography
  • the terms“combination” or“pharmaceutical combination,” as used herein mean a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients.
  • the term“fixed combination” means that the active ingredients, by way of example, a compound of the invention and one or more additional therapeutic agent, are administered to a subject simultaneously in the form of a single entity or dosage.
  • the term“non-fixed combination” means that the active ingredients, by way of example, a compound of of the invention and one or more additional therapeutic agent, are administered to a subject as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the active ingredients in the body of the subject.
  • cocktail therapy e.g. the administration of 3 or more active ingredients.
  • composition refers to a mixture of a compound of the invention with at ieast one and optionally more than one other pharmaceutically acceptable chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • pharmaceutically acceptable chemical components such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • an optical isomer or“a stereoisomer”, as used herein, refers to any of the various stereo isomeric configurations which may exist for a given compound of the present invention and includes geometric isomers it is understood that a substituent may be attached at a chiral center of a carbon atom.
  • the term “chiral” refers to molecules which have the property of non-superimposability on their mirror image partner, while the term “achiral” refers to molecules which are superimposabie on their mirror image partner. Therefore, the invention includes enantiomers, diastereomers or racemates of the compound. “Enantiomers” are a pair of stereoisomers that are non- superimposabie mirror images of each other.
  • a 1 :1 mixture of a pair of enantiomers is a "racemic” mixture.
  • the term is used to designate a racemic mixture where appropriate.
  • "Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other.
  • the absolute stereochemistry is specified according to the Cahn-lngold- Prelog R-S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon may be specified by either R or S Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (d extra- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line.
  • Certain compounds described herein contain one or more asymmetric centers or axes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed Mack Printing Company, 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • pharmaceutically acceptable salt refers to a salt which does not abrogate the biological activity and properties of the compounds of the invention, and does not cause significant irritation to a subject to which it is administered.
  • subject encompasses mammals and non-mammals.
  • mammals include, but are not limited to, humans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, swine; rabbits, dogs, cats, rats, mice, guinea pigs, and the like.
  • non-mammals include, but are not limited to, birds, fish and the like. Frequently the subject is a human.
  • a subject in need of such treatment refers to a subject which would benefit biologically, medically or in quality of life from such treatment.
  • STING refers to STtimulator of INterferon Genes receptor, also known as TMEM173, ERIS, MITA, MPYS, SAVi, or NET23).
  • STING and STING receptor are used interchangeably, and include different isoforms and variants of STING.
  • the RNA and protein sequences for human STING isoform 1 are:
  • TMEM173 Homo sapiens transmembrane protein 173 (TMEM173), transcript variant 1 , mRNA
  • gagccccagc agaagaatgg agaggaggag gaggctgagt ttggggtatt gaatcccccg
  • the mRNA and protein sequences for human STING isoform 2, a shorter isoform, are:
  • TMEM173 Homo sapiens iransme brane protein 173 (TMEM173), transcript variant 2, mRNA
  • Homo sapiens stimulator of interferon genes protein isoform 2 [NP_GG1288667 1 ]
  • hSTING G238A/R293Q Reference SNR (refSNP) Cluster Report: rs1131769 rs7380824 rs78233829
  • hSTING R71 H/G230A/R2S3Q Reference SIMP (refSNP) Cluster Report:
  • STING agonist refers to a compound or antibody conjugate capable of binding to STING and activating STING Activation of STING activity may include, for example, stimulation of inflammatory cytokines, including interferons, such as type 1 interferons, including IFN-a, IFN-b, type 3 interferons, e.g., IRNl, IP10, TNF, !L ⁇ 8, CXCL9,
  • interferons such as type 1 interferons, including IFN-a, IFN-b, type 3 interferons, e.g., IRNl, IP10, TNF, !L ⁇ 8, CXCL9,
  • STING agonist activity may also include stimulation of TANK binding kinase (TBK) 1 phosphorylation, interferon regulatory factor (IRF) activation (e.g , IRF3 activation), secretion of interferon-y-inducible protein (IP-10), or other inflammatory proteins and cytokines.
  • TNK TANK binding kinase
  • IRF interferon regulatory factor
  • IP-10 interferon-y-inducible protein
  • STING Agonist activity may be determined, for example, by the ability of a compound to stimulate activation of the STING pathway as detected using an interferon stimulation assay, a reporter gene assay (e.g., a hSTING wt assay, or a THP-1 Dual assay), a TBK1 activation assay, IP-10 assay, a STING Biochemical [SHjcGAMP Competition Assay, or other assays known to persons skilled in the art.
  • STING Agonist activity may also be determined by the ability of a compound to increase the level of transcription of genes that encode proteins activated by STING or the STING pathway. Such activity may be detected, for example, using an RNAseq assay.
  • an assay to test for activity of a compound in a STING knock-out cell line may be used to determine if the compound is specific for STING, wherein a compound that is specific for STING would not be expected to have activity in a cell line wherein the STING pathway is partially or wholly deleted.
  • the terms“treat,”“treating,” or“treatment” of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof ⁇ in another embodiment,“treat,”“treating,” or“treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient in yet another embodiment,“treat,”“treating,” or“treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
  • the term“prevent”,“preventing” or“prevention” of any disease or disorder refers to the prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder
  • the term“therapeutically effective amount” or“therapeutically effective dose” interchangeably refers to an amount sufficient to effect the desired result (i.e., reduction or inhibition of an enzyme or a protein activity, amelioration of symptoms, alleviation of symptoms or conditions, delay of disease progression, a reduction in tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal or parasitic infection) in some embodiments, a therapeutically effective amount does not induce or cause undesirable side effects in some embodiments, a therapeutically effective amount induces or causes side effects but only those that are acceptable by the healthcare providers in view of a patient’s condition.
  • a therapeutically effective amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved.
  • a “prophylacticaliy effective dose” or a“prophylactically effect amount”, of the molecuies of the invention can prevent the onset of disease symptoms, including symptoms associated with cancer.
  • A“therapeutically effective dose” or a“therapeutically effective amount” of the molecuies of the invention can result in a decrease in severity of disease symptoms, including symptoms associated with cancer.
  • the compound names provided herein were obtained using ChemDraw Ultra version 14.0 (CambridgeSoft®).
  • any formula given herein is also intended to represent uniabeied forms as well as ssotopicaiiy labeled forms of the compounds isotopically labeled compounds have structures depicted by the formulae given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number.
  • Isotopes that can be incorporated into compounds of the invention include, for example, isotopes of hydrogen.
  • the conjugates or Drug moieties of the present invention refer to compounds of any of formulae (AA-a) through (FF-g) or formulae (A) through (F) or subformulae thereof and exemplified compounds, and salts thereof, as weii as all stereoisomers (including diastereoisomers and enantiomers), rotamers, tautomers and isotopically labeled compounds (including deuterium substitutions), as well as inherently formed moieties.
  • the Drug moiety (D) of the immunoconjugates of the invention is a compound which binds to Stimulator of Interferon Genes (STING) receptor and which comprises one or more reactive moieties each of which is capable of forming a covalent bond with a linker (L).
  • Drug moiety (D) of the immunoconjugates of the invention is a dinucleotide which binds to Stimulator of Interferon Genes (STING) which comprises one or more reactive moieties capable of forming a covalent bond with a linker (L)
  • Drug moiety (D) of the immunoconjugates of the invention is a cyclic dinucleotide which binds to Stimulator of interferon Genes (STING) which comprises one or more reactive moieties capable of forming a covalent bond with a linker (L).
  • STING Stimulator of interferon Genes
  • the Drug moiety (D) of the immunoconjugates of the invention is a compound having the structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or Formula (F) or stereoisomers or pharmaceutically acceptable salts thereof,
  • each Gi is independently selected from
  • XB is C, and each Z 2 is N;
  • Y 6 is -CHr, -NH-, -O- or -S;
  • Y 7 is O or S
  • Y 8 is O or S
  • Y 9 is -CH2-, -NH-, -O- or -S;
  • Y10 is -CH2r. -NH-. -O- or -S;
  • q is 1 , 2 or 3;
  • R 1 is a partially saturated or aromatic monocyclic heteroeycly! or partially saturated or aromatic fused bicyclic heierocyciyi containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R 1 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH 2 , -NHNH 2 , -NHGH, F, Cl, Br, CrC 6 alkyl and a CrC s a!kyl substituted with 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N 3 ;
  • R 1a is a partially saturated or aromatic monocyclic heteroeycly! or partially saturated or aromatic fused bicyclic heterocyciyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R 1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH 2 , -NHNH 2 , -NHOH, F, Cl, Br, Ci-C 6 alkyl and a Ci-C 3 alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 1 b is a partially saturated or aromatic monocyclic beterocyciyi or partially saturated or aromatic fused bicyclic heterocyciyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R 1 b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH 2 , ⁇ NHNH 2 , -NHOH, F, Cl, Br, Ci-C 6 alkyl and CrCealky! substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 2 is independently selected from H and CrCsaikyl
  • each R 3 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 and a CrCsaikyl substituted with a NHOH, NHNH 2 or NH 2 ;
  • each R 4 is independently selected from H, CrCsaikyl and CrC 6 aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 5 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 and a C Csalkyl substituted with a NHOH, NHNH 2 or NH 2 ;
  • each R 6 is independently selected from H, CrCsaikyl and CrCsaikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 7 is independently selected from H, CrCsaikyl and CrCsaikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 8 is independently selected from H, CrCsaikyl and CrCsaikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 9 is independently selected from H, CrCsaikyl and CrCsaikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 2a is independently selected from H and CrC 6 alkyl
  • R 3a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 and a C r Cealkyi substituted with a NHOH, NHNH 2 or NH 2 ;
  • R 4a is independently selected from H, CrC 6 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 5a is independently selected from H, -OH, -SH, F, Cl, Br, !, NHOH, NHNH 2 , NH 2 and a Cr Csalky! substituted with a NHOH, NHNH 2 or NH 2 ;
  • R 6a is independently selected from H, CrC 6 alkyl and CrC 6 alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 7a is independently selected from H, CrC 6 alkyl and C i-C 6 alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • R 8a is independently selected from H, CrC 6 alkyl and CrC 6 alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N 3 ;
  • R 9a is independently selected from H, CrC 6 alkyl and CrC 6 alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Gi, Br, I, OH, CN, and N 3 ;
  • each R 10 is independently selected from the group consisting yi, Ci-
  • R 11 is independently selected from H and Ci-C 3 alkyl
  • R 12 is independently selected from H and Ci-C 3 alkyl
  • R 3 and R 6 are connected to form -0-CrC 6 alkylene, such that when R 3 and R 6 are connected, the O is bound at. the R 3 position
  • R 3a and R 6a are connected to form -0 ⁇ CrC 6 aikyiene, such that when R 3a and R Sa are connected, the O is bound at the R 3a position;
  • R 2 and R 3 are connected to form -O-CrCgalkylene, such that when R 2 and R 3 are connected, the O is bound at the R 3 position;
  • R 2a and R 3a are connected to form -0-Ci-C 3 aikyiene, such that when R 2a and R 3a are connected, the O is bound at the R 3a position;
  • R 4 and R 3 are connected to form -G-CrC 6 alkylene, such that when R 4 and R 3 are connected, the O is bound at the R 3 position;
  • R 4a and R 3a are connected to form -Q-Ci-G 6 aikyiene, such that when R 4a and R 3a are connected, the O is bound at the R 3a position;
  • R 5 and R 6 are connected to form -Q-CrCgaikylene, such that when R 5 and R 6 are connected, the O is bound at the R 5 position; optionally R 5a and R 6a , are connected to form -0-C r C B alkylene, such that when R 5a and R 6a are connected, the O is bound at the R 5a position:
  • R 5 and R 7 are connected to form -Q-CrC 6 aiky!ene, such that when R 5 and R 7 are connected, the O is bound at the R 5 position,
  • R 5a and R 7a are connected to form -OCrC 6 aikylene, such that when R 5a and R 7a are connected, the O is bound at the R 5a position.
  • Embodiment 1 The compound of Formula (A-1), Formula (B-1), Formula (C-1), Formula (D-1), Formula (E ⁇ 1) or Formula (F-1), or siereoisomers or pharmaceutically acceptable salts thereof wherein:
  • Y 7 e the * of Gi indicates the point of attachment to -CR S R 9 -: e the * of G 2 indicates the point of attachment to ⁇ CR 8a R 93 -; , SR 10 , SeH, Se , BH 3 , SH or S ;
  • Y 5 is -CH 2 -, -NH-, -O- or -S;
  • Y 6 is -CH 2 -, -NH-, -O- or -S;
  • Y 7 is O or S
  • Y 8 is O or S
  • Y 9 is -CH ! -, -NH-, -O- or -S, and
  • Yio is -CHr, -NH-, -O- or -S.
  • Embodiment 2 A compound of Formula (A-1), Formula (B-1), Formula (C-1), Formula (D- 1), Formula (E-1) or Formula (F-1), or stereoisomers or pharmaceutically acceptable salts thereof,
  • R 1 , R 1a , R 1 b , R 2 , R 2a , R 3 , R 3a , R 4 , R 4a , R 5 , R 5a , R 6 , R 6a , R 7 , R 7a , R 8 , R Sa , R 9 , Y, , Y 2 , Y 3 , Y4 Y 5> Y 6 , Y 7 , Y 8 , U b , Y and Y are as defined above for compounds of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) and Formula (F).
  • Embodiment 3 A compound of Formulae A), Formula (B), Formula (C), Formula (D), Formula (A- 1 ) , Formula (B-1), Formula (C-1), Formula (D-1 ), Formula (E-1), or Formula (F- 1 ), wherein R 1 is pyrimidine or purine nucleic acid base or analogue thereof, R 1a is pyrimidine or purine nucieic acid base or analogue thereof, and R 1b is a pyrimidine or purine nucleic acid base or analogue thereof, each of which is substituted as described in R ⁇ R 1a or R 1b for Formula (A), Formula (BB, Formula (C), Formula (D), Formula (A- 1 ) , Formula (B-1), Formula (C-1), Formula (D-1), Formula (E-1), or Formula (F-1).
  • Formula (E-2) Formula (F-2) wherein R ⁇ R 1a , R 1 b , R 2 , R 2a , R 3 , R 3a , R 4 , R 4a , R 5 , R 5a , R 6 , R 6a , R 7 , R 7a , R 8 , R Sa , R 9 , Y, , Y 2 , Y 3 , Y 4 , Y 5> Y 6 , Y 7 , Y 8 , Ye, Yio and Y are as defined above for compounds of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) and Formula (F).
  • Embodiment S A compound of Formula (A), Formula (A-1) or Formula (A-2) of
  • Embodiments 1 , 2, 3 or 4 wherein:
  • R 3a is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 or a Gi-C 6 aikyi substituted with a NHOH, NHNH 2 or NH 2 ;
  • R 8 , R 9 , R 8a and R 9a are independently H or Ci-C 5 alkyl.
  • Embodiment 6 A compound of Formula (A), Formula (A-1) or Formula (A-2) of
  • Embodiment 1 2, 3, 4 or 5 wherein:
  • Yi and Y 2 are independently selected from O, NH, CH 2 and S;
  • Y 3 is OH, O , NH 2, SH or S ;
  • Y 4 is OH, O , NH 2 , SH or S ;
  • Y 5 and Y 6 are independently selected from O and S;
  • Y 7 and Y s are independently selected from O and S;
  • Y 9 and Yio are independently selected from O and S;
  • R 2 , R 2a , R 4 , R 4a , R 6 , R 6a , R 7 and R 7a are H;
  • R 3a is H, ⁇ NH 2 , -NHNH 2 , -NHOH, OH or F;
  • R 3 is H, -NH 2 , -NHNH 2 -NHOH, OH or F;
  • R 8a , R 9a , R 8 and R 9 are independently selected from H or CrC s aikyL
  • Embodiment 7 A compound of Formula (A), Formula (A-1) or Formula (A-2) of
  • Embodiment 1 2, 3, 4 or 5 wherein:
  • Yi and Y 2 are independently selected from O, NH, CH 2 and S;
  • Y 3 is OH, O , NH 2 , SH or S ;
  • Y 4 is OH, O , NH 2 , SH or S ;
  • Ys and Y 6 are independently selected from O and S;
  • Y 7 and Y 8 are independently selected from O and S;
  • Y 9 and Yio are independently selected from O and S;
  • R 2 , R 2a , R 4 , R 4a , R 6 , R 6a , R 7 and R 7a are H;
  • R 3a is H, -NHNHz, OH or F:
  • R 3 is H, -NHNH 2 , OH or F
  • R 8a , R 9a , R 8 and R 9 are independently selected from H or Ci-C 3 aikyl.
  • Embodiment 8 A compound of Formula (B), Formula (B-1) or Formula (B-2) of Embodiment 1 , 2, 3 or 4 wherein:
  • R 2 , R 2a , R 4 , R 4a , R 6 , R 6a , R 7 and R 7a are H;
  • R 3a is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 or a G-i-Ceaikyl substituted with a NHOH, NHNH 2 or NH 2 ;
  • R 5 is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2, NH 2 or a CrC 5 alkyl substituted with a NHOH, NHNH 2 or NH 2 ;
  • R 8 , R s , R Sa and R 9a are independently H or Ci-C 3 alkyi.
  • Embodiment 9 A compound of Formula (B), Formula (B-1) or Formula (B-2) of
  • Embodiment 1 2, 3, 4 or 8 wherein:
  • Yi and Y 2 are independently selected from O, NH, CH 2 and S;
  • Y 3 is OH, O , NH 2 , SH or S-;
  • Y 4 is OH, O , NH 2 , SH or S-;
  • Y 5 and Y 6 are independently selected from O and S;
  • Y 7 and Y s are independently selected from O and S;
  • Y 9 and Y10 are independently selected from O and S;
  • R 2 , R 2a , R 4 , R 4a , R s , R 5a , R 7 and R 7a are H;
  • R 3a is H, -NH 2 , -NHNH2, -NHOH, OH or F;
  • R 5 is H, -NH 2 , -NHNH 2I -NHOH, OH or F;
  • R 8a , R 9a , R s and R 9 are independently selected from H or Ci-C 3 a!kyi
  • Embodiment 10 A compound of Formula (B), Formula (B-1) or Formula (B-2) of
  • Embodiment 1 2, 3, 4 or 8 wherein:
  • Yi and Y 2 are independently selected from O, NH, CH 2 and S;
  • Y 3 is OH, O , NH 2 , SH or S ;
  • Y 4 is OH, O , NH 2 , SH or S ;
  • Y 5 and Y 6 are independently selected from O and S;
  • Y 7 and Y 8 are independently selected from O and S;
  • Y 9 and Y10 are independently selected from O and S;
  • R 83 , R 9a , R 8 and R 9 are independently selected from H or Ci-C 6 alkyl.
  • Embodiment 11 A compound of Formula (C), Formula (C-1) or Formula (C-2) of
  • Embodiment 1 2, 3 or 4 wherein:
  • R 2 , R 2a , R 4 , R 43 , R 6 , R 63 , R 7 and R 7a are H;
  • R 8 , R 9 , R 8a and R 93 are independently H or Ci-C 5 alkyl.
  • Embodiment 12 A compound of Formula (C), Formula (C-1) or Formula (C-2) of
  • Embodiment 1 1, 2, 3 or 1 1 wherein:
  • Yi and Y 2 are independently selected from O, NH, CH 2 and S;
  • Y 3 is OH, O , NH 2, SH or S ;
  • Y 4 is OH, O , NH 2, SH or S ;
  • Y 5 and Y 6 are independently selected from O and S;
  • Y 7 and Y s are independently selected from O and S;
  • Y 9 and Yio are independently selected from O and S;
  • R 2 , R 2a , R 4 , R 43 , R 6 , R 63 , R 7 and R 7a are H;
  • R 3 is H, -NH 2 , -NHNH 2I -NHOH, OH or F;
  • R 53 is H, ⁇ NH 2 , -NHNH 2I -NHOH, OH or F;
  • R 83 , R 9a , R 8 and R 9 are independently selected from H or CrC 6 a!kyi
  • Embodiment 13 A compound of Formula (C), Formula (C-1) or Formula (C-2) of
  • Embodiment 1 1, 2, 3 or 1 1 wherein:
  • Yi and Y 2 are independently selected from O, NH, CH 2 and S;
  • Y 3 is OH, O , NH 2 , SH or S ;
  • Y 4 is OH, O , NH 2 , SH or S ;
  • Ys and Y 6 are independently selected from O and S;
  • Y 7 and Y 8 are independently selected from O and S;
  • Y 9 and Yic are Independently selected from O and S;
  • R 2 , R 2a , R 4 , R 43 , R 6 , R 63 , R 7 and R 7a are H;
  • R 3 is H, -NHNHz, OH or F
  • R 5a is H, -NHNHz, OH or F
  • R 8a , R 9a , R 8 and R 9 are independently selected from H or Ci-C 6 alkyl.
  • Embodiment 14 A compound of Formula (D), Formula (D-1) or Formula (D-2) of
  • Embodiment 1 2, 3 or 4 wherein:
  • R 2 , R 2a , R 4 , R 43 , R 6 , R 63 , R 7 and R 73 are H;
  • R 5a is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 or a Ci-C 6 alkyl substituted with a NHOH, NHNH 2 or NH 2 ;
  • R 5 is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 or a Ci-C 6 alkyi substituted with a NHOH, NHNH 2 or NH 2
  • R 8 , R 9 , R Sa and R 9a are independently H or CrC s alkyl.
  • Embodiment 15 A compound of Formula (D), Formula (D-1) or Formula (D-2) of
  • Embodiment 1 2, 3, 4 or 14 wherein:
  • Y 1 and Y 2 are independently selected from O, NH, CH 2 and S;
  • Y 3 is OH, O , NH 2 , SH or S :
  • Y 4 is OH, O , NH 2 , SH or S ;
  • Y 5 and Y 6 are independently selected from O and S;
  • Y 7 and Y s are independently selected from O and S;
  • Y 9 and Yio are independently selected from O and S;
  • R 2 , R 2a , R 4 , R 4a , R 6 , R Sa , R 7 and R 7a are H;
  • R 5a is H, -NH 2 , -NHNH 2 , -NHOH, OH or F;
  • R 5 is H, ⁇ NH 2 , -NHNH 2 , -NHOH, OH or F;
  • R 8 , R s , R Sa and R 9a are independently H or Ci-C 3 alkyi.
  • Embodiment 16 A compound of Formula (D), Formula (D-1) or Formula (D-2) of
  • Embodiment 1 2, 3, 4 or 14 wherein:
  • Y 1 and Y 2 are independently selected from O, NH, CH 2 and S;
  • Y 3 is OH, O , NH 2 , SH or S-;
  • Y 4 is OH, O , NH 2 , SH or S-;
  • Y 5 and Y 6 are independently selected from O and S;
  • Y ? and Y s are independently selected from O and S;
  • Y 9 and Yio are independently selected from O and S;
  • R 8 , R 9 , R 8a and R Sa are independently H or Ci-C 3 alkyl
  • Embodiment 17 A compound of Formula (E), Formula (E-1) or Formula (E-2) of
  • Embodiment 1 , 2 or 3 wherein:
  • R 2 , R 2a , R 4 , R 4a , R 6 , R 6a , R 7 and R 7a are H;
  • R 3a is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 or a C C 6 aikyi substituted with a NHOH, NHNH 2 or NH 2 ;
  • R 3 is H, -OH, -SH, F, Ci, Br, I, NHOH, NHNH 2 , NH 2 or a C C 6 alkyi substitifted with a NHOH, NHNH 2 or NH 2 ;
  • R 5 is H, -OH, -SH, F, Ci, Br, I, NHOH, NHNH 2 , NH 2 or a Ci-C 6 alkyl substitifted with a NHOH, NHNH 2 or NH 2 ;
  • R 8 , R 9 , R 8a and R 9a are independently H or Ci-C 3 alkyl H.
  • Embodiment 18 A compound of Formula (E), Formula (E-1) or Formula (E-2) of Embodiment 1 , 2, 3, 4 or 17 wherein:
  • Y 1 and Y 2 are independently selected from O, NH, CH 2 and S;
  • Y 3 is OH, O , NH 2 SH o S :
  • Y 5 is O or S
  • Y 7 is O or S
  • Y 9 is O or S
  • R 2 , R 2a , R 4 , R 4a , R 53 , R 6 , R 63 , R 7 and R 7a are H
  • R 3a is H, -NH 2 , ⁇ NHNH 2S -NHOH, OH or F;
  • R 3 is H, ⁇ NH 2 , -NHNH 2 , -NHOH, OH or F;
  • R 5 is H, ⁇ NH 2 , -NHNH 2 , -NHOH, OH or F;
  • R 8 , R 9 , R Sa and R 9a are independently H or C -C 3 alkyi
  • Embodiment 19 A compound of Formula (E), Formula (E-1) or Formula (E-2) of
  • Embodiment 1 1, 2, 3 or 12 wherein:
  • Y 1 and Y 2 are independently selected from O, NH, CH 2 and S;
  • Y 3 is OH, O , NH 2 , SH or S ;
  • Y s is O or S
  • Y 9 is O or S:
  • R 2 , R 2a , R 4 , R 43 , R 5a , R 6 , R 6a , R 7 and R 7a are H
  • R 3a is H, -NHNHs, OH or F:
  • R 3 is H, -NHNH 2 OH or F
  • R 5 is H, -NHNH 2 OH or F
  • R 8 , R s , R 8a and R 9a are independently H or Ci-C 6 alkyl.
  • Embodiment 20 A compound of Formula (F), Formula (F-1) or Formula (F-2) of
  • Embodiment 1 , 2 or 3 wherein:
  • R 2 , R 2a , R 4 , R 43 , R 6 , R 63 , R 7 and R 7a are H;
  • R 8 , R 9 , R 8a and R 9a are independently H or Ci-C e alkyl.
  • Embodiment 21 A compound of Formula (F), Formula (F-1) or Formula (F-2) of
  • Embodiment 1 , 2, 3 or 12 wherein: Y 1 and Y 2 are independently selected from O, NH, CH 2 and S;
  • each Y 3 is OH, O , NH 2 , SH or S ;
  • each Y 5 is independently selected from O and S;
  • each Y ? is independently selected from O and S;
  • each Y 9 is independently selected from O and S;
  • R 2 , R 2a , R 4 , R 4a , R 6 , R Sa , R 7 and R 7a are H;
  • R 3a is H, -NH 2 , ⁇ NHNH 2 , -NHOH, OH or F;
  • R 3 is H, ⁇ NH 2 , -NHNH 2I -NHOH, OH or F;
  • R 5 is H, ⁇ NH 2 , -NHNH 2I -NHOH, OH or F, and
  • R 8 , R s , R Sa and R 9a are independently H or Ci-C 3 alkyl.
  • Embodiment 22 A compound of Formula (F), Formula (F ⁇ 1) or Formula (F-2) of Embodiment 1 , 2, 3 or 12 wherein:
  • Y 1 and Y 2 are independently selected from O, NH, CH 2 and S;
  • each Y 3 is OH, O , NH 2 , SH or S ;
  • each Y 5 is independently selected from O and S;
  • each Y ? is independently selected from O and S;
  • each Yg is independently selected from O and S;
  • R 8 , R s , R 8a and R 9a are independently H or Ci-C 6 alkyi.
  • Embodiment 23 A compound of any one of Embodiments 1 to 22 wherein:
  • R 1a is substituted with 0,
  • R 1 is substituted with 0, 1 ,
  • each R 2 is independently selected from H and CrC s aikyi;
  • each R 3 Is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 and a Ci-G 6 alkyi substituted with a NHOH, NHNH 2 or NH 2 ;
  • each R 4 Is independently selected from H, Ci-C 6 alkyl and Gi-C 6 aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Gi, Br, I, OH, CN, and N 3 ;
  • each R 5 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 and a Ci-C 3 aikyl substituted with a NHOH, NHNH 2 or NH 2 ;
  • each R 6 is independently selected from H, CrC 6 alky! and CrC 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 7 is independently selected from H, Ci ⁇ C 6 alkyl and Ci-C 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R ® is independently selected from H, CrC 6 alky! and Ci-C 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R s is independently selected from H, CrC 6 alkyl and CrC 6 aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 2a is independently selected from H and C i-C 6 aikyl
  • each R 3a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 and a Ci-Csalkyi substituted with a NHOH, NHNH 2 or NH 2 :
  • each R 4a is independently selected from H, Ci-C 3 alkyl and Ci-C 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N 3 ;
  • each R 5a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH 2 , NH 2 and a Ci-C 6 alkyi substituted with a NHOH, NHNH 2 or NH 2 ;
  • each R 6a is independently selected from H, C -C 3 alkyl and Ci-Cealkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N 3 ;
  • each R 7a is independently selected from H, C -C 3 alkyl and Ci-Cealkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N 3 ;
  • each R Sa is independently selected from H, Ci-C 3 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 9a is independently selected from H, Ci-C 3 alkyl and CrC 6 alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N 3 ;
  • each R 11 is independently selected from H and CrC 6 alkyl
  • each R 12 is independently selected from H and CrC 6 alkyl
  • R 3 and R 6 are connected to form -Q-G i-C 6 aikylene, such that when R 3 and R s are connected, the O is bound at the R 3 position
  • R 3a and R 6a are connected to form -0-Ci-C 6 alkylene, such that when R 38 and R 6a are connected, the O is bound at the R 3a position;
  • R 2 and R 3 are connected to form -0-CrC 6 alkylene, such that when R 2 and R 3 are connected, the O is bound at the R 3 position;
  • R 2a and R 3a are connected to form -0-Ci-C 6 alkylene, such that when R 2a and R 3a are connected, the O is bound at the R 3a position;
  • R 4 and R 3 are connected to form -0-CrC 6 alkylene, such that when R 4 and R 3 are connected, the O is bound at the R 3 position;
  • R 4a and R 3a are connected to form -G-CrC s a!kylene, such that when R 4a and R 3a are connected, the O is bound at the R 3a position;
  • R 5 and R 6 are connected to form -O-CrCeaikylene, such that when R 5 and R s are connected, the O is bound at the R 5 position;
  • R 5a and R 6a are connected to form -G-Ci-G 6 alkylene, such that when R 5a and R Sa are connected, the O is bound at the R 5a position;
  • R 5 and R 7 are connected to form -0-Ci-C 6 alkylene, such that when R 5 and R 7 are connected, the O is bound at the R 5 position,
  • R 5a and R 7a are connected to form -Q-CrCga!kyiene, such that when R 5a and R 7a are connected, the O is bound at the R 5a position.
  • Embodiment 25 The compound Formula (A-3) , or a pharmaceutically acceptable salt thereof, having the structure of Formula (A-4), or a pharmaceutically acceptable salt thereof:
  • R 1 , R ia , R 3 , R 3a , R 6 , R Sa , Y 3 and Y 4 are as defined in Embodiment 24.
  • Embodiment 26 The compound of Formula (A-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (A-4a), Formula A-4b), Formula A-4c) or Formula A- 4d), or a pharmaceutically acceptable salt thereof:
  • R 1 , R 1a , R 3 , R 3a , R 6 and R 6a are as defined in Embodiment 24;
  • Y 3 is OH, NH 2 , SH or S .
  • Y 4 is OH, NH 2 , SH or S .
  • Embodiment 27 The compound of Formula (A-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (A-4e), Formula (A-4f), Formula (A-4g), Formula (A-4h), Formula (A-4i), Formula (A-4j), Formula (A-4k), Formula (A-41), Formula (A-4m), Formula (A-4n), Formula (A-4o) or Formula (A-4p), or a pharmaceutically acceptable salt thereof:
  • R ⁇ R 1a , R 3 , R 3a , R s and R 6a are as defined In Embodiment 24;
  • Y 3 is OH, NH 2 , SH or S .
  • Y 4 is OH, NH 2 , SH or S .
  • Embodiment 28 The compound of Formula (B-3) having the structure of Formula (B-4), or a pharmaceutically acceptable salt thereof:
  • R 1 , R 1a , R 3 , R 3a , R 5 , R 6a , Y 3 and Y 4 are as defined in Embodiment 24.
  • R 1 , R 18 , R 3a , R 5 and R 68 are as defined in Embodiment 24;
  • Y 3 is OH, NH 2 , SH or S .
  • Y 4 is OH, NH 2 , SH or S .
  • Embodiment 30 The compound of Formula (B-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (B-4e), Formula (B-4f), Formula (B-4g) or Formula (B-4h), or a pharmaceutically acceptable salt thereof:
  • Y 3 is OH, NH 2 , SH or S .
  • Y 4 is OH, NH 2 , SH or S .
  • Embodiment 31 The compound of Formuia (C-3) having the structure of Formula (C-4), or a pharmaceutically acceptable salt thereof:
  • R 1 , R 1a , R 3 , R 5a , R 6 , R 6a , Y 3 and Y 4 are as defined in Embodiment 24.
  • Embodiment 32 The compound of Formula (C-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (C-4a), Formula (C-4b), Formula (C-4c) or Formula (C-4d), or a pharmaceutically acceptable salt thereof:
  • R 1 , R 1a , R 3 , R 5a and R 6 are as defined in Embodiment 24;
  • Y 3 is OH, NH 2 , SH or S .
  • Y 4 is OH, NH 2 , SH or S .
  • Embodiment 33 The compound of Formula (C-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (C-4e), Formula (C-4f), Formula (C-4g) or Formula (C-4h), or a pharmaceutically acceptable salt thereof:
  • R ⁇ R 1a and R 5a are as defined in Embodiment 24;
  • Y 3 is OH, NH 2 , SH or S .
  • Y 4 is OH, NH 2 , SH or S .
  • Embodiment 34 The compound of Formula (D-3), or a pharmaceutically acceptable salt thereof, having the structure of Formula (D-4), or a pharmaceutically acceptable salt thereof:
  • R 1 , R 1a , R 5 , R 5a , Y 3 and Y 4 are as defined in Embodiment 24.
  • Embodiment 35 The compound of Formula (D-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (D-4a), Formula (D ⁇ 4b), Formula (D-4c) or Formul; (D-4d), or a pharmaceutically acceptable salt thereof:
  • R 1 , R ia , R 5 and R £,a are as defined in Embodiment 24;
  • Y3 is OH, NH2, SH or S .
  • Y 4 is OH, NH2, SH or S
  • Embodiment 38 The compound of Formula (E-3), or a pharmaceutically acceptable salt thereof, having the structure of Formula (E-4), or a pharmaceutically acceptable salt thereof:
  • R 1 , R 1a , R 3 , R 3a , R 4 , R 4a , R 5 and R' are as defined in Embodiment 24.
  • Embodiment 37 The compound of Formula (E-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (E-4a) or Formula (E-4b), or a pharmaceutically acceptable salt thereof:
  • R 1 , R 1 a , R 3 , R 3a , R 4 , R 4a , R 5 and R 7 are as defined in Embodiment 24;
  • Y 3 is OH, NH 2I SH or S .
  • Embodiment 38 The compound of Formula (F-3), or a pharmaceutically acceptable salt thereof, having the structure of Formula (F-4), or a pharmaceutically acceptable salt thereof:
  • R 1 , R 1a , R 1b , R 3 , R 3a , R 4 , R 4a , R 5 and R 7 are as defined in Embodiment 24.
  • Embodiment 39 The compound of Formula (F-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (F-4a), Formula (F-4b), Formula (F-4c), or Formula (F-4d), or a pharmaceutically acceptable salt thereof:
  • Formula (F-4c) Formula (F-4d) wherein: R 1 , R 1 a , R 1 b , R 3 , R 3a , R 4 , R 4a , R 5 and R 7 are as defined in Embodiment 24; and
  • each Y 3 is independenlty selected from Y 3 is OH, NH 2 , SH or S .
  • Embodiment 40 The compound of any one of Embodiments 1 to 39, wherein R 1 is
  • Embodiment 44 The compound of any one of Embodiments 1 to 39, wherein R 1a is
  • Embodiment 45 The compound of any one of Embodiments 1 to 39, wherein R 1 b is
  • Embodiment 46 The compound of any one of Embodiments 1 to 39, wherein R 1 is
  • Embodiment 47 The compound of any one of Embodiments 1 to 39, wherein R 1 a is
  • Embodiment 48 The compound of any one of Embodiments 1 to 39, wherein R 1 b is
  • Embodiment 49 The compound of any one of Embodiments 1 to 39, wherein R ! is
  • Embodiment The compound of any one of Embodiments 1 to 39, wherein R 1a is d of any one of Embodiments 1 to 39, wherein R 1b is
  • Embodiment 52 The compound of any one of Embodiments 1 to 39, wherein R 1 is
  • Embodiment 53 The compound of any one of Embodiments 1 to 39, wherein R 1a is
  • Embodiment 55 The compound of any one of Embodiments 1 to 39, wherein R 1 is
  • Embodiment SS The compound of any one of Embodiments 1 to 39, wherein R ia is
  • Embodiment 57 The compound of any one of Embodiments 1 to 39, wherein R 1b is ound of any one of Embodiments 1 to 39, wherein R 1 is
  • Embodiment S9 The compound of any one of Embodiments 1 to 39, wherein R 1 is
  • Embodiment 73 The compound of any one of Embodiments 1 to 39, wherein R 1 is
  • Embodiment 74 The compound of any one of Embodiments 1 to 39, wherein R 1 is
  • Embodiment 75 The compound of any one of Embodiments 1 to 39, wherein R 1 is
  • Embodiment 76 The compound of any one of Embodiments 1 to 39, wherein R 1 is
  • Embodiment 77 The compound of any one of Embodiments 1 to 39, wherein R 1 is
  • Embodiments 1 to 39 wherein R 1 is one of R 3 , R 3a , R 5 or R 5a is -NHNH 2 , and the others are independently selected from H, -OH and F.
  • R 1 is one of R 3 , R 3a , R 5 or R 5a is -NHNH 2 , and the others are independently selected from H, -OH and F
  • R 1 is one of R 3 , R 3a , R 5 or R 5a is -NHNH 2 , and the others are independently selected from H, -OH and F.
  • Embodiment 82 The compound of any one of Embodiments 1 to 39, wherein R ! is the others are independently selected from H, -OH and F.
  • Embodiments 1 to 39 wherein R 1 is , one of R 3 , R 3a , R 5 or R 5a is -NHOH, and the others are independently selected from H, -OH and F.
  • Embodiment 85 The compound of any one of Embodiments 1 to 39, wherein R 1 is one of R 3 , R 3a , R 5 or R 5a is -NHGH, and the others are independently selected from H, -OH and F.
  • Embodiment 86 The compound of any one of Embodiments 1 to 85, wherein:
  • Y 3 is OH, O-, SH or S ⁇ , and
  • Y 4 is OH, O , SH or S ⁇
  • Embodiment 87 The compound of any one of Embodiments 1 to 85, wherein:
  • Y 3 is OH or O
  • Y 4 is OH or O .
  • Embodiment 88 The compound of any one of Embodiments 1 to 85, wherein:
  • Y 3 is SH or S ⁇ .
  • Y is OH or O .
  • Embodiment 89 The compound of any one of Embodiments 1 to 85, wherein:
  • Y 3 is OH or O
  • Y is SH or S ⁇ .
  • Y 3 is SH or S .
  • Y 4 is SH or S .
  • Embodiment 91 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 2 , R 2a , R 4 , R 4a , R 6 , R 6a , R 7 and R 7a are each H.
  • Embodiment 92 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 3 is -NH 2 , -NHNH 2 , -NHOH, OH or F.
  • Embodiment 93 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 3 is -NHNH 2 , OH or F.
  • Embodiment 94 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 3 is -OH or F.
  • Embodiment 95 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 3a is -NH 2 , -NHNH 2 , -NHOH, OH or F.
  • Embodiment 9S The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 3a is -NHNH 2 , OH or F.
  • Embodiment 97 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 3a is -OH or F.
  • Embodiment 98 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 5 is -NH2, -NHNH2, -NHOH, OH or F.
  • Embodiment 99 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 5 is -NHNH2, OH or F.
  • Embodiment 100 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 5 is -OH or F.
  • Embodiment 101 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 83 is -NHNH 2 , OH or F.
  • Embodiment 102 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 83 is -NH 2 , -NHNH 2 , -NHOH, OH or F.
  • Embodiment 103 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R 8a is -OH or F.
  • Embodiment 104 The compound of any one of Embodiments 1 to 103, wherein: R 8 , R 9 , R 8a and R 9a are H.
  • Embodiment 105 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
  • Embodiment 106 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
  • Embodiment 107 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
  • R 2 , R 2a , R 4 , R 4a , R 6 , R Sa , R 7 , R 7a R 8 , R 8a , R 9 and R 9a are each H;
  • R 3 is F
  • R 3a is F.
  • Embodiment 108 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
  • R 3a is -OH.
  • Embodiment 109 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
  • R 2 , R 2a , R 4 , R 4a , R 6 , R 6a , R 7 , R 7a R 8 , R 83 , R 9 and R 9a are each H;
  • R 3 is -NHNH 2 , and
  • R 3a is F.
  • Embodiment 110 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90. wherein, when present:
  • Embodiment 111 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
  • Embodiment 112 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
  • Embodiment 113 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
  • Embodiment 114 The compound of any one of Embodiments 1 io 77 or Embodiments 86 to 90, wherein, when present:
  • Embodiment 115 The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:

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Abstract

Provided herein are STING agonists and immunoconjugates comprising STING agonists. Also disclosed are methods of making the STING agonists and immunoconjugates and methods of treating cancer using them.

Description

ANTIBODY CONJUGATES COMPRISING STING AGONIST
FIELD OF THE INVENTION
The invention provides antibody conjugates, also known as immunconjugates, comprising agonists of STING (stimulator of interferon genes) receptor, and the use of such conjugates for the treatment of cancer. BACKGROUND OF THE INVENTION
Innate immunity is a rapid nonspecific immune response that fights against
environmental insults including, but not limited to, pathogens such as bacteria or viruses.
Adaptive immunity is a slower but more specific immune response, which confers long-lasting or protective immunity to the host and involves differentiation and activation of naive T lymphocytes into CD4+ T helper ceils and/or CD8+ cytotoxic T cells, to promote cellular and humoral immunity. Antigen presentation cells of the innate immune system, such as dendritic ceils or macrophages, serve as a critical link between the innate and adaptive immune systems by phagocytosing and processing the foreign antigens and presenting them on the cell surface to the T cells, thereby activating T cell response.
STING (stimulator of interferon genes) is an endoplasmic reticulum adaptor that facilitates innate immune signaling (Ishikawa and Barber, Nature 2008, 455(7213):674~678). It was reported that STING comprises four putative transmembrane regions (Ouyang et. a!., immunity (2012) 36, 1073), predominantly resides in the endoplasmic reticulum and is able to activate NF-kB, STAT6, and IRF3 transcription pathways to induce expression of type I interferon (e.g , !FN-Q and IFN-b) and exert a potent anti-viral state following expression
(Ishikawa and Barber, Nature 2008, 455(7213):674-678; Chen et af , Cell (2011) 147, 436-446). in contrast, loss of STING rendered murine embryonic fibroblasts extremely susceptible to negative stranded virus infection, including vesicular stomatitis virus. (Ishikawa and Barber, Nature. 2008, 455(7213):674-678).
There remains a need for new immunotherapies for the treatment of diseases, in particular cancer.
SUMMARY OF THE INVENTION
The invention provides immunoconjugates comprising antibodies conjugated with STING agonists, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof and combinations thereof, which are useful for the treatment of diseases, in particular, cancer. The invention further provides methods of treating, preventing, or ameliorating cancer comprising administering to a subject in need thereof an effective amount of an immunoconjugate of the invention. The terms“immunoconjugate” and“antibody conjugate” are used interchangeably herein. The invention also provides compounds comprising STING agonists and a linker which are useful to conjugate to an antibody and thereby make the immunostimmuiatory conjugates (or immune Stimulator Antibody Conjugates (ISACs)) of the invention. Various embodiments of the invention are described herein.
In one embodiment, this application discloses an immunoconjugate comprising an antibody (Ab), or a functional fragment thereof, coupled to an agonist of Stimulator of Interferon Genes (STING) receptor (D) via a linker (L), wherein the linker optionally comprises one or more cleavage elements.
In one embodiment, the immunoconjugate comprises Formula (I):
Ab— (L— (D)m)n (Formula (I))
wherein:
Ab is an antibody or a functional fragment thereof;
L is a linker comprising one or more cleavage elements;
D is a drug moiety that has agonist activity against STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20.
In another embodiment, the immunoconjugate comprises Formula (I):
Ab— (L— (D)m)„ (Formula (I))
wherein:
Ab is an antibody or a functional fragment thereof;
L is a linker;
D is a drug moiety that binds to STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20:
wherein D, or a cleavage product thereof, that is released from the immunoconjugate has STING agonist activity.
In another embodiment, the immunconjugate comprises Formula (I):
Ab— (L— (D)m)n (Formula (I))
wherein:
Ab is an antibody or a functional fragment thereof;
L is a linker;
D is a drug moiety that binds to STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20;
wherein the immunoconjugate delivers D, or a cleavage product thereof, to a cell targeted by the Ab, and wherein D, or the cleavage product thereof, has STING agonist activity.
In another embodiment, the immunoconjugate comprises Formula (I):
Ab— (L— (D)m)n (Formula (I))
? wherein:
Ab is an antibody or a functional fragment thereof;
L is a linker comprising one or more cleavage elements;
D is a drug moiety that binds to STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20;
wherein the immunoconjugate releases D, or a cleavage product thereof, in a cell targeted by the Ab, and wherein D, or the cleavage product thereof, has STING agonist activity.
In another embodiment, the immunoconjugate comprises Formula (I):
Ab— (L— (D)m)n (Formula (I))
wherein:
Ab is an antibody or a functional fragment thereof;
L is a linker comprising one or more cleavage elements;
D is a drug moiety that has agonist activity against STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20:
wherein the immunoconjugate releases D, or a cleavage product thereof, in a cell targeted by the Ab, and wherein D, or the cleavage product thereof, has STING agonist activity in the cell.
In a further embodiment, the present application discloses an immunoconjugate for delivery of a STING receptor agonist to a ceil, the immunoconjugate comprising Formula (I):
Ab— (L— (D)m)n (Formula (I))
wherein:
Ab is an antibody or a functional fragment thereof;
L is a linker comprising one or more cleavage elements;
D is a drug moiety that binds to STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20;
wherein the immunoconjugate specifically binds to an antigen expressed on the ceil surface and is internalized into the cell, and wherein D, or a cleavage product thereof, is cleaved from L and has STING agonist activity as determined by one or more STING agonist assays selected from: an interferon stimulation assay, a hSTING wt assay, a THP1~Dua! assay, a TANK binding kinase 1 (TBK1) assay, or an interferon-y-inducible protein 10 (IP-10) secretion assay.
In some embodiments, D, or the cleavage product thereof, has STING agonist activity if it. binds to STING and is able to stimulate production of one or more STING-dependent cytokines in a STING-expressing cell at least 1 .1 -fold, 1.2-fold, 1.3-fold, 1 .4-fold, 1.5-fold, 1.6- fold, 1 7-fold, 1.8-fold, 1.9-fold, 2-fold or greater than an untreated STING-expressing cell. In another embodiment, the STING-dependent cytokine is selected from interferon, type 1 interferon, IFN-a, !FN-b, type 3 interferon, IRNl, IP10, TNF, !L-6, CXCL9, CCL4, CXCL1 1 ,
CCL5, CGL3, or CCL8. In other embodiments, D, or the cleavage product thereof, has STING agonist activity if it binds to STING and is able to stimulate phosphorylation of TBK1 in a STING- expressing cell at least 1.1-fold, 1.2-fold, 1.3-fo!d, 1.4-fold , 1 .5-fold, 1 .8-fold, 1.7-fold, 1 .8-foid, 1 .9-foid, 2-foid or greater than an untreated STING-expressing cell. In another embodiment, D, or the cleavage product thereof, has STING agonist activity if it binds to STING and is able to stimulate expression of a iuciferase reporter gene controlled by interferon (IFN)-stimu!ated response elements in a STING-expressing cell at an EC5o of 20 micromolar (mM), 15 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less. In other embodiments, D, or the cleavage product thereof, has STING agonist activity if it binds to STING and is able to stimulate expression of a iuciferase reporter gene controlled by interferon (IFN)-stimuiated response elements in a STING-expressing cell to a level equal to or greater than the level of stimulation of 50 mM of 2’3’-cGAIVSP. In some embodiments, the STING-expressing cell is THP1 -Dual cell, and the Iuciferase reporter gene is the IRF-Lucia reporter gene in THP1 -Dual cell, and optionally the STING agonist activity is determined by the THP1 ~Duai assay described herein in another embodiment, the iuciferase reporter gene is the 5xlSRE-m!FNb~GL4 reporter gene and the STING-expressing ceil is a ceil expressing wild-type human STING protein, and optionally the STING agonist activity is determined by the hSTING wt assay described in Table 3A. in other embodiments, the immunoconjugate stimulates IP-10 secretion from a STING- expressing cell targeted by the Ab at an EC5o of 5 nanomoiar (nM) or less in an IP-10 secretion assay.
In some embodiments disclosed herein, the immunoconjugate is parentera!ly administered.
In other embodiments, the immunoconjugate comprises an Ab that specifically binds a target antigen. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the Ab is human or humanized. In other embodiments, the Ab is a monoclonal antibody.
In some embodiments of the immunconjugate disclosed herein, the Ab comprises a modified Fc region in one embodiment, the Ab comprises cysteine at one or more of the following positions, which are numbered according to EU numbering:
(a) positions 152, 360 and 375 of the antibody heavy chain, and
(b) positions 107, 159, and 165 of the antibody light chain.
In some embodiments, L is attached to the Ab via conjugation to one or more modified cysteine residues in the Ab. in one embodiment, L is conjugated to the Ab via modified cysteine residues at positions 152 and 375 of the heavy chain of the Ab, wherein the positions are determined according to EU numbering. In some embodiments, L is conjugated via a maieimide linkage to the cysteine. In one embodiment of the immunoconjugates disclosed herein, D is a dinucleotide in some cases, D is a cyclic dinucieotide (CDN). In other embodiments, D is a compound selected from any one of the compounds of Table 1 and Table 2.
In some embodiments disclosed herein, D is a compound selected from
Figure imgf000006_0001
Figure imgf000007_0001
In one embodiment, the present application discloses immunconjugates wherein L is a cleavable linker comprising one or more cleavage elements. In some embodiments, L comprises two or more cleavage elements, and each cleavage element is independently selected from a self-immoiative spacer and a group that is susceptible to cleavage. In some embodiments, the cleavage is selected from acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphodiesterase- induced cleavage, phosphatase-induced cleavage, protease- induced cleavage, lipase-induced cleavage, or disulfide bond cleavage. In one embodiment of the immunconjugates disclosed herein the Linker-Drug Moiety (- (L-(D)m)), wherein m is 1 , has a structure selected from:
Figure imgf000008_0001
wherein:
Lc is a linker component and each Lc is independently selected from a linker component as disclosed herein;
x is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 and 20; y is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 and 20; p is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10;
D is the compound selected from any one of embodiments 1 to 186;
and each cleavage element (CE) is independently selected from a self-immolative spacer and a group that is susceptible to cleavage selected from acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage.
In one embodiment of the immunconjugates disclosed herein the Linker (L) ot the Linker- Drug Moiety (-(L-(D) )), wherein m is 1 , has a structure selected from:
Figure imgf000008_0002
wherein:
Le is a linker component and each Lc is independently selected from a linker component as disclosed herein;
x is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 and 20; y is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 and 20; p is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10;
and each cleavage element (C£) is independently selected from a self-immoiatlve spacer and a group that is susceptible to cleavage selected from acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage.
In some embodiments, L comprises a structural component selected from: ;
Figure imgf000008_0003
Figure imgf000009_0001
the ** denotes the point of attachment of L to Drug moiety (D).
In some embodiments disclosed herein, the immunoconjugate is selected from the following:
Figure imgf000009_0002
Formula (AA-e) Formula (AA-d)
Figure imgf000010_0001
Formula (BB-c) Formula (BB-d)
Figure imgf000010_0002
Formula (CC-c) Formula (CC-d)
Figure imgf000011_0001
Formula (DD-c) Formula (DD-d)
Figure imgf000011_0002
Formula (EE~a) Formula (EE-b)
Figure imgf000012_0001
Formula (EE-e) Formula (FF-a)
Figure imgf000013_0001
Formula (FFd) Formula (FF-e)
Figure imgf000014_0001
Formula (FF-f) Formula (FF-g) wherein:
each Gi is independently selected from
Figure imgf000014_0002
the * of Gi indicates the point of attachment to -CR8R9-;
XA is C(=0)-, -C(=S)- or-C(=NR11)- and each Zi is NR12;
XB is C, and each Z2 is N;
Figure imgf000014_0003
Y5 is -CH2-,-NH-,-0-or-S;
Ys is -CH2-,-NH-,-0-or-S;
Y-/ is O or S;
Ys is O or S; Yg is -GH2-, -NH-, -0- or -S;
Yio is -CH2-, -NH-, -O- or -S;
Yu is -O-, -NH-, -S-, -S(=0)-, -SOr, -CH2-, or -CF2-;
q is 1 , 2 or 3:
each R1 is independently a partially saturated or aromatic monocyclic heterocyclyl or
partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms Is independently selected from O, N or S, or a tautomer thereof, wherein R1 is substituted with Q, 1 , 2, 3 or 4 substituents independently selected from ~NH2, ~NHNH2, -NHOH, F, Cl, Br, Ci-C3alkyl and a Ci-C6alkyi substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3, or wherein when Ab is attached to R1 then R1 is substituted with -NHLiR115, -NHNHLiR1 15, -NHOLiR115- or -NHN=CR12iCH2),iL,R1 15- and Ab is attached to the R115 moiety;
each R1a is independently a partially saturated or aromatic monocyclic heterocyclyl or
partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -NHGH, F, Cl, Br, Ci-Csalkyl and a Ci-G6alkyi substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3, or wherein when Ab is attached to R1a then R1a is substituted with -NHLiR115, -NHNHLiR115, -NHOL1R115- or - NHN=CR (CH2)r.LiR115- and Ab is attached to the R115 moiety;
each R1b is independently a partially saturated or aromatic monocyclic heterocyclyl or
partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R,b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -NHOH, F, Cl, Br, CrC6a!kyi and a GrGsalkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3, or wherein when Ab is attached to R1b then R1b is substituted with -NHLiR115, -NHNHLiR115, -NHOL1R115- or - attached to the R115 moiety;
Figure imgf000015_0001
each R2 is independently selected from H and Ci-C6alkyl;
each R3 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-G6a!kyi substituted with a NHOH, NHNH2 or NH2; or when Ab is attached to R3, then R3 is -NHL1R115, -NHNHLiR115, -NHOL1R115- or -NHN=CR12(CH2)nLiR115- and Ab is attached to the R115 moiety; each R4 is independently selected from H, CrC6alkyl and Ci-C6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R5 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-Geaikyi substituted with a NHOH, NHNH2 or NH2; or when Ab is attached to R5, then R5 is -NHL1R115, -NHNHL1R115, -NHOL1R115- or -NHN=CR12(CH2)nLiR1 15- and Ab is attached to the R115 moiety;
each R6 is independently selected from H, Ci-C6alkyl and C i-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7 is independently selected from H, CrC6alky! and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R8 is independently selected from H, CrC6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R9 is independently selected from H, CrC6alkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R2a is independently selected from H and CrC6aikyl;
each R3a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-CBalkyi substituted with a NHOH, NHNH2 or NH2: or when Ab is attached to R3a, then R3a is -NHL1R115, -NHNHL1R115, -NHOL1R115- or -NHN=CR12(CH2)nLiR115- and Ab is attached to the R115 moiety;
each R4a is independently selected from H, Ci-C5alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R5a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a CrC6alkyi substituted with a NHOH, NHNH2 or NH2; or when Ab is attached to R5a, then R5a is -NHL1R115, -NHNHL1R115, -NHOL1R115- or -NHN=CR (CH2)nLiR115- and Ab is attached to the R115 moiety;
each R6a is independently selected from H, C -C3alkyi and CrCgalkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7a is independently selected from H, C -C3alkyi and CrCgalkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each RSa is independently selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R9a is independently selected from H, Ci-C6alkyi and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3; eac yl, Ci
Figure imgf000017_0001
wherein the Cr
Cl2aikyi and Ci-Csheteroalkyi of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, Ci-C 2aikoxy, -S-C(=0)Ci-G6aikyl, halo, -CN, Gr
Ci2aikyl, -O-aryl, -Q-heteroaryl, -O-cycloalkyi, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryi, -0C(0)0CrCsaikyland C(0)0Ci-C3alkyl, wherein each alkyl, cycloalkyi, heterocyclyl, aryl, and heteroaryi is substituted by 0,1 , 2 or 3 substituents independently selected from C1-C12 alkyl, O-Cr0i2alkyl, Ci~Ci2heteroalkyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heferoaryl, -C(=0)Ci-Ci2alkyl, -0C(=0)Ci-Ci2alkyl, -C(=0)0C Ci2alkyi, -0C(=0)0Ci-Ci2aikyl, -C(=0)N(Ri 1)-Ci-Ci2alkyl, ~N(R11)C(=0)~C Ci2aikyl; - OC(=0)N(Rn)-Ci-Ci2aikyl, -C(=Q)-aryl, -G(=0)-heteroaryl, -GC(=0)-aryi, -C(=G)G-aryl, - OC(=0)-heteroaryl, -C(=0)0-heteroaryl, -C(=0)0-aryi, -C(=Q)G-heteroaryl, - C(=Q)N(Ri 1)-aryl, -C(=Q)N(Ri 1)-heteroaryl, -IM(R11)C(G)-aryl, -N(R11)2C(0)-aryi, - N(R11)C(Q)-heteroaryi, and S(0)2N(R11)-aryl;
each R11 is independently selected from H and CrC6aikyl;
each R12 is independently selected from H and CrC6aikyl;
optionally R3 and R6 are connected to form -Q-G i-C6aikylene, such that when R3 and Rs are connected, the O is bound at the R3 position
optionally R3a and R6a, are connected to form -0-Ci-C6alkylene, such that when R3a and RSa are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form -0-CrC6alkyiene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form -0~CrC6alkylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form -O-CrCeaikylene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -0-CrGsalkylene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form -O-CrCeaikylene, such that when R5 and R6 are connected, the O is bound at the R5 position;
optionally R5a and R6a, are connected to form -0-Ci-G6alkylene, such that when R5a and RSa are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form -O-CrCeaikylene, such that when R5 and R7 are connected, the O is bound at the R5 position; optionally R5a and R73, are connected to form -0-CrCBalkyiene, such that when R53 and R73 are connected, the O is bound at the R53 position:
Li is a linker;
Figure imgf000018_0002
Figure imgf000018_0001
Figure imgf000019_0001
of R1 indicates the point of attachment to Ab;
R13 is H or methyi;
R14 is H, -CHs or phenyl;
R110 is independently selected from H, Gi-C6aikyl, F, Cl, and -OH;
R111 is independently selected from H, Gi-C6alkyi, F, Cl, -NH2, -OCHs, -OCH2CH3,
N(CH3)2, -GN, -NO2 and -OH;
each R112 is independently selected from H, Ci-Salkyl, fiuoro, benzyloxy substituted with - G(=0)0H, benzyl substituted with -C(=0)0H, C^alkoxy substituted with -C(=0)0H and Gi-4alkyl substituted with -C(=0)0H;
each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 and 18;
Ab is an antibody or fragment thereof; and
y is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In some embodiments disclosed herein, the immunconjugates comprise a structure selected from:
Figure imgf000019_0002
Figure imgf000020_0001
WO 2020/089815
Figure imgf000021_0001
Figure imgf000022_0001
In some embodiments, the immunoeonjugate has in vivo anti-tumor activity.
The present application also discloses a pharmaceutical composition comprising an i munconjugate as disclosed herein and a pharmaceutically acceptable excipient.
The present application also discloses an immunoeonjugate as disclosed herein for use in combination with one or more additional therapeutic agents. In one embodiment, the additional therapeutic agent is selected from the group consisting of an inhibitor of a co- inhibitory molecule, an activator of a co-stimulatory molecule, a cytokine, an agent that reduces cytokine release syndrome (CRS), a chemotherapy, a targeted anti-cancer therapy, an oncolytic
zi drug, a cytotoxic agent, an immune-based therapy, a vaccine, or a ceil therapy in another embodiment, the additional therapeutic agent is an inhibitor of a co-inhibitory molecule, an activator of a co-stimulatory molecule, or a cytokine, wherein:
(i) the co-inhibitory molecule is selected from Programmed death-1 (PD-1), Programmed death- ligand 1 (PD-L1), Lymphocyte activation gene-3 (LAG-3), or T-celi immunoglobulin domain and mucin domain 3 (TIM-3),
(ii) the co-stimulatory molecule is Glucocorticoid-induced TNFR-related protein (G!TR), and (ill) the cytokine is IL-15 complexed with a soluble form of IL-15 receptor alpha (IL-15Ra).
The present application also discloses a method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of an
immunconjugate, a pharmaceutical composition thereof, or a composition comprising an immunoconjugate in combination with one or more additional therapeutic agents, as disclosed herein.
The present application also discloses use of an immunconjugate, a pharmaceutical composition thereof, or a composition comprising an immunoconjugate in combination with one or more additional therapeutic agents, as disclosed herein for treatment of a cancer in a subject in need thereof.
In another embodiment, this application discloses an immunconjugate, a pharmaceutical composition thereof, or a composition comprising an immunoconjugate in combination with one or more additional therapeutic agents, as disclosed herein for use in the treatment of cancer.
In yet another embodiment, disclosed herein is the use an immunconjugate, a pharmaceutical composition thereof, or a composition comprising an immunoconjugate in combination with one or more additional therapeutic agents, as disclosed herein in the manufacture of a medicament for use in the treatment of cancer.
In some embodiments, the cancer is selected from sarcomas, adenocarcinomas, blastemas, carcinomas, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, lymphoid cancer, colon cancer, renal cancer, urothelial cancer, prostate cancer, cancer of the pharynx, rectal cancer, renal cell carcinoma, cancer of the small intestine, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, colorectal cancer, cancer of the anal region, cancer of the peritoneum, stomach or gastric cancer, esophageal cancer, salivary gland carcinoma, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, penile carcinoma, glioblastoma,
neuroblastoma, cervical cancer , Hodgkin lymphoma, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small iniesiine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias inciuding acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CMS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, neuroendocrine tumors (inciuding carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, leukemia, lymphoma, acute
myelogenous leukemia (AML), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphoid leukemia (CLL), myelodysplastic syndromes, B-cell acute lymphoid leukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), B cell prolymphocytic leukemia, blast ic plasmacytoid dendritic ceil neoplasm, Burkitt's lymphoma, diffuse large B ceil lymphoma, Follicular lymphoma, Hairy cell leukemia, small ceil- or a large cell-follicular lymphoma, malignant lymphopro!iferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, piasmablastic lymphoma, plasmacytoid dendritic ceil neoplasm, and Waldenstrom macroglobullnemia.
In some embodiments, the immunoconjugate is administered to the subject
intravenously, intratumoraliy, or subcutaneously.
The present application also discloses an Immunconjugate, a pharmaceutical composition thereof, or a composition comprising an immunoconjugate in combination with one or more additional therapeutic agents, as disclosed herein for use as a medicament.
This application also discloses a method of manufacturing any of the immunoconjugates as disclosed herein comprising the steps of:
a) Reacting D and L to form L— (D)m; and
b) Reacting L— (D)mwith Ab to form the immunoconjugate Ab— (L— (D) )n (Formula (I))
In another embodiment, this application discloses a compound having a structure selected from Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or Formula (F) or stereoisomers or pharmaceutically acceptable salts thereof,
Figure imgf000024_0001
Formula (A) Formula (B)
Figure imgf000025_0001
Formula (E) Formula (F)
wherein:
Y7 each Gi is independently selected from
Figure imgf000025_0002
where the * of Gi indicates the point of attachment to ~CR8R9~;
XA is C(=0)-, -C(=S)- or ~C(=NR11)- and each Zi is NR12;
XB is C, and each Z2 is N ;
Y8
Figure imgf000025_0003
Xc is C(=0)-, -C(=S)- or ~C(=NR11)- and each Z3 is NR12;
XD is C, and each Z4 is N;
Yi is -0-, -NH-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2-; Y2 is -O, -NH-, -S-, -S(=0)-, -SO2-, -CHr, or -CF2-;
Figure imgf000026_0001
Ys is -CH2-, -NH-, -G- or -S;
Y6 is -CH2-, -NH-, -G- or -S;
Y7 is O or S;
Y8 is O or S;
Y9 is -CHr, -NH-, -O- or -S;
Y10 is -CH2-, -NH-, -O- or -S;
Y11 is -O-, -NH-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2~;
q is 1 , 2 or 3;
R1 is a partially saturated or aromatic monocyclic heterocyclyl or partially saturated or aromatic fused bicyciic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatorns, and each heteroatorns is independently selected from O, N or S, or a tautomer thereof, wherein R1 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHLR15, -NHNHL,R15, -NHOLiR15, -NHN=CR12(CH2)nL,R15, NH2 -NHNH2, - NHOH, F, Cl, Br, CrC6alkyl and CrCBalkyi substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N3;
R18 is a partially saturated or aromatic monocyclic heterocyclyl or partially saturated or aromatic fused bicyciic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatorns, and each heteroatorns is independently selected from O, N or S, or a tautomer thereof, wherein R18 is substituted with Q, 1 , 2, 3 or 4 substituents independently selected from -NHL R15, -NHNHL1R15, -NHOLiR15, -NHN=CR12(CH2),iLiR15, ~NH2, -NHNH2, - NHOH, F, Cl, Br, CrC6alkyl and Ci-C6alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N3;
R1 b is a partially saturated or aromatic monocyclic heterocyclyl or partially saturated or aromatic fused bicyciic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatorns, and each heteroatorns is independently selected from O, N or S, or a tautomer thereof, wherein R1 b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHL1 R15, -NHNHL1R15, -NHOL,R15, -NHN=CR12(CH2)nLiR15, -NH2, -NHNH2, - NHOH, F, Cl, Br, CrC6aikyl and CrCsaikyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R2 is independently selected from H and Ci-C6alkyi;
each R3 is independently selected from -NHL1R15, -NHNHL1R15, -NHOL1R15, -
NHN=CR12(CH2)r.LiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, -NHNH2, NH2 and a CrC6alkyi substituted with a NHOH, NHNH2 or NH2; each R4 is independently selected from H, CrC6alkyl and Ci-C6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R5 is selected from -NHL,R15, -NHNHLiR15, -NHOLiR15, -NHN=CR12(CH2)nL,R15, H, -OH, -SH,
F, Cl, Br, I, NHOH, -NHNH2 NH2 and a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH2; each R6 is independently selected from H, Ci-C6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, !, OH, CN, and N3;
each R7 is independently selected from H, Ci-C6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R8 is selected from H, Ci-C3alkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents
independently selected from F, Cl, Br, I, OH, CN, and N3;
each R9 is selected from H, Ci-C3alkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents
independently selected from F, Cl, Br, !, OH, CN, and N3;
R2a is selected from H and Ci-C6alkyl;
R3a is selected from -NHL1R15, -NHNHLiR15, -NHOLiR15, -NHN=CR12(CH2)r,LiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, -NHNH2, NH2 and a C C6alkyl substituted with a NHOH, NHNH2 or NH2; R4a is selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents
independently selected from F, Cl, Br, !, OH, CN, and N3;
R5a is selected from -NHL1R15, -NHNHLiR15, -NHOLiR15, -NHN=CR12(CH2)nL,R15, H, -OH, -SH, F, Cl, Br, I, NHOH, -NHNH2, NH2 and a C C6alkyl substituted with a NHOH, NHNH2 or NH2; R6a is selected from H, Ci-C6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents
independently selected from F, Cl, Br, I, OH, CN, and N3;
R78 is selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents
independently selected from F, Cl, Br, I, OH, CN, and N3;
R8a is selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents
independently selected from F, Cl, Br, I, OH, CN, and N3;
R9a is selected from H, Ci-Csalkyl and Ci-C6alkyl substituted by 1 , 2 or 3 substituents
independently selected from F, Cl, Br, I, OH, CN, and N3;
each R10 is independently selected from the group consisting of H, Ci-Csalkyl, Cr
Csheteroalkyl,
Figure imgf000027_0001
wherein the Cr
Ci2aikyl and CpCsheteroaikyl of R10 is substituted by Q, 1 , 2 or 3 substituents independentiy selected from -OH, Ci-Ci2alkoxy, -S-C(=Q)Ci-C6aikyl, halo, -CN, CrCi2alkyl, -O-ary!, -G- heteroaryl, -O-cycloalky!, oxo, cycioalkyl, heterocydyl, aryl, or heteroaryi, -0C(0)0Ci- C6alkyiand C(OjOCi-C3alkyl, wherein each alkyl, cycloalkyl, heterocydyl, aryl, and heteroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from C1-C12 alkyl, O-Ci-C^alkyl, Cr-C^heteroalkyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroary!, -
C(=0)CrCi2alkyl, -OC(=0)CrCi2alkyl, -C(=0)OCi-Ci2alkyl, -OC(=0)OCi-Ci2alkyl, - C(=0)N(R11)-Ci-Ci2alkyl, -N(R11)C(=0)-Ci-Ci2alkyl; -OC(=0)N(R11)-Ci-Ci2alkyl, -C(=0)-aryl, -C(=0)-heteroaryl, -OC(=0)-aryl, -G(=0)0-aryl, -OC(=0)-heteroaryl, -G(=0)0heteroaryl, - C(=0)0-aryl, -C(=0)0-heteroaryl, -C(=0)N(R11)-aryi, -C(=0)N(R11)-heteroaryl, -N(R11)C(0)- aryl, -N(R11)2C(0)-aryl, -N(R11)C(0)-heteroai l, and S(G)2N(R11)-aryi;
R11 is selected from H and Ci-C6alkyl;
R12 is selected from H and CrC6alkyi;
optionally R3 and R6 are connected to form -0-Ci-C6alkylene, such that when R3 and R6 are connected, the O is bound at the R3 position
optionally R38 and R6a, are connected to form -0-Ci-C3aikylene, such that when R3a and R6a are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form -G-Ci-C6aikylene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form -0-C rCBalkyiene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form -Q-CrC6aikyiene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -0-Gi-C6alkylene, such that when R4a and R38 are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form -0-C i-C6aikylene, such that when R5 and Rs are connected, the O is bound at the R5 position;
optionally R5a and R6a, are connected to form -0-Ci-C3alkylene, such that when R38 and R68 are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form ~0-Ci~C6aikylene, such that when R5 and R7 are connected, the O is bound at the R5 position;
optionally R5a and R7a, are connected to form -0-Ci-C6alkylene, such that when R£,a and R7a are connected, the O is bound at the R5a position;
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
C(=0)NH(CH2)mNHC(=0)X5C(=0)((CH2)rriO)n(CH2)mX3(CH2)m-ii; - C(=0)NH(CH2)mNHC(=0)X5C(=0)(CH2)mNHC(=0)((CH2)mO)n(CH2)m-**; - C(=0)NH(CH2)mNHC(=0)X5C(=0)(CH2)mNHC(=0)((CH2)mO)n(CH2)mX3(CH2)m-**; - C(=0)NH(CH2)mNHC(=0)X5(CH2)mX3(CH2)rr,-M; -C(=0)NH(CH2)mNHC(=0)Xs((CH2)m0)n(CH2)m- **; -C(=0)NH(CH2)mNHC(=0)X5((CH2)m0)n(CH2)mNHC(=0)(CH2)m-**; - C(=0)NH(CH2)mNHC(=0)X5((CH2)m0)r!(CH2)mNHC(=0)(CH2)mX3(CH2)m-,t,t; - C(=0)NH(CH2)mNHC(=0)X5((CH2)m0)r!(CH2)mX3(CH2)m-M; - C(=0)NH(CH2)mNHC(=0)X5(CH2)mNH((CH2)m0)n(CH2)m-**; - C(=0)fMH(CH2)mNHC(=0)X5C(=0)(CH2)rr!NH((CH2)rr!0),1(CH2)mX3(CH2)m-i'i'; - C(=0)fMH(CH2)mNHC(=0)X5(CH2)nr44; -G(=0)XiC(=0)NH(CH2)mNHG(=0)(CH2)m-“; - C(=0)X1C(=0)NH(CH2)mX3(CH2)m-i'i'; -C(=0)NH(CH2)mNHC(=0)(CH2)mX3(CH2)m-A'A'; - C(=0)NH(CH2)mNHC(=0)-**; -G(=0)NH((GH2)m0)n(CH2)mX3(CH2)m-** or
-C(=0)XiC(=0)(CH2)mNHC(=0)(CH2)m-**; where the ** of Li indicates the point of attachment to R15;
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000032_0002
the * of X indicates the point of attachment to X2;
Figure imgf000032_0003
Figure imgf000033_0002
indicates orientation toward R15;
R17 is 2-pyridyl or 4-pyridyl;
each R11 is independently selected from H and CrCealkyi;
each R12 is independently selected from H and CrC6alkyl;
each m is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10; and
each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 and 18;
each R110 is independently selected from H, Ci-C6aikyl, F, Cl, and ~-GH;
each R111 is independently selected from H, CrCealkyi, F, Cl, -NH2, -OCH3, -QCH2CH3, -
N(CH3)2, -CN, -NQ2 and -OH;
each R112 is independently selected from H, C^alkyl, fiuoro, benzyloxy substituted with - C(=0)0H, benzyl substituted with -C(=0)0H, Ci-4alkoxy substituted with -C(=0)0H and Cr 4alkyl substituted with -C(=0)0H;
and provided at least one of R1 , R1a or R1b Is substituted with -NHL R15, -NHNHL R15, - least one of R3, R5, R33 or R53 is -NHL1R15, -
Figure imgf000033_0001
2)nLiR15. In some embodiments Li is -C(=0)(CH2)m-**; -C(=0)(CH2)mO(CH2)m-**; -
Figure imgf000034_0001
where the ** of Li indicates the point of attachment to R15.
In some embodiments, the compound is selected from:
Figure imgf000034_0002
In some embodiments, the compound is selected from:
Figure imgf000035_0001
In another embodiment, this application discloses a compound having a structure selected from Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or Formula (F) or stereoisomers or pharmaceutically acceptable salts thereof, wherein:
Y7
Figure imgf000035_0003
XA is C(=0)-, -C(=S)- or ~-C(=NR11)- and each Z^ is NR12;
XB is C, and each Z2 is N;
Figure imgf000035_0004
Figure imgf000035_0002
Y6 is -CH2-, -NH-, -O- or -S;
Y7 is O or S;
Y8 is O or S;
Y9 is -CH2-, -NH-, -O- or -S;
Y10 is -CH2-, -NH-, -O- or -S;
Y11 is -O-, -NH-, -S-, -S(=0)-, -SO2-, -CH2-, or -CF2-;
q is 1 , 2 or 3;
R1 is a partially saturated or aromatic monocyclic heterocyclyi or partially saturated or aromatic fused bicyclic heterocyclyi containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -NHOH, F, Cl, Br, Ci-C6aikyi and a Ci-Csalkyi substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N3;
R18 is a partially saturated or aromatic monocyclic heterocyclyi or partially saturated or aromatic fused bicyclic heterocyclyi containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R18 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -NHOH, F, Cl, Br, Ci-C6alkyl and a Ci-C3alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N3;
R1b is a partially saturated or aromatic monocyclic heterocyclyi or partially saturated or aromatic fused bicyclic heterocyclyi containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -NHOH, F, Cl, Br, CrC6alkyl and Ci-Cealkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R2 is independently selected from H and Ci-C6alkyl;
each R3 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6alkyi substituted with a NHOH, NHNH2 or NH2;
each R4 is independently selected from H, CrC6alkyl and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R5 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C Cealkyi substituted with a NHOH, NHNH2 or NH2;
each R6 Is independently selected from H, CrCgalkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7 is independently selected from H, CrC6alkyl and Ci-C6aiky! substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R8 is independently selected from H, CrC6aikyi and CrCBalky! substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R9 is independently selected from H, Gi-C6aikyl and CrC6alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Gi, Br, I, OH, CN, and N3;
R28 is independently selected from H and Ci-C6alkyl
R38 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C Csa!ky! substituted with a NHOH, NHNH2 or NH2; R4a is independently selected from H, CrC6alkyl and CrC6alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R5a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Cr Cealkyi substituted with a NHOH, NHNH2 or NH2;
R6a is independently selected from H, Ci-C6alkyl and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R7a is independently selected from H, Ci-C6alkyl and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R8a is independently selected from H, CrC6alky! and CrC6alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R98 is independently selected from H, CrC6alky! and CrC6alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
each R1C is independently selected from the group consisting yl, Cr
Cgheteroalkyi, -(CH2CH2G)nCH2CH2C(=G)GCi-C3alkyl, a
Figure imgf000037_0001
wherein the Cr
Ci2alkyi and CrCBheteroalkyi of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, Ci-Ci2aikoxy, -S-C(=0)Ci-C6alkyl, halo, -CN, Cr Ci2alkyl, -O-aryl, -O-heteroaryl, -O-cycloaikyl, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, -0C(0)0CrC6alkyiand C(0)0CrC6alkyi, wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from C1-C12 alkyl, 0-Ci-Ci2alkyi, Ci-C^heteroalkyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroaryl, -C(=0)Ci-Ci2alkyl, -0C(=0)Ci-Ci2alkyl, -C(=0)0Ci- Ci2aikyi, -0C(=0)0Ci-Ci2alkyl, -C(=0)N(R11)-Ci-Ci2aikyl, -N(Rl 1)C(=0)-CrCi2aikyl; - 0C(=0)N(R11)-CrC12aikyl, -C(=0)-aryl, -C(=0)-he†eroaryl, -0C(=0)-aryl, -C(=0)0-aryl, - OC(=0)-heteroaryl, -C(=0)0-heteroaryl, -C(=0)0-aryl, -C(=0)0-heteroaryl, - C(=0)N(R1 1)-aryi, -C(=0)N(R1 1)-heteroaryl, -N(R11)C(0)-aryl, -N(R11)2C(0)-aryl, - N(R11)C(0)-heteroaryl, and S(G)2N(R11)-aryi;
R11 is Independently selected from H and CrCBalkyi;
R12 is independently selected from H and CrCBalkyi;
optionally R3 and R6 are connected to form -0-CrCBalkylene, such that when R3 and R6 are connected, the O is bound at the R3 position
optionally R3a and R6a, are connected to form -G-Ci-C6a!kyiene, such that when R3a and RSa are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form -0-Ci-C6alkylene, such that when R2 and R3 are connected, the O is bound at the R3 position; optionally R2a and R3a, are connected to form -OCr-CBalkyiene, such that when R2a and R3a are connected, the O is bound at the R3a position:
optionally R4 and R3 are connected to form -Q-Ci-C6aiky!ene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -0-CrC6aikylene, such that when R4a and R3a are connected, the O is bound at the R3a position:
optionally R5 and R6 are connected to form -Q-G i-C6aikylene, such that when R5 and Rs are connected, the O is bound at the R5 position;
optionally R5a and R6a, are connected to form -0-Ci-C6alkylene, such that when R58 and R6a are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form -0-CrC6alkylene, such that when R5 and R7 are connected, the O is bound at the R5 position,
and
optionally R£,a and R7a, are connected to form -O-GrCsalkylene, such that when R5a and R7a are connected, the O is bound at the R5a position.
In some embodiments, the compound is selected from any one of the compounds of
Table 1 and Table 2.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a line graph showing the anti-HER2 mAb1 -C4 conjugate inhibits N87 gastric tumor growth in mice.
FIG. 2 is a line graph showing the anti-HER2 mAb1 -C4 conjugate is well tolerated in the N87 gastric tumor xenograft mice.
DETAILED DESCRIPTION OF THE INVENTION
Various enumerated embodiments of the invention are described herein. It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.
Throughout the text of this application, should there be a discrepancy between the text of the specification (e.g., Table 8) and the sequence listing, the text of the specification shall prevail.
Definitions
The term "alkyl", as used herein, refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation. The term "Cr Csaikyi", as used herein, refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms, and which is attached to the rest of the molecuie by a single bond. Non-iimiting examples of "Ci-C6aiky!" groups include methyi, ethyi, 1 -methyiethyi , n-propyi, isopropyl, n- butyi, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl and hexyl.
The term“alkenyl”, as used herein, refers to a straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one double bond. The term“C2-C6alkenyr, as used herein, refers to a straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one double bond, having from two to six carbon atoms, which is attached to the rest of the molecuie by a single bond. Non-limiting examples of "C2-C3alkenyi" groups include ethenyl, prop-1 -enyi, but-1-enyl, pent-1 -enyl, pent-4-enyl and penta-1 ,4-dienyl.
The term“alkynyl”, as used herein, refers to a straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one triple bond, The term“C2-C6alkynyi”, as used herein, refers to a straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one triple bond, having from two to six carbon atoms, and which is attached to the rest of the molecuie by a single bond. Non-limiting examples of "C2-CBalkynyr groups include ethynyl, prop-1 -ynyl, but-1-ynyi, pent-1-ynyl, pent-4-ynyl and penta-1 ,4-diynyl.
The term "alkylene", as used herein, refers to a bivalent straight or branched hydrocarbon chain radical. The term "Ci-C6alkylene", as used herein, refers to a bivalent straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms.
The term“alkenyiene”, as used herein, refers to a bivalent straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one double bond. The term“C2-C3aikenylene”, as used herein, refers to a bivalent straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one double bond, and having from two to six carbon atoms.
The term“aikynylene”, as used herein, refers to a bivalent straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one triple bond. The term“C2-C3alkynyiene”, as used herein, refers to a bivalent straight or branched hydrocarbon chain radical group consisting soieiy of carbon and hydrogen atoms, containing at least one triple bond and having from two to six carbon atoms.
The term "Ci-C5alkoxyalkyiene", as used herein, refers to a radical of the formula -Ra-Q- Ra, where each Ra is independently a Creaikyi radical as defined above. The oxygen atom may be bonded to any carbon atom in either alkyl radical. Examples of Ci.5alkoxy include, but are not limited to, methoxy-methyl, methoxy-ethyl, ethoxy-ethyl, 1 -ethoxy-propyl and 2-methoxy-butyl.
The term“cycloalkyl,” or“Cs-Cscyc!oa!kyi,” as used herein, refers to a saturated, monocyclic, fused bicyciic, fused tricyclic or bridged polycyclic ring system. Non-limiting examples ef fused bicyciie or bridged polycyclic ring systems include bieyc!o[1 1 1 ]pentane, bicyclo[2.1 1]hexane, bicyclo[2.2.1]heptane, bieycio[3.1.Ijheptane, bicy o[3.2.1 joctane, bicycio[2.2 2]octane and adamantanyl. Non-limiting examples monocyclic C3-C8eycioaikyl groups include cyclopropyl, cyciobutyi, cyciopentyl and cyclohexyl groups.
The term "heteroalkyi", as used herein, refers to an "alkyl" moiety wherein at least one of the carbon atoms has been replaced with a heteroatom such as O S, or N.
The term“heterocycloalkyl,” or“C4-C8 heterocycloalkyl as used herein refers to a monocyclic ring structure having 3 to 6 ring members, wherein one to two of the ring members are independently selected from N, NH, NR16, O or ~S~, wherein R16 is Ci-C6alkyl. Non-limiting examples of 3-6 membered heterocycloalkyl groups, as used herein, include aziridin-1 -yi, aziridin-2-yl, aziridin-3-yl, azetadinyl, azetadin-1 -yl, azetadin-2-yl, azetadin-3-yl, oxetanyl, oxetan-2-yl, oxetan-3-yl, oxetan-4-yi, thietanyl, thietan-2-yl, thietan-3-yi, thietan-4-yl, pyrrolidinyl, pyrrolidin-1 -yl, pyrro!idin-2-yl, pyrro!idin-3-yl, pyrrolidin-4-yl, pyrro!idin-5-yl, tetrahydrofuranyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrofuran-4-yl, tetrahydrofuran-5-yl, tetrahydrothienyl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, tetrahydrothien-4-yl, tetrahydrothien-
5-yl, piperidinyl, piperidin-1 -yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, piperidin-5-yl, piperidin-6-yl, tetrahydropyranyl, tetrahydropyran-2-yi, tetrahydropyran-3-yl, tetrahydropyran-4- yl, tetrahydropyran-5-yl, tetrahydropyran-6-yl, tetrahydrothiopyranyi, tetrahydrothiopyran-2-yl, tetrahydrothiopyran-3-yl, tetrahydrothiopyran-4-yl, tetrahydrothiopyran-5-yl, tetrahydrothiopyran-
6-yl, piperazinyl, piperazin-1 -yl, piperazin-2-yi, piperazin-3-yi, piperazin-4-yl, piperazin-5-yi, piperazin-6-yi, morphoiinyi, morpholin-2-yi, morpholin-3-yl, morphoiin-4-yl, morphoiin-5-yl, morpholin-6-yl, thiomorpholinyl, thiomorphoiin-2-yl, thiomorpholin-3-yi, thiomorpholin-4-yl, thiomorpholin-5-yi, thiomorpholin-6-yl, oxathianyi, oxatbian-2-yi, oxatbian-3-yl, oxathian-5-yl, oxathian-6-yl, dithianyl, dithian-2-yi, dithian-3-yl, dithian-5-yi, dithian-6-yl, dioxolanyl, dioxolan-2- yl, dioxolan-4-yl, dioxolan-5-yl, thioxanyl, thioxan-2-yl, thioxan-3-yi, thioxan-4-yl, thioxan-5-yl, dithiolanyl, dithiolan-2-yl, dithiolan-4-yl, dithioian-5-yi, pyrazolidinyl, pyrazolidin-1 -yl, pyrazolidin- 2-yl, py razolid in-3-yl , pyrazolidin-4-yl and pyrazolidin-5-yl.
The term“heterocyclyl”, as used herein, includes partially saturated or aromatic monocyclic or fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S. in a preferred embodiment, the heteroatoms are nitrogen. Non-limiting examples of substituents include oxo, halo, Ci.6alkyl, Ci-6alkoxy, amino, Ci-5alkyiamino, di-Ci-5alkyiamino. The heterocyclic group can be attached at a heteroatom or a carbon atom.
For fused bicyclic heterocyclyl system, the system can be fully aromatic (i.e. both rings are aromatic). When fully aromatic, the heterocyclyl can be referred to as heteroaryl. Examples of aromatic bicyclic heteroaryl include 9-10 membered fused bicyclic heteroaryl having 2-5 heteroatoms, preferably nitrogen atoms. Non-limiting examples are: pyrrolo[2,3-bjpyrldlnyl, pyrroio[3,2-c]pyridinyL pyrrolo[3,2-c]pyridinyl, pyrrolo[3,2-b]pyridinyl, imidazo[4,5-b]pyridinyl, imidazo[4,5-c]pyridinyl, pyrazolo[4,3-d]pyridinyl, pyrazo!Q[4,3-c]pyridinyi, pyrazolo[3,4- cjpyridiny!, pyrazolo[3,4-d]pyridinyl, pyrazolo[3,4-b]pyridinyl, imidazo[1 ,2-a]pyridinyl,
pyrazolojd ,5-ajpyridinyl, pyrrolo[1 ,2-bjpyridazinyl, imidazo[1 ,2-cjpyrimidinyl, pyrido[3,2- djpyrimidinyl, pyrido[4,3-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrido[2,3-d]pyrimidinyl, pyrido[2,3-b]pyrazinyl, py!ido[3,4-b]pyrazinyl, pyrimido[5,4-d]pyrimidinyl, pyrazino[2,3- b]pyrazinyl, or pyrimido[4,5-d]pyrimidinyl. Other non-limiting examples of fused bicyciic
Figure imgf000041_0003
Additionally, bicyciic heierocyclyl ring systems include heierocyclyl ring systems wherein one of the fused rings is aromatic but the other is non-aromatic. For such systems, the heierocyclyl is said to be partially saturated. Examples of partially saturated bicyciic system are for example dihydropurinones such as 2-amino- 1 ,9-dihydro-6H-purin-9-yl-6-one and 1 ,9-
dihydro-6H-purin-9-y!-6-one. Other examples of partially saturated bicyciic system are
Figure imgf000041_0001
Figure imgf000041_0002
Heterocyclyi also includes a 5- or 6- membered ring aromatic heterocyciyl having 2 to 3 heteroatom (preferably nitrogen) (also referred to as 5- to 6-membered heteroaryl). Examples of monocyclic heteroaryl are: imidazolyi, pyrazolyl, thiazolyl, isothiazoiyi, 1 , 2, 3-oxadiazolyi, 1 ,2,4- oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazolyi, 1 ,2,4-th iadiazolyl, 1 ,2,5- thiadiazoiyl, 1 ,3,4-thiadiazo!yi, isothiazol-3-yl, isothiazol-4-yl, isothiazo!-5-yl, oxazol-2-yi, oxazo!- 4-yl, Qxazol-5-yi, isoxazol-3-yl, isoxazol-4-yl, isoxazol-S-yi, 1 ,2,4-triazoi-3-yi, 1 ,2,4-triazol-S-yl, 1 ,2, 3-t riazo l-4-y 1 , 1 ,2, 3-triazoi-5-yl, tetrazolyl, pyrid-2-yl, pyrid-3-yi, or pyridyl-4-yl, pyridazin-3- yl, pyridazin-4-yl, pyrazin-3-yl, 2-pyrazin-2-yl, pyrazin-4-yl, pyrazin-5-yl, 2-, 4-, or 5-pyrimidin-2- yl, pyrimidin-4-yl, pyrimidin-5-yl. Heterocyclyi also includes 6-membered monocyclic partially saturated ring having 1-3 heteroatoms (preferably nitrogen). Examples of partially saturated monocyclic heterocyclyi are pyrimidine-one and pyrimidine-dione, specifically pyrimidin-2(1 H)-one and pyrimidin-1 -yl-2,4(1 /-/, 3H)-dione.
Heterocyclyi can exist in various tautomeric forms. For example, when a heterocyclyi moiety is substituted with an oxo group next to a nitrogen atom, the invention also pertains to its hydroxy tautomeric form. For example, 2-amino- 1 ,9-dihydro-6H-purin-6-one can tautomerize into 2-amino-9H-purin-6-ol. The tautomerization is represented as foliow:
Figure imgf000042_0001
As used herein, the term tautomer is used to designate 2 molecules with the same molecular formula but different connectivity, which can interconvert in a rapid equilibrium.
Additional examples of tautomers are phosporothioic acid which can exist in an equilibrium as shown below.
Figure imgf000042_0002
Similarly, phosphoric acid exists as 2 tautomeric forms which interconvert in an equilibrium.
The term“Drug moiety”, as used herein, refers to a compound which binds to Stimulator of interferon Genes (STING) receptor and which comprises one or more functional groups each of which is capable of forming a covalent bond with a linker. Examples of such functional groups include, but are not limited to, primary amines, secondary amines, hydroxyls, thiols, aikenes, aikynes and azides in certain embodiments, such functional groups include reactive groups of Table 4 provided herein.
The term“sugar moiety” or“sugar moieties”, as used herein, refers to the following ring structures of the compounds of the invention
Figure imgf000042_0003
,
wherein Yi, Y2 and Yu are each independently selected from -0-, -S-, -S(=0)-, -SO2-, -CH2-, or -CF2-.
As used herein, when partial structures of the compounds are illustrated a wavy line ( 'LLL ) indicates the point of attachment of the partial structure to the rest of the molecule.
As used herein,“HER2” ( also known as ERBB2; NEU; NGL; TKR1 ; CD340; p185; MLN19; HER-2/neu) refers to a transmembrane tyrosine kinase receptor of the epidermal growth factor (EGF) receptor family. HER2 comprises an extracellular binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. HER2 does not have a ligand binding domain of its own and therefore cannot bind growth factors. However, HER2 binds tightly to other ligand-bound EGF receptor family members such as HER1 or HER3, to form a heterodimer, stabilizing ligand binding and enhancing kinase-mediated activation of downstream signalling pathways. The human HER2/NEU gene is mapped to chromosomal location 17q 12, and the genomic sequence of HER2/NEU gene can be found in GenBank at NG_007503.1. in human, there are five HER2 isoforms: A, B, C, D, and E: the term“HER2” is used herein to refer collectively to all HER2 isoforms. As used herein, a human HER2 protein also encompasses proteins that have over its full length at least about 70%, 71 %, 72%, 73%, 74%, 75%, 78%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 88%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with HER2 isoforms: A, B, C, D, and E, wherein such proteins still have at least one of the functions of HER2 The mRNA and protein sequences for human HER2 isoform A, the longest isoform, are:
Homo sapiens erfo-fo2 receptor tyrosine kinase 2 (ERBB2), transcript variant 1 , mR!MA [HM__004448.3]
1 gcttgctccc aatcacagga gaaggaggag gtggaggagg agggctgctt gaggaagtat 61 aagaatgaag ttgtgaagct gagattcccc tccattggga ccggagaaac caggggagcc 121 ceccgggcag cegcgcgccc cttcccacgg ggccctttac tgcgccgcgc gcccggcccc 181 cacccctcgc agcaccccgc gccccgcgcc ctcccagccg ggtccagccg gagccatggg 241 gccggagccg cagtgagcac catggagctg gcggccttgt gccgctgggg gctcctcctc 3Q1 gccctcttgc cccccggagc egcgagcacc caagigigca ccggcacaga catgaagctg 381 cggctcccig ccagicccga gacccacctg gacatgctcc gccacctcia ccagggctgc 421 caggiggtgc agggaaacct ggaactcacc tacctgccca ccaatgccag ccigicciic 481 ctgcaggata tccaggaggt gcagggctac gtgctcatcg ctcacaacca agtgaggcag 541 gicccactgc agaggctgcg gaitgtgcga ggcacccagc tctttgagga caactatgcc 6Q1 ctggccgtgc iagacaatgg agacccgctg aacaatacca ccccigicac aggggcctcc 681 ccaggaggcc tgcgggagct gcagcitcga agcctcacag agatctigaa aggaggggic 721 iigatccagc ggaaccccca gctctgctac caggacacga tttigiggaa ggacatcttc 781 cacaagaaca accagctggc tctcacactg atagacacca accgctctcg ggcctgccac 841 ccctgttctc cgatgtgtaa gggctcccgc tgctggggag agagttctga ggattgtcag
901 agcctgacgc gcactgtctg tgccggtggc tgtgcccgct gcaaggggcc actgcccact 961 gactgctgcc atgagcagtg tgctgceggc tgcacgggcc ccaagcactc tgactgcctg 1021 gcctgcctcc acttcaacca cagtggcatc tgtgagctgc actgcccagc cctggtcacc
1081 tacaacacag acacgtttga gtccatgccc aatcccgagg gccggtatac attcggcgcc
1141 agctgtgtga ctgcctgtcc ctacaactac ctttctacgg acgtgggatc ctgcaccctc
1201 gtctgccccc tgcacaacca agaggtgaca gcagaggatg gaacacagcg gtgtgagaag 1281 tgcagcaagc cctgtgcccg agtgtgctat ggtctgggca tggagcactt gcgagaggtg
1321 agggcagtta ccagtgccaa tatccaggag tttgctggct gcaagaagat ctttgggagc
1381 ctggcatttc tgccggagag ctttgatggg gacccagcct ccaacactgc cccgctccag
1441 ccagagcagc iccaagtgtt tgagactctg gaagagatca caggitacci atacatctca
1501 gcaiggccgg acagcctgcc tgaccicagc gtcitccaga acctgcaagt aaiccgggga
1581 cgaattctgc acaatggcgc ctactegctg acccigcaag ggctgggcat cagctggctg
1821 gggctgcgct cactgaggga actgggcagt ggactggccc tcatccacca taacacccac
1881 ctctgcttcg igcacacggi gccctgggac cagcicittc ggaacccgca ccaagctctg
1741 ctccacactg ccaaccggcc agaggacgag tgtgtgggcg agggcctggc ctgccaccag 1801 ctgtgcgccc gagggcacig ctggggtcca gggcccaccc agtgigicaa cigcagccag
1881 ticcttcggg gccaggagig cgiggaggaa tgccgagtac tgcaggggct ccccagggag
1921 iatgtgaatg ccaggcacig tttgccgtgc cacccigagt gtcagcccca gaatggctca 1981 gtgacctgtt ttggaccgga ggctgaccag tgtgtggcct gtgcccacta taaggaccct 2041 cccttctgcg tggcecgctg ccccagcggt gtgaaacctg acctctccta catgcccatc 2101 tggaagtttc cagatgagga gggcgcatgc cagccttgcc ccatcaactg cacccactcc 2181 tgtgtggacc tggatgacaa gggctgcccc gccgagcaga gagccagccc tctgacgtcc 2221 atcatctctg cggtggttgg cattctgctg gtcgtggtct tgggggtggt ctttgggatc
2281 ctcatcaagc gacggcagca gaagatccgg aagtacacga tgcggagact gctgcaggaa 2341 acggagctgg tggagccgct gacacctagc ggagcgatgc ccaaccaggc gcagatgcgg 2401 atcctgaaag agacggagct gaggaaggtg aaggtgcttg gatctggcgc ttttggcaca 2481 gtctacaagg gcatctggat ccctgatggg gagaatgtga aaattccagt ggccatcaaa 2521 gtgttgaggg aaaacacatc ccccaaagcc aacaaagaaa tcttagacga agcatacgtg 2581 aiggciggtg igggcicccc ataigicicc cgccttctgg gcaiclgcct gacaiccacg 2841 gtgcagctgg igacacagci iatgccciai ggctgccici iagaccatgl ccgggaaaac 27Q1 cgcggacgcc igggciccca ggacclgclg aaciggtgia tgcagattgc caaggggatg 2781 agciacctgg aggaigigcg gctcgtacac agggacttgg ccgcicggaa cglgclggic 2821 aagaglccca accatgtcaa aattacagac tlcgggcigg ctcggctgct ggacatlgac 2881 gagacagagi accaigcaga tgggggcaag gigcccaica agtggaiggc gctggagtcc 2941 attctccgcc ggcggttcac ccaccagagt gatgtgtgga gttatggtgt gactgtgtgg 3001 gagctgatga cttttggggc caaaccttac gatgggatcc cagcccggga gatccctgac 3061 ctgctggaaa agggggagcg gctgccccag ccccccatct gcaccattga tgtctacatg 3121 atcatggtca aatgttggat gattgactct gaatgtcggc caagattccg ggagttggtg 3181 tctgaattct cccgcatggc cagggacccc cagcgctttg tggtcatcca gaatgaggac 3241 ttgggcccag ccagtccctt ggacagcacc ttctaccgct cactgctgga ggacgatgac 3301 atgggggacc tggtggatgc tgaggagtat ctggtacccc agcagggctt cttctgtcca 3361 gaccctgccc cgggcgctgg gggcatggtc caccacaggc accgcagctc atctaccagg 3421 agtggcggtg gggacctgac actagggctg gagccctctg aagaggaggc ccccaggtct 3481 ccactggcac cctccgaagg ggctggctcc gatgtatttg atggtgacct gggaatgggg 3541 gcagccaagg ggctgcaaag cctccccaca catgacccca gccctctaca gcggtacagt 3601 gaggacccca cagtacccct gccctctgag acigaiggct acgiigcccc cctgacctgc 3661 agcccccagc ctgaatatgt gaaccagcca gatgttcggc cccagccccc ttcgccccga 3721 gagggccctc tgcctgctgc ccgacctgct ggtgccactc tggaaaggcc caagactctc 3781 tccccaggga agaatggggt cgtcaaagac gtttttgcct ttgggggtgc cgtggagaac 3841 cccgagtact tgacacccca gggaggagcl gccccicagc cccacccicc tcctgccttc 3901 agcccagcct tcgacaacci ctattactgg gaccaggacc caccagagcg gggggcicca 3961 cccagcacci icaaagggac acciacggca gagaacccag agiacciggg tctggacgtg 4021 ccagigigaa ccagaaggcc aagiccgcag aagcccigat gtgtcctcag ggagcaggga 4081 aggcctgact tctgctggca tcaagaggtg ggagggccct ccgaccactl ccaggggaac 4141 ctgccatgcc aggaacctgt cctaaggaac cttccttcct gcttgagttc ccagatggct 4201 ggaaggggtc cagcctcgtt ggaagaggaa cagcactggg gagtctttgt ggattctgag 4281 gccctgccca atgagactct agggtccagt ggatgccaca gcccagcttg gccctttcct 4321 tccagatcct gggtactgaa agccttaggg aagctggcct gagaggggaa gcggcectaa 4381 gggagtgtct aagaacaaaa gcgacccatt cagagactgt ccctgaaacc tagtactgcc 4441 ccccatgagg aaggaacagc aatggtgtca gtatccaggc tttgtacaga gtgcttttct 4501 gtttagtttt tacttttttt gttttgtttt tttaaagatg aaataaagac ccagggggag
4581 aatgggtgtt gtatggggag gcaagtgtgg ggggtccttc tccacaccca ctttgtccat 4821 ttgcaaatat attttggaaa acagctaaaa aaaaaaaaaa aaaa (SEQ ID HO: 25)
Receptor tyrosine-protein kinase erbB-2 isoform a precursor [Homo sapiens] EMP_ 004439,2]
MELAALCRWG LLLALLPPGA ASTGVCTGTD MKLRLPASPE THLDMLRHLY GGCGVVGGNL ELTYLPTNAS LSFLQDIGEV QGYVLIAHNG VRGVPLGRLR IVRGTGLFED !MYALAVLDNG DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLiQRNPQ LCYQDTILWK DIFHKNNGLA LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC AAGCTGPKHS DCLACLHFNH SG!CELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTGR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN IGEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF
ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGA YSLTLGGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDGLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTGCVN CSQFLRGGEC VEECRVLQGL PREYVNARHC LPCHPECQPG NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVG ILLVVVLGVV FGIL!KRRGG KIRKYTMRRL LGETELVEPL
TPSGA PNGA GMRILKETEL RKVKVLGSGA FGTVYKGIW! PDGENVKIPV
AIKVLRENTS PKANKEILDE AYVMAGVGSP YVSRLLGICL TSTVGLVTQL MPYGGLLDHV RENRGRLGSQ DLLNWCMQiA KG SYLEDVR LVHRDLAARN VLVKSPNHVK ITDFGLARLL DIDETEYHAD GGKVPIKWMA LESILRRRFT HQSDVWSYGV TVWELMTFGA KPYDGIPARE IPDLLEKGER LPQPP!CTID
VYM!MVKCWM IDSECRPRFR ELVSEFSRMA RDPQRFVVIG NEDLGPASPL
DSTFYRSLLE DDDMGDLVDA EEYLVPGGGF FCPDPAPGAG GMVHHRHRSS STRSGGGDLT LGLEPSEEEA PRSPLAPSEG AGSDVFDGDL GMGAAKGLGS LPTHDPSPLQ RYSEDPTVPL PSETDGYVAP LTCSPQPEYV NGPDVRPGPP SPREGPLPAA RPAGATLERP KTLSPGKNGV VKDVFAFGGA VENPEYLTPG GGAAPQPHPP PAFSPAFDNL YYWDQDPPER GAPPSTFKGT PTAENPEYLG LDVPV
The mRNA and protein sequences of the other human HER2 isoforms can be found in GeneBank with the following Accession Nos:
HER2 isoform B: NMJJ01005862.2 (mRNA)--> NPJJ01005862.1 (protein);
HER2 isoform C: NMJ3G1289936 1 (mRNA)— > NPJJ01276865.1 (protein):
HER2 isoform D: NM_001289937.1 (mRNA)— > NP__G01276866.1 (protein):
HER2 isoform E: NMJJ01289938.1 (mRNA)— NPJ3Q1276887.1 (protein).
The term“antibody,” as used herein, refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. A naturally occurring“antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region
(abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CHS. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl- terminus in the following order: FR1 , GDR1 , FR2, GDR2, FRS, CDRS, FR4. The variable regions of the heavy and light chains contain a binding domain that Interacts with an antigen. The constant regions of the antibodies may mediate the binding of the Immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1 q) of the classical complement system. An antibody can be a monoclonal antibody, human antibody, humanized antibody, camelised antibody, or chimeric antibody. The antibodies can be of any isotype (e.g., igG, IgE, IgM, IgD, IgA and IgY), class (e.g., igG1 , lgG2, lgG3, igG4, lgA1 and !gA2) or subclass.
The term“antibody fragment” or“antigen-binding fragment” or“functional fragment” refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), came!id VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, naxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v~NAR and bis-scFv (see, e.g., Holiinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Patent No.: 6,703,199, which describes fibronectin polypeptide minibodies). The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-ierminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-iinker-VL.
The terms“complementarity determining region” or“CDR,” as used herein, refer to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., HCDR1 , HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1 , LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991),“Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Ai-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme), or a combination thereof, and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-138 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (“IMGT” numbering scheme) in a combined Kabat and Chothia numbering scheme for a given CDR region (for example, HC CDR1 , HC CDR2, HC CDRS, LC CDR1 , LC CDR2 or LC CDR3), In some embodiments, the CDRs correspond to the amino acid residues that are defined as part of the Kabat CDR, together with the amino acid residues that are defined as part of the Chothia CDR. As used herein, the CDRs defined according to the“Chothia” number scheme are also sometimes referred to as“hypervariabie loops.”
For example, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1) (e.g., insertion(s) after position 35), 50-65
(HGDR2), and 95-102 (HCDR3): and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1) (e.g., insertion(s) after position 27), 50-56 (LCDR2), and 89-97 (LCDR3). As another example, under Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1) (e.g., insertion(s) after position 31), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1) (e.g., inseriion(s) after position 30), 50-52 (LCDR2), and 91-95 (LCDR3) By combining the CDR definitions of both Kabat and Chothia, the CDRs comprise or consist of, e.g., amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and a ino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Under !MGT, the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDRS) (numbering according to“Kabat”). Under I GT, the CDR regions of an antibody can be determined using the program IMGT/DomainGap Align.
The term“epitope” includes any protein determinant capable of specific binding to an immunoglobulin or otherwise interacting with a molecule. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be“linear” or “conformational.” Conformational and linear epitopes are distinguished in that the binding to the former but not. the latter is lost in the presence of denaturing solvents.
The phrases“monoclonal antibody” or“monoclonal antibody composition” as used herein refers to polypeptides, including antibodies, bispeeific antibodies, etc., that have substantially identical amino acid sequence or are derived from the same genetic source. This term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
The phrase“human antibody,” as used herein, includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences, e.g., human ger iine sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik, et ai (2000.
J Mol Biol 296, 57-86). The structures and locations of immunoglobulin variable domains, e.g., CDRs, may be defined using weii known numbering schemes, e.g , the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia, and
ImMunoGenTics (IMGT) numbering (see, e.g., Sequences of Proteins of Immunological interest, U.S. Department of Health and Human Services (1991), eds. Kabat et al.; AI Lazikani et al., (1997) J. Mol. Bio. 273:927 948); Kabat et al., (1991) Sequences of Proteins of immunological Interest, 5th edit., N!H Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901 -917; Chothia et al., (1989) Nature 342:877-883; Al-Lazikani et ai., (1997) J. Mai. Biol. 273:927-948; and Lefranc, M.-P., The immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)).
The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or
manufacturing). However, the term“human antibody” as used herein, is not intended to include antibodies in which CDR sequences derived from the germiine of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The phrase“recombinant human antibody” as used herein, includes ail human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DMA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences in certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, whiie derived from and related to human germiine VH and VL sequences, may not naturally exist within the human antibody germiine repertoire in vivo.
The term“Fc region” as used herein refers to a polypeptide comprising the CHS, CH2 and at least a portion of the hinge region of a constant domain of an antibody. Optionally, an Fc region may include a CH4 domain, present in some antibody classes. An Fc region may comprise the entire hinge region of a constant domain of an antibody. In one embodiment, the invention comprises an Fc region and a CH1 region of an antibody. In one embodiment, the invention comprises an Fc region CH3 region of an antibody in another embodiment, the invention comprises an Fc region, a CH1 region and a Ckappa/lambda region from the constant domain of an antibody in one embodiment, a binding molecule of the invention comprises a constant region, e.g., a heavy chain constant region in one embodiment, such a constant region is modified compared to a wild-type constant region. That is, the polypeptides of the invention disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1 , CH2 or CHS) and/or to the light chain constant region domain (CL). Example modifications include additions, deletions or substitutions of one or more amino acids in one or more domains. Such changes may be included to optimize effector function, half-life, etc.
The term“binding specificity” as used herein refers to the ability of an individual antibody combining site to react with one antigenic determinant and not with a different antigenic determinant. The combining site of the antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. Binding affinity of an antibody is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody it is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody.
The term“affinity” as used herein refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody“arm” interacts through weak non-covaient forces with antigen at numerous sites: the more interactions, the stronger the affinity.
The term“conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site- directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoieucine, proline, phenylalanine, methionine), beta- branched side chains (e.g., threonine, valine, isoieucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within an antibody can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested using the functional assays described herein. The term“homologous” or“identity” refers to the subunit sequence identity between two polymeric molecules, e.g , between two nucleic acid molecules, such as, two DMA molecules or two RNA molecules, or between two polypeptide molecules. Wien a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 1 Q), are matched or homologous, the two sequences are 90% homologous. Percentage of“sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical 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 resuit by 100 to yield the percentage of sequence identity. The output is the percent identity of the subject sequence with respect to the query sequence. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm in a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.corn), using either a Biossum 62 matrix or a PAM25Q matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.corn), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Biossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the aigorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11 -17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.nGbi.nim.nih.gov
The terms“cancer” and“cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, solid tumors and hematological cancers, including carcinoma, lymphoma, biastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovia! cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), 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 cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, neuroblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract 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, testicular cancer, esophageal cancer, tumors of the biliary tract, as well as head and neck cancer. Additional cancer indications are disclosed herein.
The terms“tumor antigen” or“cancer associated antigen” interchangeably refer to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of a cancer ceil, either entirely or as a fragment (e.g., MHC/peptide), and which is useful for the preferential targeting of a pharmacological agent to the cancer ceil. In some embodiments, a tumor antigen is a marker expressed by both normal ceils and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a ceil surface molecule that is
overexpressed in a cancer cell in comparison to a normal cell, for instance, 1 -fold over expression, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell. In some embodiments, a tumor antigen is a ceil surface molecule that is inappropriately synthesized in the cancer ceil, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal ceil. In some embodiments, a tumor antigen will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment (e.g., MHC/peptide), and not synthesized or expressed on the surface of a normal cell. Normally, peptides derived from endogenous proteins fill the pockets of Major histocompatibility complex (MHC) class I molecules, and are recognized by T ceil receptors (TCRs) on CDS + T lymphocytes. The MHC class I complexes are constitutively expressed by all nucleated cells in cancer, virus-specific and/or tumor-specific peptide/MHC complexes represent a unique class of cell surface targets for immunotherapy.
The terms“tumor-supporting antigen’’ or“cancer-supporting antigen” interchangeably refer to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of a ceil that is, itself, not cancerous, but supports the cancer cells, e.g , by promoting their growth or survival e.g., resistance to immune cells. The tumor-supporting antigen itself need not play a role in supporting the tumor cells so long as the antigen is present on a cell that supports cancer ceils.
A“HER2-positive cancer” or“HER2-expressing cancer” is a cancer comprising ceils that have HER2 protein present at their ceil surface. Many methods are known in the art for detecting or determining the presence of HER2 on a cancer ceil. For example, in some embodiments, the presence of HER2 on the cell surface may be determined by
immunohistochemistry (IHC), flow cytometry, Western blotting, immunofluorescent assay, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), homogeneous time resolved fluorescence (HTRF), or positron emission tomography (PET).
The terms“combination” or“pharmaceutical combination,” as used herein mean a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term“fixed combination” means that the active ingredients, by way of example, a compound of the invention and one or more additional therapeutic agent, are administered to a subject simultaneously in the form of a single entity or dosage. The term“non-fixed combination” means that the active ingredients, by way of example, a compound of of the invention and one or more additional therapeutic agent, are administered to a subject as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the active ingredients in the body of the subject. The latter also applies to cocktail therapy, e.g. the administration of 3 or more active ingredients.
The terms“composition” or“pharmaceutical composition,” as used herein, refers to a mixture of a compound of the invention with at ieast one and optionally more than one other pharmaceutically acceptable chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
The term“an optical isomer” or“a stereoisomer”, as used herein, refers to any of the various stereo isomeric configurations which may exist for a given compound of the present invention and includes geometric isomers it is understood that a substituent may be attached at a chiral center of a carbon atom. The term "chiral" refers to molecules which have the property of non-superimposability on their mirror image partner, while the term "achiral" refers to molecules which are superimposabie on their mirror image partner. Therefore, the invention includes enantiomers, diastereomers or racemates of the compound. “Enantiomers” are a pair of stereoisomers that are non- superimposabie mirror images of each other. A 1 :1 mixture of a pair of enantiomers is a "racemic” mixture. The term is used to designate a racemic mixture where appropriate. "Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn-lngold- Prelog R-S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon may be specified by either R or S Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (d extra- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain compounds described herein contain one or more asymmetric centers or axes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-.
The term "pharmaceutically acceptable carrier", as used herein, includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed Mack Printing Company, 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
The term“pharmaceutically acceptable salt,” as used herein, refers to a salt which does not abrogate the biological activity and properties of the compounds of the invention, and does not cause significant irritation to a subject to which it is administered.
The term“subject”, as used herein, encompasses mammals and non-mammals.
Examples of mammals include, but are not limited to, humans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, swine; rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. Frequently the subject is a human.
The term“a subject in need of such treatment”, refers to a subject which would benefit biologically, medically or in quality of life from such treatment.
The term“STING” refers to STtimulator of INterferon Genes receptor, also known as TMEM173, ERIS, MITA, MPYS, SAVi, or NET23). As used herein, the terms“STING” and “STING receptor” are used interchangeably, and include different isoforms and variants of STING. The RNA and protein sequences for human STING isoform 1 , the longest isoform, are:
Homo sapiens transmembrane protein 173 (TMEM173), transcript variant 1 , mRNA
[NM_198282.3]
1 tataaaaata gctcttgtta ccggaaataa ctgttcattf ttcactcctc cctcctaggi
61 cacacttttc agaaaaagaa tctgcatcct ggaaaccaga agaaaaatat gagacgggga
121 atcatcgtgt gatgtgtgtg ctgcctftgg ctgagtgtgt ggagtcctgc tcaggtgtta
181 ggtacagtgt gtttgatcgt ggtggcttga ggggaacceg ctgttcagag ctgtgactgc
241 ggctgcactc agagaagctg cccttggctg ctcgtagcgc cgggccttct ctcctcgtca
301 tcatccagag cagccagtgt ccgggaggca gaagatgccc cactccagcc tgcatccatc
361 catcccgtgt cccaggggtc acggggccca gaaggcagcc ttggttctgc tgagtgcctg
421 cctggtgacc ctttgggggc taggagagcc accagagcac actctccggt acctggtgct
481 ccacctagcc tccctgcagc tgggactgct gttaaacggg gtctgcagcc tggctgagga
541 gctgcgccac atccactcca ggtaccgggg cagctactgg aggactgtgc gggcctgcct
601 gggctgcccc ctccgccgtg gggccctgtt gctgctgtcc atctatttct actactccct
661 cccaaaigcg gtcggcccgc ccttcacttg gatgcttgcc ctcctgggcc tctcgcaggc
721 actgaacatc ctcctgggcc tcaagggcct ggccccagct gagatctctg cagtgtgtga
781 aaaagggaat ttcaacgtgg cccatgggct ggcatggtca tattacatcg gatatctgcg
841 gctgatcctg ccagagctcc aggcccggat tcgaacttac aatcagcatt acaacaacct
901 gctacggggt gcagtgagcc agcggctgta tattctcctc ccattggact gtggggtgcc
961 tgataaccig agtatggclg accccaacat tcgcitcctg gataaactgc cccagcagac
1021 cggtgaccat gciggcatca aggatcgggt ttacagcaac agcatctatg agcticigga
1081 gaacgggcag cgggcgggca cctgtgtcct ggagtacgcc acccccttgc agactttgtt
1141 tgccatgtca caatacagtc aagctggcit tagccgggag gataggcttg agcaggccaa
1201 actcttctgc cggacacttg aggacatcct ggcagaigcc cctgagtctc agaacaactg
1261 ccgcctcatt gcctaccagg aacctgcaga tgacagcagc ttctcgctgt cccaggaggt
1321 tctccggcac ctgcggcagg aggaaaagga agaggttact gtgggcagct tgaagacctc
1381 agcggtgccc agtacctcca cgatgtccca agagcctgag ctcctcatca gtggaatgga
1441 aaagcccctc cctctccgca cggatttctc ttgagaccca gggtcaccag gccagagcct
1501 Gcagtggtct ccaagcctct ggactggggg ctctcttcag tggctgaatg tccagcagag
1561 Gfatttcctt ccacaggggg ccttgcaggg aagggtccag gacttgacat cttaagatgc
1621 gtcttgtccc cttgggccag tcatttcccc tctctgagcc tcggtgtctt caacctgtga
1681 aatgggatca taatcactgc cttacctccc tcacggttgt tgtgaggact gagtgtgtgg
1741 aagtttttca taaactttgg atgctagtgt acttaggggg tgtgccaggt gtctttcatg
1801 gggccttcca gacccactcc ccacccttct ccccttcctt tgcccgggga cgccgaactc
1861 tctcaatggt atcaacaggc tccttcgccc tctggctcct ggtcatgttc cattattggg
1921 gagccccagc agaagaatgg agaggaggag gaggctgagt ttggggtatt gaatcccccg
1981 gctcccaccc tgcagcatca aggttgctat ggactctcct gccgggcaac tcttgcgtaa
2041 tcatgactat ctctaggatt ctggcaccac ttccttccct ggccccttaa gcctagctgt
2101 gtatcggcac ccccacccca ctagagtact ccctctcact tgcggtttcc ttafactcca
2161 cccctttctc aacggtcctt ttttaaagca catctcagat tacccaaaaa aaaaaaaaaa
2221 aaa [SEQ ID NO: 246]
Homo sapiens stimulator of interferon genes protein isoform 1 [NP__938023.1]
MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVC SLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWIViLALLGLSQ ALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELGARIRTYNGHYNNLLRGAV SQRLYILLPLDCGVPDNLS ADPNIRFLDKLPGGTGDHAGIKDRVYSNSIYELLENGQRAGTCV LEYATPLGTLFA SQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSS FSLSGEVLRHLRGEEKEEVTVGSLKTSAVPSTSTMSQEPELLiSGMEKPLPLRTDFS [SEQ ID NO: 247]
The mRNA and protein sequences for human STING isoform 2, a shorter isoform, are:
Homo sapiens iransme brane protein 173 (TMEM173), transcript variant 2, mRNA
[NMJJ01301738.1 ]
1 gctgcactca gagaagctgc ccttggctgc tcgtagcgcc gggccttctc tcctcgtcat
61 catccagagc agccagtgtc cgggaggcag aagatgcccc actccagcct gcatccatcc
121 atcccgtgtc ccaggggtca cggggcccag aaggcagcct tggttctgct gagtgcctgc
181 ctggtgaccc tttgggggct aggagagcca ccagagcaca ctctccggta cctggtgctc
241 cacctagcci ccctgcagct gggactgctg ttaaacgggg tctgcagcct ggctgaggag
301 ctgcgccaca tccactccag gtaccggggc agctactgga ggactgtgcg ggcctgcctg
361 ggctgccGcc tccgccgtgg ggccctgttg ctgctgtcca tctatttcta ctactccctc
421 ccaaatgcgg tcggcccgcc cttcacttgg atgcttgccc tcctgggcct ctcgcaggca
481 ctgaacatcc tcctgggcct caagggcctg gccccagctg agatctctgc agtgtgtgaa
541 aaagggaatt tcaacgtggc ccatgggctg gcatggtcat attacatcgg atatctgcgg
601 ctgatcctgc cagagctcca ggcccggatt cgaacttaca atcagcatta caacaacctg
661 ctacggggtg cagtgagcca gcggctgtat attctcctcc cattggactg tggggtgcct
721 gataacctga gtatggctga ccccaacatt cgcttcctgg ataaactgcc ccagcagacc
781 ggtgaccatg ctggcatcaa ggatcgggtt tacagcaaca gcatctatga gcttctggag
841 aacgggcagc ggaaccigca gatgacagca gcticicgct gtcccaggag gttctccggc
901 acctgcggca ggaggaaaag gaagaggtta ctgtgggcag cttgaagacc tcagcggigc
961 ccagtacctc cacgatgicc caagagcctg agctcctcat cagtggaaig gaaaagcccc
1021 iccctctccg cacggatttc tcttgagacc cagggtcacc aggccagagc ctccagtggt
1081 ctccaagcct ctggactggg ggctctcttc agtggctgaa tgtccagcag agctatttcc
1141 ttccacaggg ggcctigcag ggaagggtcc aggacttgac atcttaagat gcgtcttgtc
1201 ccctigggcc agtcatttcc ccicictgag ccicggtgtc ttcaacctgt gaaatgggat
1261 cataatcact gccttacctc ccicacggtt gttgtgagga ctgagtgtgt ggaagttitt
1321 cataaaciti ggatgctagt gtacttaggg ggtgtgccag gtgtctttca tggggccttc
1381 cagacccact ccccacccti ctccccttcc ttigcccggg gacgccgaac tcicicaaig
1441 gtatcaacag gctccttcgc cctctggctc ctggtcatgt tccattattg gggagcccca
1501 gcagaagaat ggagaggagg aggaggctga gtttggggta ttgaatcccc cggctcccac
1561 cctgcagcat caaggttgct atggactctc ctgccgggca actcttgcgt aatcatgact
1621 atctctagga ttctggcacc acttccttcc ctggcccctt aagcctagct gtgtatcggc
1681 acecccacec cactagagta ctccctctca cttgcggttt ccttatactc cacccctttc
1741 tcaacggtcc ttttttaaag cacatctcag attacccaaa aaaaaaaaaa aaaaa [SEG ID NO: 248]
Homo sapiens stimulator of interferon genes protein isoform 2 [NP_GG1288667 1 ]
PHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLGLGLLLNGVC SLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWIVILALLGLSQ ALNILLGLKGLAPAEiSAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARiRTYNGHYNNLLRGAV SQRLYILLPLDCGVPDNLS ADPNiRFLDKLPQGTGDHAGIKDRVYSNSIYELLENGQRNLGMT AASRCPRRFSGTCGRRKRKRLLWAA [SEQ ID NO: 249]
The sequences of other human STING isoforms/SNPs (single nucleotide
polymorphisms) include the following and those described in Yi, PLoS One. 2013 Oct 21 ; 8(10):e77846. tiST!IMG wt (wind type): Reference SNR (refSNP) Cluster Report: rs1 131769 atgccceactccagcctgcatccatccatcecgtgteccaggggtcacggggcceagaaggcagcctiggttctgctgagtgcctgcc tggtgacecittgggggctaggagagccaccagagcacacictccggtacctggtgctccacetagcctccctgcagctgggaetget gttaaacggggtctgeagcctggetgaggagctgcgccacatccactceaggtaccggggcagctactggaggactgtgcgggcet gcctgggcigccccctccgccgtggggccctgtigcigcigtccaictatttctactactccctcccaaatgcggtcggcccgcccttcact iggatgcttgccctcctgggccictcgcaggcactgaacatcctcctgggcctcaagggcciggccccagctgagatctctgcagtgtgi gaaaaagggaatticaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggccc ggattcgaacttacaaicagcattacaacaacctgctacggggigcagtgagccagcggcigtatattctcctcccattggacigtggg gigcctgataacctgagtatggctgaccccaacaitcgcitcctggataaactgccccagcagaccggtgaccgtgctggcatcaagg atcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgc agactttgtttgccatgtcacaatacagtcaagclggctttagccgggaggataggcttgagcaggccaaactcttctgccggacacttg aggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagaigacagcagcttctcgct gtcccaggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagt acctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga [SEQ ID
NO: 250]
hST! G R293Q: Reference SNR (refSNP) Cluster Report: rs1131769 rs7380824
atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcc tggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgct gttaaacggggtctgcagcctggctgaggagctgcgccacatccaotccaggtaccggggcagctactggaggactgtgcgggcct gcctgggctgccccGtGcgcegtggggccctgttgctgctgtGcatctatttGtactaetGcctcecaaatgcggtcggccegcGcttcact iggatgcttgcectcctgggccictcgcaggcactgaacatcctcctgggcctcaagggcciggccccagctgagatetctgcagtgtgi gaaaaagggaatttcaacgtggeccatgggctggcatggtGatattacatcggatatetgcggctgatcctgccagagctceaggcec ggattegaacttacaatcagcattacaacaacetgetaeggggtgcagtgagccagcggctgtatattctcctcccattggactgtggg gtgcctgataacetgagtatggctgaceccaacattGgcttcctggataaactgccecagcagacGggtgaccgtgctggcatcaagg atcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagiacgccacccccttgc agactttgtttgccatgicacaatacagtcaagctggctitagccgggaggataggcttgagcaggccaaactcttctgccagacacttg aggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagaigacagcagcttctcgct gtcccaggaggttctccggcaccigcggcaggaggaaaaggaagaggitactgtgggcagcitgaagacctcagcggtgcccagt acctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccciccctctccgcacggaittcicitga [SEQ ID
NO: 251]
hSTING G238A/R293Q: Reference SNR (refSNP) Cluster Report: rs1131769 rs7380824 rs78233829
atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcc tggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgct gttaaacggggtctgcagcctggctgaggagctgcgccacatccactccaggtaccggggcagctactggaggactgtgcgggcct gcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcact tggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccccagctgagatctctgcagtgtgt gaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggccc ggattcgaacttacaatcagcattaeaacaacctgctacggggtgGagtgagccagcggctgtatattctectcccattggactgtggg gtgcctgataacctgagtatggetgaccGcaaGattcgGttcctggataaactgccccagcagaecgGtgaecgtgctggcatcaagg atcgggtttacagcaacagGatGtatgagcttctggagaacgggcagGgggcgggcacetgtgtcctggagtacgccacccGcttgc agactttgtttgccatgtcacaatacagteaagctggctttagecgggaggataggcttgagGaggcGaaactcttGtgccagaeacttg aggacatcetggeagatgcccctgagtctcagaacaactgccgcctcattgcctaceaggaaGctgcagatgacagcagcttctegct gicccaggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagt acctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatitctctiga [SEQ ID
NO: 252]
hSTING R71 H/G230A/R2S3Q: Reference SIMP (refSNP) Cluster Report:
rs1131789 rs7380824 rs78233829 rs11554776
atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcc tggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgct gttaaacggggtctgeagcctggetgaggagctgcaccacatccactceaggtaccggggcagctactggaggactgtgcgggcet gcctgggctgccceciccgccgtggggccctgttgctgctgtccatctaittciactactcccteccaaatgcggtcggcccgeccticact tggatgcttgccetcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccceagctgagatctcigcagtgtgt gaaaaagggaatttcaacgtggcGcatgggGtggcatggtcatattacatcggatatctgcggctgatGctgccagagctccaggccG ggattcgaacttacaatcagcattacaaGaacctgctacggggtgGagtgagcGagcggctgtatattctcctcccattggactgtggg gtgcctgataacctgagtatggctgaccccaacattcgcitcctggataaactgccccagcagaccgcigaccgtgctggcatcaagg atcgggtttacagcaacagcatciatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgc agactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggaiaggcttgagcaggccaaactcttctgccagacacttg aggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgct gtcccaggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagt acctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga [SEQ ID
NO: 253]
The term“STING agonist”, as used herein, refers to a compound or antibody conjugate capable of binding to STING and activating STING Activation of STING activity may include, for example, stimulation of inflammatory cytokines, including interferons, such as type 1 interferons, including IFN-a, IFN-b, type 3 interferons, e.g., IRNl, IP10, TNF, !L~8, CXCL9,
CCL4, CXCL11 , CCL5, CCL3, or CCL8. STING agonist activity may also include stimulation of TANK binding kinase (TBK) 1 phosphorylation, interferon regulatory factor (IRF) activation (e.g , IRF3 activation), secretion of interferon-y-inducible protein (IP-10), or other inflammatory proteins and cytokines. STING Agonist activity may be determined, for example, by the ability of a compound to stimulate activation of the STING pathway as detected using an interferon stimulation assay, a reporter gene assay (e.g., a hSTING wt assay, or a THP-1 Dual assay), a TBK1 activation assay, IP-10 assay, a STING Biochemical [SHjcGAMP Competition Assay, or other assays known to persons skilled in the art. STING Agonist activity may also be determined by the ability of a compound to increase the level of transcription of genes that encode proteins activated by STING or the STING pathway. Such activity may be detected, for example, using an RNAseq assay. In some embodiments, an assay to test for activity of a compound in a STING knock-out cell line may be used to determine if the compound is specific for STING, wherein a compound that is specific for STING would not be expected to have activity in a cell line wherein the STING pathway is partially or wholly deleted.
As used herein, the terms“treat,”“treating,” or“treatment” of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof} in another embodiment,“treat,”“treating,” or“treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient in yet another embodiment,“treat,”“treating,” or“treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
As used herein, the term“prevent”,“preventing" or“prevention” of any disease or disorder refers to the prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder The term“therapeutically effective amount” or“therapeutically effective dose” interchangeably refers to an amount sufficient to effect the desired result (i.e., reduction or inhibition of an enzyme or a protein activity, amelioration of symptoms, alleviation of symptoms or conditions, delay of disease progression, a reduction in tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal or parasitic infection) in some embodiments, a therapeutically effective amount does not induce or cause undesirable side effects in some embodiments, a therapeutically effective amount induces or causes side effects but only those that are acceptable by the healthcare providers in view of a patient’s condition. A therapeutically effective amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved. A “prophylacticaliy effective dose” or a“prophylactically effect amount”, of the molecuies of the invention can prevent the onset of disease symptoms, including symptoms associated with cancer. A“therapeutically effective dose” or a“therapeutically effective amount” of the molecuies of the invention can result in a decrease in severity of disease symptoms, including symptoms associated with cancer. The compound names provided herein were obtained using ChemDraw Ultra version 14.0 (CambridgeSoft®).
As used herein, the term "a,” "an,” "the” and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.
Any formula given herein is also intended to represent uniabeied forms as well as ssotopicaiiy labeled forms of the compounds isotopically labeled compounds have structures depicted by the formulae given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Isotopes that can be incorporated into compounds of the invention include, for example, isotopes of hydrogen.
Unless specified otherwise, the conjugates or Drug moieties of the present invention refer to compounds of any of formulae (AA-a) through (FF-g) or formulae (A) through (F) or subformulae thereof and exemplified compounds, and salts thereof, as weii as all stereoisomers (including diastereoisomers and enantiomers), rotamers, tautomers and isotopically labeled compounds (including deuterium substitutions), as well as inherently formed moieties.
Immunostimulatorv Compounds of the Invention
Drug Moiety (D j
The Drug moiety (D) of the immunoconjugates of the invention is a compound which binds to Stimulator of Interferon Genes (STING) receptor and which comprises one or more reactive moieties each of which is capable of forming a covalent bond with a linker (L). In one aspect, Drug moiety (D) of the immunoconjugates of the invention is a dinucleotide which binds to Stimulator of Interferon Genes (STING) which comprises one or more reactive moieties capable of forming a covalent bond with a linker (L)
In one aspect, Drug moiety (D) of the immunoconjugates of the invention is a cyclic dinucleotide which binds to Stimulator of interferon Genes (STING) which comprises one or more reactive moieties capable of forming a covalent bond with a linker (L).
In one aspect the Drug moiety (D) of the immunoconjugates of the invention is a compound having the structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or Formula (F) or stereoisomers or pharmaceutically acceptable salts thereof,
Figure imgf000059_0001
Formula (C) Formula (D)
Figure imgf000059_0002
Formula (E) Formula (F) wherein:
each Gi is independently selected from
Figure imgf000060_0001
where the 4 of Gi indicates the point of attachment to -CR8R9-;
XA is C(=0)-, -C(=S)- or -C(=NRi 1)~ and each Z1 is NR12;
XB is C, and each Z2 is N;
Figure imgf000060_0003
Figure imgf000060_0002
Y6 is -CHr, -NH-, -O- or -S;
Y7 is O or S;
Y8 is O or S;
Y9 is -CH2-, -NH-, -O- or -S;
Y10 is -CH2r. -NH-. -O- or -S;
Y11 is -O-, -NH-, -S-. -S(=0)-, -SO2-, -CH2-. or -CFz-;
q is 1 , 2 or 3;
R1 is a partially saturated or aromatic monocyclic heteroeycly! or partially saturated or aromatic fused bicyclic heierocyciyi containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -NHGH, F, Cl, Br, CrC6alkyl and a CrCsa!kyl substituted with 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
R1a is a partially saturated or aromatic monocyclic heteroeycly! or partially saturated or aromatic fused bicyclic heterocyciyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -NHOH, F, Cl, Br, Ci-C6alkyl and a Ci-C3alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R1 b is a partially saturated or aromatic monocyclic beterocyciyi or partially saturated or aromatic fused bicyclic heterocyciyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1 b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, ~NHNH2, -NHOH, F, Cl, Br, Ci-C6alkyl and CrCealky! substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R2 is independently selected from H and CrCsaikyl;
each R3 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a CrCsaikyl substituted with a NHOH, NHNH2 or NH2;
each R4 is independently selected from H, CrCsaikyl and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R5 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C Csalkyl substituted with a NHOH, NHNH2 or NH2;
each R6 is independently selected from H, CrCsaikyl and CrCsaikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7 is independently selected from H, CrCsaikyl and CrCsaikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R8 is independently selected from H, CrCsaikyl and CrCsaikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R9 is independently selected from H, CrCsaikyl and CrCsaikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R2a is independently selected from H and CrC6alkyl
R3a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Cr Cealkyi substituted with a NHOH, NHNH2 or NH2;
R4a is independently selected from H, CrC6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R5a is independently selected from H, -OH, -SH, F, Cl, Br, !, NHOH, NHNH2, NH2 and a Cr Csalky! substituted with a NHOH, NHNH2 or NH2;
R6a is independently selected from H, CrC6alkyl and CrC6alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R7a is independently selected from H, CrC6alkyl and C i-C6alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3; R8a is independently selected from H, CrC6alkyl and CrC6alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N3;
R9a is independently selected from H, CrC6alkyl and CrC6alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Gi, Br, I, OH, CN, and N3;
each R10 is independently selected from the group consisting yi, Ci-
Cgheleroa!kyl, -(CH2GH20)„eH2CH2e(=0)0Ci-Cealkyl, a
Figure imgf000062_0001
wherein the C
Ci2a!kyl and Ci-Cgheieroalkyl of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, Ci-Ci2aikoxy, -S~C(=0)Ci-Cgalky!, halo, -CM, Cr Ci2alkyi, -O-aryl, -O-heteroaryl, -O-cyc!oalkyi, oxo, cycioaiky!, heterocyciyl, aryl, or heteroaryl, -OCfQlQCrCgalky!and C(0)0Ci-C6alkyl, wherein each alkyl, cycloalkyl, heterocyciyl, aryl, and heteroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from C1-C12 alkyl, O-CrC^alkyl, CrC^heteroalkyi, halo, CM, OH, oxo, aryl, heteroaryl, G-aryl, O-heteroaryl, -C(=0)CrCi2alkyl, -QC(=G)CrCi2aikyl, -C(=0)0Cr Ci2alkyi, -0C(=0)0C Ci2aikyl, -C(=0)N(R11)-Ci-Gi2alkyl, -N(R11)C(=0)-Ci-Gi2alkyl; - 0C(=0)N(R1 ^-CrC^aikyl, -C(=Oj-aryl, -C(=0)-heteroaryl, -QC(=0)-aryl, -C(=0)0-aryl, - OC(=;0)-heteroa!yi, -C ^OJO-heteroaryl, -G^O^O-aryi, -C(=Q)0-heteroaryl, - e(0)N(R11)-aryl, -C(=0)N(R11)-heteroaryl, -!M(R11)C(G)-aryl, -N(R11)2G(0)-aryl, - N(R11)C(0)-heteroaryl, and S(0)2N(R11)-aryi;
R11 is independently selected from H and Ci-C3alkyl;
R12 is independently selected from H and Ci-C3alkyl;
optionally R3 and R6 are connected to form -0-CrC6alkylene, such that when R3 and R6 are connected, the O is bound at. the R3 position
optionally R3a and R6a, are connected to form -0~CrC6aikyiene, such that when R3a and RSa are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form -O-CrCgalkylene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form -0-Ci-C3aikyiene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form -G-CrC6alkylene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -Q-Ci-G6aikyiene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form -Q-CrCgaikylene, such that when R5 and R6 are connected, the O is bound at the R5 position; optionally R5a and R6a, are connected to form -0-CrCBalkylene, such that when R5a and R6a are connected, the O is bound at the R5a position:
optionally R5 and R7 are connected to form -Q-CrC6aiky!ene, such that when R5 and R7 are connected, the O is bound at the R5 position,
and
optionally R5a and R7a, are connected to form -OCrC6aikylene, such that when R5a and R7a are connected, the O is bound at the R5a position.
Certain aspects and examples of compounds which can be incorporated as a Drug moiety (D) in the immunoconjugates of the invention are provided in the following listing of additional, enumerated embodiments. It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.
Embodiment 1. The compound of Formula (A-1), Formula (B-1), Formula (C-1), Formula (D-1), Formula (E~1) or Formula (F-1), or siereoisomers or pharmaceutically acceptable salts thereof wherein:
Y7 e the * of Gi indicates the point of attachment to -CRSR9-: e the * of G2 indicates the point of attachment to ~CR8aR93-; , SR10, SeH, Se , BH3, SH or S ;
Figure imgf000063_0001
, SR10, SeH, Se~, BH3, SH or S ;
Y5 is -CH2-, -NH-, -O- or -S;
Y6 is -CH2-, -NH-, -O- or -S;
Y7 is O or S;
Y8 is O or S;
Y9 is -CH !-, -NH-, -O- or -S, and
Yio is -CHr, -NH-, -O- or -S.
Embodiment 2, A compound of Formula (A-1), Formula (B-1), Formula (C-1), Formula (D- 1), Formula (E-1) or Formula (F-1), or stereoisomers or pharmaceutically acceptable salts thereof,
Figure imgf000064_0001
Formula (C-1) Formula (D-1)
Figure imgf000064_0002
Formula (E-1) Formula (F-1 )
wherein R1 , R1a, R1 b, R2, R2a, R3, R3a, R4, R4a, R5, R5a, R6, R6a, R7, R7a, R8, RSa, R9, Y, , Y2, Y3, Y4 Y5> Y6, Y7, Y8, Ub, Y and Y are as defined above for compounds of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) and Formula (F).
Embodiment 3. A compound of Formulae A), Formula (B), Formula (C), Formula (D), Formula (A- 1 ) , Formula (B-1), Formula (C-1), Formula (D-1 ), Formula (E-1), or Formula (F- 1 ), wherein R1 is pyrimidine or purine nucleic acid base or analogue thereof, R1a is pyrimidine or purine nucieic acid base or analogue thereof, and R1b is a pyrimidine or purine nucleic acid base or analogue thereof, each of which is substituted as described in R\ R1a or R1b for Formula (A), Formula (BB, Formula (C), Formula (D), Formula (A- 1 ) , Formula (B-1), Formula (C-1), Formula (D-1), Formula (E-1), or Formula (F-1).
ormula (D-
Figure imgf000065_0001
Formula (G-2) Formula (D-2)
Figure imgf000065_0002
Formula (E-2) Formula (F-2) wherein R\ R1a, R1 b, R2, R2a, R3, R3a, R4, R4a, R5, R5a, R6, R6a, R7, R7a, R8, RSa, R9, Y, , Y2, Y3, Y4, Y5> Y6, Y7, Y8, Ye, Yio and Y are as defined above for compounds of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) and Formula (F).
Embodiment S. A compound of Formula (A), Formula (A-1) or Formula (A-2) of
Embodiments 1 , 2, 3 or 4 wherein:
are H;
Figure imgf000066_0001
H, NHNH2, NH2 or a CrC6alkyl substituted with a NHOH, NHNH2 or NH2;
R3a is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 or a Gi-C6aikyi substituted with a NHOH, NHNH2 or NH2; and
R8, R9, R8a and R9a are independently H or Ci-C5alkyl.
Embodiment 6. A compound of Formula (A), Formula (A-1) or Formula (A-2) of
Embodiment 1 , 2, 3, 4 or 5 wherein:
Yi and Y2 are independently selected from O, NH, CH2 and S;
Y3 is OH, O , NH2, SH or S ;
Y4 is OH, O , NH2, SH or S ;
Y5 and Y6 are independently selected from O and S;
Y7 and Ys are independently selected from O and S;
Y9 and Yio are independently selected from O and S;
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are H;
R3a is H, ~NH2, -NHNH2, -NHOH, OH or F;
R3 is H, -NH2, -NHNH2 -NHOH, OH or F; and
R8a, R9a, R8 and R9 are independently selected from H or CrCsaikyL
Embodiment 7, A compound of Formula (A), Formula (A-1) or Formula (A-2) of
Embodiment 1 , 2, 3, 4 or 5 wherein:
Yi and Y2 are independently selected from O, NH, CH2 and S;
Y3 is OH, O , NH2, SH or S ;
Y4 is OH, O , NH2, SH or S ;
Ys and Y6 are independently selected from O and S;
Y7 and Y8 are independently selected from O and S;
Y9 and Yio are independently selected from O and S;
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are H;
R3a is H, -NHNHz, OH or F:
R3 is H, -NHNH2, OH or F; and
R8a, R9a, R8 and R9 are independently selected from H or Ci-C3aikyl. Embodiment 8. A compound of Formula (B), Formula (B-1) or Formula (B-2) of Embodiment 1 , 2, 3 or 4 wherein:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are H;
R3a is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 or a G-i-Ceaikyl substituted with a NHOH, NHNH2 or NH2;
R5 is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 or a CrC5alkyl substituted with a NHOH, NHNH2 or NH2; and
R8, Rs, RSa and R9a are independently H or Ci-C3alkyi.
Embodiment 9. A compound of Formula (B), Formula (B-1) or Formula (B-2) of
Embodiment 1 , 2, 3, 4 or 8 wherein:
Yi and Y2 are independently selected from O, NH, CH2 and S;
Y3 is OH, O , NH2, SH or S-;
Y4 is OH, O , NH2, SH or S-;
Y5 and Y6 are independently selected from O and S;
Y7 and Ys are independently selected from O and S;
Y9 and Y10 are independently selected from O and S;
R2, R2a, R4, R4a, Rs, R5a, R7 and R7a are H;
R3a is H, -NH2, -NHNH2, -NHOH, OH or F;
R5 is H, -NH2, -NHNH2I -NHOH, OH or F; and
R8a, R9a, Rs and R9 are independently selected from H or Ci-C3a!kyi
Embodiment 10, A compound of Formula (B), Formula (B-1) or Formula (B-2) of
Embodiment 1 , 2, 3, 4 or 8 wherein:
Yi and Y2 are independently selected from O, NH, CH2 and S;
Y3 is OH, O , NH2, SH or S ;
Y4 is OH, O , NH2, SH or S ;
Y5 and Y6 are independently selected from O and S;
Y7 and Y8 are independently selected from O and S;
Y9 and Y10 are independently selected from O and S;
are H;
Figure imgf000067_0001
R83, R9a, R8 and R9 are independently selected from H or Ci-C6alkyl.
Embodiment 11. A compound of Formula (C), Formula (C-1) or Formula (C-2) of
Embodiment 1 , 2, 3 or 4 wherein:
R2, R2a, R4, R43, R6, R63, R7 and R7a are H;
Figure imgf000068_0001
R8, R9, R8a and R93 are independently H or Ci-C5alkyl.
Embodiment 12. A compound of Formula (C), Formula (C-1) or Formula (C-2) of
Embodiment 1 , 2, 3 or 1 1 wherein:
Yi and Y2 are independently selected from O, NH, CH2 and S;
Y3 is OH, O , NH2, SH or S ;
Y4 is OH, O , NH2, SH or S ;
Y5 and Y6 are independently selected from O and S;
Y7 and Ys are independently selected from O and S;
Y9 and Yio are independently selected from O and S;
R2, R2a, R4, R43, R6, R63, R7 and R7a are H;
R3 is H, -NH2, -NHNH2I -NHOH, OH or F;
R53 is H, ~NH2, -NHNH2I -NHOH, OH or F; and
R83, R9a, R8 and R9 are independently selected from H or CrC6a!kyi
Embodiment 13. A compound of Formula (C), Formula (C-1) or Formula (C-2) of
Embodiment 1 , 2, 3 or 1 1 wherein:
Yi and Y2 are independently selected from O, NH, CH2 and S;
Y3 is OH, O , NH2, SH or S ;
Y4 is OH, O , NH2, SH or S ;
Ys and Y6 are independently selected from O and S;
Y7 and Y8 are independently selected from O and S;
Y9 and Yic are Independently selected from O and S;
R2, R2a, R4, R43, R6, R63, R7 and R7a are H;
R3 is H, -NHNHz, OH or F;
R5a is H, -NHNHz, OH or F; and
R8a, R9a, R8 and R9 are independently selected from H or Ci-C6alkyl.
Embodiment 14. A compound of Formula (D), Formula (D-1) or Formula (D-2) of
Embodiment 1 , 2, 3 or 4 wherein:
R2, R2a, R4, R43, R6, R63, R7 and R73 are H;
R5a is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 or a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH2;
R5 is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 or a Ci-C6alkyi substituted with a NHOH, NHNH2 or NH2, and R8, R9, RSa and R9a are independently H or CrCsalkyl.
Embodiment 15. A compound of Formula (D), Formula (D-1) or Formula (D-2) of
Embodiment 1 , 2, 3, 4 or 14 wherein:
Y1 and Y2 are independently selected from O, NH, CH2 and S;
Y3 is OH, O , NH2, SH or S :
Y4 is OH, O , NH2, SH or S ;
Y5 and Y6 are independently selected from O and S;
Y7 and Ys are independently selected from O and S;
Y 9 and Yio are independently selected from O and S;
R2, R2a, R4, R4a, R6, RSa, R7 and R7a are H;
R5a is H, -NH2, -NHNH2, -NHOH, OH or F;
R5 is H, ~NH2, -NHNH2, -NHOH, OH or F; and
R8, Rs, RSa and R9a are independently H or Ci-C3alkyi.
Embodiment 16. A compound of Formula (D), Formula (D-1) or Formula (D-2) of
Embodiment 1 , 2, 3, 4 or 14 wherein:
Y1 and Y2 are independently selected from O, NH, CH2 and S;
Y3 is OH, O , NH2, SH or S-;
Y4 is OH, O , NH2, SH or S-;
Y5 and Y6 are independently selected from O and S;
Y? and Ys are independently selected from O and S;
Y9 and Yio are independently selected from O and S;
are H;
Figure imgf000069_0001
R8, R9, R8a and RSa are independently H or Ci-C3alkyl
Embodiment 17, A compound of Formula (E), Formula (E-1) or Formula (E-2) of
Embodiment 1 , 2 or 3 wherein:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are H;
R3a is H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 or a C C6aikyi substituted with a NHOH, NHNH2 or NH2;
R3 is H, -OH, -SH, F, Ci, Br, I, NHOH, NHNH2, NH2 or a C C6alkyi substitifted with a NHOH, NHNH2 or NH2;
R5 is H, -OH, -SH, F, Ci, Br, I, NHOH, NHNH2, NH2 or a Ci-C6alkyl substitifted with a NHOH, NHNH2 or NH2;
and
R8, R9, R8a and R9a are independently H or Ci-C3alkyl H. Embodiment 18. A compound of Formula (E), Formula (E-1) or Formula (E-2) of Embodiment 1 , 2, 3, 4 or 17 wherein:
Y1 and Y2 are independently selected from O, NH, CH2 and S;
Y3 is OH, O , NH2 SH o S :
Y5 is O or S;
Y7 is O or S;
Y9 is O or S;
R2, R2a, R4, R4a, R53, R6, R63, R7 and R7a are H
R3a is H, -NH2, ~NHNH2S -NHOH, OH or F;
R3 is H, ~NH2, -NHNH2, -NHOH, OH or F;
R5 is H, ~NH2, -NHNH2, -NHOH, OH or F; and
R8, R9, RSa and R9a are independently H or C -C3alkyi
Embodiment 19. A compound of Formula (E), Formula (E-1) or Formula (E-2) of
Embodiment 1 , 2, 3 or 12 wherein:
Y1 and Y2 are independently selected from O, NH, CH2 and S;
Y3 is OH, O , NH2, SH or S ;
Ys is O or S;
Y? is O or S:
Y9 is O or S:
R2, R2a, R4, R43, R5a, R6, R6a, R7 and R7a are H
R3a is H, -NHNHs, OH or F:
R3 is H, -NHNH2 OH or F;
R5 is H, -NHNH2 OH or F; and
R8, Rs, R8a and R9a are independently H or Ci-C6alkyl.
Embodiment 20. A compound of Formula (F), Formula (F-1) or Formula (F-2) of
Embodiment 1 , 2 or 3 wherein:
R2, R2a, R4, R43, R6, R63, R7 and R7a are H;
a
Figure imgf000070_0001
R8, R9, R8a and R9a are independently H or Ci-Cealkyl.
Embodiment 21 , A compound of Formula (F), Formula (F-1) or Formula (F-2) of
Embodiment 1 , 2, 3 or 12 wherein: Y1 and Y2 are independently selected from O, NH, CH2 and S;
each Y3 is OH, O , NH2, SH or S ;
each Y5 is independently selected from O and S;
each Y? is independently selected from O and S;
each Y9 is independently selected from O and S;
Y11 Is O, CH2 or S;
R2, R2a, R4, R4a, R6, RSa, R7 and R7a are H;
R3a is H, -NH2, ~NHNH2, -NHOH, OH or F;
R3 is H, ~NH2, -NHNH2I -NHOH, OH or F;
R5 is H, ~NH2, -NHNH2I -NHOH, OH or F, and
R8, Rs, RSa and R9a are independently H or Ci-C3alkyl.
Embodiment 22. A compound of Formula (F), Formula (F~1) or Formula (F-2) of Embodiment 1 , 2, 3 or 12 wherein:
Y1 and Y2 are independently selected from O, NH, CH2 and S;
each Y3 is OH, O , NH2, SH or S ;
each Y5 is independently selected from O and S;
each Y? is independently selected from O and S;
each Yg is independently selected from O and S;
Figure imgf000071_0002
R8, Rs, R8a and R9a are independently H or Ci-C6alkyi.
Embodiment 23, A compound of any one of Embodiments 1 to 22 wherein:
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000074_0002
with 0, 1 , 2 or 3 substituents independently selected from halogen, CrC6alkyl and Ci-C6alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
Figure imgf000074_0003
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000077_0002
wherein: R1a is substituted with 0,
1 , 2 or 3 substituents independently selected from halogen, C rCBalkyl and CrCsalky! substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I , OH , CN, and N3;
and
Figure imgf000077_0003
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
with 0,
1 , 2 or 3 substituents independently selected from halogen, Gi-Cealkyl and CrC6alky! substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, !, OH, CN, and N3.
Figure imgf000080_0002
A compound of Formula (A-3), Formula (B-3), Formula (C-3) , Formula (D-3), Formula (E-3) or Formula (F-3):
Figure imgf000080_0003
Formula (C-3) Formula (D-3)
Figure imgf000081_0001
Formula (E-3) Formula (F~3) wherein:
Figure imgf000081_0002
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000084_0002
wherein R1 is substituted with 0, 1 ,
2 or 3 substituents independently selected from halogen, Ci~C6aikyl and CrC6alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
Figure imgf000084_0003
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
1 , 2 or 3 substituents independently selected from halogen, CrCsalkyl and CrC6alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I , OH, CN, and N3;
and
Figure imgf000087_0002
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
with 0,
1 , 2 or 3 substituents independently selected from halogen, Ci-Cealkyl and Ci~C6alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, !, OH, CN, and N3;
each R2 is independently selected from H and CrCsaikyi;
each R3 Is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-G6alkyi substituted with a NHOH, NHNH2 or NH2;
each R4 Is independently selected from H, Ci-C6alkyl and Gi-C6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Gi, Br, I, OH, CN, and N3;
each R5 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C3aikyl substituted with a NHOH, NHNH2 or NH2;
each R6 is independently selected from H, CrC6alky! and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7 is independently selected from H, Ci~C6alkyl and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R® is independently selected from H, CrC6alky! and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each Rs is independently selected from H, CrC6alkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R2a is independently selected from H and C i-C6aikyl
each R3a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-Csalkyi substituted with a NHOH, NHNH2 or NH2:
each R4a is independently selected from H, Ci-C3alkyl and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
each R5a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6alkyi substituted with a NHOH, NHNH2 or NH2;
each R6a is independently selected from H, C -C3alkyl and Ci-Cealkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
each R7a is independently selected from H, C -C3alkyl and Ci-Cealkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3; each RSa is independently selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R9a is independently selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R11 is independently selected from H and CrC6alkyl;
each R12 is independently selected from H and CrC6alkyl;
optionally R3 and R6 are connected to form -Q-G i-C6aikylene, such that when R3 and Rs are connected, the O is bound at the R3 position
optionally R3a and R6a, are connected to form -0-Ci-C6alkylene, such that when R38 and R6a are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form -0-CrC6alkylene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form -0-Ci-C6alkylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form -0-CrC6alkylene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -G-CrCsa!kylene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form -O-CrCeaikylene, such that when R5 and Rs are connected, the O is bound at the R5 position;
optionally R5a and R6a, are connected to form -G-Ci-G6alkylene, such that when R5a and RSa are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form -0-Ci-C6alkylene, such that when R5 and R7 are connected, the O is bound at the R5 position,
and
optionally R5a and R7a, are connected to form -Q-CrCga!kyiene, such that when R5a and R7a are connected, the O is bound at the R5a position.
Embodiment 25, The compound Formula (A-3) , or a pharmaceutically acceptable salt thereof, having the structure of Formula (A-4), or a pharmaceutically acceptable salt thereof:
Figure imgf000091_0001
Formula (A-4)
wherein: R1, Ria, R3, R3a, R6, RSa, Y3 and Y4 are as defined in Embodiment 24. Embodiment 26. The compound of Formula (A-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (A-4a), Formula A-4b), Formula A-4c) or Formula A- 4d), or a pharmaceutically acceptable salt thereof:
Figure imgf000092_0001
Formula (A-4c) Formula (A-4d)
wherein: R1, R1a, R3, R3a, R6 and R6a are as defined in Embodiment 24;
Y3 is OH, NH2, SH or S , and
Y4 is OH, NH2, SH or S .
Embodiment 27. The compound of Formula (A-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (A-4e), Formula (A-4f), Formula (A-4g), Formula (A-4h), Formula (A-4i), Formula (A-4j), Formula (A-4k), Formula (A-41), Formula (A-4m), Formula (A-4n), Formula (A-4o) or Formula (A-4p), or a pharmaceutically acceptable salt thereof:
Figure imgf000092_0002
Formula (A~4e) Formula (A-41) Formula (A-4g)
Figure imgf000093_0001
Formula (A-4h) Formula (A-4i) Formula (A-4j)
Figure imgf000093_0002
Formula (A-4n) Formula (A-4o) Formula (A-4p)
wherein: R \ R1a, R3, R3a, Rs and R6a are as defined In Embodiment 24;
Y3 is OH, NH2, SH or S , and
Y4 is OH, NH2, SH or S .
Embodiment 28. The compound of Formula (B-3) having the structure of Formula (B-4), or a pharmaceutically acceptable salt thereof:
Figure imgf000093_0003
Formula (B-4)
wherein: R1, R1a, R3, R3a, R5, R6a, Y3 and Y4 are as defined in Embodiment 24. The compound of Formula (B-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (B-4a), Formula (B-4b), Formula (B-4c) or Formula (B-4d), or a pharmaceutically acceptable salt thereof:
Figure imgf000094_0002
Formula (B-4d)
wherein: R1, R18, R3a, R5 and R68 are as defined in Embodiment 24;
Y3 is OH, NH2, SH or S , and
Y4 is OH, NH2, SH or S .
Embodiment 30. The compound of Formula (B-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (B-4e), Formula (B-4f), Formula (B-4g) or Formula (B-4h), or a pharmaceutically acceptable salt thereof:
Figure imgf000094_0001
Formula (B-4h) wherein: R1, R1a and R5 are as defined in Embodiment 24:
Y3 is OH, NH2, SH or S , and
Y4 is OH, NH2, SH or S .
Embodiment 31. The compound of Formuia (C-3) having the structure of Formula (C-4), or a pharmaceutically acceptable salt thereof:
Figure imgf000095_0001
Formula (C-4)
wherein: R1, R1a, R3, R5a, R6, R6a, Y3 and Y4 are as defined in Embodiment 24.
Embodiment 32. The compound of Formula (C-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (C-4a), Formula (C-4b), Formula (C-4c) or Formula (C-4d), or a pharmaceutically acceptable salt thereof:
Figure imgf000095_0002
Formula (C~4a) Formula (C-4b) Formula (C~4c)
Figure imgf000095_0003
Formula (C-4d)
wherein: R1, R1a, R3, R5a and R6 are as defined in Embodiment 24;
Y3 is OH, NH2, SH or S , and
Y4 is OH, NH2, SH or S .
Embodiment 33. The compound of Formula (C-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (C-4e), Formula (C-4f), Formula (C-4g) or Formula (C-4h), or a pharmaceutically acceptable salt thereof:
Figure imgf000096_0001
Formula (C-4e) Formula (C-4f) Formula (C-4g)
Figure imgf000096_0002
Formula (C-4h)
wherein: R\ R1a and R5a are as defined in Embodiment 24;
Y3 is OH, NH2, SH or S , and
Y4 is OH, NH2, SH or S .
Embodiment 34. The compound of Formula (D-3), or a pharmaceutically acceptable salt thereof, having the structure of Formula (D-4), or a pharmaceutically acceptable salt thereof:
Figure imgf000096_0003
Formula (D-4)
wherein: R1, R1a, R5, R5a, Y3 and Y4 are as defined in Embodiment 24.
Embodiment 35, The compound of Formula (D-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (D-4a), Formula (D~4b), Formula (D-4c) or Formul; (D-4d), or a pharmaceutically acceptable salt thereof:
Figure imgf000096_0004
Formula (D-4a) Formula (D-4b) Formula (D-4c)
Figure imgf000097_0001
Formula (D-4d)
wherein: R1, Ria, R5 and R£,a are as defined in Embodiment 24;
Y3 is OH, NH2, SH or S , and
Y4 is OH, NH2, SH or S
Embodiment 38, The compound of Formula (E-3), or a pharmaceutically acceptable salt thereof, having the structure of Formula (E-4), or a pharmaceutically acceptable salt thereof:
Figure imgf000097_0002
Formula (E-4)
wherein: R1, R1a, R3, R3a, R4, R4a, R5 and R' are as defined in Embodiment 24.
Embodiment 37. The compound of Formula (E-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (E-4a) or Formula (E-4b), or a pharmaceutically acceptable salt thereof:
Figure imgf000097_0003
Formula (E-4a) Formula (E-4b)
wherein: R1, R1 a, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 24;
and
Y3 is OH, NH2I SH or S . Embodiment 38. The compound of Formula (F-3), or a pharmaceutically acceptable salt thereof, having the structure of Formula (F-4), or a pharmaceutically acceptable salt thereof:
Figure imgf000098_0001
Formula (F-4)
wherein: R1, R1a, R1b, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 24.
Embodiment 39. The compound of Formula (F-4), or a pharmaceutically acceptable salt thereof, having the structure of Formula (F-4a), Formula (F-4b), Formula (F-4c), or Formula (F-4d), or a pharmaceutically acceptable salt thereof:
Figure imgf000098_0002
Formula (F-4a) Formula (F-4b)
Figure imgf000099_0001
Formula (F-4c) Formula (F-4d) wherein: R1, R1 a, R1 b, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 24; and
each Y3 is independenlty selected from Y3 is OH, NH2, SH or S .
Embodiment 40, The compound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000099_0002
The compound of any one of Embodiments 1 to 39, wherein R1 a is
Figure imgf000099_0003
Figure imgf000099_0004
The compound of any one of Embodiments 1 to 39, wherein R1 b is
Figure imgf000099_0005
Figure imgf000099_0006
The compound of any one of Embodiments 1 to 39, wherein R! is
Figure imgf000099_0007
Embodiment 44. The compound of any one of Embodiments 1 to 39, wherein R1a is
Figure imgf000100_0001
Embodiment 45. The compound of any one of Embodiments 1 to 39, wherein R1 b is
Figure imgf000100_0002
Embodiment 46, The compound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000100_0003
Embodiment 47. The compound of any one of Embodiments 1 to 39, wherein R1 a is
Figure imgf000100_0004
Embodiment 48. The compound of any one of Embodiments 1 to 39, wherein R1 b is
Figure imgf000100_0005
Embodiment 49, The compound of any one of Embodiments 1 to 39, wherein R! is
Figure imgf000100_0006
Embodiment
Figure imgf000100_0007
The compound of any one of Embodiments 1 to 39, wherein R1a is
Figure imgf000100_0008
d of any one of Embodiments 1 to 39, wherein R1b is
Figure imgf000101_0001
Embodiment 52. The compound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000101_0002
Embodiment 53, The compound of any one of Embodiments 1 to 39, wherein R1a is
Figure imgf000101_0006
Embodiment 55. The compound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000101_0003
Embodiment SS, The compound of any one of Embodiments 1 to 39, wherein Ria is
Figure imgf000101_0004
Embodiment 57. The compound of any one of Embodiments 1 to 39, wherein R1b is
Figure imgf000101_0005
ound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000102_0004
Embodiment S9. The compound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000102_0005
ound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000102_0001
ound of any one of Embodiments 1 to 39, wherein R! is
Figure imgf000102_0002
ound of any one of Embodiments 1 to 39 wherein R1 is
Figure imgf000102_0003
one of Embodiments 1 to 39 wherein R1 is
Figure imgf000103_0001
one of Embodiments 1 to 39 wherein R1 is
Figure imgf000103_0002
one of Embodiments 1 to 39 wherein R1 is
Figure imgf000103_0003
one of Embodiments 1 to 39 wherein R1 is
Figure imgf000103_0004
one of Embodiments 1 to 39 wherein R1 is
Figure imgf000103_0005
f any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000103_0006
f any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000103_0007
of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000104_0001
Embodiment 73. The compound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000104_0002
Embodiment 74, The compound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000104_0003
Embodiment 75. The compound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000104_0004
Embodiment 76, The compound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000104_0005
Embodiment 77, The compound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000104_0006
e independently selected from H, -OH and F. y one of Embodiments 1 to 39 wherein R1 is
Figure imgf000105_0001
one of R3, R3a, R5 or R5a is -NHNH2, and the others are independently selected from H, -OH and F.
of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000105_0002
one of R3, R3a, R5 or R5a is -NHNH2, and the others are independently selected from H, -OH and F
of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000105_0003
one of R3, R3a, R5 or R5a is -NHNH2, and the others are independently selected from H, -OH and F.
Embodiment 82, The compound of any one of Embodiments 1 to 39, wherein R! is
Figure imgf000105_0004
the others are independently selected from H, -OH and F.
y one of Embodiments 1 to 39 wherein R1 is
Figure imgf000105_0005
, one of R3, R3a, R5 or R5a is -NHOH, and the others are independently selected from H, -OH and F.
of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000105_0006
, one of R3, R3a, R5 or R5a is -NHOH, and the others are independently selected from H, -OH and F. Embodiment 85. The compound of any one of Embodiments 1 to 39, wherein R1 is
Figure imgf000106_0001
one of R3, R3a, R5 or R5a is -NHGH, and the others are independently selected from H, -OH and F.
Embodiment 86. The compound of any one of Embodiments 1 to 85, wherein:
Y3 is OH, O-, SH or S~, and
Y4 is OH, O , SH or S\
Embodiment 87. The compound of any one of Embodiments 1 to 85, wherein:
Y3 is OH or O , and
Y4 is OH or O .
Embodiment 88. The compound of any one of Embodiments 1 to 85, wherein:
Y3 is SH or S~, and
Y is OH or O .
Embodiment 89. The compound of any one of Embodiments 1 to 85, wherein:
Y3 is OH or O , and
Y is SH or S~.
Figure imgf000106_0002
The compound of any one of Embodiments 1 to 85, wherein:
Y3 is SH or S , and
Y4 is SH or S .
Embodiment 91. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H.
Embodiment 92. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R3 is -NH2, -NHNH2, -NHOH, OH or F.
Embodiment 93. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R3 is -NHNH2, OH or F.
Embodiment 94. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R3 is -OH or F.
Embodiment 95. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R3a is -NH2, -NHNH2, -NHOH, OH or F.
Embodiment 9S, The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R3a is -NHNH2, OH or F.
Embodiment 97, The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R3a is -OH or F. Embodiment 98. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R5 is -NH2, -NHNH2, -NHOH, OH or F.
Embodiment 99. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R5 is -NHNH2, OH or F.
Embodiment 100. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R5 is -OH or F.
Embodiment 101. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R83 is -NHNH2, OH or F.
Embodiment 102. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R83 is -NH2, -NHNH2, -NHOH, OH or F.
Embodiment 103. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein: R8a is -OH or F.
Embodiment 104. The compound of any one of Embodiments 1 to 103, wherein: R8, R9, R8a and R9a are H.
Embodiment 105. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000107_0001
Embodiment 106. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000107_0002
Embodiment 107. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
R2, R2a, R4, R4a, R6, RSa, R7, R7a R8, R8a, R9 and R9a are each H;
R3 is F, and
R3a is F.
Embodiment 108. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000107_0003
R3a is -OH.
Embodiment 109. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
R2, R2a, R4, R4a, R6, R6a, R7, R7a R8, R83, R9 and R9a are each H; R3 is -NHNH2, and
R3a is F.
Embodiment 110. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90. wherein, when present:
are each H:
Figure imgf000108_0001
Embodiment 111. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000108_0002
Embodiment 112. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000108_0003
Embodiment 113. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000108_0004
Embodiment 114. The compound of any one of Embodiments 1 io 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000108_0005
Embodiment 115. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000108_0006
Embodiment 116. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
R2, R2a, R4, R4a, Rs, RSa, R7, R7a R8, R83, R9 and R3a are each H:
R3a is F, and
R5 is F. Embodiment 117. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000109_0001
Embodiment 118. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000109_0002
Embodiment 119. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
R2, R2a, R4, R4a, Rs, R6a, R7, R7a R8, R8a, R9 and R9a are each H;
R3 is F, and
R5 is -NHNH2.
Embodiment 120. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000109_0003
Embodiment 121. The compound of any one of Embodimenis 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000109_0004
Embodiment 122. The compound of any one of Embodimenis 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000109_0005
Embodiment 123. The compound of any one of Embodimenis 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000109_0006
R5a is F.
Embodiment 124. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present: R2, R2a, R4, R4a R6, R6a R7, R7a R8, R8a, R9 and R9a are each H:
R3 is F, and
R5a is -OH
Embodiment 12S. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
R2, R2a, R4, R4a, Rs, RSa, R7, R7a Rs, R8a, R9 and R3a are each H:
R3 is F, and
R5a is F
Embodiment 126. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000110_0001
Embodiment 127. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000110_0002
Embodiment 128. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000110_0003
Embodiment 129. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000110_0004
Embodiment 130. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000110_0005
Embodiment 131. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000110_0006
R5a is -NHNH2.
Embodiment 132. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000111_0001
R5a is F.
Embodiment 133. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000111_0002
Embodiment 134. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
R2, R2a, R4, R4a, Rs, R6a, R7, R7a R8, R8a, R9 and R9a are each H;
R5 is F, and
R5a is F.
Embodiment 135. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000111_0003
Embodiment 136. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000111_0004
Embodiment 137. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000111_0005
Embodiment 138. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000111_0006
Embodiment 139. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000112_0001
Embodiment 140. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000112_0002
Embodiment 141. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000112_0003
Embodiment 142. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
R2, R2a, R4, R4a, R6, R6a, R7, R7a R8, R8a, R9 and R9a are each H;
R3 is F, and
R3a is -NOH.
Embodiment 143. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000112_0004
Embodiment 144. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000112_0005
Embodiment 145. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
R2, R2a, R4, R4a. Rs, RSa. R7, R7a R8, R83, R9 and R3a are each H:
R3 is -NOH, and
R3a is -NOH.
Embodiment 146. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present: R2, R2a, R4, R4a, R6, R6a R7, R7a R8, R8a, R9 and R9a are each H:
R3 is -NON, and
R5 is F.
Embodiment 147. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90. wherein, when present:
are each H:
Figure imgf000113_0001
Embodiment 148. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000113_0002
Embodiment 149. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000113_0003
Embodiment 150. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000113_0004
Embodiment 151. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000113_0005
Embodiment 152. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000113_0006
Embodiment 153. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000113_0007
R5a is OH
Embodiment 154. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000114_0001
Embodiment 155. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000114_0002
Embodiment 15S. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000114_0003
Embodiment 157. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
R2, R2a, R4, R4a, Rs, RSa, R7, R7a R8, RSa, R9 and RSa are each H;
R5 is F, and
R5a is -NOH.
Embodiment 158. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H;
Figure imgf000114_0004
Embodiment 159. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
are each H:
Figure imgf000114_0005
Embodiment 160. The compound of any one of Embodiments 1 to 77 or Embodiments 86 to 90, wherein, when present:
R2, R2a, R4, R4a. Rs, RSa, R7, R7a R8, R83, R9 and R3a are each H:
R5 is -NOH, and
R5a is -NOH. Embodiment 161. A Drug moiety (D) is a compound of Table 1 :
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0004
Embodiment 162. The Drug moiety
Figure imgf000118_0003
Embodiment 163. The Drug moiety
Figure imgf000118_0001
Embodiment 164. The Drug moiety
Figure imgf000118_0002
Embodiment 165. The Drug moiety
Figure imgf000119_0001
Embodiment 166. The Drug moiety
Figure imgf000119_0004
Embodiment 167. The Drug moiety (
Figure imgf000119_0002
Embodiment 168. The Drug moiety
Figure imgf000119_0003
Embodiment 169. The Drug moiety (
Figure imgf000119_0005
Embodiment 176. The Drug moiety (
Figure imgf000119_0006
Embodiment 171. The Drug moiety (
Figure imgf000119_0007
Embodiment 172. The Drug moiety (
Figure imgf000120_0001
Embodiment 173. The Drug moiety
Figure imgf000120_0002
Embodiment 174. The Drug moiety (
Figure imgf000120_0003
Embodiment 17S. The Drug moiety (
Figure imgf000120_0004
Embodiment 176. The Drug moiety (
Figure imgf000120_0005
Figure imgf000120_0006
Figure imgf000120_0007
Embodiment 179. The Drug moiety (
Figure imgf000121_0001
Embodiment 180. The Drug moiety (
Figure imgf000121_0002
Embodiment 181. The Drug moiety (
Figure imgf000121_0003
Embodiment 182. The Drug moiety
Figure imgf000121_0004
Embodiment 183. The Drug moiety (
Figure imgf000121_0005
Embodiment 184. The Drug moiety (
Figure imgf000121_0006
Embodiment 185. The Drug moiety (
Figure imgf000122_0001
Embodiment 188. The Drug moiety
Figure imgf000122_0002
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in (WO2017123657)
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in (WO2017123669)
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Merck & Co (WO2017/027646) Such compounds are listed in Table 2.
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Merck & Go (WO2017/027645). Such compounds are listed in Table 2. Each of the preceding applications are incorporated by reference in their entirety.
Table 2
Figure imgf000122_0003
Figure imgf000123_0002
The following Schemes describe general methods for preparing compounds of Formula (A), Formula (B), Formula (C) and Formula (D). Specific syntheses are described in the Examples.
Starting materials and intermediates are purchased from commercial sources, made from known procedures, or are otherwise illustrated in some cases the order of carrying out the steps of the reaction schemes may be varied to facilitate the reaction or to avoid unwanted reaction products.
General synthesis of compounds of Formula (A) Formula (B) Formula fC) and Formula (D)
Schemes 1 to 8 describe general methods for preparing compounds of Formula (A), Formula
Y? Ye (B), Formula (C) and Formula (D) where: Gi is
Figure imgf000123_0001
, and where Y3 is OH, a, SH or S ; Y4 is OH, O, SH or S ; Y5 is -O- or -S; Y6 is -O- or -S; Y7 is O or S; Y8 is O or S; Y9 is -O- or -S; and YiC -O- or -S.
Scheme 1 describes general methods for preparing compounds of Formula (A). Starting with a modified ribo-nucleoside (1 -1), which includes a hydroxy! at the 3’-0 position, a hydroxyl at the 5’ O-position and a nucieobase (R1a) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected), the hydroxyl at the 5’ O-position is protected using 4,4'- (ch!oro(phenyi)methy!ene)bis(methoxybenzene) (DMTC!) to give ribo-nucieoside (1 -2) Ribo- nucieoside (1-2) is then treated with 2-chloro-4H-benzo[dj[1 ,3,2]dioxaphosphinin-4-one to give ribo-nifdeoside (1 -3) with H-phosphonate functionality at the 3 -0 position. The protected hydroxyl at the 5 -0 position of ribo-nucieoside (1-3) is subsequently deprotected (using dichloroaeetic acid (DCA)) and then treated with a modified ribo-nucieoside (1 -4), which includes a phosphoramidite functionality at 3'-0 position, a DMT protected hydroxyl at the 5 -0 position and a nucleobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected), to give a modified dinucleoside (1-5). The protected hydroxyl at the 5’-0 position of dinucleoside (1-5) is subsequently deprotected (using dichloroaeetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5, 5-dimethyl- 1 ,3,2-dioxaphosphinane 2-oxide (DMOCP) wherein the resulting 5'-hydroxyl group reacts with the 3'-H-phosphonate to give a cyclized product which is then immediately oxidized with t-butyl hydroperoxide to give the cyclic dinucleotide (1-6). Treatment with triethyl-amine, or t-butylamine and methy!amine, plus fluoride anion, in case siiyl protection is used, provides the 3’3 cyclic dinucleotide (1 -7).
Alternatively, the modified dinucleoside (1-5) is immediately thiolated with (E)-N,N- dimethyl-N’-(3-thioxo-3H-1 ,2 ,4-d ithiazol-5-yl) formimidamide (DDTT) to give a modified dinucleoside (1-8), The protected hydroxyl at the 5 -0 position of dinucleoside (1 -8) is subsequently deprotected (using dichloroaeetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5,5-dimethyi-1 ,3,2-dioxaphosphinane 2-oxide (DMOCP) wherein the resulting 5'- hydroxyl group reacts with the 3'-H-phospbonate to give a cyclized product which is then thiolated with 3H-benzo[c][1 ,2]dithioi-3-one to provide the cyclic dinucleotide (1-9). Treatment with triethyi-amine, or t-butylamine and methyiamine, plus fluoride anion, in case silyi protection is used, provides the 3’3’ cyclic dinucleotide diphosphorothioate (1-10).
Scheme 1
Figure imgf000125_0001
Scheme 2 describes general methods for preparing compounds of Formula (B). Starting with a modified ribo-nucleoside (2-1). which includes a hydroxyl at the 3 -0 position, a hydroxyl at the 5 O-position and a nucleobase (R1a) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boo, Fmoc, Cbz, benzoyl, benzyi amine, trity!amine or tosylamide protected), the hydroxy! at the 5’ O-position is protected using 4,4 - (ch!oro(phenyi)methylene)bis(methoxybenzene) (DMTCi) to give ribo-nucleoside (2-2) Ribo- nucleoside (2-2) is then treated with 2-chloro-4H-benzo[d][1 ,3,2]dioxaphosphinin-4-one to give ribo-nucleoside (2-3) with H-phosphonate functionality at the 3 -0 position. The protected hydroxyl at the 5 -0 position of ribo-nucleoside (2-3) is subsequently deprotected (using dichloroacetic acid (DCA)) and then treated with a modified ribo-nucleoside (2-4), which includes a phosphoramidite functionality at 2 -0 position, a DMT protected hydroxyl at the 5 -0 position and a nucleobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected), to give a modified dinucleoside (2-5). The protected hydroxy! at the 5’-0 position of dinucleoside (2-5) is subsequently deprotected (using dichloroacetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5, 5-dimethyl- 1 ,3,2-dioxaphosphinane 2-oxide (DMGCP) wherein the resulting 5’-hydroxyl group reacts with the 3'-H-phosphonate to give a cyclized product which is then immediately oxidized with t-butyl hydroperoxide to give the cyclic dinucleotide (2-6). Treatment with triethyl-amine, or t-buty!amine and methylamine, plus fluoride anion, in case silyl protection is used, provides the 3’2’ cyclic dinucleotide (2-7).
Alternatively, the modified dinucleoside (2-5) is immediately thiolated with (E)-N,N- dirnethyl-N'-(3-thioxo-3H-1 ,2 ,4-d ithiazol-5-yl) formimidamide (DDTT) to give a modified dinucleoside (2-8). The protected hydroxyl at the 5’-Q position of dinucleoside (2-8) is subsequently deprotected (using diehloroacetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5, 5-dimethyl- 1 ,3,2-dioxaphosphinane 2-oxide (DMGCP) wherein the resulting 5'- hydroxyl group reacts with the 3'-H-phosphonate to give a cyciized product which is then thiolated with 3H-benzo[c][1 ,2]dithioi-3-one to provide the cyclic dinucleotide (2-9). Treatment with triethyl-amine, or t-butyiamine and methylamine, plus fluoride anion, in case silyl protection is used, provides the 3’2’ cyclic dinucleotide diphosphorothioate (2-10).
Scheme 2
Figure imgf000126_0001
Scheme 3 describes general methods for preparing compounds of Formula (C). Starting with a modified ribo-nucleoside (3-1), which includes a hydroxyl at the 2’-0 position, a hydroxyl at the 5’ O-position and a nucieobase (R1a) which has an appropriately protected amino group o a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, trltyiamine or tosy!amide protected), the hydroxyl at the 5’ O-position is protected using 4, 4'- (ch!oro(phenyi)methy!ene)bis(methoxybenzene) (DMTC!) to give ribo-nucleoside (3-2) Ribo- nucleoside (3-2) is then treated with 2-ehloro-4H-benzo[d][1 ,3,2]dioxaphosphinin-4-one to give ribo-niideoside (3-3) with H-phosphonate functionaiity at the 2'-0 position. The protected hydroxyl at the 5 -0 position of ribo-nucleoside (3-3) is subsequently deprotected (using dichioroaeetic acid (DCA)) and then treated with a modified ribo-nucleoside (3-4), which inciudes a phosphoramidite functionaiity at 3 -0 position, a DMT protected hydroxy! at the 5 -0 position and a nucieobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected), to give a modified dinucleoside (3-5). The protected hydroxyl at the 5’-0 position of dinucleoside (3-5) is subsequentiy deprotected (using dichioroaeetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5, 5-dimethyl- 1 ,3,2-dioxaphosphinane 2-oxide (DMOCP) wherein the resulting 5'-hydroxyl group reacts with the 2'-H-phosphonate to give a cyciized product which is then immediately oxidized with t-butyl hydroperoxide to give the cyclic dinucleotide (3-6). Treatment with triethyl-amine, or t-butylamine and methylamine, plus fluoride anion, in case siiyl protection is used, provides the 2’3 cyclic dinucleotide (3-7).
Alternatively, the modified dinucleoside (3-5) is immediately thiofated with (E)-N,N- dimethyl-N'-(3-thioxo-3H-1 ,2 ,4-d ithiazol-5-yl) formimidamide (DDTT) to give a modified dinuc!eoside (3-8), The protected hydroxy! at the 5 -0 position of dinucleoside (3-8) is subsequently deprotected (using dichioroaeetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5, 5-dimethyl- 1 ,3,2-dioxaphosphinane 2-oxide (DMOCP) wherein the resulting 5'- hydroxyl group reacts with the 2'-H-phospbonate to give a cyciized product which is then thioiated with 3H-benzo[c][1 ,2]dithiol-3-one to provide the cyclic dinucleotide (3-9). Treatment with triethyl-amine, or t-butylamine and methylamine, plus fluoride anion, in case silyi protection is used, provides the 2’3’ cyclic dinucleotide diphosphorothloate (3-10).
Scheme 3
Figure imgf000128_0001
Scheme 4 describes general methods for preparing compounds of Formula (D). Starting with a modified ribo-nucleoside (4-1), which includes a hydroxyl at the 2’-0 position, a hydroxyl at the 5’ O-position and a nucieobase (R1a) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected), the hydroxyl at the 5’ Q-positlon is protected using 4,4'- (chioro(phenyl)methyiene)bisfmeiboxybenzene) (DMTCI) to give ribo-nucieoside (4-2). Ribo- nucleoside (4-2) is then treated with 2-chloro-4H-benzo[d][1 ,3,2]dioxaphosphinin-4-one to give ribo-nucieoside (4-3) with H-phosphonate functionality at the 2 -0 position. The protected hydroxyl at the 5 -0 position of ribo-nucieoside (4-3) is subsequently deprotected (using dichloroacetic acid (DGA)) and then treated with a modified ribo-nucleoside (4-4), which includes a phosphoramidite functionality at 2 -0 position, a DMT protected hydroxyl at the 5 -0 position and a nucieobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected), to give a modified dinucieoside (4-5). The protected hydroxyl at the S’-O position of dinucleoside (4-5) Is subsequently deprotected (using dichloroacetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5,5-dimethyl-1 ,3,2-dioxaphosphinane 2-oxide (DMOCP) wherein the resulting 5'-hydroxyl group reacts with the 2'-H-phosphonate to give a cydized product which is then immediately oxidized with t-butyl hydroperoxide to give the cyclic dinucleotide (4-6). Treatment with triethyl-amine, or t-buty!amine and methylamine, plus fluoride anion, in case silyl protection is used, provides the 2’2’ cyclic dinucleotide (4-7).
Alternatively, the modified dinucleoside (4-5) is immediately thiolated with (E)-N,N- dimethyl-N'-(3-thioxo-3H-1 ,2 ,4-d ithiazol-5-yl) formimidamide (DDTT) to give a modified dinucleoside (4-8), The protected hydroxyl at the 5’-Q position of dinucleoside (4-8) is subsequently deprotected (using dichloroacetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5, 5-dimethyl- 1 ,3,2-dioxaphosphinane 2-oxide (DMGCP) wherein the resulting 5'- hydroxyl group reacts with the 2'-H-phosphonate to give a cyclized product which is then thiolated with 3H-benzo[c][1 ,2]dithioi-3-one to provide the cyclic dinucleotide (4-9). Treatment with triethyl-amine, or t-butylamine and methylamine, plus fluoride anion, in case silyl protection is used, provides the 2’2’ cyclic dinucleotide diphosphorothioate (4-10).
Scheme 4
Figure imgf000129_0001
Scheme 5 describes general methods for preparing compounds of Formula (A). Starting with a modified ribo-nucleoside (5-1), which inciudes a hydroxyl at the S’-O position, a hydroxyl at the 5’ O-position and a 6-chloro-9H-purine at the 1-position, the hydroxyl at the 5’ O-position is protected using 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (DMTCI) to give ribo- nucleoside (5-2). Ribo-nucleoside (5-2) is then treated with 2-chloro-4H- benzo[d][1 ,3,2]dioxaphosphinin-4-one to give ribo-niideoside (5-3) with H-phosphonate functionality at the 3’-G position. The protected hydroxyl at the S’-O position of ribo-nucleoside (1 -3) is subsequently deprotected (using dichioroacetic acid (DCA)) and then treated with a modified ribo-nucieoside (5-4), which includes a phosphoramidite functionality at the 3'-G position, a DMT protected hydroxyl at the 5’-Q position and a 4,5-dibydro-9H-purin-8-amine, in which the amino group has been protected with a benzoyl moiety, to give a modified dinucleoside (5-5). The protected hydroxyl at the 5’-Q position of dinucleoside (5-5) is subsequently deprotected (using dichioroacetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5,5-dimethyl-1 ,3,2-dioxaphosphinane 2-oxide (DMOCP) wherein the resulting 5‘- hydroxyl group reacts with the 3'-H-phosphonate to give a cyclized product which is then immediately oxidized with t-butyi hydroperoxide to give the cyclic dinucleotide (5-8). Treatment with hydrazine, followed by treatment with triethyl-amine, or t-butylamine and methyiamine, plus fluoride anion, in case siiyi protection is used, provides the 3’3’ cyclic dinucleotide (5-7) which has a 6-hydrazinyi-9H-purine group.
Alternatively, the modified dinucleoside (5-5) is immediately thioiated with (E)-N,N- dimethyi-N'-(3-thioxo-3H-1 ,2 ,4-d ithiazol-5-yl) formimidamide (DDTT) to give a modified dinucleoside (5-8). The protected hydroxyl at the S’-O position of dinucleoside (5-8) is subsequently deprotected (using dichioroacetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5,5-dimethyl-1 ,3,2-dioxaphosphinane 2-oxide (DMOCP), wherein the resulting 5'-hydroxyi group reacts with the 3'-H-phosphonate to give a cyclized product which is then thioiated with 3H-benzo[c][1 ,2]dithioi-3-one to provide the cyclic dinucleotide (5-9). ). Treatment with hydrazine, followed by treatment with triethyl-amine, o t-butylamine and methyiamine, plus fluoride anion, in case siiyi protection is used, provides the 3’3’ cyclic dinucieotide
dspbosphorothioate (5-10).
Scheme 5
Figure imgf000131_0001
Scheme 8 describes general methods for preparing compounds of Formula (B) Starting with a modified ribo-nucleoside (6-1), which inciudes a hydroxyl at the S’-O position, a hydroxyl at the 5’ O-position and 6-chloro-9H-purine at the 1 -position, the hydroxyl at the 5’ O-position is protected using 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (DMTCl) to give ribo- nucleoside (6-2). Ribo-nucleoside (6-2) is then treated with 2-chloro-4H- benzo[d][1 ,3,2]dioxaphosphinin-4-one to give ribo-nucleoside (6-3) with H-phosphonate functionality at the 3 -0 position. The protected hydroxyl at the 5’-G position of ribo-nucleoside (6-3) is subsequently deprotected (using dichioroacetic acid (DCA)) and then treated with a modified ribo-nucleoside (6-4), which includes a phosphoramidite functionality at the 2'-0 position, a DMT protected hydroxyl at the S’-O position and a 4,5-dihydro-9H-purin-6-amine, in which the amino group has been protected with a benzoyl moiety, to give a modified dinucieoside (6-5). The protected hydroxyl at the S’-O position of dinucleoside (6-5) is subsequently deprotected (using dichioroacetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5,5-dimethyi-1 ,3,2-dioxaphosphinane 2-oxide (DMOCP) wherein the resulting 5'- bydroxyi group reacts with the 3'-H~phospbonaie to give a cyclized product which is then immediately oxidized with t-butyl hydroperoxide to give the cyclic dinucleoiide (6-6). Treatment with hydrazine, followed by treatment with triethyl-amine, or t-butyiamine and methyla ine, plus fluoride anion, in case si!yi protection is used, provides the 3’2’ cyclic dinucleotide (8-7} which has a 6-hydrazinyl-9H-purine group.
Alternatively, the modified dinucleoside (6-5) is immediately thiolated with (E)-N,N- dimethyl-N'-(3-thioxo-3H-1 ,2,4-dithiazol-5-yi) formimidamide (DDTT) to give a modified dinucleoside (6-8). The protected hydroxyl at the 5’-Q position of dinucleoside (6-8) is subsequently deprotected (using dichloroacetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5,5-dimethy!-1 ,3,2-dioxaphosphinane 2-oxide (DMGCP) wherein the resulting 5 - hydroxyl group reacts with the 3'-H-phosphonate to give a cyciized product which is then thiolated with 3H-benzo[c][1 ,2]dithiol-3-one to provide the cyclic dinucleotide (6-9). Treatment with hydrazine, followed by treatment with triethyl-amine, or t-butylamine and methylamine, plus fluoride anion, in case silyl protection is used, provides the 3’2’ cyclic dinucieotide
diphosphorothioate (6-10) which has a 6-hydraziny!-9H~purine group.
Scheme 6
Figure imgf000132_0001
Scheme 7 describes genera! methods for preparing compounds of Formula (C). Starting with a modified ribo-nucleoside (7-1), which includes a hydroxyl at the 2’-G position, a hydroxyl at the 5’ O-position and 6-chlora-9H-purine at the 1-position, the hydroxyl at the 5’ O-position is protected using 4,4'-(chloro(pheny!)methyiene)bis(methoxybenzene) (DMTCi) to give ribo- nucleoside (7-2). Ribo-nucleoside (7-2) is then treated with 2-chloro-4H- benzo[dj[1 ,3,2]dioxaphosphinin-4-one to give ribo-nucleoside (7-3) with H-phosphonate functionality at the 2‘-G position. The protected hydroxyl at the 5’-G position of ribo-nucleoside (7-3) is subsequently deprotected (using dichloroacetic acid (DCA)) and then treated with a modified ribo-nucleoside (7-4), which includes a phosphoramidite functionality at 3'~G position, a DMT protected hydroxyl at the 5’-G position and a 4,5-dihydro-9H-purin-6-amine, in which the amino group has been protected with a benzoyl moiety, to give a modified dinucieoside (7-5). The protected hydroxyl at the 5’-Q position of dinucieoside (7-5) is subsequently deprotected (using dichloroacetic acid (DCA)) and then reacted with the coupling agent 2-chioro-5,5- dimethyl-1 ,3,2-dioxaphosphinane 2-oxide (DMOCP) wherein the resulting 5'-hydroxyl group reacts with the 2’-H-phosphonate to give a cyclized product which is then immediately oxidized with t-butyl hydroperoxide to give the cyclic dinucleotide (7-6). Treatment with hydrazine, followed by treatment with triethyl-amine, or t-buty!amine and metbyiamine, plus fluoride anion, in case silyl protection is used, provides the 2’3’ cyclic dinucieotide (7-7) which has a 6- hydrazinyi-9H-purine group.
Alternatively, the modified dinucieoside 7-5) is immediately thiolated with (E)-N,N- dimethyl-N’-(3-thioxo-3H-1 ,2,4-dithiazol-5-yi) formimidamide (DDTT) to give a modified dinucieoside (7-8), The protected hydroxyl at the 5’-G position of dinucieoside (7-8) is subsequently deprotected (using dichloroacetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5,5-dimethyi-1 ,3,2-dioxaphosphinane 2-oxide (DMOCP) wherein the resulting 5'- hydroxyl group reacts with the 2'-H-phosphonate to give a cyclized product which is then thiolated with 3H-benzo[c][1 ,2]dithiol-3-one to provide the cyclic dinucieotide (7-9). Treatment with hydrazine, followed by treatment with triethyl-amine, or t-butylamine and methylamine, plus fluoride anion, in case silyl protection is used, provides the 2’3’ cyclic dinucieotide
diphosphorothioate (7-10) which has a 6~hydrazinyi-9H-purine group.
Scheme 7
Figure imgf000134_0001
Scheme 8 describes general methods for preparing compounds of Formula (D). Starting with a modified ribo-nucleoside (8-1), which includes a hydroxyl at the 2’-Q position, a hydroxyl at the 5’ O-position and 6~ch!oro~9H-purine at the 1-position, the hydroxyl at the 5’ O-position is protected using 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (DMTC!) to give ribo- nucleoside (8-2). Ribo-nucleoside (8-2) is then treated with 2-chloro-4H- benzo[d][1 ,3,2]dioxaphosphinin-4-one to give ribo-nucleoside (8-3) with H-phosphonate functionality at the 2’-G position. The protected hydroxyl at the S’-O position of ribo-nucleoside (8-3) is subsequently deprotected (using dlchioroacetic acid (DCA)) and then treated with a modified ribo-nucleoside (8-4), which includes a phosphoramidite functionality at 2'-G position, a DMT protected hydroxyl at the 5’-0 position and a 4,5-dihydro-9H-purin-6-amine, in which the amino group has been protected with a benzoyl moiety, to give a modified dinucleoside (8-5). The proiecied hydroxyl at the 5’-Q position of dinucleoside (8-5) is subsequently deproiecied (using dichloroacetic acid (DCA)) and then reacted with the coupling agent 2-chioro-5,5- dimethyl-1 ,3,2-dioxaphosphinane 2-oxide (DMOCP) wherein the resulting 5'-hydroxy! group reacts with the 2'-H-phosphonate to give a cyciized product which is then immediately oxidized with t-butyi hydroperoxide to give the cyclic dinucleotide (8-6). Treatment with hydrazine,
1 followed by treatment with triethyl-amine, or t-butylamine and methylamine, plus fluoride anion, in case silyi protection is used, provides the 2’2’ cyclic dinucleotide (8-7) which has a 6- bydrazinyi-9H-purine group.
Alternatively, the modified dinucleoside (8-5) is immediately thiolated with (E)-N,N- dimethyl-N'-(3 thioxo-3H-1 ,2,4-dithiazo!-5-yi) formimidamide (DDTT) to give a modified diniideoside (4-8), The protected hydroxyl at the 5’-Q position of dinucleoside (8-8) is subsequently deprotected (using diehloroacetic acid (DCA)) and then reacted with the coupling agent 2-chloro-5,5-dimethyi-1 ,3,2-dioxaphosphinane 2-oxide (DMOCP) wherein the resulting 5 - hydroxyl group reacts with the 2'-H-phosphonate to give a cyclized product which is then thiolated with 3H-benzo[c][1 ,2]dithioi-3-one to provide the cyclic dinucleotide (8-9). Treatment with hydrazine, followed by treatment with triethyl-amine, or t-butylamine and methylamine, plus fluoride anion, in case silyi protection is used, provides the 2’2’ cyclic dinucieotide
diphosphorothioate (8-10) which has a 6-hydrazinyl-9H-purine group.
Scheme 8
Figure imgf000135_0001
General synthesis of compounds of Formula (A) Formula (B), Formula (C) and Formula (D) Schemes 9 to 18 describe general methods for preparing compounds of Formula (A), Formula (B), Formula (C) and Formula (D) where:
Figure imgf000136_0001
or S ; Y5 is -NH-; Ye is -NH-; Y? is O or S: Y8 is O or S; Y9 is -O-; and Y is -O-.
Scheme 9 depicts an example synthesis of cyclic dinucieotide phosphoramidates that inciude an amino linkage bonded to the 3' position of each ribose moiety. Starting with a modified ribo-nucleoside (9-1), which includes an amino moiety at the 3’ position, a hydroxyl at the 5’ G-position and a nucieobase (R1a) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, triiylamine or tosylamide protected), the amine at the 3’ position is protected using trityi chloride (TrCI) in the presence of triethylamine (TEA) to give ribo-nucleoside (9-2). Ribo-nucleoside (9-2) is then treated with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to give phosphoramidite (9-3). Subsequent water or hydrogen sulfide treatment results in phosphonate (9-4a) or phosphonothioate (9-4b), respectively. Modified ribo-nucleoside (9-5), which includes an amino moiety at the 3’ position, a hydroxyl at the 5’ O-position and a nucieobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected)can then be combined with either of compounds (9-4a) or (9-4b) with triethylamine in carbon tetrachloride to give either phosphoramidate (9-6a) or phosphoramidothioate (9-6b), respectively. Further treatment of phosphoramidate (9-6a) or phosphoramidothioate (9-6b) with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to gives the corresponding phosphoramidite (9-7a) or (9-7b). Sequential treatment of phosphoramidite (9-7a) with water and dichloroacetic acid yields phosphonate (9-8a), while sequential treatment of phosphoramidite (9-7b) with hydrogen sulfide and dichloroacetic acid yields
phosphonothioate (9-8b). Phosphonate (9~8a) or phosphonothioate (9-8b) are then taken up in triethylamine and carbon tetrachloride to enable cyclization to produce cyclic phosphoramidates (9-9a) and (9~9b), respectively. Treatment of phosphoramidates (9-9a) or (9-9b) with methylamine and triethylamine-hydrogen fluoride complex, results in cyclic dinucieotide phosphoramidates (9-10a) or (9-19b), in which the nucieobase (R1a) and nucieobase (R1) have also been deprotected.
Scheme 9
Figure imgf000137_0001
Scheme 10 depicts an example synthesis of cyclic dinucleoiide phosphoramidates that include an amino linkage bonded to the 3’ position of one ribose moiety and an amino linkage bonded to the 2' position of the other ribose moiety. Starting with a modified ribo- nucieoside (10-1), which includes an amino moiety at the 3’ position, a hydroxyl at the 5’ exposition and a nucleobase (R18) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, trityiamine or tosylamide protected), the amine at the 3’ position is protected using trity! chloride (TrCi) in the presence of triethylamine (TEA) to give ribo-nucleoside (10-2). Ribo-nucleoside (10-2) is then treated with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trif!uoracetic acid to give phosphoramidite (10-3). Subsequent water or hydrogen sulfide treatment results in phosphonate (10-4a) or phosphonothioate (10-4b), respectively. Modified ribo-nucleoside (10- SI, which includes an amino moiety at the 2’ position, a hydroxyl at the 5’ G-position and a nucleobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, trityiamine or tosylamide protected)can then be combined with either of compounds (10-4a) or (10-4b) with triethylamine in carbon tetrachloride to give either phosphoramidate f10-8a) or phosphoramidothioate (1G-6b), respectively. Further treatment of phosphoramidate (10~6a) or phosphoramidothioate (1 Q~6b) with 3-((bis(diisopropyiamino)phosphanyl)oxy)propaneniirile, pyridine and trifluoracetic acid to gives the corresponding phosphoramidite (10~7a) or (10-7b) Sequential treatment of phosphoramidite (10-7a) with water and dichioroacetic acid yields phospbonate (10-8a), while sequential treatment of phosphoramidite (10-7b) with hydrogen sulfide and dichioroacetic acid yields phosphonothioate (10-8b). Phospbonate (10-8a) or phosphonothioate (10-8b) are then taken up in iriethylamine and carbon tetrachloride to enable cyc!ization to produce cyclic phosphoramidates (10-9a) and (19~9b), respectively. Treatment of phosphoramidates (10-9a) or (18-9b) with methylamine and triethylamine-hydrogen fluoride complex, results in cyclic dinucleotide phosphoramidates (10-1 Da) or (10-10b), in which the nucieobase (R13) and nucleobase (R1) have also been deproiecied.
Figure imgf000138_0001
Scheme 11 depicts an example synthesis of cyclic dinucleotide phosphoramidates that include an a ino linkage bonded to the 2’ position of one ribose moiety and an amino linkage bonded to the 3’ position of the other ribose moiety. Starting with a modified ri bo- nucleoside (11-1), which includes an amino moiety at the 2’ position, a hydroxyl at the 5’ exposition and a nucieobase (R1a) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, trity!amine or tosy!amide protected), the amine at the 3 position is protected using trityi chloride (TrCI) in the presence of triethylamine (TEA) to give ribo-nucleoside (11-2). Ribo-nucleoside (11 -2) is then treated with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifiuoracetic acid to give phosphoramidite (11 -3). Subsequent water or hydrogen sulfide treatment results in phosphonate (11 -4a) or phosphonothioate (11 -4b), respectively. Modified ribo-nuc!eoside (11- 5), which includes an amino moiety at the 3 position, a hydroxyl at the 5’ O-position and a nu eobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected)can then be combined with either of compounds (11 -4a) or (11 -4b) with triethyiamine in carbon tetrachloride to give either phospboramidate (11 -6a) or phosphoramidothioate (11 -6b), respectively. Further treatment of phospboramidate (11 -6a) or phosphoramidothioate (11 -6b) with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to gives the corresponding phosphoramidite (11~7a) or (11 -7b). Sequential treatment of
phosphoramidite (11 -7a) with water and dicbioroaceiic acid yields phosphonate (11 ~8a), while sequential treatment of phosphoramidite (11 -7b) with hydrogen sulfide and dichloroacetic acid yields phosphonothioate (11 -8b) Phosphonate (11 ~8a) or phosphonothioate (11 -8b) are then taken up in triethyiamine and carbon tetrachloride to enable cyclization to produce cyclic phosphoramidates (11 -9a) and (11~9b), respectively. Treatment of phosphoramidates (11 -9a) or (11 -9b) with methy!amine and trietbylamine-hydrogen fluoride complex, results in cyclic dinucleotide phosphoramidates (11 -10a) or (11 -10b), in which the nudeobase (R1a) and nudeobase (R1) have also been deprotected.
Scheme 11
Figure imgf000139_0001
Scheme 12 depicts an example synthesis of cyclic dinucleotide phosphoramidates that include an amino linkage bonded to the 2’ position of each ribose moiety. Starting with a modified ribo-nucleoside (12-1), which includes an amino moiety at the 2’ position, a hydroxyl at the 5’ G-position and a nucleobase (R1a) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected), the amine at the 3’ position is protected using trityl chloride (TrCI) in the presence of triethy!amine (TEA) to give ribo-nucleoside (12-2). Ribo-nucleoside (12-2) is then treated with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to give phosphoramidite (12-3) Subsequent water or hydrogen sulfide treatment results in phosphonate (12-4a) or phosphonothioate (12~4b), respectively. Modified ribo-nucleoside (12- 5), which includes an amino moiety at the 2’ position, a hydroxyl at the 5' O-position and a nucleobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e.g Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected)can then be combined with either of compounds (12-4a) or (12~4b) with triethylamine in carbon tetrachloride to give either phosphoramidate (12-6a) or phosphoramidothioate (12-6b), respectively. Further treatment of phosphoramidate (12-6a) or phosphoramidothioate (12-6b) with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to gives the corresponding phosphoramidite (12-7a) or (12-7b). Sequential treatment of phosphoramidite (12-7a) with water and dichloroacetic acid yields phosphonate (12-Sa), while sequential treatment of phosphoramidite (12-7b) with hydrogen sulfide and dichloroacetic acid yields phosphonothioate (12-Bb). Phosphonate (12-Sa) or phosphonothioate (12-Bb) are then taken up in triethylamine and carbon tetrachloride to enable cyclization to produce cyclic phosphoramidates (12~9a) and (12-9b), respectively. Treatment of phosphoramidates (12-9a) or (12~9b) with methylamine and triethylamine-hydrogen fluoride complex, results in cyclic dinucieotide phosphoramidates (12-10a) or (12-10b), in which the nucleobase (R1a) and nucleobase (R1) have also been deprotected.
Scheme 12
Figure imgf000141_0001
Scheme 13 depicts an example synthesis of cyclic dinucleotide phosphoramldates
that include an amino linkage bonded to the 3' position of each ribose moiety. Starting with a modified ribo-nucleoside (13-1), which includes an amino moiety at the 3’ position, a hydroxyl at the 5’ O-position and S-ch!oro-9H~purine at the 1 -position, the amine at the 3’ position is protected using trityl chloride (TrC!) in the presence of triethylamine (TEA) to give ribo- nucleoside (13-2). Ribo-nucleoside (13-2) is then treated with 3-
((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to give phosphoramidite (13-3) Subsequent water or hydrogen sulfide treatment results in
phosphonate (13-4a) or phosphonothioate (13~4b), respectively Modified ribo-nucleoside (13- 5), which includes an amino moiety at the 3’ position, a hydroxy! at the 5’ O-position and a 4,5- dihydro-9H-purin-0-amine, in which the amino group has been protected with a benzoyl moiety, can then be combined with either of compounds (13-4a) or (13-4b) with triethylamine in carbon tetrachloride to give either phosphoramidate (13-6a) or phosphoramidothioate (13-6b), respectively. Further treatment of phosphoramidate (13-6a) or phosphoramidothioate (13-6b) with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to gives the corresponding phosphoramidite (13-7a) or (13-7b). Sequential treatment of phosphoramidite (13-7a) with water and dichioroacetie acid yields phosphonate (13-8a), while sequential treatment of phosphoramidite (13-7b) with hydrogen sulfide and dichioroacetie acid yields phosphonothioate (13-8b). Phosphonate (13-8a) or phosphonothioate (13-8b) are then taken up in triethylamine and carbon tetrachloride to enable cyciization to produce cyclic phosphoramldates (13-9a) and (13-9b), respectively. Treatment of phosphoramldates (13-9a) or (13-Sb) with hydrazine, followed by treatment with methylamine and triethylamine-hydrogen fluoride complex, results in cyclic dinucleotide phosphoramldates (13-10a) or (13-1 Ob).
Figure imgf000142_0001
Scheme 14 depicts an example synthesis of cyclic dinucleotide phosphoramldates
that include an amino linkage bonded to the 3’ position of one ribose moiety and an amino linkage bonded to the 2' position of the other ribose moiety. Starting with a modified ribo- nucleoside (14-1), which includes an amino moiety at the 3’ position, a hydroxyl at the 5’ exposition and 8-cbioro-9H~purine at the 1 -position, the amine at the 3’ position is protected using trityl chloride (TrCI) in the presence of trieihy!amine (TEA) to give ribo-nucieoside (14-2). Ribo- nucleoside (14-2) is then treated with 3-((bis(diisopropylamina)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to give phosphoramidite (14-3). Subsequent water or hydrogen sulfide treatment results in
phosphonate (14-4a) or phosphonothioate {14-4b|, respectively. Modified ribo-nuc!eoside (14- SI, which includes an amino moiety at the 2 position, a hydroxyl at the 5’ O-position and a 4,5- dihydro-9H-purin-6-amine, in which the amino group has been protected with a benzoyl moiety, can then be combined with either of compounds (14-4a) or (14-4b) with triethylamine in carbon tetrachloride to give either phosphoramidate (14-6a) or phosphoramidothioate (14-6b), respectively. Further treatment of phosphoramidate (14~6a) or phosphoramidothioate (14~6b) with 3-((bis(diisopropylamino)phosphanyl)oxy)propaneniirile, pyridine and thfluoracetic acid to gives the corresponding phosphoramidite (14~7a) or (14-7b). Sequential treatment of phosphoramidite (14-7a) with water and dichloroaceiic acid yields phosphonate (14~8a), while sequential treatment of phosphoramidite (14-7b) with hydrogen sulfide and dichioroacetic acid yields phosphonothioate (14-8b) Phosphonate f14~Ba) or phosphonothioate (14-8b) are then taken up in triethylamine and carbon tetrachloride to enable cyclization to produce cyclic phosphoramidates (14-Sa) and (14-9fo), respectively. Treatment of phosphoramidates (14-Sa) or (14-9b) with hydrazine, followed by treatment with methylamine and triethylamine-hydrogen fluoride complex, results in cyclic dinucleotide phosphoramidates (14-10a> or (14-10b.
Scheme 14
Figure imgf000144_0001
Scheme 15 depicts an example synthesis of cyclic dinucleotide phosphoramidates that include an amino linkage bonded to the 2’ position of one ribose moiety and an amino linkage bonded to the 3' position of the other ribose moiety. Starting with a modified ribo- nucleoside (15-1}, which includes an amino moiety at the 2’ position, a hydroxyl at the 5’ exposition and 6-chioro-9H-purine at the 1 -position, the amine at the 3 position is protected using trityi chloride (TrCI) in the presence of trieihyiamine (TEA) to give ribo-nucieoside (15-2). Ribo- nucieoside (15-2) is then treated with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to give phosphoramidite (15-3). Subsequent water or hydrogen sulfide treatment results in
phosphonate (15~4a) or phosphonothioate (15-4b), respectively. Modified ribo-nuc!eoside (15- SI, which includes an amino moiety at the 3’ position, a hydroxyl at the 5’ O-position and a 4,5- dihydro-9H-purin-6-amine, in which the amino group has been protected with a benzoyl moiety, can then be combined with either of compounds (15~4a) or (15-4b) with trieihyiamine in carbon tetrachloride to give either phospboramidate (15-6a> or phosphoramidothioate (15-8b), respectively. Further treatment of phospboramidate (1 S-6a) or phosphoramidothioate (1 S-6b) with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to gives the corresponding phosphoramidite (15-7a) or (15-7fo). Sequential treatment of phosphoramidite (15-7a) with water and dichloroacetic acid yields phospbonate (15~8a), while sequential treatment of phosphoramidite (15-7b) with hydrogen sulfide and dichloroacetic acid yields phosphonothioate (15-8b>. Phosphonate {1S-8a| or phosphonothioate (15-8b> are then taken up in triethylamine and carbon tetrachloride to enable cyclization to produce cyclic phosphoramidates (15~9a) and (15~9b), respectively. Treatment of phosphoramidates (15~9a) or (1S~9b) with hydrazine, followed by treatment with methylamine and triethylamine-hydrogen fluoride complex, results in cyclic dinucleoiide phosphoramidates (15-iOa) or (1 S~1 Ob).
Scheme 15
Figure imgf000145_0001
Scheme 16 depicts an example synthesis of cyclic dinucleotide phosphoramidates that include an amino linkage bonded to the 2’ position of each ribose moiety. Starting with a modified ribo-nucleoside (16-1), which includes an amino moiety at the 2’ position, a hydroxyl at the 5’ G-position and 6-ch!oro-9H-purine at the 1 -position, the amine at the 3’ position is protected using trityl chloride (TrC!) in the presence of triethylamine (TEA) to give ribo- nucleoside (16-2). Ribo-nucleoside (16-2) is then treated with 3-
((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifiuoracetic acid to give phosphoramidite (16-3). Subsequent water or hydrogen sulfide treatment results in
phosphonate (16-4a) or phosphonothioate (16~4b), respectively. Modified ribo-nucleoside (16- SI, which includes an amino moiety at the 2’ position, a hydroxyl at the 5' O-position and a 4,5- dihydro-9H-purin-6-amine, in which the amino group has been protected with a benzoyl moiety, can then be combined with either of compounds (16-4a) or (16~4b) with triethylamine in carbon tetrachloride to give either phosphoramidate (1S-6a) or phosphoramidothioate (16-6b), respectively. Further treatment of phosphoramidate (16-6a) or phosphoramidothioate (16-6b) with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifiuoracetic acid to gives the corresponding phosphoramidite (16-7a) or (16-7b). Sequential treatment of phosphoramidite (16-7a) with water and dichloroacetic acid yields phosphonate (16-8a), while sequential treatment of phosphoramidite (16-7b) with hydrogen sulfide and dichloroacetic acid yields phosphonothioate (16-Bb). Phosphonate (16-8a) or phosphonothioate (16-Bb) are then taken up in triethylamine and carbon tetrachloride to enable cyclization to produce cyclic phosphoramidates (16-Sa) and (16-9b), respectively. Treatment of phosphoramidates (16-Sa) or (16-9b) with hydrazine, followed by treatment with methylamine and triethy!amine-hydrogen fluoride complex, results in cyclic dinucleotide phosphoramidates (16-1 Qa) or (16-16b).
Scheme 16
Figure imgf000147_0002
General synthesis of compounds of Formula (A), Formula (B). Formula (C) and Formula (D)
Schemes 17 to 24 describe general methods for preparing compounds of Formula (A), Formula (B), Formula (C) and Formula (D) where:
Figure imgf000147_0001
SH or S ; Y£, is -O-; YB is -O-; Y7 is O or S; Y8 is O or S; Y8 is -NH-; and Yio is -NH-.
Scheme 17 depicts an example synthesis of cyclic dinucieotide phosphoramidates that include an amino linkage bonded to the 5’ position of each ribose moiety and an ether linkage at the 3’ O-positions of each ribose moiety. Starting with a modified ribo-nucieoside (17- 1 ), which includes a hydroxyl at the 3’ O-position, an amino moiety at the 5’ position and a nucleobase (Ria) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boo, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosy!amide protected), the amine at the 5’ position is protected using trityi chloride (TrCI) in the presence of triethyiamine (TEA) to give ribo-nucleoside (17-2). Ribo-nucleoside (17-2) is then treated with 3- ((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifiuoracetic acid to give phosphoramidite (17-3). Subsequent water or hydrogen sulfide treatment results in
phosphonate (17-4a) or phosphonothioate (17-4b), respectively. Modified ribo-nucleoside (17- SI, which includes a hydroxyl at the 3’ O-position, an amino moiety at the 5’ position and a nucleobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boo, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected) can then be combined with either of compounds (17-4a) or (17~4b) with triethyiamine in carbon tetrachloride to give either phosphoramidate (1 -6a) or phosphoramidothioate (17-6b), respectively. Further treatment of phosphoramidate (17~6a) or phosphoramidothioate (17-6b) with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifiuoracetic acid to gives the corresponding phosphoramidite (17-7a) or (17-7b). Sequential treatment of phosphoramidite (17-7a) with water and dichloroacetic acid yields phosphonate (1 -Ba), while sequential treatment of phosphoramidite (17-7b> with hydrogen sulfide and dichloroacetic acid yields phosphonothioate (17-8b). Phosphonate (17-Sa) or phosphonothioate (17-8b) are then taken up in triethyiamine and carbon tetrachloride to enable cyclization to produce cyclic phosphoramidates (17-Sa) and (17-9b), respectively. Treatment of phosphoramidates (17-Sa) or (17-9b) with methylamine and triethylamine-hydrogen fluoride complex, results in cyclic dinucieotide phosphoramidates (17-10a) or (17-10b), in which the nucleobase (R1a) and nucleobase (R1) have also been deprotected.
Scheme 17
Figure imgf000149_0001
Scheme 18 depicts an example synthesis of cyclic dinucleotide phosphoramidates that include an amino linkage bonded to the 5’ position of each ribose moiety and an ether linkage at the 3’ O-positions of one ribose moiety and an ether linkage at the 2’ O-position of the other ribose moiety. Starting with a modified ribo-nucleoside (18-1), which includes a hydroxyi at the 3’ O-position, an amino moiety at the 5’ position and a nucleobase (R1a) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected), the amine at the 5’ position is protected using trityi chloride (TrCi) in the presence of triethyiamine (TEA) to give ribo- nucleoside (18-2). Ribo-nudeoside (18-2) is then treated with 3-
((bis(diisopropyiamino)phosphanyl)oxy)propanenitrile, pyridine and trif!uoracetic acid to give phosphoramidite (18-3). Subsequent water or hydrogen sulfide treatment results in
pbosphonate (18~4a) or phosphonothioate (18-4b), respectively. Modified ribo-nudeoside (18- 5), which includes a hydroxyl at the 2’ O-position, an amino moiety at the 5’ position and a nucleobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boe, Fmoe, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected) can then be combined with either of compounds (18-4a) or (18-4b) with iriethyiamine in carbon tetrachloride to give either phosphoramidate (18-6a) or phosphoramidothioaie (18-6b), respectively. Further treatment of phosphoramidate (18-6a) or phosphoramidothioate (18-6b) with 3-((bis(diisopropylamino)phosphanyi)oxy)propanenitrile, pyridine and trifluoracetic acid to gives the corresponding phosphoramidite (18~7a) or (18-7b). Sequential treatment of phosphoramidite (18-7a) with water and dichioroacetic acid yields phosphonate (18~8a), while sequential treatment of phospboramidite (18-7b> with hydrogen sulfide and dichioroacetic acid yields phosphonothioate (18-8b>. Phosphonate (1 S-8a) or phosphonothioate (18-8b> are then taken up in triethylamine and carbon tetrachloride to enable eyclization to produce cyclic phosphoramidates (18-9a) and (18-9b), respectively. Treatment of phosphoramidaies (18-9a) or (18-Sb) with methylamine and triethylamine-hydrogen fluoride complex, results in cyclic dinucleotide phosphoramidates (18-10a) or {18-10b), in which the nucieobase (R13) and nucieobase (R1) have also been deprotected.
Scheme 18
Figure imgf000150_0001
Scheme 19 depicts an example synthesis of cyclic dinucleotide phosphoramidates that include an amino linkage bonded to the 5' position of each rlbose moiety and an ether linkage at the 2’ O-positlons of one ribose moiety and an ether linkage at the 3’ O-position of the other ribose moiety. Starting with a modified ribo-nucleoside (19-1), which includes a hydroxyl at the 2’ O-position, an amino moiety at the 5’ position and a nucieobase (R1a) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, trityiamine or tosyiamide protected), the amine at the 5’ position is protected using trityl chloride (TrC!) in the presence of triethylamine (TEA) to give ribo- nucleoside (19-2). Ribo-nucleoside (19-2) is then treated with 3-
((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to give phospboramidite (19-3) Subsequent water or hydrogen sulfide treatment results in
phosphonate {19-4a| or phosphonothioate (19-4b), respectively. Modified ribo-nucleoside {19- SI, which includes a hydroxyl at the 3 O-position, an amino moiety at the 5’ position and a nu eobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e g. Boe, Fmoe, Cbz, benzoyl, benzyl amine, trity!amine or tosy!amide protected) can then be combined with either of compounds (19-4a) or (19-4b) with triethyiamine in carbon tetrachloride to give either phosphoramidate (1S-6a) or phosphoramidothioaie (19-6b>, respectively. Further treatment of phosphoramidate (19~6a) or phosphora idothioate (19-6b> with 3-((bis(diisopropylamino)phosphanyi)oxy)propanenitriie, pyridine and trifluoracetic acid to gives the corresponding phosphoramidite (19-7a) or (1S-7fo). Sequential treatment of phosphoramidite (19-7a) with water and dicbloroacetic acid yields phosphonate (19~8a), while sequential treatment of phosphoramidite (19~7b) with hydrogen sulfide and dichioroacetic acid yields phosphonothioate (19-8b). Phosphonate (19~8a) or phosphonothioate (19-8b) are then taken up in triethyiamine and carbon tetrachloride to enable eyc!ization to produce cyclic phosphoramidates (19-9a) and (19~9b), respectively. Treatment of phosphoramidates f19-9a) or (19-9b) with methyiamine and triethyia ine-hydrogen fluoride complex, results in cyclic dinucleotide phosphoramidates (19-10a) or (19~10b), in which the nudeobase (R1a) and nudeobase (R1) have also been deprotected.
Figure imgf000151_0001
Scheme 20 depicts an example synthesis of cyclic dinucleotide phosphoramidates that include an amino linkage bonded to the 5‘ position of each ribose moiety and an ether linkage at the 2’ O-positions of each ribose moiety. Starting with a modified ribo-nucleoside (20- 1 ), which includes a hydroxyl at the 2’ O-position, an a ino moiety at the 5’ position and a nucleobase (Ria) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boo, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosy!amide protected), the amine at the 5’ position is protected using trityi chloride (TrC!) in the presence of triethy!amine (TEA) to give ribo-nucleoside (20-2). Ribo-nucleoside (2Q-2) is then treated with 3- ((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifiuoracetic acid to give phosphoramidite (20-3). Subsequent water or hydrogen sulfide treatment results in
phosphonate (20-4a) or phosphonothioate (20-4b), respectively. Modified ribo-nucleoside (20- SI, which includes a hydroxyl at the 2’ O-position, an amino moiety at the 5’ position and a nucleobase (R1) which has an appropriately protected amino group or a protected hydrazine group (e.g. Boc, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected) can then be combined with either of compounds (20-4a) or (2Q~4b) with triethyiamine in carbon tetrachloride to give either phosphoramidate (20-6a) or phosphoramidothioaie (20-6b), respectively. Further treatment of phosphoramidate (20~6a) or phosphoramidothloate (20-6b) with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifiuoracetic acid to gives the corresponding phosphoramidite (20-7a) or (20-7b). Sequential treatment of phosphoramidite (20-7a) with water and dichloroacetic acid yields phosphonate (20-Ba), while sequential treatment of phosphoramidite (20-7b> with hydrogen sulfide and dichloroacetic acid yields phosphonothioate (20-8b). Phosphonate (20-Sa) or phosphonothioate (20-8b) are then taken up in triethyiamine and carbon tetrachloride to enable cyclization to produce cyclic phosphoramidates (20-Sa) and (20-9b), respectively. Treatment of phosphoramidates (20-Sa) or (20-9b) with methylamine and triethylamine-hydrogen fluoride complex, results in cyclic dinucieotide phosphoramidates (20-10a) or (20-10b), in which the nucleobase (R1a) and nucleobase (R1) have also been deprotected.
Scheme 20
Figure imgf000153_0001
Scheme 21 depicts an example synthesis of cyclic dinucleotide phosphoramidates that include an amino linkage bonded to the 5’ position of each ribose moiety and an ether linkage at the 3’ O-positions of each ribose moiety. Starting with a modified ribo-nucieoside (Sil l, which includes a hydroxyl at the 3’ O-position, an amino moiety at the 5’ position and 6- chloro-9H-purine at the 1 -position, the amine at the 5’ position is protected using trityl chloride (TrCi) in the presence of triethy!amine (TEA) to give ribo-nucieoside (21-2). Ribo-nucieoside (21-2) is then treated with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to give phosphoramidite (21-3). Subsequent water or hydrogen sulfide treatment results in phosphonate (21~4a) or phosphonothioate (21 -4b), respectively. Modified ribo-nucieoside (21-5), which includes a hydroxy! at the 3’ O-position, an amino moiety at the 5’ position and a 4,5-dihydro-9H-purin-6-amine, in which the amino group has been protected with a benzoyl moiety, can then be combined with either of compounds (21 -4a) or (21 -4b) with triethylamine in carbon tetrachloride to give either phosphoramidate (21 -6a) or
phosphoramidothioate (21 -6b), respectively. Further treatment of phosphoramidate (21 -6a) or phosphoramldothioate (21 -6b) with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to gives the corresponding phosphoramidite (21 -7a) or (21 -7b). Sequential treatment of phosphoramidite (21 -7a) with water and dichloroacetic acid yields phosphonate (21 -Sa), while sequential treatment of phosphoramidite (21 -7b) with hydrogen sulfide and dichloroacetic acid yields phosphonothioate (21 ~8b). Phosphonate (21 -Sa) or phosphonothioate (21 -8b) are then taken up in triethylamine and carbon tetrachloride to enable cyclization to produce cyclic phosphoramidates (21 -9a) and (21 -9b), respectively. Treatment of phosphoramidates (21 -9a) or (21 -Sb) with hydrazine, followed by treatment with methylamine and triethyiamine-hydrogen fluoride complex, rests Its in cyclic dinucleotide phosphoramidates (21 -10a) or (21 -10b).
Figure imgf000154_0001
Scheme 22 depicts an example synthesis of cyclic dinucleoiide pbosphoramidates that include an amino linkage bonded to the 5' position of each ribose moiety and an ether linkage at the 3’ O-positions of one ribose moiety and an ether linkage at the 2’ O-position of the other ribose moiety. Starting with a modified ribo-nucleoside (22-1), which includes a hydroxyl at the 3’ O-position, an amino moiety at the 5’ position and 6-chloro-9H-purine at the 1 -position, the amine at the 5’ position is protected using trityi chioride (TrCi) in the presence of
triethylamine (TEA) to give ribo-nucleoside (22-2). Ribo-nucleoside (22-2) is then treated with 3- ((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to give phosphoramidite (22-3) Subsequent water or hydrogen sulfide treatment results in
phosphonate (22-4a) or phosphonothioate (22~4b), respectively Modified ribo-nucleoside (22- SI, which includes a hydroxyl at the 2’ O-position, an a ino moiety at the 5’ position and a 4,5- dihydro-9H-purin-0-amine, in which the amino group has been protected with a benzoyl moiety, can then be combined with either of compounds (22-4a) or (22-4b) with triethyiamine in carbon tetrachloride to give either phospboramidate (22-6a) or phosphoramidothioate (22-8b), respectively. Further treatment of phospboramidate (22-8a) or phosphoramidothioate (22-6b) with 3-((bis(diisopropy!amino)phosphanyi)oxy)propanenitrile, pyridine and trifluoracetic acid to gives the corresponding pbosphoramidite (22-7a) or (22-7b). Sequential treatment of phosphoramidite (22-7a) with water and dicbloroaceisc acid yields phosphonate (22~8a), while sequential treatment of phosphoramidite (22-7b) with hydrogen sulfide and dichloroacetic acid yields phosphonothioate (22-8b). Phosphonate (22~8a) or phosphonothioate (22-8b) are then taken up in triethyiamine and carbon tetrachloride to enable cyclization to produce cyclic phosphoramidates (22-9a) and (22~9b), respectively. Treatment of phosphoramidates (22-9a) or (22-9b) with hydrazine, followed by treatment with methylamine and triethylamine-hydrogen fluoride complex, results in cyclic dinucleotide phosphoramidates (22-10a) or (22-10b).
Scheme 22
Figure imgf000155_0001
Scheme 23 depicts an example synthesis of cyclic dinucleotide phosphoramidates that include an a ino linkage bonded to the 5' position of each ribose moiety and an ether linkage at the 2’ O-positions of one ribose moiety and an ether linkage at the 3’ O-position of the other ribose moiety. Starting with a modified ribo-nucleoside (23-1), which includes a hydroxyl at the 2’ O-position, an amino moiety at the 5’ position and 6-chioro-9H-purine at the 1 -position, the amine at the 5’ position is protected using trityi chioride (TrCi) in the presence of triethyiamine (TEA) to give ribo-nucleoside (23-2). Ribo-nucleoside (23-2) is then treated with 3- ((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifiuoracetic acid to give pbosphoramidite (23-3). Subsequent water or hydrogen sulfide treatment results in
phosphonate (23-4a) or phosphonothioate (23~4b), respectively. Modified ribo-nucleoside (23- SI, which includes a hydroxyl at the 3’ O-position, an amino moiety at the 5’ position and a 4,5- dihydro-9H-purin-6-amine, in which the amino group has been protected with a benzoyl moiety, can then be combined with either of compounds (23-4a) or (23~4b) with triethyiamine in carbon tetrachloride to give either phosphoramidate (23-8a) or phosphoramidothioate (23-6b), respectively. Further treatment of phosphoramidate (23~6a) or phosphoramidothioate (23~6b) with 3-((bis(diisopropylamino)phosphanyl)oxy)propaneniirile, pyridine and trifiuoracetic acid to gives the corresponding phosphoramidite (23~7a) or (23-7b). Sequential treatment of
phosphoramidite (23~7a) with water and dichloroacetic acid yields phosphonate (23~8a), while sequential treatment of phosphoramidite (23-7b) with hydrogen sulfide and dichloroacetic acid yields phosphonothioate (23-8b). Phosphonate (23-8a) or phosphonothioate (23-8b) are then taken up in triethyiamine and carbon tetrachloride to enable cyclization to produce cyclic phosphoramidates (23-Sa) and (23-9fo), respectively. Treatment of phosphoramidates (23-Sa) or (23-9b) with hydrazine, followed by treatment with methylamine and triethy!amine-hydrogen fluoride complex, results in cyclic dinucleotide phosphoramidates (23-10a) or (23-10b).
Scheme 23
Figure imgf000157_0001
Scheme 24 depicts an example synthesis of cyclic dlnucleoilde phosphoramidates that include an amino linkage bonded to the 5’ position of each ribose moiety and an ether linkage at the 2’ O-positions of each ribose moiety. Starting with a modified ribo-nucleoside (24- 1 ), which includes a hydroxyl at the 2’ O-position, an amino moiety at the 5’ position and 6- chloro-9H-purine at the 1 -position, the amine at the 5’ position is protected using trityl chloride (TrCi) in the presence of triethylamine (TEA) to give ribo-nucleoside (24-2). Ribo-nucleoside (24-2) is then treated with 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine and trifluoracetic acid to give phosphoramidite (24-3). Subsequent water or hydrogen sulfide treatment results in phosphonate (24~4a) or phosphonothioate (24-4b), respectively. Modified ribo-nucleoside (24-5), which includes a hydroxyl at the 2’ O-position, an amino moiety at the 5’ position and a 4,5-dihydro-9H-purin-6-amine, in which the amino group has been protected with a benzoyl moiety, can then be combined with either of compounds (24-4a) or (24-4b) with triethyiamine in carbon tetrachloride to give either phosphoramidate (24-6a) or
phosphoramidothloate (24~6b), respectively. Further treatment of phosphoramidate (24-6a) or phosphoramidothloate (24~6b) with 3-((bis(diisopropyiamino)phosphanyl)oxy)propanenitriie, pyridine and trifluoracetic acid to gives the corresponding phosphoramidite (24-7a) or (24~7b). Sequential treatment of phosphoramidite (24-7a) with water and dichioroacetic acid yields phosphonate (24-Sa), while sequential treatment of phosphoramidite (24-7b) with hydrogen sulfide and dicbioroacetic acid yieids phosphonothioate (24-8b) Phosphonate (24-8a) or phosphonothioate (24-8b) are then taken up in iriethylamine and carbon tetrachloride to enabie cyclization to produce cyclic pbosphoramidates (24-9a) and (24-9b), respectively. Treatment of phosphoramidates (24-9a) or (24-Sb) with hydrazine, followed by treatment with methylamine and triethyiamine-hydrogen fluoride complex, results in cyclic dinucleotide phosphoramidates (24-1 Da) or (24-1 Ob).
Figure imgf000158_0001
In Schemes 1 to 24, the substituents R1 , R1a, R2, R2a, R3, R3a, R4, R4a, R5, R5a, R7 and R7a are as defined herein. Also, one of skill would readily recognize that the 6-chloro-9H-purine and 4,5- dihydro-9H-purin-6-amine can be replaced with a group selected from R1 of Embodiment 23 wherein the amino or hydrazine moieties have been replaced with a halogen (e.g. Ci) or the amino or hydrazine moieties are protected (e.g. Boo, Fmoc, Cbz, benzoyl, benzyl amine, tritylamine or tosylamide protected).
Corresponding bydroxy!amine containing compounds can be synthesized using the methods described in Schemes 1 -24, wherein hydrazine is replaced with hydroxyiamine. By way of example the synthesis of compounds CDN-9 and CDN-10 in Exampie 3-3 given below. With respect to the embodiments wherein Gi is selected from
Figure imgf000159_0001
Figure imgf000159_0002
are as defined herein, and compounds of Formula (E and Formula (F), example syntheses can be found in
US2015158886, WO2017011622, WG2Q17004499 and W02007070598, the disclosures of which are herein incorporated by references with regards to the synthetic schemes disclosed.
The activity of exemplary Drug moieties as tested in a hSTING wt assay are given in Tabie 3A (see below for description of assays). hST!MG reporter assay:
A human STING cDNA expression plasmid (mammalian gene collection clone
BCQ47779 with position 232 mutated to Arg) was cotransfected with a 5x!SRE-!uciferase reporter plasmid into HEK-293T cells using FuGENE6 transfection reagent. Cells were plated into a 384 well plate (14,000 cells/well in 40 uL of culture medium) and allowed to attach for 12 hours. Compounds were added to wells (serial 1 :3 dilutions starting at 100 uM compound concentration in triplicate) and incubated for 12 hours. Bright-glo reagent (Promega, 12 uL) was added to each well and plates were read in luminescence mode on an Envision plate reader.
The ECso values (triplicates) were calculated by performing logistic regression on measured dose-response curves. Data were curve fitted with the following formula to obtain
ECso values:
Figure imgf000159_0003
where Y is the observed value, Bottom is the lowest observed value, Top is the highest observed value, and the Hill coefficient gives the largest absolute value of the slope of the curve. EC5o value characterizes the concentration of the compound for a 50% activation, i.e., Yjx-Ecso = (Top+Bottom)/2. The curve fitting is carried out by a curve fitting program using
Matlab. The maximum activity was compared to 2’,3’-cGAMP (100 mM) treated cells to determine %Efficacy. Values reported are the average of two independent experiments.
Figure imgf000160_0001
Figure imgf000161_0002
THP1 -Dua! assay:
THRΊ -Dual cells were purchased from Invlvogen. THRΊ -Dual cells were plated In 384 well plates (22,500 cells/well In 45 pL of culture medium) and compounds were added to wells (serial 1 :3 dilutions starting at. 20 mM compound concentration In triplicate) and Incubated for 48 hours Lucia reporter signal was read out by adding Quantliue reagent from Invlvogen (20 pL) followed by reading on an Envision plate reader. The fold change over background was calculated and normalized to the fold-change induced by 2’3’-cGAMP at 50 uM. Plates were run in triplicate.
The EG so values (triplicates) were calculated by performing logistic regression on measured dose-response curves. Data were curve fitted with the following formula to obtain
ECso values:
Figure imgf000161_0001
where Y is the observed value, Bottom is the lowest observed value, Top is the highest observed value, and the Hill coefficient gives the largest absolute value of the slope of the curve. ECso value characterizes the concentration of the compound for a 50% activation, i.e., Y|X=EC5O = (Top+Bottom)/2. The curve fitting is carried out by a curve fitting program using Matiab. The maximum activity was compared to 2’,3’-cGAMP (50 mM) treated cells to determine % Efficacy.
The activity of exemplary Drug moieties as tested in the THP1 Dual assay are given in
Table 3B.
THP1 -Dua!/STI G-KO assay
Guide RNA (gRNA) oiigo (TCCATCCATCCCGTGTCCCA) for human STING was cloned into Lentivirus vector pNGx_LV_gGG3 and transduced into THP1 -Duai_Cas9 cells. FACS sorted single clones were then plated into 384 well plate (22,500 ceils/well in 45 uL of culture medium) and compounds were added to wells (serial 1 :3 dilutions starting at 20 mM compound concentration in triplicate) and incubated for 48 hours. Each single well also contained 500 THP1-Dual parental cells as supporting cells. After 30 days 1 ug/ml puromycin was added to each well to eliminate supporting ceils. Each individual THP1-Dual/STING-KO clone was tested using western blotting and NGS to confirm loss of STING expression and non-sense nucleotide insertion/deletion in both alleles. Six confirmed clones were then pooled and tested with cGAMP, and any of compounds CDN-1 to CDN-20, using the methods described in the THP1- Dua! assay above.
The activity of exemplary Drug moieties as tested in the THP1 Dual/STING-KO assay are given in Table 3B.
Table 3B
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000164_0002
Figure imgf000164_0001
Linker
As used herein, a“linker” is any chemical moiety that is capable of linking an antibody, antibody fragment (e.g., antigen binding fragments) or functional equivalent to another moiety, such as a drug moiety, (e.g. a cyclic dinucleotide or cyclic dinucleoside), which binds to Stimulator of Interferon Genes (STING) receptor. Linkers of the immunoconjugates of the invention may be a cieavabie linker or a non-cleavable linker.
Linkers of the immunoconjugates of the invention may comprise one or more cleavage elements and in certain embodiments the linkers of the immunoconjugates of the invention comprise two or more cleavage elements, wherein each cleavage element is independently selected from a seif-immoiative spacer and a group that is susceptible to cleavage (such as a group which is susceptible to acid-induced cleavage, peptidase-induced cleavage, esterase- induced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage).
In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxyiic acid based linker.
Acid-labile linkers are linkers cieavabie at acidic pH. For example, certain intracellular compartments, such as endosomes and lysosomes, have an acidic pH (pH 4-5), and provide conditions suitable to cleave acid-labile linkers.
Some linkers can be cleaved by peptidases, i.e., peptidase cieavabie linkers. Only certain peptides are readily cleaved Inside or outside cells, see e.g., Trout et a!., 79 Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umernoto et al. 43 Int. J Cancer, 677-684 (1989). Furthermore, peptides are composed of a-amino acids and peptidic bonds, which chemicaily are amide bonds between the carboxylate of one amino acid and the amino group of a second amino acid. Other amide bonds, such as the bond between a carboxylate and the e-amino group of lysine, are understood not to be peptidic bonds and are considered non-cleavable.
Some linkers can be cleaved by esterases, i.e., esterase cleavable linkers. Again, only certain esters can be cleaved by esterases present inside or outside of cells. Esters are formed by the condensation of a carboxylic acid and an alcohol. Simple esters are esters produced with simple alcohols, such as aliphatic alcohols, and small cyclic and small aromatic alcohols.
Cleavable linkers, such as those containing a hydrazone, a disulfide, and a dipeptide (e.g. Vai-Cit), are well known in the art, and can be used. See, e.g., Ducry, et al., Bioconiugate
Chem.. vol. 21 , 5-13 (2010).
In addition, cleavable linkers containing a giucuronidase-cieavabie moiety, are well known in the art, and can be used. See, e.g., Ducry, et al., Bioconiugate Chem., vol. 21 , 5-13 (2010).
For the immunoeonjugates of the invention comprising a cleavable linker, the linker is substantially stable in vivo until the immunoconjugate binds to or enters a ceil, at which point either intracellular enzymes or intracellular chemical conditions (pH, reduction capacity) cleave the linker to free the Drug moiety.
Procharged linkers are derived from charged cross-linking reagents that retain their charge after incorporation into an antibody drug conjugate. Examples of procharged linkers can be found in US 2009/0274713.
The linker (L) can be attached to the antibody, antigen binding fragment or their functional equivalent at any suitable available position on the antibody, antigen binding fragment or their functional equivalent: typically, linker (L) is attached to an available amino nitrogen atom (i.e., a primary or secondary amine, rather than an amide) or a hydroxylic oxygen atom, or to an available sulfhydryl, such as on a cysteine.
The linker (L) of the immunoeonjugates of the invention can be divalent, where the linker is used to link only one drug moiety per linker to an antibody, antigen binding moiety or functional equivalent, or the linker (L) of the immunoeonjugates of the invention can be trivaient and is able to link two drug moieties per linker to an antibody, antigen binding moiety or functional equivalent. In addition, the linker (L) of in the immunoeonjugates of the invention can also polyvalent and is able to link multiple drug moieties per linker to an antibody, antigen binding moiety or functional equivalent.
The linker (L) of the immunoeonjugates of the invention is a linking moiety comprising one or more linker components. Some preferred linkers and linker components are described herein.
A linker component of linker (L) of the immunoeonjugates of the invention can be, for example,
a) an alky!ene group: -(CH2)n- (where in this instance n is 1-18); b) an alkenyiene group;
c) an alkynylene group;
d) an alkenyl group;
e) an alkynyl group;
f) an ethylene glycol unit: -CH2CH2G-;
g) an polyethylene glycol unit: (-CH2CH20-)X (where x in this instance is 2-20);
h) -0-;
i) -S-;
j) a carbonyl: -C(=0)-;
k) an ester: -C(=0)-0- or -0-C(=0)-;
L) a carbonate: -0C(=0)0-;
rn) an amine: -NH-;
n) an amides: -C(=0)-NH-, -NH-C(=0)- or -C(=0)N(Ci^alkyl)-;
0) a carbamate: -0C(=0)NH- or -NHC(=0)0-;
p) a urea: -NHC(=0)NH-;
q) an alkylene substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide, phosphate and phosphonate); r) an alkenyiene substituted with one or more groups independently selected from
carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide, phosphate and phosphonate);
s) an alkynylene substituted with one or more groups independently selected from
carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide, phosphate and phosphonate);
t) a Ci-Cioalkylene in which one or more methylene groups is replace by one or more -
S-, -NH- or -O- moieties;
u) a ring systems having two available points of attachment such as a divalent ring
selected from phenyl (including 1 ,2- 1 ,3- and 1 ,4- di-substituted phenyls), a C5-C3 heteroaryl, a C3-C8 cycloalkyl (including 1 ,1 -disubstituted cyclopropyl, cyclobutyl, cyclopentyi or cyclohexyl, and 1 ,4-disubstituted cyclohexyl), and a C4-C8 heterocycioaikyl;
v) a residue of an amino acid selected from alanine (Ala), cysteine (Cys), aspartic acid
(Asp), glutamic acid (Giu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (lie), lysine (Lys), leucine (Leu), ethionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citruliine (Cit), norvaiine (Nva), norleucune (Nle), selenocysieine (Sec), pyrrolysine (Pyi), homoserine, homocysteine, and desmethyl pyrrolysine; a combination of 2 or more amino acid residues where each residue is independently selected from a residue of an amino acid selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoieucine (lie), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citruliine (Cit), norvaiine (Nva), norleucune (Hie), seienocysteine (Sec), pyrroiysine (Pyl), homoserine, homocysteine, and desmethyl pyrroiysine, for example Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit;
Figure imgf000167_0001
and
w) a self-immolative spacer, wherein the self-immolative spacer comprises one or more protecting (triggering) groups which are susceptible to acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, giycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage Non-limiting examples of such self-immolative spacer include:
Figure imgf000167_0002
where:
PG is a protecting (triggering) group;
Xa is O, NH or S;
Xb is O, NH, NCH3 or S;
Xc is O or NH;
Ya is CH2, O or NH; Yb is a bond, CH2, O or NH, and
LG is a leaving group such as a Drug moiety (D) of the
immunoconjugates of the invention.
Additional non-limiting examples of such self-immolative spacers are described n Angew. Cbem. Ini. Ed. 2015, 54, 7492 - 7509.
By way of example only, certain self-immolative spacers used in the immunoconjugates of the invention are:
Figure imgf000168_0001
!n addition, a linker component can be a chemical moiety which is readily formed by reaction between two reactive groups. Non-limiting examples of such chemical moieties are given in Table 4.
Table 4
Figure imgf000168_0002
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
where: R32 in Table 4 is H, C alkyl, phenyl, pyrimidine or pyridine; R35 in Table 4 is H, Ci- 6aikyl, phenyl or Ci-4aikyl substituted with 1 to 3 -OH groups; each R36 in Table 4 is independently selected from H, Ci 6alkyl, fluoro, benzyloxy substituted with -C(=0)0H, benzyl sifbstituted with -C(=0)0H, Ci-4aikoxy substituted with -C(=0)0H and Ci-4alkyl substituted with -G(=0)0H; R37 in Table 4 is independently selected from H, phenyl and pyridine; n in Table 4 is 0, 1 , 2 or 3; R13 in Table 4 is H or methyl; R50 in Table 4 is H or nitro; and R14 in Table 4 Is H, -GH3 or phenyl.
In some embodiments, a linker component of linker, L, of the immunoconjugates of the invention is a group formed upon reaction of a reactive functional group with a side chain of an amino acid residue commonly used for conjugation, e.g., the thiol of a cysteine residue, or the free -NH2 of a lysine residue. In other embodiments a linker component of linker, L, of the immunoconjugates of the invention is a group formed upon reaction of a reactive functional group with a side chain of an amino acid residue of an non-naturaily occuring amino acid, such as para-acetyl Phe or para-azido-Phe. In other embodiments a linker component of linker, L, of the immunoconjugates of the invention is a group formed upon reaction of a reactive functional group with a side chain of an amino acid residue which has been engineered into the antibody, antigen binding fragment or their functional equivalent, e.g. the thiol of a cysteine residue, the hydroxyl of a serine residue, the pyrroline of a pyrrolysine residue or the pyrroline of a desmethyl pyrrolysine residue engineered into an antibody. See e.g., Ou, et al., PNAS 108(26), 10437-42 (2011).
A linker component formed by reaction with the thiol of a cysteine residue of the antibody, antigen binding fragment or their functional equivalent includes, but are not limited to
Figure imgf000173_0001
linker components formed by reaction with the amine of a lysine residue of the antibody, antigen binding fragment or their o functional equivalent include, but are not limited to,
Figure imgf000173_0002
R
Figure imgf000173_0003
emu wherein each p is 1-10, and each R is independently H or C alkyl (preferably methyl). A linker component formed by reaction with a pyrrolysine residue or desmethy! pyrrolysine
residue includes, but are not limited to,
Figure imgf000174_0008
Figure imgf000174_0001
A , wherein R13 is H or methyl, and R14 is H, methyl or phenyl.
In some embodiments, a linker component of linker, L, of immunoconjugat.es of the invention
O
Figure imgf000174_0002
which is formed upon reaction of a hydroxylamine and
Figure imgf000174_0003
moiety, where o
,
the "' Ά moiety is formed by reduction of an interchain disulfide bridge of the antibody and re-bridging using a 1 ,3-dibaloacetone (e.g. 1 ,3-dichioroaeetone, 1 ,3-dibromoacetone, 1 ,3- diiodoacetone) and bissulfonate esters of 1 , 3-dihydroxyacetone In some embodiments, a linker
component of linker, L, of immunoconjugates of the invention
Figure imgf000174_0004
which is formed o o
. .
Figure imgf000174_0006
S 5
upon reaction of a hydrazine and a X moiety, where the
Figure imgf000174_0005
moiety is formed by reduction of an interchain disulfide bridge of the antibody and re-bridging using a 1 ,3- dihaioacetone (e.g. 1 ,3-dichloroacetone, 1 ,3-dibromoacetone, 1 ,3-diiodoacetone) and bissulfonate esters of 1 , 3-dihydroxyacetone.
In some embodiments, a linker component of linker, L, of immunoconjugates of the invention is selected from the groups shown in Table 5 below:
Figure imgf000174_0007
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0002
The linker, L, in the immunoconjugates of the invention typically contain two or more linker components, which may be selected for convenience in assembly of the conjugate, or they may be selected to impact properties of the conjugate.
Linkers of the immunoconjugates of the invention comprise one or more cleavage elements and in certain embodiments the linkers of the immunoconjugates of the invention comprise two or more cleavage elements. In certain embodiments one of the cleavage elements is directly attached to a Drug moiety which, after the cleavage process, allows for release of a Drug moiety which does not comprise a fragment of the cleaved linker. By way of example, the Linker-Drug Moiety (-(L-(D)n)), wherein m is 1 , of the immunoconjugates of the invention is designed to have one of the following structures:
Figure imgf000177_0001
wherein:
Lc is a linker component and each Lc is independently selected from a linker component described herein;
x is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 and 20;
y is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 and 20;
p is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10;
D is a Drug moiety described herein;
and each cleavage element (CE) is independently selected from a self-immolative spacer and a group that is susceptible to cleavage (such as a group which is susceptible to acid-induced cleavage, peptidase-induced cleavage, esterase- induced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage).
In one embodiment of the immunconjugates disclosed herein the Linker (L) ot the Linker- Drug Moiety (-(L-(D)m)), wherein m is 1 , has a structure selected from:
Figure imgf000178_0001
wherein:
Lc is a linker component and each Lc is independently selected from a linker component as disclosed herein;
x is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 18, 17, 18, 19 and 20: y is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 18, 17, 18, 19 and 20: p is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10:
and each cleavage element (CE) is independently selected from a seif-immo!aiive spacer and a group that is susceptible to cleavage selected from acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage.
Certain aspects and examples of the linkers and linker components of the
immunoconjugates of the invention are provided in the following listing of enumerated embodiments it will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention. Embodiment 187. A linker component of linker, L, or combinations thereof, of the
Figure imgf000178_0003
Figure imgf000178_0002
l??
Figure imgf000179_0001
Embodiment 188. A linker, L, of -(L-(D)m) selected from:
-**XEC(=0)(CH2)mXGM-; -**XEC(=0)(CH2)mG(CH2)mXCM-; -MXEC(=0)XiX2C(=0)(CH2)mXCM-: - i*XEC(=0)XiX2C(=0)(CH2)-0(CH2)mXGM-; -*iXEC(=0)XlX2C(=G)(CH2)m0(CH2)mG(=0)XCM-; - i*XEC(=0)X4G(=0)NH(CH2)mNHC(=G)(CH2)m0(CH2)mXCM-; - 'J,'A'XEC(=0)X5C(=0)(CH2)mNHC(=0)(CH2)mXCM-; - 'AAXEC(=0)(CH2)mNHC(=Q)XiX2C(=G)(CH2)mXCM-; - **XEC (= O) (C H2) m N H C (= 0)X2C (= O) (C H 2) mXCIV1- ; - **XEC(=Q)Q(CH2)mNHC(=Q)XiX2C(=Q)(CH2)mXCM-; -
**XEC (= O) (C H2) m N H C (= O) ((C H 2) mO) » (C H2) mXCM- ; -iiXEC(=G)0(CH2)mNHC(=0)(CH2)mXCM-; - **XEC(=0)G(CH2)mNHC(=0)(CH2)m0(CH2)rpXCM-; -
*AXEC(=0)G(CH2)mNHC(=0)XiX2C(=G)(CH2)m0(CH2)mXCM-; -**X£X2C(=0)(CH2)mXCM-; - **XEC(=0)X5C(=0)(CH2)mNHC(=G)(CH2)rr CM-; - **XEC(=0)(CH2)rnNHC(=0)XiX2C(=0)(CH2)mXCM-; - **XEC(=0)0(CH2)mNHC(=0)XiX2C(=0)(CH2)mO(CH2)mC(=0)XCM-: - **XEC(=0)0(CH2)mNHC(=0)X4C(=G)NH(CH2)mNHC(=0)(CH2)m0(CH2)rrXCM-; - ^EC(=0)0((CH2)m0)n(CH2)mNHC(=0)X5G(=0)(CH2)mXCM-; - **XEC(=0)0((CH2)m0)l1(CH2)mNHC(=0)X5C(=0)(CH2)r,iNHG(=0)(CH2)mXCM-; - **XEC(=0)0((CH2)m0)l1(CH2)mNHC(=0)X5C(=0)(CH2)r,iX3(CH2)mXCM-: - **XEC(=0)0((CH2)m0)„(CH2)mNHC(=0)X5C(=0)((CH2)m0)„(CH2)mXCM-; - 4*X£C(=0)0((CH2)m0)n(CH2)mNHC(=0)X5C(=0)((CH2)m0)„(CH2)rriNHC(=0)(CH2)mXCM-; - 44X£C(=0)0((CH2)m0)11(CH2)mNHC(=0)X5C(=0)((CH2)m0)n(CH2)rr!NHC(=0)(CH2)mX3(CH2)mXCM-; -**X¾(=0)0((CH2)m0)n(CH2)mNHC(=0))X5C(=0)((CH2)mG),i(CH2)n1X3(CH2)mXCM-; - iiX£C(=0)0((CH2)mG)n(CH2)mNHC(=0)X5C(=0)(CH2)mNHC(=G)((CH2)rpO)n(CH2)mXCM-; - iiX£C(=0)0((CH2)mG)n(CH2)mNHC(=0)X5C(=0)(CH2)mNHC(=G)((CH2)rpO)n(CH2)mX3(CH2)mXCM-; -**XiiC(=0)0((CH2)m0) (CH2)rpNHC(=0)X5(CH2)rpX3(CH2)rpXCM-; - AAX£C(=0)0((CH2)m0)n(CH2)mNHC(=0)X5((CH2)rp0)p(CH2)mXCM-; -
Figure imgf000180_0001
*iXEC(=0)((CH2)m0)n(CH2)r!,NHC(=0)X5((CH2)m0)n(CH2)mX3(CH2)mXCM-; - *iXEC(=0)((CH2)m0)n(CH2)r!,NHC(=0)X5(CH2)r!,NH((CH2)r!,0)n(CH2)mXCM-: - *iXEC(=0)((CH2)m0)n(CH2)r!,NHC(=0)X5C(=0)(CH2)mNH((CH2)m0)ii(CH2)rriX3(CH2)rriXCM-; - i*XEC(=0)((CH2)m0)n(CH2)r!,NHC(=0)X5(CH2)r!,XCM-; **XEC (=0) (C H2) FTIC (=0) N H (C H 2) mXCM ; - i*XEC(=0)XiX2C(=0)(CH2)-NHC(=0)(CH2)mXCM-; -MXEC(=0)X!X2C(=0)(CH2)mX3(CH2)mXGM-; - i*XEC(=0)XiX2C(=0)((CH2)m0)n(CH2)mXCM-; - i*XEC(=0)XiX2C(=0)((CH2)m0)n(CH2)mNHC(=0)(CH2)mXGM-; - -X¾(=0)X1X2C(=0)((CH2)m0)n(CH2)rr!NHC(=0)(CH2)mX3(CH2)mXCM-; - -X¾(=0)X1X2C(=0)((CH2)m0)n(CH2)rr!X3(CH2)rr!XCM-; - **XEC(=0)XiX2C(=0)(CH2)mNHC(=0)((CH2)rr!0)n(CH2)mXCM-; - **XEC(=0)XiX2C(=0)(CH2)mNHC(=0)((CH2)rr!0)n(CH2)mX3(CH2)mXCM-; - **X£C (= 0)Xi X2 (C H2) mXa (C H2) XCM- ; -44X£C(=0)XiX2((CH2)m0)„(CH2)mXCM-; - **XEC(=0)XiX2((CH2)rri0)r!(CH2),nNHC(=:0)(CH2)rriXCM-; - **XEC(=0)XiX2((CH2)rri0)n(CH2)mNHC(=0)(CH2)mX3(CH2)mXCM-; -
**XEC(=0)XiX2((CH2)fFi0)n(CH2)mX3(CH2)mXCM-; -**X£C(=0)XiX2(CH2)fFiNH((CH2)fFi0)n(CH2)mXCM-;**XEC(=0)XiX2C(=0)(CH2)mNH((CH2)m0)F,(CH2)mX3(CH2)mXGM-; - **XEC(=0)XiC(=0)NH(CH2)Fr,X5(CH2)Fr,XCM-; -**XEC(=0)XIX2(CH2)F^Xcm-: - **XEC(=0)XiC(=0)(CH2)mNHC(=0)((CH2)m0)n(CH2)FFXGM-; -**XEC(=0)NH(CH2)mXCM-; - **X£C (= O) N H (C H2) F Xs (C H2) Fi,XCM- ; -**XEC(=0)N H(CH2)F11NHC(=0)XIX2C(=0)(CH2)FT1Xcm-; - **XEC(=0)NH(CH2)FiFNHC(=0)0(CH2)mXGM-; -**XEC(=0)M H(CH2)F^NHC(=0)XIX2Xgm-; - **XEC(=0)NH (CH2)F?FNHC(=0)X5Xcm-; -**XEC(=0)NH(CH2)mNHC(=0)(CH2)F11X5(CH2)F11XCM-; - 44X£C(=0)NHC(=0)(CH2)mNHC(=0)XiX2C(=0)(CH2),11XCM-;
-i'*XEC(=0)fslH(CH2)mNHC(=0)(CH2)Fr!XCIV1-; -44X£C(=0)NH(CH2)m^lHC(=0)(CH2)mO(CH2)Fr!Xclv,-; - 4*X£C(=0)NH(CH2)mNHC(=0)XiX2C(=0)(CH2)m0(CH2)mXCM-; - iiX£C(=0)NH(CH2)mNHC(=0)XiX2C(=0)(CH2)m0(CH2)F!,C(=0)XGM-; - iiX£C(=0)NH(CH2)mNHC(=0)X4C(=0)NH(CH2)mNHC(=0)(CH2)„10(CH2)FpXCM-; - iiX£C(=0)NH(CH2)mNHC(=0)X5C(=0)(CH2)FpXCM-; - iiX£C(=0)NH(CH2)mNHC(=0)X5C(=0)(CH2)FpNHC(=0)(CH2)mXCM-; - *iX£C(=0)NH(CH2)mNHC(=0)X5C(=0)(CH2)FpX3(CH2)fpXCM-; - *iX£C(=0)NH(CH2)mNHC(=0)X5C(=0)((CH2)FT,0)n(CH2)FpXGM-; - *iX£C(=0)NH(CH2)mNHC(=0)X5C(=0)((CH2)FT,0)n(CH2)FpiMHC(=0)(CH2)FT,XCM-; - i*X£C(=0)NH(CH2)FpNHC(=0)X5C(=0)((CH2)m0)n(CH2)FpNHC(=0)(CH2)mX3(CH2)mXGM-; - i*X£C(=0)NH(CH2)FpNHC(=0)X5C(=0)((CH2)m0)n(CH2)FpX3(CH2)FpXGM-; - i*X£C(=0)NH(CH2)FpNHC(=0)X5C(=0)(CH2)linNHC(=0)((CH2)Fp0)l1(CH2)mXCM-; - i*X£C(=0)NH(CH2)FpNHC(=0)X5C(=0)(CH2)linNHC(=0)((CH2)Fp0)l1(CH2)mX3(CH2)mXGM-; - **XEC(=0)NH(CH2)rpfMHC(=0)X5(CH2)rpX3(CH2)rpXCM-; - **XEC(=0)NH(CH2)rpfMHC(=0)X5((CH2)m0)11(CH2)mXCM-; - *iXEC(=0)NH(CH2)mNHC(=0)X5((CH2)m0)i,(CH2)mNHC(=0)(CH2)mXCM-; - *iXEC(=0)NH(CH2)mNHC(=0)X5((CH2)m0)i,(CH2)mNHC(=0)(CH2)mX3(CH2)mXCM-; - *iXEC(=0)NH(CH2)mNHC(=0)X5((CH2)m0)i,(CH2)mX3(CH2)mXCM-; - i*XEC(=0)NH(CH2)mNHC(=0)X5(CH2)mNH((CH2)m0)n(CH2)rriXCM-; - i*XEC(=0)NH(CH2)mNHC(=0)X5C(=0)(CH2)mNH((CH2)m0)n(CH2)mX3(CH2)mXCM-; - i*XEC(=0)NH(CH2)mNHC(=0)X5(CH2)mXCM-: -MXEC(=0)XiC(=0)NH(CH2)mNHC(=0)(CH2)rT1XCM-: -**XEC (=0)X 1 C (= O) N H (C H2) mXs (C H2) mXCM- ; -**XEC(=0)NH(CH2)mNHC(=0)(CH2)mX3(CH2)mXCM- ; -**XeG (= Q) N H (C H2) m N H C (=0)XCM- ; -**XEC(=0)NH((CH2)m0)n(CH2)mX3(CH2)mXCM- and -**XEC (=0)Xi C(=0) (CH2)mN H C (=0) (C H2)mXCIV1- , wherein the ** of L indicates the point of attachment to the drug moiety (D);
wherein:
Figure imgf000182_0001
-**~NHG- or -**NHN=CR2(CH2)n-, wherein the ** of XE indicates the point of attachment to the drug moiety (D);
XCM is a group selected from any one of the chemical moiety groups in Table 4;
Figure imgf000182_0002
Xi is H or u , wherein the * of Xi indicates the point of attachment to X2;
Figure imgf000182_0003
Figure imgf000183_0001
; where the of X2 indicates the point of attachment to X1 or to XE
Figure imgf000183_0002
the ** of X5 indicates orientation toward XE;
each R11 is independently selected from H and Ci-C3alkyl;
each R12 is independently selected from H and Ci-Csaiky!;
each rn is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10, and
each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16,17 and 18.
Embodiment 189. A linker, L, of -(L-(D)m) selected from:
Figure imgf000183_0003
44XEC(=0)((CH2)m0)n(CH2)rpNHC(=0)X5C(=0)((CH2)m0)n(CH2)mNHC(=0)(CH2)mXCM-; - 44XEC(=0)((CH2)m0)n(CH2)rpNHC(=0)X5C(=0)((CH2)m0)n(CH2)mNHC(=0)(CH2)mX3(CH2)mXCM-; - 44XEC(=0)((CH2)m0)n(CH2)rpNHC(=0))X5C(=0)((CH2)rri0)n(CH2)mX3(CH2)mXCM-; - 4*XEC(=0)((CH2)m0)n(CH2)r!,NHC(=0)X5C(=0)(CH2)mNHC(=0)((CH2)rri0)n(CH2)mXCM-; - 4*XEC(=0)((CH2)m0)n(CH2)r NHC(=0)X5C(=0)(CH2)mNHCY0)((CH2)m0)n(CH2)mX3(CH2)mXCM-; - 4*XEC(=0)((CH2)m0)n(CH2)r NHC(=0)X5(CH2)r X3(CH2)r XCM-: - 4*XEC(=0)((CH2)m0)n(CH2)r NHC(=0)X5((CH2)m0)n(CH2)mXCM-; - 44XEC(=0)((CH2)mO)n(CH2)mNHC(=0)X5((CH2)mO)n(CH2)rr!NHC(=0)(CH2)mXCM-; - 44XEC(=0)((CH2)mO)n(CH2)mNHC(=0)X5((CH2)mO)n(CH2)rr!NHC(=0)(CH2)mX3(CH2)mXCM-; - 44XEC(=0)((CH2)mO)n(CH2)mNHC(=0)X5((CH2)mO)n(CH2)rrX3(CH2)rr!XCM-; - 4LXEC(=0)((CH2)m0)n(CH2)mNHC(=0)X5(CH2)mNH((CH2)m0)11(CH2)rr!Xclv,-; -
**XEC(=0)((CH2)m0)ri(CH2)mNHC(=0)X5C(=0)(CH2)mNH((CH2)rp0)n(CH2)mX3(CH2)mXCM-; -
44XEC(=0)((CH2)m0)ri(CH2)mNHC(=0)X5(CH2)mXCM-; and -44XiiC(=0)(CH2)mC(=0)NH(CH2)mXCM-, vvhere Xi , X2, X3, X4, X5, XE, XCM, R12, n and m are as defined in Embodiment 188, and the 44 of L indicates the point of attachment to the drug moiety (D).
Embodiment 190. A linker, L, of -(L-(D)m) selected from:
Figure imgf000184_0001
Figure imgf000185_0001
where the ** of L indicates the point of attachment to the drug moiety (D).
In one aspect, the Linker-Drug moiety of the immunoconjugates of the invention comprises one or more Drug moieties (D) as described herein.
In one aspect, the Linker-Drug moiety of the immunoconjugates of the invention comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a compound which binds to Stimulator of interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with a linker (L).
In one aspect, the Linker-Drug moiety of the immunoconjugates of the invention comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a compound which binds to Stimulator of interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with a linker (L), wherein linker (L) is a cleavable linker.
In one aspect, the Linker-Drug moiety of the immunoconjugates of the invention comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a dinucleotide which binds to Stimulator of interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with one or more linker(s) (L).
in one aspect, the Linker-Drug moiety of the immunoconjugates of the invention, comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a dinucleotide which binds to Stimulator of interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with one or more linker(s) (L), wherein linker (L) is a cleavable linker.
In one aspect, the Linker-Drug moiety of the immunoconjugates of the invention comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a cyclic dinucieotide which binds to Stimulator of Interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with one or more linker(s) (L).
In one aspect, the Linker-Drug moiety of the immunoconjugates of the invention, comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a cyclic dinucieotide which binds to Stimulator of Interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with one or more linker(s) (L), wherein linker (L) is a cleavable linker.
In one aspect the Linker-Drug moiety of the invention is a compound having the structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) or Formula (F) or stereoisomers or pharmaceutically acceptable salts thereof, wherein:
a) one or more linkers is attached to one or more sugar moieties of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) or Formula (F), or b) one or more linkers is attached to one or more R1 , R13 and R1 b groups of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) or Formula (F), or
c) one or more linkers is attached to one or more sugar moieties of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) or Formula (F) and one or more linkers is attached to one or more R1 , R1a and R1 b groups of Formula (A), Formula (B), Formula
(C), Formula (D), Formula (E) or Formula (F).
Gertaln aspects and examples of the Linker-Drug moiety of the invention are provided in the following listing of additional, enumerated embodiments. It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.
Embodiment 191. A compound having the structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) or Formula (F), or stereoisomers or pharmaceutically acceptable salts thereof,
wherein:
each Gi is Independently selected from
Figure imgf000186_0001
,
the * of G1 indicates the point of attachment to -~CR8R9-;
XA is C(=0)-, -C(=S)- or -C(=NR11)- and each Z^ is NR12;
XB is C, and each Z2 is N;
Figure imgf000186_0002
Figure imgf000186_0003
Xc is C(=0)-, -C(=S)- or -C(=NR11)- and each Z3 is NR12;
X[) is C, and each Z4 is N;
Y, is -0-, -NH-, -S-, -S(=0)-, -SO2-, -CH2-, or -CF2-;
Y2 is -0-, -NH-, -S-, -S(=0)-, -SO2-, -CH2-, or -CF2-;
Y3 is OH, O , OR10, N(R i0)2, SR10, SeH, Se , BH3, SH or S ;
Y4 is OH, O , OR10, N(R i0)2, SR10, SeH, Se , BH3, SH or S ;
Y5 is -CH2-, -NH-, -O- or -S;
Y6 is -CH2-, -NH-, -O- or -S;
Y7 is O or S;
Ys is O or S;
Y3 is -CH2-, -NH-, -O- or -S;
Y10 is -CH2-, -NH-, -O- or -S;
Y11 is -O-, -NH-, -S-, -S(=0)-, -SO2-, -CH2-, or -CF2-; q is 1 , 2 or 3;
R1 is a partially saturated or aromatic monocyclic heterocyclyl or partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1 is substituted with Q, 1 , 2, 3 or 4 substituents independently selected from -NHL1R15, -NHNHL-iR15, -NHGL1R15, - NHN=GR12(GH2)nLiR15, F, Cl, Br, -NHOH, -NHNH2, -NH2, Ci-C6alkyl and Ci-C6alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R18 is a partially saturated or aromatic monocyclic heterocyclyl or partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from ~NHLiR1s, -NHNHLiR16, -NHOL1R15, - NHN=CR12(CH2)nLiR15, F, Cl, Br, -NHOH, -NHNH2, -NH2, C C6aikyl and C Csalkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
Rib is a partially saturated or aromatic monocyclic heterocyclyl or partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHL1R15, -NHNHLIR15, -NHOL1 R15, - NHN=CR12(CH2),LI R15, F, Cl, Br, -NHOH, -NHNH2, ~NH2, Ci-C6aikyl and Ci~C6alky! substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R2 is independently selected from H and CrC6a!kyl;
each R3 is independently selected from -NHL1R15, -NHNHL1R15, -NHOUR15, -
NHN=CRi2(CH2)nLiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C C6aikyl substituted with a NHOH, NHNH2 or NH2;
each R4 is independently selected from H, CrC6alkyl and Ci-C6alky! substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R5 is independently selected from -NHL1R15, -NHNHL1R15, -NHOL1R15, -
NHN=GR12(GH2)nLiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH22;
each R6 is independently selected from H, Ci-C6alkyl and Gi-C6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3; each R7 is independently selected from H, CrC6alkyl and CrC6alky! substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each Rs is independently selected from H, CrC6alkyl and Ci-C6alky! substituted by 1 , 2 or 3 substituents independently selected from F, Gi, Br, I, OH, CN, and N3;
each R9 is independently selected from H, CrC6alkyl and Ci-C6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R23 is independently selected from H and CrCsalkyl;
each R38 is independently selected from -NHL1R15, -NHNHL1R15, -NHOL1R15, -
NHN=CR12(CH2),LI R15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C C6aikyl substituted with a NHOH, NHNH2 or NH2;
each R43 is independently selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R5a is independently selected from -NHLiR1£, ~NHNHLiR1£, -NHOL1R15, -
NHN=CR12(CH2)r!LiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C C6alkyl substituted with a NHOH, NHNH2 or NH2;
each R6a is independently selected from H, CrCsalkyl and CrCsalky! substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7a is independently selected from H, CrCsalky! and C Csalkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each RSa is independently selected from H, CrCsalkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R9a is independently selected from H, CrCsalkyl and CrCsalkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R10 is independently selected from the group consisting of H, CrCsalkyl, Cr
Csheteroaikyi,
Figure imgf000188_0001
, wherein the Cr
Ci2aikyi and Ci-C3heteroaikyi of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, CrC12aikoxy, -S-C(=0)CrCsaikyl, halo, -CN, Cr Ci2alkyl, -O-aryl, -O-heteroaryl, -O-cycloalkyi, oxo, cycloalkyl, heterocycly!, aryl, or heteroaryl, -0C(0)0CrC6aikyland C(0)0CrC6alkyi, wherein each alkyl, cycioaikyl, heterocyclyl, aryl, and heteroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from C Ci2 alkyl, 0-CrCi2alkyl, Ci-C12heteroalkyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroaryl, -C(=0)Ci-C 2alkyl, -0C(=;0)CrCi2aikyl, -C(=0)0Ci- Ci2aikyl, -0C(=0)0Ci-Ci2alkyl, -C(=0)N(Rl1)-Ci-Ci2alkyi, -N(R11)C(=0)-Ci-C12alkyl; - 0C(=0)N(R11)-CrC12alkyl, -C(=0)-aryl, -C(=0)-heteroaryl, -0C(=0)-aryl, -C(=0)0-aryl, - OC(=0)-heteroaryl, -C(=0)0-heieroaryl, -C(=0)0-aryi, -C(=Q)0-heieroaryl, - C(=Q)N(Ri 1)-aryl, -C(=Q)N(Ri 1)-heieroaryl, -IM(R11)C(G)-aryl, -N(R11)2C(OVaryi, - N(R11)C(Q)-beteroaryi, and S(0)2N(R11)-aryl;
each R11 is independently selected from H and Ci-Cealkyl;
each R12 is independently selected from H and CrC6alkyl;
optionally R3 and R6 are connected to form -Q-G i-C6alkylene, such that when R3 and Rs are connected, the O is bound af the R3 position
optionally R3a and R6a, are connected to form -0-Ci-C6alkyiene, such that when R38 and R6a are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form -0-CrC6alkylene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form -G~Ci-C6a!kyiene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form ~0-Ci-C6aikylene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -G-CrCsa!kylene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form -O-CrCsaikylene, such that when R5 and R6 are connected, fhe O is bound at the R5 position;
optionally R5a and R6a, are connected to form -Q-Ci-G6a!kylene, such that when R5a and RSa are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form -0-Gi-C6alkylene, such that when R5 and R7 are connected, the O is bound at the R5 position;
optionally R5a and R7a, are connected to form -0-Ci-Gsalkylene, such that when R5a and R7a are connected, the O is bound at the R5a position;
Li is a linker;
each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18;
R1S is a reactive group selected from any one of the groups RG1 in Table 4;
and provided at least one of R1 , R1a or R1b is substituted with -NHL1R15, -NHNHL1R15, - least one of R3, R5, R3a or R5a is -NHL1R15, -
Figure imgf000189_0001
2)nLiR15.
Embodiment 192. The compound of Embodiment 191 , wherein is a linker comprising one or more cleavage elements.
Embodiment 193. A compound of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), Formula (F), Formula (A-1 ), Formula (B-1), Formula (C~1), Formula (D-1), Formula (E-1) or Formula (F-1), wherein R1 is pyrimidine or purine nucleic acid base or analogue thereof, R1a is a pyrimidine or purine nucleic acid base or analogue thereof and R1 b is a pyrimidine or purine nucleic acid base or analogue thereof, each of which is substituted as described in R1 , R1a and R1 b in Embodiment 191 .
Embodiment 194. A compound of Formula (A-2), Formula (B-2), Formula (C-2), Formula (D- 2). Formula (E-2) or Formula (F-2), wherein R1 , Ria, R1 b, R2, R2a, R3, R3a, R4, R4a, R5, RSa,
R6, R6a, R7, R7a, R8, R8a, R9, RSa, R10, R11 , R12, R1S, Li, n, Yi, Y2, Y3 Y<, Y5, Ye, Y7, Y8, Yg, Y10 and Yu are as defined in Embodiment 191 , and provided at least one of R1 , R1 a or R1 b is substituted least one of R3,
Figure imgf000190_0001
LiR15.
Embodiment 19S. A compound of Formula (A), Formula (A-1) or Formula (A-2) of any one of Embodiments 191 to 194, wherein:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are
R3 is selected from -NHL R15, -NHNHL1
Figure imgf000190_0002
-SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH2;
R8, Rs, R8a and R9a are independently H or C -C3aikyl, and
R3a is selected from -NHL1R15, -NHNHL1R15, -NHOLiR15, -NHN=CR12(CH2)nLiR15, H, - OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a CrC6aikyl substituted with a NHOH, NHNH2 or NH2;
and provided at least one of R1 or R ia is substituted with is -NHL1R15, -NHNHL1R15, - least one of R3, or R3a is -NHL1R15, -
Figure imgf000190_0003
2)nLiR15.
Embodiment 196. A compound of Formula (A), Formula (A-1) or Formula (A-2) of any one of Embodiments 191 to 194, wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O-, NH2, SH or S ;
Y4 is OH, O-, NH2, SH or S ;
Y5 and Y6 are O or S;
Y7 and Y8 are O or S;
Y9 and Y10 are O or S;
Rs, R5a, R7 and R7a are H
NHNHL1 R15, H, OH or F;
-NHNHL1R15, H, OH or F; and
Figure imgf000190_0004
s are independently selected from H or C rCBalkyi,
and provided at least one of R1 or R1a is substituted with -NHL1R1 5 or -NHNHL1R15, or at least one of R3, or R3a Is -NHL1R1 5 or -NHNHL1R15. Embodiment 197. A compound of Formula (A), Formula (A-1) or Formula (A-2) of any one of Embodiments 191 to 194, wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH o S :
Y4 is OH, O , MHz, SH or S ;
Y5 and Y6 are O or S;
Y7 and Ys are O or S;
Y9 and Yio are O or S;
R2, R2a, R4, R4a, R6, R63, R7 and R7a are H
R3 is -NHNHL1R15, OH or F;
R3a is -NHNHL1R15, OH or F; and
R8a, R9a, R8 and R9 are independently selected from H or Ci-C6alkyl,
and provided at least one of R1 or R1a is substituted with -NHNHLiR15, or at least one of R3, or R3a is -NHNHL1R15.
Embodiment 198. A compound of Formula (B), Formula (B-1) or Formula (B-2) of any one of Embodiments 191 to 194, wherein:
R2 R2a^ R4_ R4a_ R0. R6a R7 a n(j R7a a fe e ach H ;
R3a is selected from -NHL1R15, -NHNHLiR15, -NHGLiR15, -NHN=CR12(CH2)nLiR15, H, - substituted with a NHOH,
Figure imgf000191_0001
R5 is selected from -NHL1R15, -NHNHL1R15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2,
NH2 and a Ci-C6alkyi substituted with a NHOH, NHNH2 or NH2, and
R8, R9, R8a and R9a are independently H or Ci-C6alkyl,
and provided at least one of R1 or R1a is substituted with -NHL1R15, -NHNHL1R15, - least one of R5, or R3a is -NHL1R15, -
Figure imgf000191_0002
2)nL1R15.
Embodiment 199. A compound of Formula (B), Formula (B-1) or Formula (B-2) of any one of Embodiments 191 to 194 or 198, wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S :
Y4 is OH, O , NH2, SH or S :
Y5 and Y6 are O or S;
Y7 and Ys are O or S;
Y9 and Y10 are O or S;
R7a are H;
, OH or F;
Figure imgf000191_0003
OH or F, and R8a, R9a, R8 and Rs are independently selected from H or C rCBalkyi, and provided at least one of R1 or R1a is substituted with -NHLiR15 or -NHNHLiR15, or at least one of R5, or R3a is -NHLiR15 or -NHNHLiR15.
Embodiment 200. A compound of Formula (B), Formula (B-1) or Formula (B-2) of any one of Embodiments 191 to 194 or 198, wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2 SH o S-;
Y4 is OH, O , NH2, SH or S-;
Ys and Y6 are O or S;
Y7 and Ys are O or S;
Y9 and Yio are O or S;
R2, R2a, R4, R4a, R5, RSa, R7 and R7a are H;
R3a is -NHNHLiR15, OH or F;
R5 is -NHNHLiR15, OH or F, and
R8a, R9a, R8 and R9 are independently selected from H or Ci-C3alkyl,
and provided at least one of R1 or R1a is substituted with -NHNHLiR15, or at least one of R5, or R3a is -NHNHLiR15.
Embodiment 201. A compound of Formula (C), Formula (C-1) or Formula (C-2) of any one of Embodiments 191 to 194, wherein:
Figure imgf000192_0001
-SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH2;
R5a is selected from -NHLiR15, -NHNHLiR15, -NHOL R15, -NHN=CR12(CH2)aLiR15, H, - OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6aikyl substituted with a NHOH, NHNHz or NH2, and
R8, R9, R8a and RSa are independently H or Ci-Cealkyl;
and provided at least one of R1 or R1a is substituted with -NHL1R15, -NHNHL1R15, - least one of R5a, or R3 Is -NHLiR15, -
Figure imgf000192_0002
2)nLiR15.
Embodiment 202. A compound of Formula (C), Formula (C-1) or Formula (C-2) of any one of Embodiments 191 to 194 or 201 , wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH o S :
Y4 is OH, O , NH2, SH o S :
Y5 and Y6 are O or S;
Y7 and Ys are O or S; Ys and Yio are O or S;
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H:
R3 is -NHL,R15, -NHNHLiR15, H, OH or F;
R5a is -NHL,R15, -NHNHLiR15, H, OH or F, and
R8a, R9a, R8 and R3 are independently selected from H or Ci-C6alkyl,
and provided at least one of R1 or R1a is substituted with -NHL1R1 5 or -NHNHLiR15, or at least one of R5a, or R3 is -NHL1R1 5 or -NHNHLiR15.
Embodiment 203. A compound of Formula (C), Formula (C-1) or Formula (C-2) of any one of Embodiments 191 to 194 or 201 , wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S-;
Y4 is OH, O , NH2, SH or S ;
Ys and Y6 are O or S;
Y7 and Ys are O or S;
Y9 and Y10 are O or S;
Figure imgf000193_0001
R8a, R9a, R8 and Rs are independently selected from H or Ci-C6alkyl,
and provided at least one of R1 or R1a is substituted with -NHNHLiR15, or at least one of R5a, or R3 is -NHNHL1R15.
Embodiment 204. A compound of Formula (D), Formula (D-1) or Formula (D-2) of any one of Embodiments 191 to 194, wherein:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each
R5a is selected from -NHL1R15, -NHNHLiR15,
Figure imgf000193_0002
substituted with a NHOH,
Figure imgf000193_0003
R5 is selected from -NHL,R15, -NHNHLiR15, -NHOL1R15, -NHN=CR,2(CH2)nL,R15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2 NH2 and a CrCealkyl substituted with a NHOH, NHNH2 or NH2, and
R8, R9, R8a and R9a are independently H or Ci-C3alkyi,
and provided at least one of R1 or R1a is substituted with -NHL1R15, -NHNHLiR15, - NHGL1R1 5 or -NHN=CR12(CH2)nL R15, or at least one of R5a, or R5 -NHL1R15, - NHNHL1R15, -NHOL1R15 or -NHN=CR12(CH2)nL R15.
Embodiment 20S. A compound of Formula (D), Formula (D-1) or Formula (D-2) of any one of Embodiments 191 to 194 or 204, wherein:
Yi and Y2 are O, CH2 or S; Y3 is OH, O , NH2, SH or S ;
Y4 is OH, O , NH2, SH or S ;
Y5 and Y6 are O or S;
Yy and Ye are O or S;
Y 9 and Yio are O or S;
R7a are each H:
, OH or F;
Figure imgf000194_0001
OH or F, and
R8, Rs, RSa and R9a are independently H or Ci-C3aikyi,
and provided at least one of R1 or R1a is substituted with -NHLiR15 or -NHNHLiR15, or at least one of R5a, or R5 is -NHL1R15 or -NHNHLiR15.
Embodiment 20S. A compound of Formula (D), Formula (D-1) or Formula (D-2) of any one of Embodiments 191 to 194 or 204, wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S ;
Y4 is OH, O , NH2, SH or S ;
Ys and Y6 are O or S;
Y? and Ys are O or S;
Y9 and Y10 are O or S;
Figure imgf000194_0002
R8, R9, R8a and R9a are independently H or Ci-C6alkyl,
and provided at least one of R1 or R1a is substituted with -NHNHLiR15, or at least one of R5a, or R5 is -NHNHLiR15
Embodiment 207. A compound of Formula (E), Formula (E-1) or Formula (E-2) of any one of Embodiments 191 to 194, wherein:
R2, R2a, R4, R4a, Rs, R5a and R7 are each H;
R3a is selected from -NHLiR15, -NHNHLiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C C6alkyi substituted with a NHOH, NHNH2 or NH2;
R3 is selected from -NHLsR15, -NHNHL,R15, -NHOL1R15, -NHN=CR12(CH2)nL,R15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH2;
R5 is selected from -NHL1R15, -NHNHL1R15, -NHOL1R15, -NHN=CR12(CH2)nLiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH2;
and R8, R9, RSa and R9a are independently H or CrCsalkyl,
and provided at least one of R1 or R1a is substituted with -NHLiR15, -NHNHLiR15, - least one of R3a, R3, or R5 is -NHL,R15, -
Figure imgf000195_0001
2)nLiRi 5.
Embodiment 208. A compound of Formula (E), Formula (E-1) or Formula (E-2) of any one of Embodiments 191 to 194 or 207, wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S-;
Y5 and Y9 are O or S;
Y7 and Ys are O or S;
R2, R2a, R4, R4a, R6, R63 and R7 are each H;
R3a is selected from -NHL1R15, -NHNHLiR15, H, OH or F;
R3 is selected from -NHLiR15, -NHNHL1R15, H, OH or F;
R5 is selected from -NHLiR15, -NHNHL1R15, H, OH or F, and
R5a is selected from -NHL1R15, -NHNHL1R15, H, OH or F, and
R8, R9, R8a and RSa are independently H or Ci-C3a!kyl,
and provided at least one of R1 or R ia is substituted with -NHL1R15 or -NHNHL1R15, or at least one of R3a, R3 or R5 is -NHL1R15 or -NHNHLiR15.
Embodiment 209. A compound of Formula (E), Formula (E-1) or Formula (E-2) of any one of Embodiments 191 to 194 or 207, wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O-, NH2, SH or S ;
Y5 and Y9 are O or S;
Y7 and Ys are O or S;
R2, R2a, R4, R4a, R6, R6a and R7 are each H;
R3a is selected from -NHNHLiR15, OH or F;
R3 is selected from -NHNHLi R15, OH or F;
R5 is selected from -NHNHLi R15, OH or F, and
R5a is selected from -NHNHL R15, OH or F, and
R8, R9, R8a and R9a are independently H or CrCsalkyl,
and provided at least one of R1 or R1a is substituted with -NHNHLiR15, or at least one of R3a, R3 or R5 is -NHNHLiR15.
Embodiment 210. A compound of Formula (F), Formula (F-1) or Formula (F-2) of any one of Embodiments 191 to 194, wherein:
R2, R2a, R4, R4a, R6, R6 RSa, R6a and R7 are each H; R3a is seiected from -NHL,R15, -NHNHLiR15, -NHOLiR15, -NHN=CR12(CH¾)nLiR15, H, - OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a CrC6aikyl substituted with a NHOH, NHNH2 or NH2;
each R3 is independently selected from -NHLiR15, -NHNHLiR15, -NHOLiR15, - NHN=CR12(CH2)nLiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Cr Csalkyl substituted with a NHOH, NHNH2 or NH2;
each R5 is independently selected from -NHLiR15, -NHNHLiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2 and NH2, and
R8, Rs, RSa and R98 are independently H or Ci-C3alkyi,
and provided at least one of R1, R1a or R1b is substituted with -NHLiR15, -NHNHLiR15, - NHOL1R15 or -NHN=CR12(CH2)nLiR15, or at least one of R3a, R3 or R5 is -NHL1R15, - NHNHL1R15, -NHOL1R15 or -NHN=CR12(CH2)nLiR15.
Embodiment 211. A compound of Formula (F), Formula (F-1) or Formula (F-2) of any one of Embodiments 191 to 194 or 210, wherein:
Y1, Y2 and Yu are O, CH2 or S;
each Y3 is OH, O , NH2, SH or S ;
each Y5 is O or S;
each Y? is independently are O or S;
each Y9 is independently O or S;
Figure imgf000196_0001
R3a is selected from -NHL1R15, -NHNHL1R15, H, OH or F;
each R3 is independently selected from -NHL1R15, -NHNHLiR15, H, OH or F;
R5 is selected from -NHLiR15, -NHNHL1R15, H, OH or F, and
each R5 is independently selected from -NHL1R15, -NHNHLiR15, H, OH or F;
R8, R9, R8a and R9a are independently H or Ci-Cealkyl,
and provided at least one of R1, R1a or R1b is substituted with -NHL1R15 or -NHNHLiR15, or at least one of R3a, R3 or R5 is -NHL1R15 or -NHNHLiR15.
Embodiment 212. A compound of Formula (F), Formula (F-1) or Formula (F-2) of any one of Embodiments 191 to 194 or 210, wherein:
Y1, Y2 and Y are O, CH2 or S;
each Y3 is OH, O , NH2, SH or S ;
each Y5 is O or S;
each Y7 is independently are O or S;
each Y9 is independently O or S;
R2, R2a, R4, R4a, R6, R6 RSa, RSa and R7 are each H;
R3a is selected from -NHNHLiR15, OH or F;
each R3 is independently selected from -NHNHLiR15, OH or F; R5 is selected from -NHNHLiR15, OH or F, and
each R5 is independently selected from -NHNHLiR15, OH or F;
R8, R9, RSa and R9a are independently H orCi-C3alkyl,
and provided at least one of R1, R1a or R1b is substituted with -NHNHLiR15, or at least one of R3a, R3or R5 is -NHNHLiR15.
Figure imgf000197_0001
A compound of any one of Embodiments 191 to 212, wherein:
Figure imgf000197_0002
Figure imgf000198_0001
Figure imgf000199_0001
Figure imgf000200_0001
Figure imgf000201_0001
Figure imgf000202_0001
wherein: R1 is substituted with Q, 1 , 2 or 3 substituents independently selected from Fs Cl, Br, Ci-C3alkyl and Ci-C3alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3; each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 and 18 and each R20 is independently selected from H and LiR15;
Figure imgf000203_0001
Figure imgf000204_0001
Figure imgf000205_0001
Figure imgf000206_0001
Figure imgf000207_0001
Figure imgf000208_0001
wherein: R1a is substituted with 0, 1 , 2 or 3 substituents independently selected from F, Cl, Br, NHOH, Nhh, Ci-Csalkyi and C-i-Cealkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3; each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 18, 17 and 18 and each R21 is independently selected from H and LiR15;
Figure imgf000208_0002
Figure imgf000209_0001
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
Figure imgf000213_0001
Figure imgf000214_0001
wherein: R1 b is substituted with 0, 1 , 2 or 3 substituents independently selected from F, Cl, Br, NHOH, NH2, Ci-C3alkyl and CrC6alkyi substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and Ns; each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 and 1 8 and each R21 is independently selected from H and Li R1s.
Embodiment 214. A compound of Formula (A-3), Formula (B-3), Formula (C-3) , Formula
(D-3), Formula (E-3) or Formula (F-3), wherein :
Figure imgf000214_0002
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
Figure imgf000219_0001
wherein: R1 is substituted with 0, 1 , 2 or 3 substituents independently selected from F, Cl, Br, CrC6alkyi and Ci-C3alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N3, and 18and each R20 is independently selected from H and LiR15;
Figure imgf000220_0001
Figure imgf000221_0001
Figure imgf000222_0001
Figure imgf000223_0001
Figure imgf000224_0001
Figure imgf000224_0002
Figure imgf000224_0003
Figure imgf000224_0004
Figure imgf000225_0001
wherein: R1a is substituted with 0, 1 , 2 or 3 substituents independently selected from F, Cl, Br, Ci-Ceaikyi and Cs-Csalkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3, and each R21 is independently selected from H and LiR15;
Figure imgf000225_0002
Figure imgf000226_0001
Figure imgf000227_0001
Figure imgf000228_0001
Figure imgf000229_0001
Figure imgf000230_0001
Figure imgf000231_0001
wherein: R1b is substituted with 0, 1 , 2 or 3 substituents independently selected from F, Cl, Br, Ci-C6alkyi and Ci-C3alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and Ns, and each R21 is independently selected from H and UR15;
each R2 is independently selected from H and Ci-C6alkyl;
each R3 is independently selected from -NHL1R15, -NHNHL1R15, -NHOLiR15, -
NHN=CRi2(CH2)nLiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C C6afkyl substituted with a NHOH, NHNH2 or NH2;
each R4 is independently selected from H, CrC6alkyl and Ci-C6aikyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R5 is independently selected from -NHL1R15, -NHNHL1R15, -NHOL1R15, -
NHN=CR12
substituted
Figure imgf000231_0002
each R6 is independently selected from H, CrC6alkyi and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7 is independently selected from H, CrC6alkyi and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R® is independently selected from H, CrC6alkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R9 is independently selected from H, CrC6alkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R2a is independently selected from H and CrC6aiky!;
each R3a is independently selected from -NHL1R15, -NHNH R15, -NHOL1R15, -
NHN=CR12(CH2)nLiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH2;
each R4a is independently selected from H, Ci-C5alkyi and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R5a is independently selected from -NHL1R15, -NHNHL1R15, -NHOLIR15, -
NHN=CRl2(CH2),LiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C C6alkyl substituted with a NHOH, NHNH2 or NH2;
each R6a is independently selected from H, Ci-C3alkyi and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3; each R7a is independently selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each RSa is independently selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R9a is independently selected from H, Ci-G6alkyi and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R10 is independently selected from the group consisting yl, Cr
Ceheteroalky!, -(CH2CH20)nCH2CH2C(=0)0Ci-G6alkyl, an
Figure imgf000232_0001
wherein the Ci-
Csaikyi and Ci~CBheteroalkyi of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, CrCi2alkoxy, -S-C(=0)CrC6alkyl, halo, -CN, Cr Ci2aikyi, -O-aryi, -O-heteroaryl, -O-cycloaikyl, oxo, cycloalkyl, heterocyciyl, aryl, or heteroaryl, -0C(0)0CrCBalkyland C(0)0CrCBalkyl, wherein each alkyl, cycioaikyl, heterocyciyl, aryl, and heteroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from C1-C12 alkyl, 0-Ci-Ci2alkyl, CrCi2heteroaikyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroaryl, -C(=0)Ci-Ci2aikyi, -0C(=0)Ci-Ci2alkyl, -C(=0)0Ci- Ci2aikyi, -0C(=0)0C1-Ci2alkyl, -C(=0)N(R11)-Ci-C !2alkyl, -N(R11)C(=0)-CrC 2aikyl; - 0C(=;0)N(R11)-Ci-C 2alkyl, -C(=0)-aryl, -C^C -heteroaryl, -0C(=:0)-aryl, -C(=G)G-aryl, - OC(=;0)-heteroa!yl, -C ^OJO-heteroaryl, -C(=:0)0-aryl, -C(=Q)0-heteroaryl, - C(=0)N(R11)-aryl, -C(=0)N(R11)-heteroaryl, -N(R11)C(G)~aryl, ~N(R11)2C(Q)~aryi, - N(R11)C(0)-heteroaryi, and S(0)2N(R11)-aryl;
each R11 is independently selected from H and Ci-C6alkyl;
each R12 is independently selected from H and Ci-CBalkyl;
optionally R3 and R6 are connected to form ~0-CrC6aikylene, such that when R3 and R6 are connected, the O is bound at the R3 position
optionally R3a and R6a, are connected to form -0-CrC6alkylene, such that when R3a and RSa are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form -0-CrC6alkylene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form -0-CrC6alkylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form -0-CrC6alkylene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -0-Ci-C3alkylene, such that when R4a and R3a are connected, the O is bound at the R3a position; optionally R5 and R6 are connected to form -Q-CrC6aikylene, snch that when R5 and R6 are connected, the O is bound at the R5 position;
optionally R5a and R63, are connected to form -0-CrCBalkyiene, such that when R53 and R63 are connected, the O is bound at the R53 position:
optionally R5 and R7 are connected to form -Q-G i-C6aikylene, such that when R5 and R7 are connected, the O is bound at the R5 position;
optionally R53 and R73, are connected to form -0-CrC6alkylene, such that when R53 and R73 are connected, the O is bound at the R53 position;
Li is ~C(=0)(CH2)m-**; -C(=0)(CH2)m0(CH2)m-'J‘A'; -G(=0)XiX2C(=0)(CH2)m-**; - C(=0)XiX2C(=0)(CH2)m0(CH2)m-**; -C(=Q)XiX2C(=0)CCH2)m0(CH2)mG(=Q)~**; - C(=0)X4C(=0)NH(CH2) NHC(=0)(CH2)mO(CH2)m-**; -
C(=0)X5C(=0)(CH2)mNHC(=0)(CH2)m-ii; -C(=0)(CH2)mNHC(=0)XiX2C(=0)(CH2)m-i*; - C(=0)0(CH2)mNHC(=0)X1X2C(=0)(CH2)nr **; -C(=0)(CH2)mNHC(=0)((CH2)mO)„(CH2)m- **; -C(=0)0(GH2)mNHe(=0)(CH2)m-**; -C(=0)0(CH2)mNHC(=0)(GH2)m0(CH2)m-**; - C(=0)0(CH2)rriNHC(=0)XiX2C(=0)(CH2)m0(CH2)m~**; -C20(=O)(0H2n**-; - C(=0)X5C(=0)(CH2)mNHC(=0)(CH2)m-**; - C(=0)0(CH2)rriNHC(=0)XiX2C(=0)(CH2)m0(CH2)mC(=0)-**; ··
C(=0)0(CH2)rriNHC(=0)X4C(=0)NH(CH2)rr,NHC(=0)(GH2)m0(CH2)m-“; - C(=0)0((GH2)m0)n(CH2)mNHG(=0)X5C(=0)(CH2)rn-**; - C(=0)0((CH2)m0)n(GH2)mNHG(=0)X5C(=0)(CH2)mNHC(=0)(CH2)m-**; - C(=0)0((GH2)m0)n(CH2)mNHG(=0)X5C(=0)(CH2)mX3(GH2)m-**; - C(=0)0((CH2)m0)n(CH2)mNHC(=0)X5C(=0)((CH2)rr!0),1(CH2)m-A*; - C(=0)0((CH2)m0)n(CH2)mNHC(=0)X5C(=0)((CH2)rr!0),1(CH2)mfslHC(=0)(CH2)rrr*'J‘; - G(=0)0((GH2)m0)n(CH2)mNHC(=0)X5C(=0)((CH2)m0)n(CH2)mNHC(=0)(CH2)mX3(CH2)m- -C(=0)0((CH2)m0)n(CH2)mNHC(=0))X5C(=0)((CH2)m0)n(CH2)mX3(CH2)m-ii; - C(=0)0((CH2)m0)„(CH2)mNHC(=0)X5C(=0)(CH2)mNHC(=0)((CH2)m0)„(CH2)nr **; - C(=0)0((CH2)m0)„(CH2)mNHC(=0)X5C(=0)(CH2)mNHC(=0)((CH2)m0)„(CH2)mX3(CH2)m- “; -C(=0)0((CH2)m0)„(CH2)mNHC(=0)X5(CH2)mX3(CH2)m-**; - C(=0)0((CH2)m0)„(CH2)mNHC(=0)X5((CH2)m0)n(GH2)m-**; - C(=0)0((CH2)m0)„(CH2)mNHC(=0)X5((CH2)m0)n(GH2)mNHC(=0)(CH2)m-**; - C(=0)0((CH2)m0)„(CH2)mNHC(=0)X5((CH2)m0)n(CH2)mNHC(=0)(CH2)mX3(CH2)m-*i; - C(=0)0((CH2)m0)n(CH2)mNHC(=0)X5((CH2)m0)n(CH2)mX3(CH2)m-**; - C(=0)0((CH2)m0)n(CH2)mNHC(=0)X5(CH2)rT1NH((CH2)rT10)n(CH2)m-M; - C(=0)0((CH2)m0)n(CH2)mNHC(=0)X5C(=G)(CH2)mNH((CH2)m0)n(CH2)mX3(CH2)m-A*; - C(=0)0((CH2)m0)n(CH2)mNHC(=0)X5(CH2)m-**; -C(=0)0(CH2)m-**; - C(=0)0((GH2)mO)n(CH2)m-**; -C(=0)0(CH2)rr!NH(CH2)m-A'A'; - C(=0)0(CH2)mNH(CH2)mC(=0)X1X2C(=0)-'A'A; -G(=0)0(CH2)mX3(CH2)m-**; -
Figure imgf000234_0001
Figure imgf000235_0001
C(=0)XiC(=0)(CH2)r iNHC(=0)(CH2)m-, where the ** of Li indicates the point of attachment to R15:
Figure imgf000236_0001
Figure imgf000237_0001
Xi is
Figure imgf000237_0002
where the * of Xi indicates the point of attachment to X2;
Figure imgf000237_0003
the of X2 indicates the point of attachment to Xi , -NH-, -NHNH-, -NHO- Qr -NHN=GR12(CH2)n-
Figure imgf000238_0001
R17 is 2-pyridyi or 4-pyridyl;
each R, ! is independently selected from H and CrC6aikyl;
each R12 is independently selected from H and CrC6aikyl;
each m is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10; and
each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18;
each R110 is independently selected from H, CrCsalkyl, F, Cl, and -OH;
each R111 is independently selected from H, CrCsalkyl, F, Cl, -NH2, -OCH3, -GGH2CH3, -
N(CH3)2, -CM, -NO2 and -OH;
each R112 is independently selected from H, Ci-6alkyl, fluoro, benzyloxy substituted with - C(=0)0H, benzyl substituted with -G(=0)0H, Ci-4aikoxy substituted with -C(=0)0H and Ci-4ai yl substituted with ~-C(=0)0H;
and provided at least one of R20 or R21 is -L1R15, or at least one of R3, R5, R3a or R5a is -NHL1R15, -NHNHUR15, -NHOL1R15 or -NHH=CR12(CH2)nLiR15.
Embodiment 215. A compound of Formula (A-4), or a pharmaceutically acceptable salt thereof, wherein:
R1, R1a, R3, R3a, R6 and R6a are as defined in Embodiment 214:
Y3 is O , OH, NH2, SH or S , and
Y4 is O , OH, NH2, SH or S\
Embodiment 218. A compound of Formula (A-4a), Formula (A-4b), Formula (A-4c) or
Formula (A-4d), or a pharmaceutically acceptable salt thereof, wherein:
R1, R13, R3, R33, R6 and RSa are as defined in Embodiment 214:
Y3 is O , OH, NH2, SH or S , and
Y4 is O , OH, NH2, SH or S .
Embodiment 217. A compound of Formula (A-4e), Formula (A-4f), Formula (A-4g), Formula (A-4h), Formula (A-4I), Formula (A-4j), Formula (A-4k), Formula (A-4I), Formula (A-4m), Formula (A-4n), Formula (A-4o) or Formula (A-4p), or a pharmaceutically acceptable salt thereof, wherein:
R1, Ria, R3, R3a, R6 and R63 are as defined in Embodiment 214;
Y3 is O , OH, NH2, SH or S , and
Y4 is O , OH, NH2, SH or S .
Embodiment 218. A compound of Formula (B-4), or a pharmaceutically acceptable salt thereof, wherein:
R1, R1a, R33, R5 and R6a are as defined in Embodimeni 214;
Y3 is O , OH, NH2, SH or S , and
Y4 is O , OH, NH2, SH or S .
Embodiment 219. A compound of Formula (B~4a), Formula (B~4b), Formula (B-4c) or Formula (B-4d), or a pharmaceutically acceptable salt thereof, wherein:
R1, R1a, R3a, R5 and R5a are as defined in Embodiment 214;
Y3 is O , OH, NHs, SH or S , and
Y4 is O , OH, NH2, SH or S .
Embodiment 220. A compound of Formula (B-4e), Formula (B-4f>, Formula (B-4g) or Formula (B-4h), or a pharmaceutically acceptable salt thereof, wherein:
R1, R1a and R5 are as defined In Embodiment 214;
Y3 is O , OH, NH2I SH or S , and
Y4 is O , OH, NH2I SH or S-.
Embodiment 221. A compound of Formula (C-4), or a pharmaceutically acceptable salt thereof, wherein:
R1, R13, R3, R5a, R6, Y3 and Y4 are as defined in Embodiment 214;
Y3 is O , OH, NH2, SH or S , and
Y4 is O , OH, NH2, SH or S .
Embodiment 222. A compound of Formula (C-4a), Formula (C-4b), Formula (C-4c) or Formula (C-4d), or a pharmaceutically acceptable salt thereof, wherein:
R1, R13, R3, R53 and R6 are as defined in Embodiment 214;
Y3 is O , OH, NH2, SH or S , and
Y4 is O , OH, NH2, SH or S .
Embodiment 223. A compound of Formula (C-4e), Formula (C-4f), Formula (G-4g) or Formula (C-4h), or a pharmaceutically acceptable salt thereof, wherein:
R1, R1a and R5a are as defined in Embodiment 214;
Y3 is O , OH, NH2, SH or S , and
Y4 is O , OH, NH2, SH or S .
Embodiment 224. A compound of Formula (D-4), or a pharmaceutically acceptable salt thereof, wherein: R1, Ria, R5, R5a, Y3 and Y4 are as defined in Embodiment 214:
Y3 is O , OH, NH2, SH or S , and
Y4 is O , OH, NH2, SH or S .
Embodiment 22S. A compound of of Formula (D-4a), Formula (D-4b), Formula (D-4c) or Formula (D-4d), or a pharmaceutically acceptable salt thereof, wherein:
R1, R1a, R5 and R5a are as defined in Embodiment 214;
Y3 is O , OH, NH2, SH or S , and
Y4 is O , OH, NH2, SH or S .
Embodiment 226. A compound of Formula (E-4), or a pharmaceutically acceptable salt thereof, wherein:
R1, R1a, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 214;
and
Y3 is O , OH, NH2, SH or S .
Embodiment 227. A compound of Formula (E-4a) or Formula (E-4b), or a pharmaceutically acceptable salt thereof, wherein:
R1, R1a, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 214;
and
Y3 is O , OH, NH2, SH or S\
Embodiment 228. A compound of Formula (F-4), or a pharmaceutically acceptable salt thereof, wherein:
R1, R1a, R1b, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 214, and
each Y3 is independently selected from O , OH, NH2, SH or S.
Embodiment 229. The compound of Formula (F~4a), Formula (F~4b), Formula (F-4c), or Formula (F-4d), or a pharmaceutically acceptable salt thereof, wherein:
R1, R1a, R1b, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 214;
and
each Y3 is independently selected from O , OH, NH2, SH or S .
Embodiment 230. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000241_0001
L1R15.
Figure imgf000241_0002
The compound of any one of Embodiments 214 to 229, wherein R ia is
Figure imgf000241_0003
iR15.
Figure imgf000241_0004
The compound of any one of Embodiments 214 to 229, wherein R1b is
Figure imgf000241_0005
iR1£
Figure imgf000241_0006
The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000242_0001
,
Figure imgf000242_0002
The compound of any one of Embodiments 214 to 229, wherein R1a is
Figure imgf000242_0003
Embodiment 235. The compound of any one of Embodiments 214 to 229, wherein R1 b is
Figure imgf000242_0004
,
Figure imgf000242_0005
The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000242_0006
wherein R20 is -L1R15.
Figure imgf000243_0001
The compound of any one of Embodiments 214 to 229, wherein R13 is
Figure imgf000243_0002
wherein R21 is - R15.
Figure imgf000243_0003
The compound of any one of Embodiments 214 to 229, wherein R1 b is
Figure imgf000243_0004
wherein R21 is - R15.
Embodiment 239. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000243_0005
, wherein R2Q is -Li R15.
Figure imgf000243_0006
The compound of any one of Embodiments 214 to 229, wherein R13 is
Figure imgf000243_0007
wherein R21 is -Li R15.
Figure imgf000243_0008
The compound of any one of Embodiments 214 to 229, wherein R18 is
Figure imgf000243_0009
nd of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000243_0010
wherein R20 is -LR15.
Figure imgf000244_0002
Figure imgf000244_0003
,
Figure imgf000244_0001
wherein R2C is -LiR16 and R21 is H. und of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000245_0001
wherein R21 is -L iR15 and R20 is H
Embodiment 2S0. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000245_0002
Embodiment 2S1 The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000245_0004
,
, wherein R1 is
Figure imgf000245_0005
, wherein R1 is
Figure imgf000245_0003
wherein R20 is -LiR15 and R21 is H. ne of Embodiments 214 to 229, wherein R1 is
Figure imgf000246_0001
, wherein R21 is -L1R15 and R20 is H.
of Embodiments 214 to 229, wherein R1 is
Figure imgf000246_0002
wherein R20 is -L1 R15 and R21 is -L1R15.
e of Embodiments 214 to 229, wherein R1 is
Figure imgf000246_0003
wherein R20 is -LiR15 and R21 is H.
ne of Embodiments 214 to 229, wherein R1 is
Figure imgf000246_0004
, wherein R21 is -LiR15 and R20 is H.
of Embodiments 214 to 229, wherein R1 is
Figure imgf000246_0007
wherein R20 is -U R15 and R21 is -UR15.
ny one of Embodiments 214 to 229, wherein R1 is
Figure imgf000246_0005
wherein R20 is -UR15 and R21 is H.
any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000246_0006
wherein R21 is -LiR15 and R20 is H. Embodiment 262. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000247_0001
, wherein R2Q is -LiR15 and R21 is H.
Embodiment 263. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000247_0002
, wherein R21 is -LiR15 and R20 is H.
Embodiment 264. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000247_0003
, wherein R20 is -UR15 and R21 is H.
Embodiment 265. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000247_0004
, wherein R21 is -LiR15 and R20 is H.
Embodiment 266. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000247_0005
wherein R20 is -LiR15 and R21 is H.
Embodiment 267. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000247_0006
, wherein R21 is -LiR15 and R20 is H. y one of Embodiments 214 to 229, wherein R1 is
Figure imgf000248_0001
wherein R20 is -LiR15 and R21 is -LiR15.
ny one of Embodiments 214 to 229, wherein R1 is
Figure imgf000248_0006
, wherein R20 is -LiR15 and R21 is -Li R15.
ny one of Embodiments 214 to 229, wherein R1 is
Figure imgf000248_0002
, wherein R20 is -Li R15.
ny one of Embodiments 214 to 229, wherein R18 is
Figure imgf000248_0003
, wherein R21 is -Li R1
ny one of Embodiments 214 to 229, wherein R1 b is
Figure imgf000248_0004
. wherein R21 is -L R15.
pound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000248_0005
wherein R20 is -LiR15.
Figure imgf000249_0002
214 to 229, wherein R1 is
Figure imgf000249_0003
214 to 229, wherein R1 is
Figure imgf000249_0001
to 229, wherein R1 is
Figure imgf000249_0004
to 229, wherein R1 is
Figure imgf000249_0005
one of Embodiments 214 to 229, wherein R1 is
Figure imgf000250_0001
wherein R20 is LiR15 and R21 is LiR15
of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000250_0002
wherein R20 is H and R21 is UR15.
f any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000250_0003
wherein R20 is UR15 and R21 is H.
of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000250_0004
wherein
Figure imgf000250_0005
f any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000250_0006
wherein R20 is H and R21 is UR15.
f any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000250_0007
wherein R20 is UR15 and R21 is H. , wherein R1 is
Figure imgf000251_0002
, wherein R1 is
Figure imgf000251_0003
mbodiments 214 to 229, wherein R1 is
Figure imgf000251_0001
wherein R20 is UR15 and each R21 is H.
Figure imgf000251_0004
Embodiments 214 to 229, wherein R1 is
Figure imgf000252_0001
, wherein R20 is H, R21 of R1 b is H and R21 of
R| ; is LiR15
Embodiment 294. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000252_0002
wherein R20 is H, and one of R3, R3a, R5 or R5a is -
NHL R15 or -NHNHL iR15 and the others are independently selected from H, -OH and F.
ny one of Embodiments 214 to 229, wherein R1a is
Figure imgf000252_0003
, wherein R21 is H, and one of R3, R3a, R5 or R5a is - NHLiR15 or -NHNHLiR15 and the others are independently selected from H, -OH and F.
ny one of Embodiments 214 to 229, wherein R1 b is
Figure imgf000252_0004
wherein R21 is H, and one of R3, R3a, R5 or R58 is
NHL R15 or -NHNHLiR15 and the others are independently selected from H, -OH and F.
pound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000252_0005
wherein R2Q is H, and one of R3, R3a, R5 or R5a is -NHLiR15 or -
NHNHLiR15 and the others are independently selected from H, -OH and F. mpound of any one of Embodiments 214 to 229, wherein R13 is
Figure imgf000253_0001
wherein R21 is H, and one of R3, R3a, R£ or R5a is --NHLiR15 or ·
NHNHL R15 and the others are independently selected from H, -OH and F.
pound of any one of Embodiments 214 to 229, wherein R1 b is
Figure imgf000253_0002
, wherein R21 is H, and one of R3, R3a, R5 or R5a is -NHL1R15 or
NHNHL R15 and the others are independently selected from H, -OH and F.
Figure imgf000253_0004
,
Figure imgf000253_0005
of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000253_0003
, wherein R20 is H, R21 is H and one of R3, R3a, R5 or R5a is -NHLiR 15 or -NHNHLiR15 and the others are independently selected from H, -OH and F. Embodiment 303. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000254_0001
wherein R20 is H, R21 is H and one of R3, R3a, R5 or R5a is -~NHLiR1s or -NHNHLiRi5 and the others are independently selected from H, -OH and F. Embodiment 304. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000254_0002
, wherein R20 is H, R2i is H and one of R3, R3a, R£ or R5a is -
NHLiR15 or -NHNHLiR1s and the others are independently selected from H, -OH and F Embodiment 305. The compound of any one of Embodiments 214 to 229, wherein R1 is
Figure imgf000254_0003
from H, -OH and F.
Embodiment 308. The compound of any one of Embodiments 214 to 305, wherein:
Y3 is OH, O-, SH or S-, and
Y4 is OH, O-, SH or S\
Embodiment 307. The compound of any one of Embodiments 214 to 305, wherein:
Y3 is OH or Q\ and
Y4 is OH or Q\
Embodiment 308. The compound of any one of Embodiments 214 to 305, wherein:
Y3 is SH or S , and
Y4 is OH or O .
Embodiment 309. The compound of any one of Embodiments 214 to 305, wherein:
Y3 is OH or O , and
Y4 is SH or S .
Embodiment 310. The compound of any one of Embodiments 214 to 305, wherein:
Y3 is SH or S , and
Y4 is SH or S .
Embodiment 311. The compound of any one of Embodiments 214 to 293 or Embodiments 308 to 310, wherein R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H. Embodiment 312. The compound of any one of Embodiments 214 to 293 or Embodiments 308 to 310, wherein R3 is -OH, F or -Nhh.
Embodiment 313. The compound of any one of Embodiments 214 to 293 or Embodiments 308 to 310, wherein R3 is -OH or F.
Embodiment 314. The compound of any one of Embodiments 214 to 293 or Embodiments 308 to 310, wherein R3a is -OH, F or -NH2.
Embodiment 31 S. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein R3a is -OH or F.
Embodiment 316. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein R5 is -OH, F or -NH2.
Embodiment 317. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein R5 is -OH or F.
Embodiment 318. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein R5a is -OH, F or -NH2.
Embodiment 319. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein R5a is -OH or F.
Embodiment 320. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein, when present:
are each H:
Figure imgf000255_0001
Embodiment 321. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein, when present:
are each H;
Figure imgf000255_0002
Embodiment 322. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein, when present:
R2, R2a, R4, R4a, R6, RSa, R7 and R7a are each H;
R3 is F, and
R3a is F.
Embodiment 323. The compound of any one of Embodiments 214 to 293 or Embodiments 308 to 310, wherein, when present:
R2, R2a, R4, R4a, Rs, RSa, R7 and R7a are each H;
R3 is -OH, and
R3a is -OH. Embodiment 324. The compound of any one of Embodiments 214 to 293 or Embodiments 308 to 310, wherein, when present:
are each H;
Figure imgf000256_0001
Embodiment 32S. The compound of any one of Embodiments 214 to 293 or Embodiments 308 to 310, wherein, when present:
are each H;
Figure imgf000256_0002
Embodiment 326. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein, when present:
R2, R2a, R4, R4a, Rs, R6a, R7 and R7a are each H;
R3a is F, and
R5 is F.
Embodiment 327. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein, when present:
are each H:
Figure imgf000256_0003
Embodiment 328. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 31 Q, wherein, when present:
are each H;
Figure imgf000256_0004
Embodiment 329. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein, when present:
are each H;
Figure imgf000256_0005
Embodiment 330. The compound of any one of Embodiments 214 to 293 or Embodiments 308 to 310Q, wherein, when present:
R2, R2a, R4, R4a, Rs, RSa, R7 and R7a are each H;
R3 is F, and
R5a is F.
Embodiment 331. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein, when present: are each H;
Figure imgf000257_0001
R5a is -OH
Embodiment 332. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein, when present:
R2, R2a, R4, R4a, Rs, RSa, R7 and R7a are each H;
R5 is -OH, and
R5a is F
Embodiment 333. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein, when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R5 is F, and
R5a is -OH.
Embodiment 334. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein, when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H:
R5 is F, and
R5a is F.
Embodiment 335. The compound of any one of Embodiments 214 to 293 or Embodiments 306 to 310, wherein, when present:
are each H:
Figure imgf000257_0002
Embodiment 336. The compound of any one of Embodiments 214 to 335, wherein:
Figure imgf000257_0003
C(=0)((CH2)m0)n(CH2)mNHC(=0)X5C(=0)(CH2)mX3(CH2)m-ii; -
C(=0)((CH2)m0)n(CH2)mNHC(=0)X5C(=0)((CH2)m0)n(CH2)m-**; - C(=0)((CH¾)m0)n(CH2)mNHC(=0)X5C(=0)((CH2)m0)n(CH2)mNHC(=0)(CH2)m-**; - C(=0)((CH2)m0)n(CH2)mNHC(=0)X5C(=0)((CH2)m0)n(CH2)mNHC(=0)(CH2)mX3(CH2)m- **; -C(=0)((CH2)m0)n(CH2)mNHC(=0))X5C(=0)((CH2)m0)r!(CH2)mX3(CH2)m-M; - C(=0)((CH2)m0)n(CH2)mNHC(=0)X5C(=0)(CH2)mNHC(=0)((CH2)m0)n(CH2)m-**; - C(=0)((CH2)m0)n(CH2)mNHC(=0)X5C(=0)(CH2)mNHC(=0)((CH2)m0)n(CH2)mX3(CH2)m- **; -C(=0)((CH2)m0)n(CH2)mNHC(=0)X5(CH2)mX3(CH2)nr**; - C(=0)((CH2)m0)ri(CH2)mNHC(=0)X5((CH2)m0)ri(CH2)m-44; - C(=0)((CH2)m0)n(CH2)mNHC(=0)X5((CH2)m0)n(CH2)mNHC(=0)(CH2)m-**; - C(=0)((CH2)m0)ri(CH2)mNHC(=0)X5((CH2)m0)ri(CH2)mNHC(=0)(CH2)mX3(CH2)nr*4; - C(=0)((CH2)m0)n(CH2)mNHC(=0)X5((CH2)m0)n{CH2)mX3(CH2)m-ii; - C(=0)((CH2}m0)n(CH2)mNHC(=0)X5(CH2)mNH((CH2)m0)„(CH2)m-**; - C(=0)((CH2)m0)n(CH2)mNHC(=0)X5C(=0)(CH2)mNH((CH2)m0)n(CH2)mX3(CH2)„riti; -
C(=0)((CH2)mO)n(CH2)mNHC(=0)X5(CH2)m-**; or -C(=0)(CH2)mC(=0)NH(CH2)m-**, where the ** of indicates the point of attachment to R15 and where R12, Xs , X2, X3, X5 m and n are as defined in Embodiment 214.
Figure imgf000258_0001
The compound of any one of Embodiments 214 to 335, wherein:
Figure imgf000258_0002
Figure imgf000259_0001
where the * of Li denotes the point of attachment to -NH-, -NHNH-, -MHO- or -NHN=CRi2-, and where the ** of Li indicates the point of attachment to R15.
Embodiment 338. A compound of Formula (A) selected from:
Figure imgf000259_0002
.
Embodiment 339. A compound of Formula (B) selected from:
Figure imgf000259_0003
Methods of Conjugation
The present invention provides various methods of conjugating Linker-Drug moieties to antibodies or antibody fragments to produce antibody drug conjugates, also referred to as immunconjugates.
A general reaction scheme for the formation of immunostimmulator antibody conjugates of Formula (I) Is shown in Scheme 25 below:
Scheme 25
Figure imgf000260_0001
where: RG2 is a reactive group which reacts with a compatible R15 group to form a
corresponding R115 group (such groups are illustrated in Table 4). D, R15, Li, Ab, y, m, n and R115 are as defined herein.
Scheme 26 further illustrates this general approach wherein the antibody comprises reactive groups (RG2) which react with an R15 group (as defined herein) to covalently attach the Linker-Drug moiety to the antibody via an R115 group (as defined herein). For illustrative purposes only Scheme 26 shows the antibody having four RG2 groups.
Scheme 26
Figure imgf000260_0002
In one aspect, Linker-Drug moieties are conjugated to antibodies via modified cysteine residues in the antibodies (see for example WO2014/124316). Scheme 27 illustrates this approach wherein a free thiol group generated from the engineered cysteine residues in the antibody react with an R15 group (where R15 is a ma!eimide) to covalently attach the Linker-Drug moiety to the antibody via an R115 group (where R115 is a succinimide ring). For illustrative purposes only Scheme 27 shows the antibody having four free thiol groups.
Scheme 27
Figure imgf000261_0001
In another aspect, Linker-Drug moieties are conjugated to antibodies via lysine residues in the antibodies. Scheme 28 illustrates this approach wherein a free amine group from the lysine residues in the antibody react with an R15 group (where R15 is an NHS ester, a
pentafluorophenyl or a tetrafluorophenyl) to covalently attach the Linker-Drug moiety to the antibody via an R115 group (where R115 is an amide). For illustrative purposes only Scheme 28 shows the antibody chaving four amine groups.
Scheme 28
Figure imgf000261_0002
In another aspect, Linker-Drug moieties are conjugated to antibodies via formation of an oxime bridge at the naturally occurring disulfide bridges of an antibody. The oxime bridge is formed by initiaiiy creating a ketone bridge by reduction of an interchain disulfide bridge of the antibody and re-bridging using a 1 ,3-dihaloacetone (e.g. 1 ,3-dichioroacetone). Subsequent reaction with a Linker-Drug moiety comprising a hydroxyl amine thereby form an oxime linkage (oxime bridge) which attaches the Linker-Drug moiety to the antibody (see for example WO2Q14/0835G5). Scheme 29 illustrates this approach. Scheme 29
Figure imgf000262_0001
In yet another aspect, Linker-Drug moieties are conjugated to antibodies by inserting a peptide tag containing a serine residue, such as an So, ybbR, or A1 tag, into the sequence of an antibody as described in Bioconjugate Chemistry, 2015, 26, 2554-2562. These tags acts as a substrate for 4’-phosphopanteiheinyl transferases (PPTase) enzymes wherein the PPTase posttransiationai!y modifies the serine residue to covalently attach a linker derived from coenzyme A (CoA) or from CoA analogues. The linker comprises a pendent ketone which is subsequently reacted with a Linker-Drug moiety comprising a hydroxyl amine thereby forming an oxi e linkage which attaches the Linker-Drug moiety to the antibody. Scheme 30 illustrates this approach.
Scheme 30
Figure imgf000263_0001
Immunoconiugates of the Invention
The present invention provides immunoconjugates, also referred to as antibody drug conjugates, where an antibody, or a functional fragment thereof, is coupled to an agonist of STING via a linker.
in one aspect, the antibodies, antigen binding fragments or their functional equivalents of the invention are linked, via covalent attachment by a linker, to one or more compounds that are agonists of Stimulator of Interferon Genes (STING) receptor.
In one aspect, the antibodies, antigen binding fragments or their functional equivalents of the invention are linked, via covalent attachment by a linker, to one or more compounds that are cyclic dinucleotides which bind to Stimulator of Interferon Genes (STING) receptor.
In one aspect, the antibodies, antigen binding fragments or their functional equivalents of the invention are linked, via covalent attachment by a linker, to one or more compounds that are cyclic dinucleotides which are agonists of Stimulator of interferon Genes (STING) receptor.
In one aspect, the immunoconjugates of the invention comprises one or more Drug moieties (D) as described herein.
In one aspect, the immunoconjugates of the invention comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a compound which binds to Stimulator of Interferon Genes
(STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with one or more iinker(s) (L).
In one aspect, the immunoconjugates of the invention comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a compound which binds to Stimulator of Interferon Genes (STING) receptor and which a comprises one or more reactive moieties capable of forming a covalent bond with one or more linker(s) (L), wherein linker (L) is a eieavable iinker.
In one aspect, the immunoconjugates of the invention comprise one or more Drug moieties (D), wherein the Drug moiety (D) is a dinucieotide which binds to Stimulator of Interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with one or more linker(s) (L).
In one aspect, the immunoconjugates of the invention, comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a dinucleotide which binds to Stimulator of interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with one or more linker(s) (L), wherein iinker (L) is a eieavable iinker.
In one aspect, the immunoconjugates of the invention comprise one or more Drug moieties (D), wherein the Drug moiety (D) is a cyclic dinucieotide which binds to Stimulator of interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with one or more iinker(s) (L).
In one aspect, the immunoconjugates of the invention, comprise one or more Drug moieties (D), wherein the Drug moiety (D) is a cyclic dinucieotide which binds to Stimulator of Interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with one or more iinker(s) (L), wherein iinker (L) is a eieavable Iinker.
In one aspect, the invention provides an immunoconjugate of Formula (i):
Ab— (L— (D)m)n (Formula (I))
wherein:
Ab is an antibody or fragment thereof;
L is a Iinker comprising one or more cleavage elements;
D is a compound which binds to Stimulator of interferon Genes (STING) receptor;
m is an integer from 1 to 8; and
n is an integer from 1-20.
In another aspect, the invention provides an immunoconjugate of Formula (II):
Ab-— (L— D)n (Formula (II))
wherein:
Ab is an antibody or fragment thereof;
L is a iinker comprising one or more cleavage elements;
D is a compound which binds to Stimulator of interferon Genes (STING) receptor;
and
n is an integer from 1-20.
In another aspect, the invention provides an immunoconjugate of Formula (I):
Ab— (L— (D)m)n (Formula (I)
wherein: Ab is an antibody or fragment thereof;
L is a iinker comprising two or more cleavage elements;
D is a compound which binds to Stimulator of Interferon Genes (STING) receptor; m is an integer from 1 to 8; and
n is an integer from 1-20.
In an embodiment of Formula (!) or Formula (II), D is an agonist of Stimulator of Interferon Genes (STING) receptor.
In an embodiment of Formula (!) or Formula (II), D is a cyclic dinucleotides which bind to Stimulator of interferon Genes (STING) receptor.
In an embodiment of Formula (!) or Formula (II), D is a cyclic dinucleotide which is an agonist of Stimulator of Interferon Genes (STING) receptor.
In one aspect, the immunoconjugafes of the invention comprise one or more Drug moieties (D) as described herein.
In one aspect, the immunoconjugafes of the invention comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a compound which binds to Stimulator of Interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with a Iinker.
In one aspect, the immunoconjugaies of the invention comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a compound which binds to Stimulator of Interferon Genes (STING) receptor and which a comprises one or more reactive moieties capable of forming a covalent bond with a Iinker, wherein iinker (L) is a cleavabie iinker.
In one aspect, the immunoconjugates of the invention comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a dinucleotide which binds to Stimulator of Interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with a Iinker.
In one aspect, the immunoconjugates of the invention, comprises one or more Drug moieties (D), wherein the Drug moiety (D) is a dinucleotide which binds to Stimulator of Interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with a Iinker, wherein iinker (L) is a cleavabie Iinker.
In one aspect, the immunoconjugates of the invention comprise one or more Drug moieties (D), wherein the Drug moiety (D) is a cyclic dinucleotide which binds to Stimulator of Interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with a iinker.
In one aspect, the immunoconjugates of the invention, comprise one or more Drug moieties (D), wherein the Drug moiety (D) is a cyclic dinucleotide which binds to Stimulator of Interferon Genes (STING) receptor and which comprises one or more reactive moieties capable of forming a covalent bond with a Iinker, wherein iinker (L) is a cleavabie iinker. The term “eieavage product”, as used herein, refers to a drug moiety (D) linked to a fragment of the linker wherein the fragment comprises one or more linker components (Lc). The cleavage product is formed upon cleavage of Linker (L) from Ab— (L— (D)m)n, wherein a fragment of the Linker (L) remains attached to the drug moiety (D).
In one embodiment, the immunoconjugates of the invention comprise Formula (I):
Ab— (L— (D)m)n (Formula (I))
wherein:
Ab is an antibody or a functional fragment thereof;
L is a linker comprising one or more cleavage elements;
D is a drug moiety] that has agonist activity against STING receptor; m is an integer from 1 to 8; and
n is an integer from 1 to 20.
In one embodiment, the immunoconjugates of the invention comprise Formula (I):
Ab— (L— (D)m)n (Formula (I))
wherein:
Ab is an antibody or a functional fragment thereof;
L is a linker;
D is a drug moiety that binds to STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20;
and wherein D, or a cleavage product thereof, that is released from the immunoconjugaie has STING agonist activity.
In one embodiment, the immunoconjugates of the invention comprise Formula (I):
Ab— (L— (D)m)n (Formula (I))
wherein:
Ab is an antibody or a functional fragment thereof;
L is a linker;
D is a drug moiety that binds to STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20;
wherein the immunoconjugaie delivers D, or a cleavage product thereof, to a ceil targeted by the Ab, and wherein D, or the cleavage product thereof, has STING agonist activity.
In one embodiment, the immunoconjugates of the invention comprise Formula (I):
Ab— (L— (D)m)n (Formula (I))
wherein: Ab is an antibody or a functional fragment thereof;
L is a linker comprising one or more cleavage elements;
D is a drug moiety that binds to STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20;
and wherein the immunoconjugate releases D, or a cleavage product thereof, in a cell targeted by the Ab, and wherein D, or the cleavage product thereof, has STING agonist activity.
In one embodiment, the immunoconjugaies of the invention comprise Formula (I):
Ab— (L— (D)m)n (Formula (I))
wherein:
Ab is an antibody or a functional fragment thereof;
L is a linker comprising one or more cleavage elements;
D is a drug moiety that has agonist activity against STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20;
wherein the immunoconjugate releases D, or a cleavage product thereof, in a cell targeted by the Ab, and wherein D, or the cleavage product thereof, has STING agonist activity in the cell.
In one embodiment, the immunconjugates of the invention comprise Formula (I):
Ab— (L— (D)m)n (Formula (I))
wherein:
Ab is an antibody or a functional fragment thereof;
L is a linker comprising one or more cleavage elements;
D is a drug moiety that binds to STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20;
wherein the immunoconjugate specifically binds to an antigen expressed on the ceil surface and is internalized into the cell, and wherein D, or a cleavage product thereof, is cleaved from L and has STING agonist activity as determined by one or more STING agonist assays selected from: an interferon stimulation assay, a hSTING wt assay, a THP1-Duai assay, a TANK binding kinase 1 (TBK1) assay, or an inierferon-y-inducihle protein (IP-10) secretion assay.
In one aspect the immunoconjugate of the invention, the immunoconjugate is selected from the following:
Figure imgf000268_0001
Formula (AA-c) Formula (AA-d)
Figure imgf000268_0002
Formula (BB-c) Formula (BB-d)
Figure imgf000269_0001
Formula (CC-c) Formula (CC-d)
Figure imgf000269_0002
Formula (DD-c) Formula (DD-d)
Figure imgf000270_0001
Formula (EE-c) Formula (EE-d)
Figure imgf000270_0002
Formula (EE-e) Formula (FF-a)
Figure imgf000271_0001
Formula (FFd) Formula (FF-e)
Figure imgf000272_0001
Formula (FF-f) Formula (FF-g) wherein:
g7 each Gi is independently selected from
Figure imgf000272_0002
where the * of Gi indicates the point of attachment to ~CR8R3 XA is C(=0)-, -C(=S)- or -C(=NR11)- and each Z-\ is NR12;
XB is C, and each Z2 is N;
Y8
Figure imgf000272_0003
Yi is -0-, -S-, -S(=0)-, -SOz-, -CHr, or -CF2-;
Y2 is -0-, -S-, -S(=0)-, -SOz-, -CHr, or -CF2-;
Y3 is OH, O , OR10, N(R10)2, SR1C, SeH, Se , BH3, SH or S
Y4 is OH, O , OR10, N(R10)2, SR1C, SeH, Se , BH3, SH or S
Y5 is -CHr, -NH-, -O- or -S;
Y6 is -CHr, -NH-, -O- or -S;
Y7 is O or S;
Ys is O or S; Y9 is -CHr, -NH-, -O- or -S;
Yio is -CH2-, -NH-, -O- or -S:
Yu is -O-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2-;
q is 1 , 2 or 3;
each R1 is independently a partially saturated or aromatic monocyclic heterocyclyl or
partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms Is independently selected from O, N or S, or a tautomer thereof, wherein R1 is substituted with Q, 1 , 2, 3 or 4 substituents independently selected from -NHL1R115, -NHNHL1R115, - NHOL1R115, -NHN=CRl2(CH2),LiR115, F, Cl, Br, -NHOH, ~NHNH2, -NH2, C C6alkyl and Ci-C3aikyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R1a is independently a partially saturated or aromatic monocyclic heterocyclyl or
partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHL1R115, -NHNHL1R115, - NHOL1R115, -NHN=CR12(CH2)nLiR115, F, Cl, Br, -NHOH, -NHNH2, -NH2, Ci-C6alkyl and Ci-C6alkyi substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R1b is independently a partially saturated or aromatic monocyclic heterocyclyl or
partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHL1R115, -NHNHLiR115, - NHOL1 R1 15, -NHN=CR12(CH2)r!LiR11s, F, Cl, Br, -NHOH, -NHNH2, -NH2, C C6alkyl and CrC6aikyi substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R2 is independently selected from H and CrCsaikyi;
each R3 is independently selected from -NHL1R115, -NHNHL1R115, -NHOLi R115, - NHN=CR12(CH2)gLiR115, H, -OH, -SH , F, Cl, Br, I, NHOH, NHNH2, and NH2;
each R4 is independently selected from H, Ci-C6alkyl and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R5 is independently selected from -NHL1R115, -NHNHL1R115, -NHGL1R115, - NHN=CR12(CH2)nLi R115, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, and NH2;
each R6 is independently selected from H, CrC6alky! and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3; each R7 is independently selected from H, CrC6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each Rs is independently selected from H, CrC6alkyl and Ci-C6alky! substituted by 1 , 2 or 3 substituents independently selected from F, Gi, Br, I, OH, CN, and N3;
each R9 is independently selected from H, CrC6alkyl and Ci-C6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R23 is independently selected from H and CrC6alkyl;
each R38 is independently selected from -NHL1R115, -NHNHL1R115, -NHOL1 R115, - NHN=CR12(CH2),LI R115, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, and NH2;
each R43 is independently selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R5a is independently selected from -NHL1R115, -NHNHL1R115, -NHOL,R1 i5, - NHN=CR12(CH2)r!LiR115, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, and NH2;
each R6a is independently selected from H, Ci-C3alkyi and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7a is independently selected from H, CrCsalky! and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R8a is independently selected from H, Ci-Csalkyl and Ci-Csalkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R9a is independently selected from H, Ci-Csalkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R10 is independently selected from the group consisting yi, C1-
Csheferoaikyi, -(CH2CH20)nCH2CH2C(=0)0C,-Csalkyl, a
Figure imgf000274_0001
wherein the C Ci2aikyi and Ci-Cgheteroaikyi of R1C is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, CrCi2aikoxy, -S-C(=:0)Ci-C8aikyi, halo, -CN, Cr C^aikyl, -O-aryl, -O-heteroaryl, -O-cycloalkyi, oxo, cycloalkyl, heterocyciyl, aryl, or heteroaryl, -0C(0)0CrCsalkyiand C(0)0CrCsalkyl, wherein each alkyl, cycioaikyl, heterocyciyl, aryl, and heteroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from Ci-Ci2 alkyl, 0-CrCi2alkyl, Ci-C12heieroalkyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroaryl, -C(=0)Ci-C 2alkyl, -0C(=;0)C!-Ci2aikyl, -C(=0)0Ci- Ci2aikyi, -0C(=0)0Ci-Ci2aikyl, -C(=0)N(R11)-Ci-Ci2alkyf, -N(R11)C(=0)-Ci-Ci2aikyl; - 0C(=0)N(R11)-C!-Ci2aikyl, -C(=:0)-aryi, -C(:=0)-heteroaryl, -O0(=O)-3! ΐ, -C(=;0)0-aryl, - OC(=0)-heteroaryi, -C(=0)0-heteroaryl, -C(=0)0-aryl, -C(=0)0-heteroaryi, - C(=Q)N(Ri 1)-aryl, -C(=Q)N(Ri 1)-heteroaryl, -N(R11)C(G)-aryl, -N(R11)2C(0)-aryi, - N(R11)C(Q)-heteroaryi, and S(0)2N(R11)-aryl;
each R11 is independently selected from H and CrC6aikyl;
each R12 is independently selected from H and Ci-Cealkyl;
optionally R3 and R6 are connected to form -Q-G i-C6alkylene, such that when R3 and Rs are connected, the O is bound at the R3 position
optionally R3a and R6a, are connected to form -0-CrC6alkylene, such that when R3a and RSa are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form -0-CrC6alkylene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form -0-Ci-C6alkylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form ~0-Ci-C6aikylene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -G-CrCsa!kylene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form -0-CrC6alkylene, such that when R5 and R6 are connected, the O is bound at the R5 position;
optionally R5a and R6a, are connected to form -G-Ci-G6a!kylene, such that when R5a and RSa are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form -O-CrCeaikylene, such that when R5 and R7 are connected, the O is bound at the R5 position;
optionally R5a and R7a, are connected to form -0-Ci-C3aikylene, such that when R5a and R7a are connected, the O is bound at the R5a position;
Li is a linker;
Figure imgf000275_0002
Figure imgf000275_0001
Figure imgf000276_0001
icates the point of attachment to Ab;
R13 is H or methyl;
R is H, -CH3 or phenyl;
each R1 10 is independently selected from H, CrC6alkyi, F, Cl, and -OH;
each R1 1 1 is independently selected from H, CrCealkyi, F, Cl, -NH2, -OCH3, -OCH2CH3, N(CH3)2, -CN, -NO2 and -OH; each R1 12 is independently selected from H, Chalky!, fluoro, benzyloxy substituted with - C(=0)0H, benzyl substituted with -C(=0)0H, Ci-4aikoxy substituted with -C(=0)GH and Chalky! substituted with -C(=0)0H;
each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 and 18;
Ab is an antibody or fragment thereof; and
y is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10,
and provided at least one of R1 , R1a or R1 b is substituted with -NHL1R115, -NHNHL1R1 15, - least one of R3, R5, R3a or R5a is -NHL1R1 15,
Figure imgf000277_0001
2)nL1R1 15.
Gertain aspects and examples of the Immunoconjugates of the invention are provided in the following listing of additional, enumerated embodiments. It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.
Embodiment 340. The immunoconjugate of Formulas (AA-a to AA-d), Formulas (BB-a to BB-d), Formulas (CC-a to CC-d, Formulas (DD-a to DD-d), Formulas (EE-a to EE-e) or Formulas (FF-a to FF-g), or stereoisomers or pharmaceutically acceptable salts thereof, wherein is a linker comprising one or more cleavage elements;
Embodiment 341. An immunoconjugate of Formulas (AA-a to AA-d), Formulas (BB-a to BB- d), Formulas (CC-a to CC-d), Formulas (DD-a to DD-d), Formulas (EE-a to EE-e) or Formulas (FF-a to FF-g), or stereoisomers or pharmaceutically acceptable salts thereof selected from:
Figure imgf000277_0002
Formula (AA-1c) Formula (AA-1d)
Figure imgf000278_0001
Formula (BB-1c) Formula (BB-1d)
Figure imgf000278_0002
Formula (CC-1c) Formula (CG-1d)
Figure imgf000279_0001
Formula (DD-1c) Formula (DD-1d)
Figure imgf000279_0002
Formula (EE-1 a) Formula (EE-1 b)
Figure imgf000280_0001
Formula (EE-1e) Formula (FF-1a)
Figure imgf000281_0001
Formula (FF-1 d) Formula (FF-1e)
Figure imgf000282_0003
for imunoconjugates of Formulas (AA-a to AA-d), Formulas (BB-a to BB-d), Formulas (GC-a to CC-d), Formulas (DD-a to DD-d), Formulas (EE-a to EE-e) and Formulas (FF-a to FF-g), and provided at least one of R1 , R1a or R1 b is substituted with -NHL-iR115, -NHNHL1R115, - least one of R3, R5, R3a or R5a is -NHL R115, -
Figure imgf000282_0001
),lL1R115.
Embodimeiit 342. An immunoconjugate of Embodiment 259 selected from:
Figure imgf000282_0002
Formula (AA-2a) Formula (AA~2b)
Figure imgf000283_0001
Formula (BB-2a) Formula (BB-2b)
Figure imgf000283_0002
Formula (CC-2a) Formula (CC-2h)
Figure imgf000284_0001
Formula (DD-2a) Formula (DD-2b)
Figure imgf000284_0002
Formula (EE-2a) Formula (EE-2b)
Figure imgf000285_0001
Formula (EE-2e) Formula (FF-2a)
Figure imgf000286_0001
Formula (FF-2d) Formula (FF-2e)
Figure imgf000287_0001
Formula (FF~2f) Formula (FF~2g)
wherein y, Ab, R1 , R1a, R18, R2, R: R3, R3a, R4, R4a, R5, R5a, R6, R6a, R7, R7a, R8, RSa , R9, R9a,
R10, R11 , R12, R115, Li , n, Yi , Y2, Y3, Y4, Y5, Ye, Y7, Y8, Ub, Y10 and Yu are as defined above for imunoconjugates of Formulas (AA-a to AA-d), Formulas (BB-a to BB-d), Formulas (CC-a to CC- d), Formulas (DD-a to DD-d), Formulas (EE-a to EE-e) and Formulas (FF-a to FF-g), and provided at least one of R1 , R1a or R1 b is substituted with -NHL1R115, -NHNHL1R115, -NHOL1R1 15 or -NHN=CR12(CH2)nLi R115, or at least one of R3, R5, R3a or R5a is -NHL1 R115, -NHNHL1 R115, - NHOL1 R1 15 or -NHN=CR12(CH2)r.Li R115.
Embodiment 343. The immunoconjugate of Embodiment 341 or Embodiment 342, wherein R1 is pyrimidine or purine nucleic acid base or analogue thereof, R1a is pyrimidine or purine nucleic acid base or analogue thereof, and R1 b is a pyrimidine or purine nucleic acid base or analogue thereof, each of which is substituted as described in R1 , R1a or R18 for
imunoconjugates of Formulas (AA-a to AA-d), Formulas (BB-a to BB-d), Formulas (CC-a to CC-d), Formulas (DD-a to DD-d), Formulas (EE-a to EE-e) and Formulas (FF-a to FF-g).
Embodiment 344. An immunoconjugate of any one of immunoconjugates of Formulas (AA- a) to (AA-d), Formulas (AA~1 a) to (AA-1 d) or Formulas (AA-2a) to (AA-2d), wherein:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are
R3 is selected from -NHL, R1 15, -NHNHL
Figure imgf000287_0002
substituted with a NHOH,
Figure imgf000287_0003
R3a is selected from -NHL1R115, -NHNHLiR115, -NHOL,R115, -NHN=CR12(CH2)nLiR115,H, - OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH2; and
R8, R9, RSa and R9a are independently H or CrCsalkyl,
and provided at least one of R1 or R1a is substituted with -NHL1R115, -NHNHLiR115, - least one of R3, or R3a is -NHL1R115, -
Figure imgf000288_0001
2)r!LiR115.
Embodiment 34S. An immunoconjugate of any one of immunoconjugates of Formulas (AA- a) to (AA-d), Formulas (AA-1 a) to (AA-1 d) or Formulas (AA-2a) to (AA-2d), wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S-;
Y4 is OH, O , NH2, SH or S-;
Y5 and Y6 are O or S;
Y7 and Ys are O or S;
Y9 and Y10 are O or S;
R5, RSa, R7 and R7a are H;
, -NHNHL1R115, H, OH or F;
-NHNHLiR115, H, OH or F; and
Figure imgf000288_0002
R9 are independently selected from H or CrCsaikyl,
and provided at least one of R1 or R1a is substituted with -NHL1R1 1 5 or -NHNHL1R115, or at least one of R3, or R3a Is -NHL1R115 or -NHNHL1R115.
Embodiment 346. An immunoconjugate of any one of immunoconjugates of Formulas (AA- a) to (AA-d), Formulas (AA-1 a) to (AA-1 d) or Formulas (AA-2a) to (AA-2d), wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S ;
Y4 is OH, O , NH2, SH or S ;
Ys and Y6 are O or S;
Y7 and Y8 are O or S;
Y9 and Y10 are O or S;
R2, R2a, R4, R4a, Rs, R5a, R7 and R7a are H;
R3a is -NHNHL1R115, OH or F;
R3 is -NHNHLiR115, OH or F; and
R8a, R9a, R8 and R9 are independently selected from H or C rCBalkyi,
and provided at least one of R1 or R1a is substituted with -NHNHLiR115, or at least one of R3, or R3a is -NHNHLiR115.
Embodiment 347. An immunoconjugate of any one of immunoconjugates of Formulas (BB- a) to (BB-d), Formulas (BB-1 a) to (BB-1 d) or Formula (BB-2a) to (BB-2d), wherein:
R2, R2a, R4, R4a, R6, R63, R7 and R7a are H; R3a is seiected from -NHLiR115, -NHNHLiR115, -NHOL,R115, -NHN=CR12(CH2)nLiR115, H, - OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6aikyl substituted with a NHOH, NHNH2 or NH2;
R5 is seiected from -NHLiR115, -NHNHLiR115, -NHOL1R115, -NHN=CR12(CH )nLiR115, H, - substituted with a NHOH,
Figure imgf000289_0001
and
R8, Rs, R8a and R98 are independently H or Ci-C3alkyi,
and provided at least one of R1 or R1a is substituted with -NHL1 R115, -NHNHLi R115, - least one of R5 or R3a is -NHL1R115, -
Figure imgf000289_0002
2)nLi R115.
Embodiment 348. An immunoconjugate of any one of immunoconjugates of Formulas (BB- a) to (BB-d), Formulas (BB-1 a) to (BB~1 d) or Formula (BB-2a) to (BB-2d), wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S ;
Y4 is OH, O , NH2, SH or S ;
Ys and Y6 are O or S;
Y? and Ys are O or S;
Y9 and Y10 are O or S;
R6, RSa, R7 and R7a are H;
, -NHNHL1R115, H, OH or F;
-NHNHLiR115, H, OH or F, and
Figure imgf000289_0003
R9 are independently selected from H or Ci-C3a!kyl,
and provided at least one of R1 or R18 is substituted with -NHL1R115 or -NHNHLiR115, or at least one of R5 or R3a is -NHL1R1 15 or -NHNHLiR115
Embodiment 349. An immunoconjugate of any one of immunoconjugates of Formulas (BB- a) to (BB-d), Formulas (BB-1 a) to (BB-Ή1) or Formula (BB-2a) to (BB-2d), wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S :
Y4 is OH, O , NH2, SH or S ;
Y5 and Y6 are O or S;
Y7 and Ys are O or S;
Y9 and Y10 are O or S;
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are H;
R3a is -NHNHL1R115, OH or F;
R5 is -NHNHL1R115, OH or F, and
R8a, R98, R8 and R9 are independently selected from H or Ci-C6alkyi, and provided at least one of R1 or R1a is substituted with -NHNHL1R115, or at least one of R5 or R3a is -NHNHL1R115.
Embodiment 350. An immunoconjugate of any one of immunoconjugates of Formulas (CC- a) to (CC-d), Formulas (CC-1 a) to (CC-1 d) or Formulas (GC-2a) to (CC-2d), wherein:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are
R3 is selected from -NHLiR115, -NHNHL
Figure imgf000290_0001
OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH2;
R5a is selected from -NHLiR115, -NHNHL1R115, -NHOL1 R115, -NHN=CR12(CH2),LI R115, H, - OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a CrC6alkyi substituted with a NHOH, NHNH2 or NH2I
and
R8, R9, R8a and R9a are independently H or C -C3alkyi;
and provided at least one of R1 or R,a is substituted with -NHL1R115, -NHNHLiR115, - least one of R5a or R3 is -NHLiR1 15, -
Figure imgf000290_0002
2)nLiR115.
Embodiment 351. An immunoconjugate of any one of Immunoconjugates of Formulas (CC- a) to (CG-d), Formulas (CC-1 a) to (GC-1 d) or Formulas (CC-2a) to (CC-2d), wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S ;
Y4 is OH, O , NH2, SH or S ;
Y5 and Y6 are O or S;
Y7 and Ys are O or S;
Y9 and Y10 are O or S;
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are H;
R3 is -NHLiR1 15, -NHNHLiR1 15, H, OH or F;
R5a is -NHLiR1 15, -NHNHLiR115, H, OH or F, and
R8a, R9a, R8 and Rs are Independently selected from H or CrCgaikyi,
and provided at least one of R1 or R1a is substituted with -NHL1R1 15 or -NHNHLiR115, or at least one of R5a or R3 is -NHL,R115 or -NHNHL1R115.
Embodiment 352. An Immunoconjugate of any one of immunoconjugates of Formulas (CC- a) to (CC-d), Formulas (CC-1 a) to (CC-1 d) or Formulas (GC-2a) to (CC-2d), wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S ;
Y4 is OH, O , NH2, SH or S ;
Y5 and Y6 are O or S;
Y7 and Ys are O or S; Ys and Yio are O or S;
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are H;
R3 is -NHNH R115, OH or F;
R5a is -NHNHUR115, OH or F, and
R8a, R9a, R8 and R3 are independently selected from H or Ci-C6alkyl,
and provided at least one of R1 or R1a is substituted with -NHNHUR115, or at least one of R5a or R3 is -NHNHUR115.
Embodiment 3S3. An immunoconjugate of any one of immunoconjugates of Formulas (DD- a) to (DD-d), Formulas (DD-1 a) to (DD-1 d) or Formulas (DD-2a) to (DD-2d), wherein:
R2, R2a, R4, R4a, R6, R63, R7 and R7a are
R5a is selected from -NHUR115, -NHNH
Figure imgf000291_0001
-OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-Cealkyl substituted with a NHOH, NHNH2
R5 is selected from
Figure imgf000291_0002
substituted with a NHOH,
Figure imgf000291_0003
and
R8, R9, R8a and RSa are independently H or Ci-Csalkyl,
and provided at least one of R1 or R1a is substituted with -NHUR115 or -NHNHUR115, or at least one of R5a or R5 is -NHUR115 or -NHNHUR115.
Embodiment 354. An immunoconjugate of any one of immunoconjugates of Formulas (DD- a) to (DD-d), Formulas (DD-1 a) to (DD~1 d) or Formulas (DD-2a) to (DD-2d), wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S ;
Y< is OH, O , NH2, SH or S ;
Ys and Y6 are O or S;
Y7 and Y8 are O or S;
Y9 and Y10 are
R2, R2a, R4, R4a 7a are H;
R5a is -NHUR11 , OH or F;
R5 is -NHUR115 OH or F, and
Figure imgf000291_0004
R8, R9, RSa and R9a are independently H or Ci-Csalkyl,
and provided at least one of R1 or R1a is substituted with -NHUR115, -NHNHUR115, - least one of R5a or R5 is -NHUR115, -
Figure imgf000291_0005
2)r!LiR115.
Embodiment 355. An immunoconjugate of any one of immunoconjugates of Formulas (DD- a) to (DD-d), Formulas (DD-1 a) to (DD-1 d) or Formulas (DD-2a) io (DD~2d), wherein: Yi and Y2 are O, CH2 or S;
Y3 is OH, O , !MH2 SH or S-;
Y4 is OH, O , NH2 SH or S-;
Ys and Ye are O or S;
Y7 and Ys are O or S;
Y 9 and Yio are O or S;
are H;
Figure imgf000292_0001
R8, Rs, RSa and R9a are independently H or Ci-C3aikyi,
and provided at least one of R1 or R1a is substituted with -NHNHL1R115, or at least one of R5a or R5 is -NHNHL1R"5
Embodiment 35S. An immunoconjugate of any one of immunoconjugates of Formulas (EE- a) to (EE-e), Formulas (EE-1 a) to (EE~1 e) or Formulas (EE-2a) to (EE-2e), wherein:
R2, R2a, R4, R4a, R5, RSa, R7 and R7a are
R3a is selected from -NHL1R115, -NHNH
Figure imgf000292_0002
OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-Cealkyl substituted with a SMHGH, NHNH2 or NH2;
R3 is selected from -NHL1R115, -NHNHL1R115, -NHOLiR115, -NHN=CR12(CH2)nLiR115, H, - OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C C6alkyl substituted with a NHOH, NHNH2 or NH2;
R5 is selected from -NHL1R115, -NHNHL1R115, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-C6alkyi substituted with a NHOH, NHNH2 or NH2,
and
R8, R9, R8a and R9a are independently H or Ci-Cealkyl,
and provided at least one of R1 or R1a is substituted with -NHL1R115, -NHNHLiR115, - least one of R3a, R3 or Rs is -NHL1R115, -
Figure imgf000292_0003
2)r!LiR115.
Embodiment 357. An immunoconjugate of any one of immunoconjugates of Formulas (EE- a) to (EE-e), Formulas (EE-1 a) to (EE-1 e) or Formulas (EE-2a) to (EE-2e), wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2 SH o S :
Y5 is O or S;
Y7 is O or S; 7a are H;
Figure imgf000292_0004
, OH or F; R3 is -NHL, R115, -NHNHLs R115, H , OH or F;
R5 is -NHL, R115, -NHNHLs R115, H , OH or F, and
R8, R9, RSa and R9a are independentiy H or Cs-C3alky!,
and provided at least one of R1 or R1a is substituted with -NHL1 R115 or -NHNHLs R115, or at least one of R3a, R3 or R5 is -NHLs R1 15 or -NHNHLs R115.
Embodiment 3S8. An immunoconjugate of any one of immunoconjugates of Formulas (EE- a) to (EE-e), Formulas (EE-1 a) to (EE-1 e) or Formulas (EE-2a) to (EE-2e), wherein:
Yi and Y2 are O, CH2 or S;
Y3 is OH, O , NH2, SH or S-;
Y5 is O or S;
Y7 is O or S;
Y9 is O or S;
R2, R2a, R4, R4a, R5, R6a, R7 and R7a are H;
R3a is -NHNHLsR1 15, OH or F;
R3 is -NHNHLs R115, OH or F;
R5 is -NHNHLs R115, OH or F, and
R8, R9, R8a and RSa are independently H or Ci-C3a!kyl,
and provided at least one of R1 or R1a is substituted with -NHNHLs R115, or at least one of R3a, R3 or R5 is -NHNHLs R115.
Embodiment 359. An immunoconjugate of any one of immunoconjugates of Formulas (FF- a) to (FF-g), Formulas (FF-1 a) to (FF-1 g) or Formulas (FF-2a) to (FF-2g), wherein:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are
R3a is selected from -NHLs R115, -NHNH
Figure imgf000293_0001
OH, -SH, F, Cl, Br, i, NHOH, NHNH2, NH2 and a Cs-C6aikyl substituted with a NHOH, NHNHz or NH2;
each R3 is independentiy selected from -NHLs R115, -NHNHLs R115, -NHOLs R115, - NHN=CR12(CH2)„Ls R115, H, -OH, -SH , F, Cl, Br, I, NHOH, NHNH2, NH2 and a Cs- Csalkyl substituted with a NHOH, NHNH2 or NH2;
each R5 is independentiy selected from -NHLs R115, -NHNHLs R115, H, -OH, -SH , F, Cl, Br, I , NHOH , NHNHz, NH2 and a C,-C6alkyi substituted with a NHOH, NHNH2 or NH2,
R8, R9, R8a and R9a are independently H or Cs-Cgalkyi,
and provided at least one of R1 , R1a or R1b is substituted with -NHLs R115, -NHNHLsR115, - least one of R3a, R3 or R5 -NHLs R115, -
Figure imgf000293_0002
2)r!Ls R115.
Embodiment 360. An immunoconjugate of any one of immunoconjugates of Formulas (FF- a) to (FF-g), Formulas (FF-1 a) to (FF~1 g) or Formulas (FF-2a) to (FF~2g), wherein:
Ys and Y2 are O, CH2 or S; each Y3 is independently OH, O , NH2, SH or S ;
each Y5 is independently O or S;
each Y-/ is independently O or S;
each Yg is independently O or S; 7a are H;
Figure imgf000294_0001
, OH or F;
each R3 is independently selected from -NHLiR115, -NHNHLiR115, H, OH or F;
each R5 is independently selected from -NHLiR115, -NHNHLiR115, H, OH or F, and R8, Rs, RSa and R9a are independently H or Ci-C3aikyl,
and provided at least one of R1 , R1a or R1b is substituted with -NHLiR115 or -NHNHLiR115, or at least one of R3a, R3 or R5 is -NHLiR115 or -NHNHLiR115
Embodiment 3S1. An immunoconjugate of any one of immunoconjugates of Formulas (FF- a) to (FF-g), Formulas (FF-1 a) to (FF-1 g) or Formulas (FF-2a) to (FF-2g), wherein:
Yi and Y2 are O, CH2 or S;
each Y3 is independently OH, O , NH2, SH or S ;
each Y5 is independently O or S;
each Y? is independently O or S;
each Y9 is independently O or S;
Yu is O, GH2 or S;
R2, R2a, R4, R4a, R6, RSa, R7 and R7a are H;
R3a is -NHNHLiR115, H, OH or F;
each R3 is independently selected from -NHNHLiR115, H, OH or F;
each R5 is independently selected from -NHNHLiR115, H, OH or F, and
R8, R9, R8a and R9a are independently H or Ci-Cealkyl,
and provided at least one of R1 , R1a or R1b is substituted with -NHNHLiR115, or at least one of R3a, R3 or R5 is -NHNHLiR115
Embodiment 362. An immunoconjugate of Formula (AA-a to AA-d), Formula (BB-a to BB-d), Formula (CC-a to CC-d), Formula (DD-a to DD-d), Formula (EE-a to EE-e), Formula (FF-a to FF-g) or an immunoconjugate of any one of Embodiments 340 to 361 , wherein:
Figure imgf000295_0001
Figure imgf000296_0001
Figure imgf000297_0001
Figure imgf000298_0001
Figure imgf000299_0002
Figure imgf000299_0001
Figure imgf000300_0001
Figure imgf000300_0002
, , , stituents independently selected from F, Cl, Br, NHOH, NH2, CrC6aikyl and Ci-C6alkyl
substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and Ns; each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 18,17 and 18 and each R2C0 is independently selected from H and UR115;
Figure imgf000301_0001
Figure imgf000302_0001
Figure imgf000303_0001
Figure imgf000304_0001
Figure imgf000305_0001
Figure imgf000306_0001
Figure imgf000306_0002
, wherem: Rla is substituted with 0, 1 , 2 or 3 substituents independently selected from F, Cl, Br, NHGH, NH2, C -e5alkyi and Cr C5alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3; each n is independently selected from 1, 2, 3, 4, 5, 8, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17 and 18 and each R210 is independently selected from H and LiR115, and
Figure imgf000307_0001
Figure imgf000308_0001
Figure imgf000309_0001
Figure imgf000310_0001
Figure imgf000311_0001
Figure imgf000312_0001
Figure imgf000312_0002
, wherem: RID is substituted with 0, 1 , 2 or 3 substituents independently selected from F, Cl, Br, NHGH, NH2, C -e5alkyi and Cr C5alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3; each n is independently selected from 1, 2, 3, 4, 5, 8, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17 and 18 and each R210 is independently selected from H and LiR115.
Figure imgf000312_0003
An immunoconjugate selected from:
Figure imgf000313_0001
Formula (BB-3c) Formula (BB-3d)
Figure imgf000314_0001
Formula (CC-3c) Formula (CC-3d)
Figure imgf000314_0002
Formula (DD-3c) Formula (DD-3d)
Figure imgf000315_0001
Formula (EE-3c) Formula (EE-3d)
Figure imgf000315_0002
Formula (EE-3e) Formula (FF-3a)
Figure imgf000316_0001
Formula (FF-3d) Formula (FF-3e)
Figure imgf000317_0001
Formula (FF-3f) Formula (FF-3g) wherein:
Figure imgf000317_0002
Figure imgf000318_0001
Figure imgf000318_0002
Figure imgf000319_0001
Figure imgf000320_0001
Figure imgf000321_0001
Figure imgf000322_0001
Figure imgf000323_0001
, , , stituents independently selected from F, Cl, Br, NHOH, NH2 CrC6alkyl and CrC6aikyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and Ns, and each R200 is independently selected from H and L R115;
Figure imgf000323_0002
Figure imgf000324_0001
32:
Figure imgf000325_0001
Figure imgf000326_0001
Figure imgf000327_0001
Figure imgf000328_0001
Figure imgf000329_0001
, wherein : R1 a is substituted with Q, 1 , 2 or 3 substituents independently selected from F, Cl, Br, NHQH, NH2, Ci-C3alkyl and Cr Csalkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, i , OH , CN, and Na, and each R210 is independently selected from H and Li R115,
Figure imgf000329_0002
Figure imgf000330_0001
Figure imgf000331_0001
Figure imgf000332_0001
Figure imgf000333_0001
Figure imgf000334_0001
Figure imgf000334_0002
Figure imgf000334_0003
Figure imgf000334_0004
Figure imgf000334_0005
Figure imgf000334_0006
L
Figure imgf000334_0007
Figure imgf000334_0008
wherein: R10 is substituted with Q, 1 , 2 or 3
5 3 substituents independently selected from F, Cl, Br, NHOH, NH2, CrCsalkyl and C¾- Cgalkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3, and each R210 is independently selected from H and R115;
each R2 is Independently selected from H and Ci-Csalkyl;
each R3 is Independently selected from -NHL1R115, -NHNHL1R115, -NHGL1R115, -
NHN=CR12(CH2).LI R115, H, -OH, -SH , F, Cl, Br, I, NHOH, NHNH2, NH2 and a Cr C6alkyl substituted with a NHOH, NHNH2 or NH2;
each R4 is independently selected from H, CrC6alky! and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
each R5 is independently selected from -NHL1R115, -NHNHL1R115, -NHOL1R115, -
NHN=CR12(CH2)r,LiR115, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci- Csalkyl substituted with a NHOH, NHNH2 or NH2;
each R6 is independently selected from H, CrC6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7 is independently selected from H, CrC6alkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
each R8 is independently selected from H, CrC6alkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
each R9 is independently selected from H, CrC6alkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
each R2a is Independently selected from H and C i-C6aikyl;
each R3a is independently selected from -NHL1R115, -NHNHL1R115, -NHOL1R115, -
NHN^CR^iCH^nLiR115, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C Csalkyl substituted with a NHOH, NHNH2 or NH2;
each R4a is independently selected from H, C -C3alkyi and CrCgalkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
each RSa is independently selected from -NHL1R115, -NHNHLiR115, -NHOL R115, -
NHN=CR12(CH2)„LiR115, H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C Csalkyl substituted with a NHOH, NHNH2 or NH2;
each R6a is independently selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
each R7a is independently selected from H, Ci-C6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
each RSa is independently selected from H, Ci-C6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3;
each R98 is independently selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Ci, Br, I, OH, CN, and N3; each R10 is independently selected from the group consisting r
Csheteroalkyl, -(GH2CH20)nCH2GH2C(=0)0Gi-C6alky
Figure imgf000336_0001
wherein the
CrG ^aikyi and Ci-Gsheieroaikyi of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, Ci-C^aikoxy, -S-C(=;0)CrC6alkyi, halo, -CM, Cr Ci2alkyi, -O-aryl, -O-heteroaryl, -O-cycioalkyl, oxo, cycloalkyl, heierocyciyl, aryl, or heteroaryl, -QC(0)OCi-C6a!kyland C(OjOCi-C3alkyi, wherein each alkyl, cycioaikyl, heierocyciyl, aryl, and heieroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from C -C alkyl, Q~Ci-Ci2alkyi, CrCi2heteroalkyi, halo, CN, OH, oxo, aryl, heieroaryl, Q~aryi, Q-heteroaryi, -C(=0)Ci~Ci2alkyi, -0C(=0)Ci- N(Rn)-Ci-Ci2alkyl, -
Figure imgf000336_0002
)-aryl, -C(=0)-heteroaryl, -GC(=0)-aryi, -C(=G)0-aryl, -OC(=0)-heteroaryi, -C(=0)Q-heteroaryi, -C(=0)0-aryi, -C(=0)0-heteroaryl, -G(=0)N(R11)-aryl, -G(=0)IM(R11)-heteroaryl, -N(R11)C(0)-aryl, - N(R11)2C(0)-aryl, -N(R11)C(0)-heteraaryl, and S(G)2N(R11)-aryi;
each R11 is independently selected from H and CrC6aikyl;
each R12 is independently selected from H and CrC6aikyl;
optionally R3 and R6 are connected to form -Q-Gi-C6aikylene, such that when R3 and Rs are connected, the O is bound at the R3 position
optionally R3a and R6a, are connected to form -0-Ci-C6aikyiene, such that when R3a and RSa are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form -0-CrC6alkylene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form -0-Gi-C6alkylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form ~0-Ci-C6alkylene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -Q-Ci-C3aikyiene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form -0-CrC6alkylene, such that when R5 and R6 are connected, the O is bound at the R5 position;
optionally R5a and R6a, are connected to form -Q-Ci-G6a!kyiene, such that when R5a and RSa are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form -0-Ci-C6alkylene, such that when R5 and R7 are connected, the O is bound at the R5 position; optionally R5a and R7a, are connected to form -0-CrCBalkyiene, such that when R5a and R7a are connected, the O is bound at the R5a position;
Figure imgf000337_0001
Figure imgf000338_0001
C(=0)XiX2((CH2)m0)n(CH2)rrNHC(=0)(CH2)m-44; - C(=0)XiX2((CH2)mO)n(CH2)mNHC(=0)(CH2)mX3(CH2)m-**;
Figure imgf000339_0001
C(=0)XiC(=0)(CH2)mNHC(=0)(CH2)m-**; where the ** of Li indicates the point of attachment to R115;
Figure imgf000340_0001
Figure imgf000341_0001
f R115 indicates the point of attachment to Ab;
X
Figure imgf000341_0002
where the * of Xi indicates the point of attachment to X2:
Figure imgf000341_0003
the * of X2 indicates the point of attachment to Xi, -NH-, -NHNH-, -NHO- or -NHN=CR12(CH2)„
Figure imgf000342_0001
Figure imgf000342_0003
each R1 1 is independently selected from H and CrC6aikyl;
each R12 is independently selected from H and CrC6aikyl;
R13 is H or methyl;
R14 is H, -CH3 or phenyl;
each R1 10 is independently selected from H, CrCsa!kyi, F, Cl, and -OH;
each R111 is independently selected from H, CrCsalkyi, F, Cl, -NH2, -GCH3, -GCF^CFb, -
N(CH3)2, -CN, -NG2 and -OH;
each R1 12 is independently selected from H, Ci-6alkyl, fluoro, benzyloxy substituted with -
C(=G)OH, benzyl substituted with -0(=O)OH, Ci-4alkoxy substituted with -G^OjOH and Ci 4aikyl substituted with -0(=O)OH;
each m is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10;
each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 Q, 1 1 , 12, 13, 14, 15, 18, 17
Ah is an antibody or fragment thereof, and
each y is independently selected from 1 , 2, 3, 4, 5, 8, 7, 8, 9 or 10,
and provided at least one of R200 or R21C is -LiR11s, or at least one of R3, R5, R3a or R5a is - NHL1R115, -iMHNHLiR115, -NHQLiRi 1£' or -SMHN=CR12(CH2)nLiRn5
Embodiment 384. An immunoconjugate selected from:
Figure imgf000342_0002
Formula (AA-4a) Formula (AA-4a)
Figure imgf000343_0001
Formula (AA-4c) Formula (AA-4d) wherein: Ab, y, R1 , R1a, R3, R3a, R6, R6a, Y3 and Y4 are as defined in Embodiment 383.
Embodiment 36S. An immunoconjugate selected from:
Figure imgf000343_0002
Formula (AA-4Ϊ) Formula (AA-4j)
Figure imgf000344_0001
Formula (AA-4s) Formula (AA-4t) wherein: Ab, y, R1, R1a, R3, R3a, R6 and R6a are as defined in Embodiment 363; Y3 is O, OH, NH2, SH or S, and
Y4 is O, OH, NH2, SH orS .
Embodiment 366. An immunoconjugate selected from:
Figure imgf000345_0001
Formula (AA-5g) Formula (AA-5h)
Figure imgf000346_0001
Formula (AA-5o) Formula (AA-5p)
Figure imgf000347_0001
Formula (AA-5s) Formula (AA-5t)
Figure imgf000347_0002
Formula (AA~5w) Formula (AA-5x)
Figure imgf000347_0003
Formula (AA-5y) Formula (AA-5z)
Figure imgf000348_0001
Formula (AA-5ec) Formula (AA-5dd)
Figure imgf000348_0002
Formula (AA-5ee) Formula (AA-5ff) wherein: Ab, y, R1 , R1a, R3, R3a, R6 and R6a are as defined in Embodiment 363;
Y3 is O , OH, NH2, SH or S , and
Y4 is O , OH, NH2, SH or S~.
Embodiment 367. An immunoconjugate selected from:
Figure imgf000348_0003
Formula (BB-4a) Formula (BB-4b)
Figure imgf000349_0001
Formula (BB~4c) Formula (BB~4d) wherein: Ab, y, R1. R1a, R3 R3a, R£ R5a, Y3 and Y4 are as defined in Embodiment 363.
Figure imgf000349_0002
An immunoconjugate selected from:
Figure imgf000349_0003
Formula (BB-4I) Formula (BB-4J)
Figure imgf000350_0001
Formula (BB-4q) Formula (BB-4r)
Figure imgf000351_0001
Formula (BB-4s) Formula (BB-41.) wherein: Ab, y, R1, R1a, R3a, Rs and R6a are as defined in Embodiment 363;
Y3 is O, OH, NH2, SH or S, and
Y4isO-.OH. NH2, SHorS.
Embodiment 369. An immunoconjugate selected from:
Figure imgf000351_0002
Formula (BB-5c) Formula (BB-5d)
Figure imgf000351_0003
Formula (BB-5e) Formula (BB-5f)
Figure imgf000352_0001
Formula (BB-5k) Formula (BB-51) wherein: Ab, y, R1, R1a and R5 are as defined in Embodiment 363;
Y3 is O, OH, NH2, SH or S, and
Y4 is O, OH, NH2, SH orS .
Embodiment 370. An Immunoeonjugate selected from:
Figure imgf000352_0002
Formula (GC-4a) Formula (CC-4b)
Figure imgf000353_0001
Formula (CC~4c) Formula (CC-4d) wherein: Ab, y, R1 , R1a, R3, R5a, R6, R6a, Y3 and Y4 are as defined In Embodiment 363
Embodiment 371. An immunoconjugate selected from:
Figure imgf000353_0002
Formula (CC-41) Formula (CC~4j)
Figure imgf000354_0001
Formula (CC-4s) Formula (CC-4t) wherein: Ab, y R\ R1a, R3, R5a, R6 and R6a are as defined In Embodiment 363; Y3 is O, OH, NH2, SH or S, and Y4 is O, OH, NH2, SH orS .
Embodiment 372. An immunoconjugate selected from:
Figure imgf000355_0001
Formula (CC-51) Formula (CC-5J)
Figure imgf000356_0001
Formula (CC-5k) Formula (CC-5I) wherein: Ab, y, R1 , R1a, R5a and R6a are as defined in Embodiment 363;
Y3 is O , OH, NH2, SH or S , and
Y4 is O , OH, NH2, SH or S .
Embodiment 373. An immunoconjugate selected from:
Figure imgf000356_0002
Formula (DD-4c) Formula (DD-4d) wherein: Ab, y R1 , R1a, R5, R5a, Y3 and Y4 are as defined in Embodiment 363
Embodiment 374. An Immunoconjugate selected from:
Figure imgf000356_0003
Formula (DD-4e) Formula (DD-41)
Figure imgf000357_0001
Formula (DD-41) Formula (DD-4j)
Figure imgf000358_0001
Formula (DD-4o) Formula (DD-4p) wherein: Ab, y, R1 , R1a, R5 and R5a are as defined in Embodiment 383;
Y3 is O , OH, NH2, SH or S~, and
Y4 is O , OH, NH2, SH or S .
Figure imgf000358_0002
An immunoconjugate selected from:
Figure imgf000358_0003
Formula (E-4a) Formula (E-4b)
Figure imgf000359_0001
Formula (EE-4e)
wherein: Ab, y, R1 , R1a, R3, R3a, R4, R43, R5, R7 and Y3 are as defined in Embodiment 363. Embodiment 376. An immunoconjugate selected from:
Figure imgf000359_0002
Formula (EE-40 Formula (EE~4g)
Figure imgf000360_0001
Formula (EE-41) Formula ( EE-4m )
Figure imgf000361_0001
Formula (EE-4p) Formula (EE-4q)
wherein: Ab, y, R 1 pia 3 3a ^4 4a ^5 p7 an(j g3 are as defined in Embodiment 363: and Y3 is O, OH, NH2, SH or S.
Figure imgf000361_0002
An immunoconjugate selected from:
Figure imgf000361_0003
Formula (FF-4a) Formula (FF-4b)
Figure imgf000362_0001
Formula (FF-4e) Formula (FF-4f)
Figure imgf000362_0002
Formula (FF-4g) wherein: Ab, y, R1 , R1a, Ri b, R3, R3a, R4, R4a, R5, R7 and Y3 are as defined in Embodiment 383, Embodiment 378. An immunoconjugate selected from:
Figure imgf000363_0001
Formula (FF-5c) Formula (FF-5d)
Figure imgf000364_0001
Formula (FF-5e) Formula (FF-5f)
Figure imgf000364_0002
7 are as defined in Embodiment 383, and each Y3 is independently selected from G , OH, NH2, SH and S\
Embodiment 379. An immunoconjugate selected from:
Figure imgf000365_0001
Formula (FF-6c) Formula (FF-6d)
Figure imgf000365_0002
Formula (FF-6e) Formula (FF-6f)
Figure imgf000366_0001
Formula (FF~8g)
wherein: Ab, y, R\ R1a, R1 b, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 363, and each Y3 is independently selected from O , OH, NH2, SH and S .
Figure imgf000366_0002
An immunoconjugaie selected from:
Figure imgf000366_0003
Formula (FF~7a) Formula (FF~7b)
Figure imgf000367_0001
Formula (FF-7e) Formula (FF-7f)
Figure imgf000367_0002
Formula (FF-7g) wherein: Ab, y, R1, R1a, R1b, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 363, and each Y3 is independently selected from O , OH, NH2, SH and S\
Figure imgf000368_0001
An immunoconjugate selected from:
Figure imgf000368_0002
Formula (FF-8c) Formula (FF-8d)
Figure imgf000369_0001
Formula (FF-8e) Formula (FF-8f)
Figure imgf000369_0002
7 are as defined In Embodiment 383, and each Y3 is independently selected from G , OH, NH2, SH and S\
Embodiment 382. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000369_0003
Embodiment 383. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000370_0001
Embodiment 384. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000370_0002
Embodiment 385. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000370_0003
is -LiR1 Embodiment 388. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000371_0001
is -LiR115.
Embodiment 387. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000371_0002
is -LiR115.
Embodiment 388. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000371_0003
wherein R200 is -LiR11
Em s 341 to 381 , wherein
Figure imgf000371_0005
wherein R210 is -L¾R115
Em
Figure imgf000371_0004
Figure imgf000372_0001
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000372_0002
wherein R20C is -L1R115.
Embodiment 392. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000372_0003
wherein R210 is -U R115.
393. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000372_0004
, wherem R2 i0 is -Li R1 ,5.
ate of any one of Embodiments 341 to 381 , wherein
Figure imgf000372_0007
, wherein R20C is -LiR11s.
e of any one of Embodiments 341 to 381 , wherein
Figure imgf000372_0005
wherein R210 is -LiR115.
Embodiment 396. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000372_0006
wherein R210 is -LiR11 Embodiment 397. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
H
-R2C0
HN HN
N~i¾ A
¾ z> c--,
( NL N
!
R1 is or i , wherein R200 is -L1R115.
Embodiment 398. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
N R210 P-r210
HN HN
G 5
R1a is T or T' , wherein R210 is -UR115
Embodiment 399. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
.N~-R210
H HN
:N
N N— ¾
N A
R ss i or ! , wherein R213 is -LiR1
Embodiment 400. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
HN HN
N- Ί
i \\
N
! y
R1 is i ' and 1a is "· , wherein R200 is -LR115 and R210 is H.
Embodiment 401. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
R200 H
n-R 21
HN
\
IN
i .\^-N
R1 is and R1a is , wherein R210 is -LiR115 and R200 is H
Embodiment 402. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000373_0001
, wherein R200 is -LiR115 and R210 is -L1R 15
1 Embodiment 403. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000374_0001
, wherein R2C0 is -LiR, i5 and R2 i0 is H.
Embodiment 404. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000374_0002
Embodiment 405. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000374_0003
wherein R200 is -LiR115 and R210 is -LiR115.
Embodiment 40S. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000374_0004
wherein R200 is -LiR115 and R210 is H.
Embodiment 407. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000374_0005
, wherein R210 is -LiR115 and R200 is H.
Embodiment 408. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000374_0007
Embodiment 409. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000374_0006
, wherein R200 is -LiR115 and R210 is H.
375
Figure imgf000375_0001
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000375_0002
wherein R210 is -Li R115 and R200 is H.
Figure imgf000375_0003
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000375_0004
wherein R2Q0 is -Li R115 and R210 is LiR115.
any one of Embodiments 341 to 381 , wherein
Figure imgf000375_0005
wherein R200 is -U R115 and R21C is H.
any one of Embodiments 341 to 381 , wherein
Figure imgf000375_0006
, wherein R210 is -Li R115 and R20tl is H.
Figure imgf000375_0007
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000375_0008
wherein R200 is -Li R115 and R210 is -LiR1 i5.
any one of Embodiments 341 to 381 , wherein
Figure imgf000375_0009
wherein R210 is -L1 R1 15 and R200 is H. any one of Embodiments 341 to 381 , wherein
Figure imgf000376_0001
, wherein R200 is -L1R1 15 and R21C is H.
any one of Embodiments 341 to 381 , wherein
Figure imgf000376_0002
, wherein R210 is -LiR115 and R2Q0 is H.
any one of Embodiments 341 to 381 , wherein
Figure imgf000376_0003
wherein R200 is -LiR115 and R210 is -UR115.
any one of Embodiments 341 to 381 , wherein
Figure imgf000376_0004
wherein R210 is -L R11s and R200 is H
Figure imgf000376_0005
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000376_0006
Figure imgf000376_0007
The immiinoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000376_0008
wherein R2Q0 is -LiR115 and R210 is -LiR115. any one of Embodiments 341 to 381 , wherein
Figure imgf000377_0009
, wherein R200 is -L R115 and R21Q is H.
any one of Embodiments 341 to 381 , wherein
Figure imgf000377_0001
, wherein R210 is -LiR115 and R200 is H.
Figure imgf000377_0002
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000377_0003
wherein R200 is -UR115.
Figure imgf000377_0004
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000377_0005
wherein R21C is -LiR11s.
Figure imgf000377_0006
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000377_0007
njugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000377_0008
, wherein R200 is -LiR115. njugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000378_0001
wherein R210 is ~L R115.
njugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000378_0002
wherein R210 is -LiR115.
ugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000378_0003
wherein R200 is L1R1 15 and R210 is H.
ugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000378_0004
, wherein R200 is H and R210 is UR1
ugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000378_0005
wherein R200 is LiR115 and R210 is L iR115.
Figure imgf000378_0006
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000378_0007
, wherein R200 is UR115 and R2UI is H.
Figure imgf000378_0008
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000378_0009
wherein R200 is H and R21C is LiR115.
Figure imgf000379_0001
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000379_0006
wherein R200 is LiR115 and R210 is L R115.
of any one of Embodiments 341 to 381 , wherein
Figure imgf000379_0007
wherein R200 is H and R210 is UR115.
of any one of Embodiments 341 to 381 , wherein
Figure imgf000379_0002
wherein R2C0 is LiR115 and R21C is H.
of any one of Embodiments 341 to 381 , wherein
Figure imgf000379_0003
wherein R200 is LiR115 and R210 is LiR115.
of any one of Embodiments 341 to 381 , wherein
Figure imgf000379_0004
, wherein R200 is H and R210 is LiR115.
of any one of Embodiments 341 to 381 , wherein
Figure imgf000379_0005
, wherein R2C0 is LiR115 and R21C is H. of any one of Embodiments 341 to 381 , wherein
Figure imgf000380_0009
, wherein R200 is LiR115 and R210 is L,R1 i5
ugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000380_0001
, wherein R200 is LiR115 and R210 is H
ugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000380_0002
, wherein R200 is H and R210 is LiR115.
ugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000380_0003
wherein
Figure imgf000380_0004
Figure imgf000380_0005
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000380_0006
wherein R200 is LiR115 and each R210 is H.
Figure imgf000380_0007
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000380_0008
wherein R200 is H R210 of R1 b is
LiR115 and R210 of R ia is H. Embodiment 447. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000381_0005
Embodiment 448. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000381_0006
and F.
Figure imgf000381_0001
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000381_0002
, wherein R210 is H, and one of R3, R3a, R5 or
R5a is -NHLiR115 or -NHNHL1R1 1 5 and the others are independently selected from H, -OH and F.
Figure imgf000381_0003
The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000381_0007
and F.
Embodiment 451. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000381_0004
, wherein R200 is H, and one of R3, R3a, R5 or R5a is -
NHL1R1 15 or -NHNHLsR1 15 and the others are independently selected from H, -OH and F. njugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000382_0001
wherein ?210 :c H, and one of R3, R3a, R5 or R5a is
NHLiR1 15 or -NHNHLiR115 and the others are independently selected from H, -OH and F.
ugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000382_0002
wherein R210 is H, and one of R3, R3a, R5 or R£,a is
NHLiR115 or -NHNHLiR1 15 and the others are independently selected from H, -OH and F.
ugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000382_0003
, wherein R200 is H, R210 is H and one of R3, R3a, R5 or
R£,a is -~NHLiR1 15 or -NHNHLiR115 and the others are independently selected from H, -OH and F.
55. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000382_0004
, wherein R2Q0 is H, R210 is H and one of R3,
R3a, R5 or R5a is -NHLiR115 or -NHNHLiR115 and the others are independently selected from H, -OH and F.
of any one of Embodiments 341 to 381 , wherein
Figure imgf000382_0005
wherein R200 is H, R210 is H and one of R3, R3a, R5 or R5a is -NHL-iR115 or -NHNHLiR115 and the others are independently selected from H, -OH and F. Embodiment 457. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000383_0001
, wherein R200 is H, R210 is H and one of R3, R3a, R5 or R5a is -NHL1R1 15 or -NHNHL1R1 15 and the others are independently selected from H, -OH and F.
Embodiment 458. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000383_0002
, wherein R20C is H, R210 is H and one of R3, R3a, R5 or
R5a is -NHLiR115 or -NHNHL1R1 15 and the others are independently selected from H, -OH and F.
Embodiment 459. The immunoconjugate of any one of Embodiments 341 to 381 , wherein
Figure imgf000383_0003
ntly selected from H, -OH and F.
Embodiment 480. The immunoconjugate of any one of Embodiments 341 to 458, wherein:
Y3 is OH, O , SH or S , and
Y4 is OH, O , SH or S .
Embodiment 461. The immunoconjugate of any one of Embodiments 341 to 458, wherein:
Y3 is OH or O , and
Y4 is OH or O .
Embodiment 462. The immunoconjugate of any one of Embodiments 341 to 458, wherein:
Y3 is SH or S , and
Y4 is OH or O .
Embodiment 463. The immunoconjugate of any one of Embodiments 341 to 458, wherein:
Y3 is OH or O , and
Y4 is SH or S .
Embodiment 464. The immunoconjugate of any one of Embodiments 341 to 458, wherein:
Y3 is SH or S- , and
Y4 is SH or S . Embodiment 465. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein:
R2, R2a, R4, R4a, R6, R6a R7 and R7a are each H.
Embodiment 466. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein:
R3 is -OH, F or -NHz.
Embodiment 467. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein:
R3 is -OH or F.
Embodiment 468. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein:
R3a is -OH, F or -NH2.
Embodiment 4S9. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein:
R3a is -OH or F.
Embodiment 470. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein:
R5 is -OH, F or -NH2.
Embodiment 471. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein:
R5 is -OH or F.
Embodiment 472. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein:
R5a is -OH, F or -NHz.
Embodiment 473. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein:
R5a is -OH or F.
Embodiment 474. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein, when present:
are each H;
Figure imgf000384_0001
R3a is F.
Embodiment 47S. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein, when present:
R2, R2a, R4, R4a, Rs, RSa, R7 and R7a are each H;
R3 is F, and
R3a is -OH. Embodiment 476. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein, when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is F, and
R3a is F.
Embodiment 477. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein, when present:
are each H;
Figure imgf000385_0001
Embodiment 478. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein, when present
are each H;
Figure imgf000385_0002
Embodiment 479. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein, when present:
are each H:
Figure imgf000385_0003
Embodiment 480. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein, when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3a is F, and
R5 is F.
Embodiment 481. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein, when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3a is -OH, and
R5 is -OH.
Embodiment 482. The immunoconjugate of any one of Embodiments 341 to 447 or Embodiments 460 to 464, wherein, when present:
R2, R2a, R4, R4a, Rs, R6a, R7 and R7a are each H;
R3 is -OH, and
R5a is F.
Embodiment 483. The immunoconjugate of any one Embodiments 341 to 447 or Embodiments 460 to 464, wherein, when present: are each H;
Figure imgf000386_0001
Embodiment 484. The immunoconjugate of any one of Embodiments 341 to 447 or
Embodiments 460 to 464, wherein, when present:
R2, R2a, R4, R4a, Rs, RSa, R7 and R7a are each H;
R3 is F, and
R5a is F
Embodiment 48S. The immunoconjugate of any one of Embodiments 341 to 447 or
Embodiments 460 to 464, wherein, when present:
are each H;
Figure imgf000386_0002
Embodiment 48S. The immunoconjugate of any one Embodiments 341 to 447 or
Embodiments 460 to 464, wherein, when present:
are each H:
Figure imgf000386_0003
Embodiment 487. The immunoconjugate of any one of Embodiments 341 to 447 or
Embodiments 460 to 464, wherein, when present:
are each H:
Figure imgf000386_0004
Embodiment 488. The immunoconjugate of any one of Embodiments 341 to 447 or
Embodiments 460 to 464, wherein, when present:
R2, R2a, R4, R4a, R6, RSa, R7 and R7a are each H;
R5 is F, and
R5a is F.
Embodiment 489. The immunoconjugate of any one of Embodiments 341 to 447 or
Embodiments 460 to 464, wherein, when present:
are each H;
Figure imgf000386_0005
R5a is -OH.
Embodiment 490. The immunoconjugate of any one of Embodiments 341 to 489, wherein:
Figure imgf000386_0006
Figure imgf000387_0001
where the ** of Li indicates the point of attachment to R, 1£ and where R12, Xi , X2, X3, X5, m and n are as defined in Embodiment 363
Embodiment 491. An immunoconjugate selected from:
Figure imgf000387_0002
Figure imgf000388_0001
Figure imgf000389_0001
Figure imgf000390_0001
Provided are also protocols for some aspects of analytical methodology for evaluating antibody conjugates of the invention. Such analytical methodology and results can demonstrate that the conjugates have favorable properties, for example properties that would make them easier to manufacture, easier to administer to patients, more efficacious, and/or potentially safer for patients. One example is the determination of molecular size by size exclusion
chromatography (SEC) wherein the amount of desired antibody species in a sample is determined relative to the amount of high molecular weight contaminants (e.g., dimer, multimer, or aggregated antibody) or low molecular weight contaminants (e.g., antibody fragments, degradation products, or individual antibody chains) present in the sample. In general, It is desirable to have higher amounts of monomer and lower amounts of, for example, aggregated antibody due to the impact of, for example, aggregates on other properties of the antibody sample such as but not limited to clearance rate, i munogeniciiy, and toxicity. A further example is the determination of the hydrophobicity by hydrophobic interaction chromatography (HIC) wherein the hydrophobicity of a sample is assessed relative to a set of standard antibodies of known properties in general, it is desirable to have low hydrophobicity due to the impact of hydrophobicity on other properties of the antibody sample such as but not limited to aggregation, aggregation over time, adherence to surfaces, hepatotoxicity, clearance rates, and pharmacokinetic exposure. See Damle, N.K., Nat Biotechnol. 2008; 28(8):884~885; Singh, S.K., Pbarm Res. 2015; 32(11):3541 -71. When measured by hydrophobic interaction
chromatography, higher hydrophobicity index scores (i.e elution from HIC column faster) reflect lower hydrophobicity of the conjugates. As shown in Examples below, a majority of the tested antibody conjugates showed a hydrophobicity index of greater than 0.8 in some embodiments, provided are antibody conjugates having a hydrophobicity index of 0.8 or greater, as determined by hydrophobic interaction chromatography.
Anti-HER2 Antibody
In some embodiments antibody conjugates provided herein include an antibody or antibody fragment thereof (e.g., antigen binding fragment) that specifically binds to human HER2 (anti-HER2 antibody). HER2 overexpression is observed in many types of cancers, such as gastric cancer, esophageal cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, bladder cancer, pancreatic cancer, lung cancer, prostate cancer, osteosarcoma, neuroblastoma, or head and neck cancer.
Antibody conjugates comprising an anti-HER2 antibody can be specifically targeted to HER2- positive cancers or tumors.
In some embodiments, antibody conjugates provided herein include a monoclonal antibody or antibody fragment thereof that specifically binds to human HER2, e.g., a human or humanized anii-HER2 monoclonal antibody. In some embodiments, the antibody or antibody fragment thereof that specifically binds to human HER2 can be selected from trastuzumab, pertuzumab, margetuximab, or HT-19, or an antibody fragment thereof or a site-specific cysteine mutant thereof.
Trastuzumab (trade name Herceptin or Herclon) is a humanized monoclonal antibody that binds to the juxtamembrane portion of the extracellular domain of the HER2 receptor (Hudis CA, N Engl J Med. 20Q7; 357(1 ):39-51 ). The amino acid sequences of trastuzumab heavy chain and light chain variable regions were described in U.S. Patent No. 5,821 ,337. Trastuzumab interacts with three loop regions formed by residues 557--561 , 570-573, and 593-803 of human HER2 (Cho et ai„ Nature 421 : 756-780, 2003). Trastuzumab interferes with HER2 signaling possibly by prevention of HER2-receptor dimerization, facilitation of endocytotic destruction of the HER2 receptor, inhibition of shedding of the extracellular domain (Hudis CA, N Eng! J Med. 2007; 357(1):39-51). Another important mechanism of action of an anti-HER2 antibody is the mediation of Antibody Dependent Cellular Cytotoxicity (ADCC). In ADCG, the anti-HER2 antibody binds to tumor ceils and then recruits immune cells, such as macrophages, through Fey receptor (FcyR) interactions. Trastuzumab has a conserved human IgG Fc region, and is capable of recruiting immune effector cells that are responsible for antibody-dependent cytotoxicity (Hudis CA, N Engl J Med. 2007; 357(1):39-51). Trastuzumab gained U.S. FDA approval in September 1998 for the treatment of rnetastic breast cancer in patients whose tumors overexpress HER2 and who received one or more chemotherapy regimens for their metastatic disease.
Pertuzumab (also called 2C4, Gmnitarg, Perjeta) is a humanized monoclonal antibody that binds to the the extracellular domain of the HER2 receptor and inhibits dimerization of HER2 with other HER receptors. The amino acid sequences of pertuzumab heavy chain and light chain were described in U.S. Patent No. 7,560,1 11. Pertuzumab mainly interact with residues within region 245-333 of human HER2, particularly residues His 245, Val 286, Ser 288, Leu 295, His 296, or Lys 31 1 (Franklin et at. , Cancer Cell 5: 317-328, 2004). Pertuzumab was shown to be more effective than trastuzumab in disrupting the formation of HER1 -HER2 and HER3-HER2 complexes in breast and prostate cancer cel! lines (Agus et a!., J Clin Oncol. 2005; 23(1 1):2534-43. Epub Feb 7, 2005). Pertuzumab does not require antibody-dependent cellular cytotoxicity for efficacy because an intact Fc region is not required for its activity (Agus et a!., J Clin Oncol. 2005; 23(11):2534-43. Epub Feb 7 2005). Pertuzumab received U.S. FDA approval for use in combination with trastuzumab and docetaxe! for the treatment of patients with HER2-positive metastatic breast cancer who have not received anti-HER2 therapy or chemotherapy for rnetastic disease in June 2012.
Margetuximab (also called MGAH22) is another anti-HER2 monoclonal antibody (See http://www. acrogenics.com/products-margetuximab.html). The Fc region of margetuximab was optimized so that it has increased binding to the activating FcyRs but decreased binding to the inhibitory FcyRs on immune effector cells. Margetuximab is currently under clinical trial for treating patients with relapsed or refractory advanced breast cancer whose tumors express HER2 at the 2+ Level by immunohistochemistry and lack evidence of HER2 gene amplification by FISH.
HT-19 is another anti-HER2 monoclonal antibody that binds to an epitope in human HER2 distinct from the epitope of trastuzumab or pertuzumab and was shown to inhibit HER2 signaling comparable to trastuzumab and enhance HER2 degradation in combination with trastuzumab and pertuzumab (Bergstrom D. A. et ai., Cancer Res. 2015; 75:LB-231). Other suitable anti-HER2 monoclonal antibodies include, but are not limited to, the anti- HER2 antibodies described in US Patent Nos.: 9,096,877; 9,017,671 ; 8,975,382; 8,974,785; 8,968,730; 8,937,159; 8,840,896; 8,802,093; 8,753,829; 8,741 ,586; 8,722,362; 8,697,071 ; 8,652,474; 8,652,466; 8,609,095; 8,512,967; 8,349,585; 8,241 ,630; 8,217,147; 8,192,737; 7,879,325; 7,850,966; 7,560,1 11 ; 7,435,797; 7,306,801 ; 6,399,063; 6,387,371 ; 6,165,464; 5,772,997; 5,770,195; 5,725,856; 5,720,954; 5,677,171.
In some embodiments, the anti-HER2 antibody or antibody fragment (e.g., an antigen binding fragment) comprises a VH domain having an amino acid sequence of any VH domain described in Table 6. Other suitable anti-HER2 antibodies or antibody fragments (e.g., antigen binding fragments) can include amino acids that have been mutated, yet have at least 80, 85,
90, 95, 96, 97, 98, or 99 percent identity in the VH domain with the VH regions depicted in the sequences described in Table 6 The present disclosure in certain embodiments also provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to HER2, wherein the antibodies or antibody fragments (e.g., antigen binding fragments) comprise a VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 6. in particular embodiments, the invention provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to HER2, comprising (or alternatively, consist of) one, two, three, four, five or more VH CDRs having an amino acid sequence of any of the VH CDRs listed in Table 6.
In some embodiments, the anti-HER2 antibody or antibody fragment (e.g., antigen binding fragments) comprises a VL domain having an amino acid sequence of any VL domain described in Table 6. Other suitable anti-HER2 antibodies or antibody fragments (e.g., antigen binding fragments can include amino acids that have been mutated, yet have at least 80, 85, 90, 95, 96, 97, 98, or 99 percent identity in the VL domain with the VL regions depicted in the sequences described in Table 6. The present disclosure also provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to HER2, the antibodies or antibody fragments (e.g., antigen binding fragments) comprise a VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 6 in particular, the invention provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to HER2, which comprise (or alternatively, consist of) one, two, three or more VL CDRs having an amino acid sequence of any of the VL CDRs listed in Table 6.
Table 6, Sequences of exemplary anti~HER2 monoclonal! antibodies
Figure imgf000393_0001
Figure imgf000394_0001
395
Figure imgf000395_0001
Figure imgf000396_0001
Figure imgf000397_0001
Figure imgf000398_0001
Figure imgf000399_0001
Figure imgf000400_0001
Figure imgf000401_0001
Figure imgf000402_0001
Figure imgf000403_0001
Other anti-HER2 antibodies or antibody fragments {e.g. , antigen binding fragments) disclosed herein include amino acids that have been mutated, yet have at least 80, 85, 90, 95, 96, 97, 98, or 99 percent identity in the CDR regions with the CDR regions depicted in the sequences described in Table 6. in some embodiments, it includes mutant amino acid sequences wherein no more than 1 , 2, 3, 4 or 5 amino acids have been mutated in the CDR regions when compared with the CDR regions depicted in the sequence described in Tabie 6.
Also provided herein are nucleic acid sequences that encode VH, VL, full length heavy chain, and full length light chain of antibodies and antigen binding fragments thereof that specifically bind to HER2, e.g., the nucleic acid sequences in Tabie 6. Such nucleic acid sequences can be optimized for expression in mammalian cells.
Other anti-HER2 antibodies disclosed herein include those where the amino acids or nucleic acids encoding the amino acids have been mutated, yet have at least 80, 85, 90 95, 96, 97, 98, or 99 percent identity to the sequences described in Tabie 6. In some embodiments, antibodies or antigen binding fragments thereof include mutant amino acid sequences wherein no more than 1 , 2, 3, 4 or 5 amino acids have been mutated in the variable regions when compared with the variable regions depicted in the sequence described in Table 6, while retaining substantially the same therapeutic activity.
Since each provided antibody binds to HER2, the VH, VL, full length light chain, and full length heavy chain sequences (amino acid sequences and the nucleotide sequences encoding the amino acid sequences) can be "mixed and matched” to create other HER2-binding antibodies disclosed herein. Such "mixed and matched" HER2-binding antibodies can be tested using binding assays known in the art (e.g., ELISAs, assays described in the Exemplification). When chains are mixed and matched, a VH sequence from a particular VH/VL pairing should be replaced with a structurally similar VH sequence. A full length heavy chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length heavy chain sequence. A VL sequence from a particular VH/VL pairing should be replaced with a structurally similar VL sequence. A full length light chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length light chain sequence.
Accordingly, in one embodiment, the invention provides an isolated monoclonal antibody or antigen binding region thereof having: a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 17: wherein the antibody specifically binds to HER2. In another embodiment, the invention provides (i) an isolated monoclonal antibody having: a full length heavy chain comprising an amino acid sequence of any of SEQ ID NOs: 9, 21 , 23, 30 or 32; and a full length light chain comprising an amino acid sequence of any of SEQ ID NQs: 19 or 34; or (ii) a functional protein comprising an antigen binding portion thereof.
In another embodiment, the present disclosure provides HER2-binding antibodies that comprise the heavy chain CDR1 , CDR2 and CDR3 and light chain CDR1 , CDR2 and CDR3 as described in Table 6, or combinations thereof. The amino acid sequences of the VH CDRi s of the antibodies are shown in SEQ ID NQs: 1 , 4, and 8. The amino acid sequences of the VH CDR2s of the antibodies and are shown in SEQ ID NOs: 2 and 5 The amino acid sequences of the VH CDR3s of the antibodies are shown in SEQ ID NO: 3. The amino acid sequences of the VL CDR1 s of the antibodies are shown in SEQ iD NOs: 1 1 and 14. The amino acid sequences of the VL CDR2s of the antibodies are shown in SEQ ID NQs 12 and 15. The amino acid sequences of the VL CDR3s of the antibodies are shown in SEQ ID NQs: 13 and 16.
Given that each of the antibodies binds HER2 and that antigen-binding specificity is provided primarily by the CDR1 , CDR2 and GDR3 regions, the VH CDR1 , CDR2 and CDR3 sequences and VL CDR1 , CDR2 and CDR3 sequences can be“mixed and matched” (i.e. ,
CDRs from different antibodies can be mixed and match, aithough each antibody must contain a VH CDR1 , CDR2 and CDRS and a VL CDR1 , CDR2 and CDR3 to create other HER2-binding binding molecules disclosed herein. Such "mixed and matched" HER2-binding antibodies can be tested using the binding assays known in the art and those described in the Examples (e.g., EL!SAs) When VH CDR sequences are mixed and matched, the CDR1 , CDR2 and/or CDRS sequence from a particular VH sequence should be replaced with a structurally similar CDR sequence(s). Likewise, when VL CDR sequences are mixed and matched, the CDR1 , CDR2 and/or CDRS sequence from a particular VL sequence should be replaced with a structurally similar CDR sequence(s). It will be readily apparent to the ordinarily skilled artisan that novel VH and VL sequences can be created by substituting one or more VH and/or VL CDR region sequences with structurally similar sequences from CDR sequences shown herein for monoclonal antibodies of the present disclosure.
Accordingly, the present disclosure provides an isolated monoclonal antibody or antigen binding region thereof comprising a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 , 4, and 6; a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 5; a heavy chain CDRS comprising an amino acid sequence of SEQ ID NO: 3; a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 1 and 14; a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 15; and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 16; wherein the antibody specifically binds HER2.
In certain embodiments, an antibody that specifically binds to HER2 is an antibody or antibody fragment (e.g., antigen binding fragment) that is described in Table 6.
In some embodiments, the antibody that specifically binds to human HER2 comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 1 ; a heavy chain complementary determining region 2 (HGDR2) comprising the amino acid sequence of SEQ iD NO: 2; a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 3; a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 1 1 : a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 12; and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 13.
In some embodiments, the antibody that specifically binds to human HER2 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 4; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 5; a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; a LCDR1 comprising the amino acid sequence of SEQ ID NO: 14; a LCDR2 comprising the amino acid sequence of SEQ ID NO: 15; and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 16.
In some embodiments, the antibody that specifically binds to human HER2 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17
In some embodiments, the antibody that specifically binds to human HER2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, the antibody that specifically binds to human HER2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 , and a light chain comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, the antibody that specifically binds to human HER2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 23, and a light chain comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, the antibody that specifically binds to human HER2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and a light chain comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, the antibody that specifically binds to human HER2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32, and a light chain comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, the antibody that specifically binds to human HER2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and a light chain comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, the antibody that specifically binds to human HER2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 23, and a light chain comprising the amino acid sequence of SEQ ID NO: 34.
In some embodiments, the present disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind an epitope in human HER2. in some embodiments, the present disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to an epitope in human HER2, wherein the epitope comprises one or more of the residues 557-501 , 570-573, and 593-603 of SEQ ID NO: 26. In some embodiments, the present disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to an epitope in human HER2, wherein the epitope comprises one or more of the residues 245-333 of SEQ ID NO: 26. in some embodiments, the present disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to an epitope in human HER2, wherein the epitope comprises one or more of the following residues: His 245, Val 286, Ser 288, Leu 295, His 296, or Lys 31 1 of SEQ ID NO: 26.
Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope, e.g., using the techniques described in the present invention. Alternatively, during the discovery process, the generation and characterization of antibodies may elucidate information about desirable epitopes. From this information, it is then possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to conduct cross-competition studies to find antibodies that competitively bind with one another, e.g., the antibodies compete for binding to the antigen. A high throughput process for“binning” antibodies based upon their cross-competition is described in International Patent Application No. WO 2003/48731. As will be appreciated by one of skill in the art, practically anything to which an antibody can specifically bind could be an epitope. An epitope can comprises those residues to which the antibody binds.
Figure imgf000407_0001
Antibodies and antibody conjugates disclosed herein may comprise modified antibodies or antigen binding fragments thereof that comprise modifications to framework residues within VH and/or VL, e.g. to improve the properties of the antibody/antibody conjugate.
In some embodiments, framework modifications are made to decrease immunogenicity of an antibody. For example, one approach is to "back-mutate" one or more framework residues to a corresponding germline sequence. Such residues can be identified by comparing antibody framework sequences to germline sequences from which the antibody is derived. To“match” framework region sequences to desired germline configuration, residues can be "back-mutated" to a corresponding germline sequence by, for example, site-directed mutagenesis. Such "back- mutated" antibodies are also intended to be encompassed by the invention.
Another type of framework modification involves mutating one or more residues within a framework region, or even within one or more CDR regions, to remove T-cell epitopes to thereby reduce potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No 20030153043 by Carr et a/. In addition or alternative to modifications made within a framework or CDR regions, antibodies disclosed herein may be engineered to include modifications within the Fc region, typically to aiter one or more functional properties of the antibody, such as serum half-iife, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
Furthermore, an antibody disclosed herein may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its
giycosyiation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below.
In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et ai The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
In some embodiments antibodies or antibody fragments (e.g., antigen binding fragment) useful in antibody conjugates disclosed herein Include modified or engineered antibodies, such as an antibody modified to introduce one or more cysteine residues as sites for conjugation to a drug moiety (Junutu!a JR, et ai : Nat Biotechnol 2008, 26:925-932). In one embodiment, the invention provides a modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with cysteine at the positions described herein. Sites for cysteine substitution are in the constant regions of the antibody and are thus applicable to a variety of antibodies, and the sites are selected to provide stable and homogeneous conjugates. A modified antibody or fragment can have two or more cysteine substitutions, and these substitutions can be used in combination with other antibody modification and conjugation methods as described herein. Methods for inserting cysteine at specific locations of an antibody are known in the art, see, e.g., Lyons et. ai, (1990) Protein Eng., 3:703-708, WO 2011/005481 , WQ2Q14/124316, WO 2015/138615. in certain embodiments a modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region selected from positions 1 17, 119, 121 , 124, 139, 152, 153, 155, 157, 164, 169, 171 , 174, 189, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, 390, 392, 398, 400 and 422 of a heavy chain of the antibody or antibody fragment, and wherein the positions are numbered according to the EU system in some embodiments a modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region selected from positions 107, 108, 109, 1 14, 129, 142, 143, 145, 152, 154, 156, 159, 161 , 165, 168, 169, 170, 182, 183, 197, 199, and 203 of a light chain of the antibody or antibody fragment, wherein the positions are numbered according to the EU system, and wherein the light chain is a human kappa light chain. In certain embodiments a modified antibody or antibody fragment thereof comprises a combination of substitution of two or more amino acids with cysteine on its constant regions wherein the combinations comprise substitutions at positions 375 of an antibody heavy chain, position 152 of an antibody heavy chain, position 360 of an antibody heavy chain, or position 107 of an antibody light chain and wherein the positions are numbered according to the ELS system. In certain embodiments a modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine on its constant regions wherein the substitution is position 375 of an antibody heavy chain, position 152 of an antibody heavy chain, position 360 of an antibody heavy chain, position 107 of an antibody light chain, position 165 of an antibody light chain or position 159 of an antibody light chain and wherein the positions are numbered according to the EU system, and wherein the iight chain is a kappa chain.
In particular embodiments a modified antibody or antibody fragment thereof comprises a combination of substitution of two amino acids with cysteine on its constant regions, wherein the modified antibody or antibody fragment thereof comprises cysteines at positions 152 and 375 of an antibody heavy chain, wherein the positions are numbered according to the EU system. in other particular embodiments a modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine at position 360 of an antibody heavy chain and wherein the positions are numbered according to the EU system.
In other particular embodiments a modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine at position 107 of an antibody light chain and wherein the positions are numbered according to the EU system, and wherein the iight chain is a kappa chain.
in additional embodiments antibodies or antibody fragments (e.g., antigen binding fragment) useful in antibody conjugates disclosed herein include modified or engineered antibodies, such as an antibody modified to introduce one or more other reactive amino acid (other than cysteine), including Pci (pyrro!ine-carhoxy-!ysine), pyrroiysine, peptide tags (such as S6, A1 and ybbR tags), and non-natural amino acids, in place of at least one amino acid of the native sequence, thus providing a reactive site on the antibody or antigen binding fragment for conjugation to a drug moiety of Formula (I) or subformu!ae thereof. For example, the antibodies or antibody fragments can be modified to incorporate Pc! or pyrroiysine (W. Ou et ai. (2011) PNAS 108 (26), 10437-10442; WO2014124258) or unnatural amino acids (J.Y. Axup, et ai. Proc Nat! Acad Sci U S A, 109 (2012), pp. 16101-16106; for review, see C.C. Liu and P.G Schultz (2010) Annu Rev Biochem 79, 413-444; C.H. Kim, et al. , (2013) Curr Opin Cbem Biol. 17, 412- 419) as sites for conjugation to a drug. Similarly, peptide tags for enzymatic conjugation methods can be introduced into an antibody (Strop P. et ai. Chem Biol. 2013, 20(2):161-7; Rabuka D., Curr Gpin Chem Biol. 2010 Dec;14(8):790-6; Rabuka D,et al., Nat Protoc. 2012,
7 (6): 1052-67). One other example is the use of 4’-phosphopantetheinyl transferases (PPTase) for the conjugation of Coenzyme A analogs (W02G13184514; Grunewald J, et al., Bioconjug Chem. 2015 Dec 16;26(12):2554-62) Methods for conjugating such modified or engineered antibodies with payloads or linker-payload combinations are known in the art.
In another embodiment, an Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the GH2-CH3 domain interface region of the Fc-binge fragment such that the antibody has impaired Staphyiococcyi Protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.
In yet other embodiments, an Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in, e.g., U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.
In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C1 q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in, e.g., U.S. Patent Nos. 6,194,551 by Idusogie et al.
In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described in, e.g., the PCT
Publication WO 94/29351 by Bodmer et a!. Allotypic amino acid residues include, but are not limited to, constant region of a heavy chain of the igG1 , lgG2, and igG3 subclasses as well as constant region of a light chain of the kappa isotype as described by Jefferis et al., MAbs. 1 :332- 338 (2009).
In a further embodiment, the Fc region is modified to“silence” the effector function of the antibody, for example, reduce or eliminate the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or antibody dependent cellular phagocytosis (ADCP). This can be achieve, for example, by introducing a mutation in the Fc region of the antibodies. Such mutations have been described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol vol. 20(6):685-691); and D265A (Baudino et al , 2008, J. Immunol. 181 : 6664-69; Strohl, W., supra). Examples of silent Fc igG1 antibodies comprise the so-called LALA mutant comprising L234A and L235A mutation in the lgG1 Fc amino acid sequence. Another example of a silent lgG1 antibody comprises the D265A mutation. Another silent lgG1 antibody comprises the N297A mutation, which results in aglycosylated/non-glycosylated antibodies.
In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or antibody dependent celluiar phagocytosis (ADCP), for example, by modifying one or more amino acid residues to increase the affinity of the antibody for an activating Fey receptor, or to decrease the affinity of the antibody for an inhibatory Fey receptor. Human activating Fey receptors include FcyR!a, FcyRIla, FeyRIIIa, and FcyRMib, and human inhibitory Fey receptor includes FcyRIlb. This approach is described in, e.g., the PCT Publication WO 00/42072 by Presta. Moreover, binding sites on human !gG1 for FcyR!, FcyRI!, FcyRII! and FcRn have been mapped and variants with improved binding have been described (see Shields et a!., J. Biol. Chem. 278:6591 -6604,
2001). Optimization of Fc-mediated effector functions of monoclonal antibodies such as increased ADCC/ADCP function has been described (see Sfrohl, W.R., Current Opinion in Biotechnology 2009; 20:685-691.) In some embodiments, an antibody conjugate comprises an immunoglobulin heavy chain comprising a mutation or combination of mutations conferring enhanced ADCC/ADCP function, e.g., one or more mutations selected from G236A, S239D, F243L, P247I, D280H, K29QS, R292P, S298A, S298D, S298V, Y300L, V3Q5I, A330L, I332E, E333A, K334A, A339D, A339Q, A339T, P396L (all positions by EU numbering).
In another embodiment, the Fc region is modified to increase the ability of the antibody to mediate ADCC and/or ADCP, for example, by modifying one or more amino acids to increase the affinity fo the antibody for an activating receptor that would typically not recognize the parent antibody, such as FcaRi. This approach is descried in, e.g , Borrok et a!., mAbs 7(4):743-751. in particular embodiments, an antibody conjugate comprises an immunoglobulin heavy chain comprising a mutation or a fusion of one or more antibody sequences conferring enhanced ADCC and/or ADCP function.
In still another embodiment, glycosylation of an antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for "antigen.” Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites fo thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et ai.
Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosyiated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such
carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host ceil with altered glycosylation machinery. Ceils with altered glycosylation machinery have been described in the art and can be used as host ceils in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, EP 1 ,176,195 by Hang et al. describes a ceil line with a functionally disrupted FUT8 gene, which encodes a fucosy! transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO ceil line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-!inked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et ai. describes cell lines engineered to express glycoprotein-modifying glycosy! transferases (e.g., beta(1 ,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting G!cNac structures which results in increased ADCC activity of the antibodies (see also Umana et ai., Nat. Biotech. 17:176-180, 1999).
In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward.
Alternatively, to Increase the biological haif-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from fvvo loops of a CH2 domain of an Fc region of an !gG, as described in U.S. Patent Nos 5,869,046 and 6,121 ,022 by Presta et ai
Figure imgf000412_0001
Antibodies and antibody fragments {e.g. , antigen binding fragments) thereof can be produced by any means known in the art, inciuding but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full-length monoclonal antibodies can be obtained by, e.g., hybridoma or recombinant production.
Recombinant expression can be from any appropriate host ceils known in the art, for example, mammalian host cells, bacterial host cells, yeast host ceils, insect host ceils, etc.
Also provided herein are polynucleotides encoding antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising complementarity determining regions as described herein in some embodiments, a polynucleotide encoding the heavy chain variable regions has at least 85%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO: 8. In some embodiments, a polynucleotide encoding the light chain variable regions has at least 85%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide that encodes an antibody.
in some embodiments, a polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide as disclosed herein in some embodiments, a polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide as disclosed herein.
Some polynucleotides disclosed herein encode a variable region of an anti-HER2 antibody. Some polynucleotides disclosed herein encode both a variable region and a constant region of an anti-HER2 antibody. Some polynucleotide sequences encode a polypeptide that comprises variable regions of both a heavy chain and a light chain of an anti-HER2 antibody. Some polynucleotides encode two polypeptide segments that respectively are substantially identical to the variable regions of a heavy chain and a light chain of any anti-HER2 antibodies disclosed herein.
Polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding an antibody or its binding fragment. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et ai., Meth. Enzymoi. 68:90, 1979; the phosphodiester method of Brown et a!. , Meth Enzymoi. 68:109, 1979; the diethylphosphoramidite method of Beaucage et ai. , Tetra. Lett., 22:1859, 1981 ; and the solid support method of U.S. Patent No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and
Applications, Innis et ai. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et ai., Nucleic Acids Res. 19:967, 1991 ; and Eckert et ai., PCR Methods and Applications 1 :17, 1991 .
Also provided are expression vectors and host ceils for producing antibodies described herein. Various expression vectors can be employed to express polynucleotides encoding antibody chains or binding fragments. Both viral-based and nonviral expression vectors can be used to produce antibodies in a mammalian host ceil.
Nonviral vectors and systems include plasmids, episornal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et ai., Nat Genet 15:345, 1997) For example, nonviral vectors useful for expression of polynucleotides and polypeptides in mammalian (e.g. , human) ceils include pThioHis A, B & C, pCDNATM3.1/His, pEBVHis A, B & C (invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Usefui viral vectors include vectors based on retroviruses, adenoviruses, adenoassociated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semiiki Forest virus (SFV). See, Brent et ai., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et ai., Cell 68:143, 1992.
Choice of expression vector depends on the intended host cells in which a vector is to be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (e g., enhancers) that are operably linked to polynucleotides encoding an antibody chain or fragment. In some embodiments, an indiicibie promoter is employed to prevent expression of inserted sequences except under inducing conditions. Inducible promoters include, e.g. , arabinose, iacZ, metaliothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under noninducing conditions without biasing the population for coding sequences whose expression products are better tolerated by host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of an antibody chain or fragment. Elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al., Results Probl. Ceil Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:518,
1987). For example, an SV40 enhancer or C V enhancer may be used to increase expression in mammalian host ceils.
Expression vectors may also provide a secretion signal sequence position to form a fusion protein with polypeptides encoded by inserted antibody sequences. More often, inserted antibody sequences are linked to a signal sequence before inclusion in the vector. Vectors to be used to receive sequences encoding antibody light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of variable regions as fusion proteins with constant regions, thereby leading to production of intact antibodies or fragments thereof. Typically, such constant regions are human.
Host cells for harboring and expressing antibody chains can be either prokaryotic or eukaryotic. E. co!i is one prokaryotic host useful for cloning and expressing polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other errferobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters typicaliy control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. Other microbes, such as yeast, can also be employed to express polypeptides, including antibodies. Insect cells in combination with baculovirus vectors can also be used.
In some particular embodiments, mammalian host ceils are used to express and produce polypeptides of the present disclosure. For example, they can be either a hybridoma cell line expressing endogenous immunoglobulin genes (e.g., myeloma hybridoma clones) or a mammalian ceil line harboring an exogenous expression vector (e.g., the SP2/Q myeloma ceils). These include any normal mortal or normal or abnormal Immortal animal or human ceil. For example, a number of suitable host ceil lines capable of secreting intact immunoglobulins have been developed, including various CHO cell lines, Cos cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas. Use of mammalian tissue ceil culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH
Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et a!., Immunol. Rev. 89:49-88, 1988), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, po!yadenyiaiion sites, and
transcriptional terminator sequences. Expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modu!atable or reguiatable. Useful promoters include, but are not limited to, a metai!othionein promoter, a constitutive adenovirus major late promoter, a dexamethasoneinducible MMTV promoter, a SV40 promoter, a MRP poll 11 promoter, a constitutive MPSV promoter, a tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), a constitutive CMV promoter, and promoter-enhancer combinations known in the art.
Methods for introducing expression vectors containing polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic ceils, whereas calcium phosphate treatment or
electroporation may be used for other cellular hosts (see generally Sambrook et a!., supra). Other methods include, e.g., electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycatiomnucieic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O'Hare, Ceil 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression will often be desired. For example, ceil lines which stably express antibody chains or binding fragments can be prepared using expression vectors disclosed herein which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following introduction of the vector, cells may be allowed to grow for 1 -2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media. Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate to the cell type.
Therapeutic Uses and Methods of Treatment
Provided antibody conjugates are useful in a variety of applications including, but not limited to, treatment of cancer in certain embodiments, antibody conjugates provided herein are useful for inhibiting tumor growth, reducing tumor volume, inducing differentiation, and/or reducing the tumorigeniciiy of a tumor. The methods of use can be in vitro , ex vivo , or in vivo methods.
In some embodiments, provided herein are methods of treating, preventing, or ameliorating a disease, e.g., a cancer, in a subject in need thereof, e.g., a human patient, by administering to the subject any of the antibody conjugates described herein. Also provided is use of the antibody conjugates of the invention to treat or prevent disease in a subject, e.g., a human patient. Additionally provided Is use of antibody conjugates In treatment or prevention of disease in a subject in some embodiments provided are antibody conjugates for use in manufacture of a medicament for treatment or prevention of disease in a subject. In certain embodiments, the disease treated with antibody conjugates is a cancer.
In one aspect, the immunoconjugates described herein can be used to treat a solid tumor. Examples of solid tumors include malignancies, e.g , sarcomas, adenocarcinomas, hiasiomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, biiiarintestinai (e.g., colon), genitourinary tract (e.g., renal, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, small ceil lung cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus in one embodiment, the cancer is a melanoma, e.g., an advanced stage melanoma. Examples of other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer .cancer of the anal region, cancer of the peritoneum, stomach or gastric cancer, esophageal cancer, salivary gland carcinoma, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, penile carcinoma , glioblastoma, neuroblastoma, cervical cancer , Hodgkin Disease, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CMS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. In another aspect, the immunoconjugates described herein can be used to treat a hematological cancer. Hematological cancers include leukemia, lymphoma, and malignant iymphoproiiferative conditions that affect blood, bone marrow and the lymphatic system.
Leukemia can be classified as acute leukemia and chronic leukemia. Acute leukemia can be further classified as acute myelogenous leukemia (AML) and acute lymphoid leukemia (ALL). Chronic leukemia includes chronic myelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Other related conditions include myelodyspiastic syndromes (IVSDS, formerly known as“preleukemia”) which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood ceils and risk of transformation to AML.
Lymphoma is a group of blood cell tumors that develop from lymphocytes. Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma.
In some embodiments, the cancer is a hematologic cancer including but is not limited to, e.g., acute leukemias including but not limited to, e.g., B-celi acute lymphoid leukemia (“BALL”), T-eei! acute lymphoid leukemia (“TALL”), acute lymphoid leukemia (ALL); one or more chronic leukemias including but not limited to, e.g., chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL): additional hematologic cancers or hematologic conditions including, but not limited to, e.g., B cell proiymphocytic leukemia, blastic plasmacytoid dendritic ceil neoplasm, Burkitt’s lymphoma, diffuse large B ceil lymphoma, Follicular lymphoma, Hairy ceil leukemia, small cell- or a large ceil-foiiicuiar lymphoma, malignant iymphoproiiferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodyspiastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom
macrogiobuiinemia, and“preieukemia” which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and the like. Further a disease associated with a tumor antigen expression includes, but not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing a tumor antigen as described herein. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the invention.
In certain embodiments, the cancer is characterized by ceils expressing a target tumor antigen to which the antibodies, or antibody fragments (e.g., antigen binding fragments) of the antibody conjugates bind in some embodiments, an immunoconjugate as described herein can comprise an antigen binding domain (e.g., antibody or antibody fragment) that binds to a tumor antigen (e.g., a tumor antigen as described herein). Methods of detecting the presence or overexpression of such tumor antigens are known to persons skilled in the art, and include methods such as immunohistocompatibility (IHC) assays using antibodies that specifically bind the tumor antigens, delecting the level of RNA expression of the tumor antigen, etc. In some embodiments, the tumor antigen is selected from one or more of the following targets: receptor tyrosine-protein kinase ERBB2 (Her2/neu); receptor tyrosine-protein kinase ERBB3 (Her3); receptor tyrosine-protein kinase ERBB4 (Her4); epidermal growth factor receptor (EGFR); E-cadherin; P-cadherin; Cadherin 6; caihepsin D; estrogen receptor;
progesterone receptor; CA125; CA15-3; CA19-9; P-glycoprotein (CD243); CD2; CD19; CD20; CD22; CD24; GD27; CD3Q; CD37; CD38; CD4Q; CD44v6; CD45; CD47; CD52; CD58; CD70; CD71 ; CD79a; GD79b; CD72; CD97; CD179a; CD123; CD137; CD171 ; CS-1 (also referred to as CD2 subset 1 , GRAGG, SLAMF7, CD319, and 19A24); C-iype lectin-like molecule-1 (GLL-1 or CLECLi); epidermal growth factor receptor variant III (EGFRviil); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGa!p(1-4)bDG!cp(1~1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); prostate- specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72);
Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCA ); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit aipha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 1 1 receptor alpha (!L-11 Ra); prostate stem ceil antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); Folate receptor alpha; neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-i receptor), carbonic anhydrase IX (GAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2);
glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abeison murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type- A receptor 2 (EphA2); Fucosyi GM1 ; sialyl Lewis adhesion molecule (sLe); ganglioside GIVIS (aNeu5Ac(2-3)bDGa!p(1 -4)bDGicp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma-associated antigen (HMW AA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1/CD248); tu or endothelial marker 7-related (TEM7R); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 81 (CXORF61); anaplastic lymphoma kinase (ALK); Poiysialic acid; piacenta-specific 1 (PLAC1); hexasaccharide portion of gioboH giycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1);
adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Olfactory receptor 51 E2 (OR51 E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2
(LAGE-1 a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding ceil surface receptor 2 (Tie 2): melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1 ; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Aceiyi giucosaminyl- transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1 ; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1 B1
(CYP1 B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the
Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Ceils 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1);
lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); iegumain; human papilloma virus E8 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (GD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module- containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican- 3 (GPC3); and immunoglobulin lambda-like polypeptide 1 (IGLL1); CD184; LGR5; AXL; RON; CD352/SLAMf6; KAAG-1 ; 5T4; c-Met; ITGA3; Endosialin; CD166; SAIL (c15orf54); NaPI2b; DLLS; CD133; FZD7; Dysadherin; PD-L1 ; SLITRK6; Nectin-4; FGFR2; FGFR3; FGFR4;
CEACAM1 ; CEACAM5; CD74; STEAP-1 ; PMEL17; Mud 6; FcRH5; TENB2; Ly6E; ETBR; 158P1 D7; 161 P2F1 GB; 191 p4d12; 162p1 e6; Notch3; PTK7; and EFNA4.
Tumor-Supporting Antigens
In some embodiments, an immunoconjugate as described herein can comprise an antigen binding domain (e.g , antibody or antibody fragment) that binds to a tumor-supporting antigen (e.g., a tumor-supporting antigen as described herein).
In some embodiments, the tumor-supporting antigen is an antigen present on a stromal ceil, an antigen presenting ceil, or a myeloid-derived suppressor cell (MDSC). Stromal cells can secrete growth factors to promote ceil division in the microenvironment. MDSC ceils can inhibit T cell proliferation and activation. In some embodiments, the stromal cell antigen is chosen from one or more of: bone marrow stromal ceil antigen 2 (BST2), fibroblast activation protein (FAR) and tenascin. in embodiments, the MDSC antigen is chosen from one or more of: CD33, CD11 b, C14, CD15, and CD66b. Accordingly, in some embodiments, the tumor-supporting antigen is chosen from one or more of: bone marrow stromal ceil antigen 2 (BST2), fibroblast activation protein (FAR) or tenascin, CD33, CD1 1 b, C14, CD15, and CD66b.
It is also contemplated that the antibody conjugates described herein may be used to treat various non-ma!ignani diseases or disorders, such as inflammatory bowel disease (!BD), gastrointestinal ulcers, Menetrier's disease, hepatitis B, hepatitis C, secreting adenomas or protein loss syndrome, renal disorders, angiogenic disorders, ocular disease such as age related macular degeneration, presumed ocular histoplasmosis syndrome, or age related macular degeneration, bone associated pathologies such as osteoarthritis, rickets and osteoporosis, hyperviscosity syndrome systemic, Osier Weber-Rendu disease, chronic occlusive pulmonary disease, or edema following bums, trauma, radiation, stroke, hypoxia or ischemia, diabetic nephropathy, Paget's disease, photoaging (e.g , caused by UV radiation of human skin), benign prostatic hypertrophy, certain microbial infections including microbial pathogens selected from adenovirus, hantaviruses, Borrelia burgdorferi, Yersinia spp., and Bordetelia pertussis, thrombus caused by platelet aggregation, reproductive conditions such as endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding, or enometrorrhagia, acute and chronic nephropathies (including proliferative glomerulonephritis), hypertrophic scar formation, endotoxic shock and fungal infection, familial adenomatosis polyposis, yelodysplastic syndromes, aplastic anemia, ischemic injury, fibrosis of the lung, kidney or liver, infantile hypertrophic pyloric stenosis, urinary obstructive syndrome, psoriatic arthritis.
Method of administration of such antibody conjugates include, but are not limited to, parenteral (e.g., intravenous) administration, e.g., injection as a bolus or continuous infusion over a period of time, oral administration, intramuscular administration, intratumoral administration, intramuscular administration, intraperitoneal administration, intracerobrospinai administration, subcutaneous administration, intra-articular administration, intrasynoviai administration, injection to lymph nodes, or intrathecal administration.
For treatment of disease, appropriate dosage of antibody conjugates of the present invention depends on various factors, such as the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, previous therapy, patient’s clinical history, and so on. Antibody conjugates can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g., reduction in tumor size). Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of a particular antibody conjugate. In some embodiments, dosage is from 0.01 mg to 20 mg (e.g., 0.01 mg, Q.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.08 mg, 0.07 mg, 0.08 mg, 0 09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6mg, 7 mg, 8 mg, 9 mg, 10 mg, 1 1 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg) per kg of body weight, and can be given once or more daily, weekly, monthly or yearly. In certain embodiments, the antibody conjugate of the present Invention is given once every two weeks or once every three weeks. In certain embodiments, the antibody conjugate of the present invention is given only once. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
Combination Therapy
In certain instances, an antibody conjugate of the present invention can be combined with other therapeutic agents, such as other anti-cancer agents, anti-allergic agents, antinausea agents (or anti-emetics), pain relievers, cytoprotective agents, and combinations thereof.
General chemotherapeutic agents considered for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyi-5- deoxy-5-fluorocytidine, carboplatin (Parapiatin®), carmustine (BiCNU®), chlorambucil
(Leukeran®), cisplatin (Plaiino!®), ciadribine (Leustatin®), cyclophosphamide (Cytoxan® or fsieosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection
(DaunoXome®), dexamethasone, docetaxei (Taxotere®), doxorubicin hydrochloride
(Adriamycin®, Rubex©), etoposide (Vepesid®), f!udarabine phosphate (Fludara®), 5- fiuorouracii (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine
(difluorodeoxycitidine), hydroxyurea (Hydrea©), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar©), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan
(Alkeran®), 6-mercaptopurine (Purinethol©), methotrexate (Folex®), mitoxantrone
(Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Veiban®), vincristine (Oncovin®), vinoreibine (Naveibine®), epirubicin (Eilence®), oxalipiatin (Eloxatin®), exemestane (Aromasin®), letrozoie (Fe ara®), and fulvestrant (Faslodex®).
The term“pharmaceutical combination” as used herein refers to either a fixed combination in one dosage unit form, or non-fixed combination or a kit of parts for the combined administration where two or more therapeutic agents may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect.
The term“combination therapy” refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration encompasses co-administration in multiple, or in separate containers (e.g., capsules, powders, and liquids) for each active ingredient. Powders and/or liquids may be reconstituted or diluted to a desired dose prior to administration. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.
The combination therapy can provide“synergy” and prove“synergistic”, i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect can be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes in general, during alternation therapy, an effective dosage of each active ingredient is administered sequentialiy, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
In one embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with one or more other anti-HER2 antibodies, e.g., trastuzumab, pertuzumab, margetuximab, or HT-19 described above, or with other anti-HER2 conjugates, e.g., ado- trastuzumab emtansine (also known as Kadcyia©, or T-DM1).
In one embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with one or more tyrosine kinase inhibitors, including but not limited to, EGFR inhibitors, Her3 inhibitors, IGFR inhibitors, and Met inhibitors.
For example, tyrosine kinase inhibitors include but are not limited to, Eriotinib hydrochloride (Tarceva®); Linifanib (N-[4-(3-amino-1 H-indazol-4-yi)pheny!j-N’-(2-fluoro-5- methylphenyl)urea, also known as ABT 889, available from Genentech); Sunitinib malate (Sutent®); Bosutinib (4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4- methylpiperazin-1 -yl)propoxy]quinoline-3-carbonitrile, also known as SKI-600, and described in US Patent No. 6,780,996); Dasatinib (Sprycel®); Pazopanib (Votrient®); Sorafenib (Nexavar®); Zactima (ZD6474); and imatinib or Imatinib mesylate (Gilvec® and Gleevec®).
Epidermal growth factor receptor (EGFR) inhibitors include but are not limited to, Eriotinib hydrochloride (Tarceva®), Gefitinib (Iressa®); N-[4-[(3-Cbloro-4-fiuorophenyl)amino]-7- [[(3"S")-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, Tovok®); Vandetanib (Caprelsa®); Lapatinib (Tykerb®); (3R,4R)-4-Amino-1 -((4-((3- methoxypheny!)amino)pyrrolo[2,1 -f][1 ,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS890514);
Canertinib dihydrochloride (CI-1033); 6-[4-[(4-Ethyl-1 -piperazinyl)methyl]phenyl]-N-[(1 R)-1 - phenylethylj- 7H-Pyrrolo[2,3-d]pyrimidin-4-amine (AEE788, CAS 497839-62-Q); Mubritinib (TAK185); Pelitinib (EKB569); Afatinib (Gi!otrif®); Neratinib (HKI-272); N-[4-[[1 -[(3- Fluoropheny!)methyl]-1 H-indazol-5-yl]amino]-5-methylpyrrolo[2,1 -f|[1 ,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinylmethyl ester (BMS599626); N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy- 7-[[(3aa,5p,6aa)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]- 4-quinazolinamine (XL647, CAS 781613-23-8); and 4-[4-[[(1 R)-1 -Phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6- yi]-phenoi (PKI106, CAS187724-61 -4).
EGFR antibodies include but are not limited to, Cetuximab (Erbitux®); Panitumumab (Veetibix®); Matuzumab (EMD-72GG0); Nimotuzumab (hR3); Zalutumumab; TheraCIM h-R3; MDXG447 (CAS 339151 -98-1 ); and ch808 (mAb-806, CAS 946414-09-1).
Other HER2 inhibitors include but are not limited to, Neratinib (HKI-272, (2E)-N-[4-[[3- chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4- (dimethylamino)but-2-enamide, and described PCT Publication No. WO 05/028443); Lapatinib or Lapatinib ditosyiate (Tykerb®); (3R,4R)-4-amino-1 -((4-((3-methoxyphenyl)amino)pyrrolo[2,1 - f][1 ,2 ,41triazi n-5-y i) methyl) pipe rid i n-3-ol (B S690514); (2E)-N-[4-[(3-Cbioro-4- fluorophenyi)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyi]-4-(dimethylamino)-2- butenamide (BIBW-2992, CAS 85014G-72-6); N-[4-[[1 -[(3-Fluorophenyl)methyl]-1 H-indazol-5- yijamino]-5-methyipyrrolo[2,1 -t][1 ,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morphoiinylmethyl ester (BMS 599626, CAS 714971 -09-2); Canertinib dihydrochioride (PD183805 or CI-1033); and N- (3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3an ,5 n,6an)-octahydro-2- methylcyelopenta[c]pyrrol-5-yi]methoxy]- 4-quinazolinamine (XL647, CAS 781613-23-8).
HERS inhibitors include but are not limited to, LJM716, MM-121 , AMG-888, RG71 16, REGN-1400, AV-203, MP-RM-1 , M-1 1 1 , and EHD-7945A.
MET inhibitors include but are not limited to, Cabozantinib (XL184, CAS 849217-68-1); Foretinib (GSK1363089, formerly XL880, CAS 849217-64-7); Tivantinib (ARQ197, CAS 10G0873-98-2); 1 -(2~Hydroxy-2-methy!propyi)-/V-(5-(7~meihoxyqu!no!in-4-yloxy)pyridin-2-yl)~5- methy!~3-oxQ-2-phenyi-2,3-dihydro-1 H~pyrazoie-4-Garboxamide (AMG 458); Cryzotinib
(Xa!kori®, PF-02341088); (3Z)-5-(2,3-Dihydro-1 H-indoi-1 -yisulfonyi)-3-({3,5-dimethyi-4-[(4- methylpiperazin-1 -yl)carbonyl]-1 H-pyrrol-2-yl}methylene)-1 ,3-dihydro-2H-indol-2-one
(SU1 1271); (3Z)-N-(3-Chlorophenyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1 -yl)carbonyl]-1 H- pyrrol-2-yl}methylene)-N-metbyl-2-oxoindoline-5-sulfonamide (SU1 1274); (3Z)-N-(3- Chlorophenyl)-3-{[3,5-dimethyl-4-(3-morpbolin-4-ylpropyl)-1 H-pyrrol-2-yl]metbylene}-N-methyl-2- oxoindoline-5-suifonamide (SU1 1608); 6-[Difluoro[6-(1 -methyl- 1 Hpyrazol-4-yl)-1 ,2,4-triazolo[4,3- b]pyridazin-3-yl]methyl]-quinoiine (JNJ38877805, CAS 943540-75-8); 2-[4-[1 -(Quinolin-6- ylmethyl)-1 H-[1 ,2,3]tnazolo[4,5-b] pyrazin-6-yl]-1 H-pyrazol-1 -yl]ethanol (PF04217903, CAS 958905-27-4); N-((2R)-1 ,4-Dioxan-2-yimethyl)~N-methyi~N’-[3-(1 ~meibyM H-pyrazoi~4~yl)~5-oxo~ 5H-benzo[4,5]eyclohepta[1 ,2-b]pyridin-7-yl]sulfamide (MK2461 , CAS 917879-39-1); 6-[[6-(1 - Methyl-1 H-pyrazol-4-yl)-1 ,2,4-triazolo[4,3-b]pyridazin 3-yl]thio]-quinoline (SGX523, CAS 1022150-57-7); and (325-5-[[(2 6-Dichlorophenyl)methyl]sulfonyl]-3-[[3,5-dimethyl-4-[[(2R)-2-(1 - pyrroiidinylmethyl)-1 -pyrro!idinyl]carbony!j-1 H-pyrrol-2-yl]methylene]-1 ,3-dihydro-2H-indoi-2-one (PHA665752, CAS 477575-56-7).
IGFR Inhibitors include but are not limited to, BMS-754807, XL-228, OSi-908,
GSK09Q4529A, A-928805, AXL1717, KW-245Q, MK0646, AMG479, IMCA12, MEDI-573, and BI838845. See e g., Yee, JNCI, 104; 975 (2012) for review.
In another embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with one or more proliferation signaling pathway inhibitors, including but not limited to, MEK inhibitors, BRAF inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and also mTGR inhibitors, and GDK inhibitors.
For example, mitogen-activated protein kinase (MEK) inhibitors include but are not limited to, XL-518 (also known as GDC-0973, Cas No. 1 Q29872-29-4, available from ACC Corp.); 2-[(2-Chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide (also known as Ci-1040 or PD184352 and described in PCI Publication No. WG20GG035436); N- [(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]- benzamide (also known as PDG3259G1 and described in PCI Publication No. W02002006213); 2,3- Bis[amino[(2-aminophenyl)thio]methylene]-butanedinitrile (also known as U0126 and described in US Patent No 2,779,780); N-[3,4-Difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6- methoxypheny!j-1 -[(2R)-2,3-dihydroxypropyi]- cyciopropanesulfonamide (also known as RDEA1 19 or BAY889766 and described in PCT Publication No. W020G7G1401 1);
(3S,4R,5Z,8S,9S,1 1 E)-14-(Ethyiamino)-8,9,18-trihydroxy-3,4-dimethyl-3,4,9, 19-tetrahydro-1 H- 2-benzoxacyclotetradecine-1 ,7(8H)-dione] (also known as E6201 and described in PCT Publication No. WG20Q3G78424); 2’-Amino-3’-methoxyflavone (also known as PD98G59 available from Biaffin GmbH & Go., KG, Germany); Vemurafenib (PLX-4032, CAS 918504-65- 1 ); (R)-3-(2,3-Dihydroxypropyl)-6-fiuoro-5-(2-fluoro-4-iodophenylamino)-8-metbylpyrido[2,3- d]pyrimidine 4,7(3H,8H)~dione (TAK-733, CAS 1035555-63-5); Pimasertib (AS-703026, CAS 1204531 -26-9); and Trametinib dimethyl sulfoxide (GSK-1 120212, CAS 1204531 -25-80).
BRAF inhibitors include, but are not limited to, Vemurafenib (or Zeiboraf®), GDC-Q879, PLX-472G (available from Symansis), Dabrafemb (or GSK21 18436), LGX 818, CEP-32496, Ul- 152, RAF 265, Regorafenib (BAY 73-4506), CCT239065, or Sorafenib (or Sorafenib Tosylate, or Nexavar®), or ipilimumab (or DX-G1 G, DX-101 , or Yervoy).
Phosphoinositide 3-kinase (P13K) inhibitors include, but are not limited to, 4-[2-(1 H- lndazol-4-yl)-6-[[4-(methyisulfonyl)piperazin-1 -yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDCQ941 , RG7321 , GNEG941 , Pictrelisib, or Picii!isib; and described in PCT Publication Nos. WO 09/036082 and WO 09/055730); 2-Methyl-2-[4-[3-meihyl-2-oxo-8- (quino!in-3-yl)-2,3-dihyd roimidazo[4,5-c]quino!in-1 -yljphenyljpropionitriie (also known as BEZ 235 or NVP-BEZ 235, and described in PCT Publication No. WO 06/122806); 4- (trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine (also known as BK 120 or NVP-BK 120, and described in PCT Publication No. W02007/084786); Tozasertib (VX68Q or MK-0457, CAS 639089-54-6); (5Z)-5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4- thiazolidinedione (GSK105961 5, CAS 958852-01 -2); (1 E,4S,4aR,5R,6aS,9aR)-5-(Acetyloxy)-1 - [(di-2-propenylamino)metbylene]-4,4a,5,6,6a,8,9,9a-octabydro-1 1 -bydroxy-4-(methoxymethyl)- 4a,6a-dimethylcyclopenta[5,6]naphtho[1 ,2-c]pyran-2,7,10(1 H)-trione (PX866, CAS 502632-66- 8); 8-Phenyl-2-(morpholin-4-yl)-chromen-4-one (LY2940Q2, CAS 154447-36-6); (S)-N1 -(4- methyl-5-(2-(1 ,1 ,1 -trifluoro-2-methyipropan-2-yi)pyridin-4-yl)thiazol-2-yi)pyrrolidine-1 ,2- dicarboxamide (also known as BYL719 or Alpelisib); 2-(4-(2-(1 -isopropyi-3-methyl-1 H-1 ,2,4- triazol-5-yi)-5,6-dihydrobenzo[f]imidazo[1 ,2-d][1 ,4]oxazepin-9-yl)-1 H-pyrazoi-1 -yl)-2- methylpropanamide (also known as GDC0Q32, RG7604, or Taseiisib).
mTOR inhibitors include but are not limited to, Temsirolimus (Torisel®); Ridaforolimus (formally known as deferolimus, (1 R,2/?,4S)-4-[(2R)-2
[(1 R,9S,12S,15R,16£,18R,19R,21 R,23S,24E,26E,28Z,30S,32S,35R)-1 ,18-dihydroxy-19,30- dimethoxy-15,17,21 ,23, 29,35-hexamethyl-2,3,10,14,20-pentaoxo-1 1 ,36-dioxa-4- azatricyclo[30.3.1 .04,9] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383); Everolimus (Afinitor® or RAD0Q1 ); Rapamycin (AY22989, Sirolimus®); Simapimod (CAS 164301 -51 -3); (5-{2,4-Bis[(3S)-3-methylmorphoiin-4-yl]pyrido[2,3-d]pyrimidin- 7-yl}-2-methoxyphenyl)methanoi (AZD8055); 2-Amino-8-[frans-4-(2-hydroxyethoxy)cyclohexyl]- 6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101 -36-4); and N2-[ 1 ,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1 -benzopyran-2-yl)morpholinium-4- yl]methoxy]butyl]-L-arginylglycyi-L-n-aspartylL-serine-, inner salt (SF1 126, CAS 936487-67-1). CDK inhibitors include but are not limited to, Paibociclib (also known as PD-G332991 , !brance®, 6-Acetyl-8-cyclopentyl-5-methyl-2-{[5-(1 -piperazinyl)-2-pyridinyl]amino}pyrido[2,3- d]pyrimidin-7(8H}-one).
In yet another embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with one or more pro-apoptotics, including but not limited to, IAP inhibitors, BCL2 inhibitors, MCL1 inhibitors, TRAIL agents, CHK inhibitors.
For examples, IAP inhibitors include but are not limited to, LCL161 , GDC-0917, AEG- 35158, AT406, and TL32711. Other examples of IAP inhibitors include but are not limited to those disclosed in WOG4/QG5284, WO 04/007529, WOQ5/097791 , WO 05/069894, WO
05/069888, WO 05/094818, US2Q08/0014700, US2006/0025347, WO 06/089083, WO
08/010118, WO 06/017295, and WOQ8/134679, ail of which are incorporated herein by reference
BCL-2 inhibitors include but are not limited to, 4-[4-[[2-(4-Chlorophenyl)-5,5-dimethyl-1- cyclohexen-1 -yl]methyl]-1 -piperazinyl]-N-[[4-[[(1 R)-3-(4-morpholinyl)-1 - [(phenyithio)methyljpropyl]amino]-3 [(trifiuoromethyi)suifonyi]phenyi]sulfonyl]benzamide (also known as ABT-263 and described in PCT Publication No. WO 09/155386): Tetrocarcin A; Antimycin; Gossypol ((-)BL-193); Obatoclax; Ethyl-2-amino-6-cyc!opentyl-4-(1-cyano-2-ethoxy- 2-oxoethyl)-4Hchromone-3-carboxylate (HA14 -1); Oblimersen (G3139, Genasense®); Bak BH3 peptide; (-)-Gossypoi acetic acid (AT -101); 4-[4-[(4'-Chloro[1 ,1 '-biphenyl]-2-yl)methyl]-1- piperazinyl]-N-[[4-[[(1 R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3- nitrophenyijsulfonylj-benzamide (ABT-737, CAS 8528G8-G4-9); and Navitoclax (ABT-263, CAS 923564-51-6).
Proapoptotic receptor agonists (PARAs) including DR4 (TRAILR1) and DR5 (TRAiLR2), including but are not limited to, Du!aner in (AMG-951 , RhApo2L/TRAIL); Mapatumumab (HRS- ETR1 , CAS 658052-09-6); Lexatumumab (HGS-ETR2, CAS 845818-02-6); Apomab
(Apornab®); Conatumumab (A G855, CAS 896731-82-1); and Tigatuzumab(CS1008, CAS 946415-34-5, available from Daiichi Sankyo).
Checkpoint Kinase (CHK) inhibitors include but are not limited to, 7- Hydroxystaurosporine (UCN-01); 6-Bromo-3-(1 -methyl-1 H-pyrazol-4-yl)-5-(3R)-3- piperidinyipyrazolo[1 ,5-a]pyrimidin-7-amine (SCH900776, CAS 891494-63-8); 5-(3- Fluorophenyi)-3-ureidothiophene-2-carboxylic acid N-[(S)-piperidin-3-yl]amide (AZD7762, CAS 860352-01 -8); 4-[((3S)-1-Azablcycio[2.2.2]oct-3-yl)aminoj-3-(1 H-benzimidazol-2-yi)-6- chloroquinoiin-2(1 H)-one (CHIR 124, CAS 405168-58-3); 7-Aminodactinomycin (7-AAD), Isogranulatimide, debromohymenialdisine; N-[5-Bromo-4-methyl-2-[(2S)-2-morpholinylmethoxy]- phenyl]-N'-(5-methyi-2-pyrazinyi)urea (LY2803818, CAS 911222-45-2); Sulforaphane (CAS 4478-93-7, 4-Methylsulfinyibutyi isothiocyanate); 9,10,11 ,12-Tetrahydro- 9,12-epoxy-1 H- diindoio[1 ,2,3-fg:3',2',1 '-/c/]pyrrolo[3,4-/][1 ,6]benzodiazocine-1 ,3(2H)-dione (SB-218078, CAS 135897-06-2); and TAT-S216A (YGRKKRRGRRRLYRSPAMPENL (SEQ ID NO: 36)), and CBP501 ((d-Bpa)sws(d-Phe-F5)(d-Cha)rrrqrr)
In a further embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with one or more immunomoduiators (e.g., one or more of an activator of a costimuiatory moiecuie or an inhibitor of an immune checkpoint moiecuie).
In certain embodiments, the i munomoduiator is an activator of a costimuiatory moiecuie. in one embodiment, the agonist of the costimuiatory molecule is selected from an agonist ( e.g an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion) of 0X40, CD2, CD27, CDS, iCAM-1 , LFA-1 (CD11 a/CD18), ICOS (CD278), 4-1 BB (CD137),
GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.
GITR Agonists
In certain embodiments, the agonist of the costimuiatory molecule is a GITR agonist in some embodiments, the GITR agonist is GWN323 (NVS), BMS-986156, MK-4166 or K-1248 (Merck), TRX518 (Leap Therapeutics), INCAGN1876 (incyte/Agenus), AMG 228 (Amgen) or INBRX-1 10 (Inhibrx).
Exemplary GITR Agonists
In one embodiment, the GITR agonist is an anti-GITR antibody moiecuie. In one embodiment, the GITR agonist is an anti-GITR antibody molecule as described in WO
2016/057846, published on April 14, 2016, entitled“Compositions and Methods of Use for Augmented immune Response and Cancer Therapy,” incorporated by reference in its entirety.
In one embodiment, the anti-GITR antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 7 (e.g., from the heavy and light chain variable region sequences of MAB7 disclosed in Table 7), or encoded by a nucleotide sequence shown in Table 7. In some embodiments, the CDRs are according to the Kabat definition (e.g., as set out in Table 7). In some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 7). In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 7, or encoded by a nucleotide sequence shown in Table 7.
In one embodiment, the anti-GITR antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 45, a VHCDR2 amino acid sequence of SEQ ID NO: 47, and a VHCDR3 amino acid sequence of SEQ ID NO: 49; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO:
50, a VLCDR2 amino acid sequence of SEQ ID NO: 52, and a VLCDR3 amino acid sequence of SEQ ID NO: 54, each disclosed in Table 7.
In one embodiment, the anti-GITR antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 37. In one embodiment, the anti-GITR antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 38. In one embodiment, the anti-GITR antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 37 and a VL comprising the amino acid sequence of SEQ ID NO: 38.
In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 41 , or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 41 . in one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 42, or a nucleotide sequence at least 85%, 90%, 95%, or 99% Identical or higher to SEQ ID NO: 42. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 41 and a VL encoded by the nucleotide sequence of SEQ ID NO: 42.
In one embodiment, the anti-GITR antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 39. In one embodiment, the anti- GITR antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 40. In one embodiment, the anti-GITR antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 39 and a light chain comprising the amino acid sequence of SEQ ID NO: 40.
In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 43, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 43. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 44, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 44. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 43 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 44.
The antibody molecules described herein can be made by vectors, host ceils, and methods described in WQ 2016/057848, incorporated by reference in its entirety.
Table 7: Amino acid and nucleotide sequences of exemplary anti-GITR antibody molecule
Figure imgf000429_0001
Figure imgf000430_0001
Figure imgf000431_0001
Other Exemplary GITR Agonists
In one embodiment, the anti-GITR antibody molecule Is BMS~986156 (Bristol-Myers Squibb), also known as BMS 986156 or BMS986156. BMS-986156 and other anti-GITR antibodies are disclosed, e.g., In US 9,228,016 and WO 2016/196792, incorporated by reference in their entirety in one embodiment, the anti-GITR antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of BMS- 986156, e.g., as disclosed in Table 8. In one embodiment, the anti-GiTR antibody molecule is MK-4166 or MK-1248 (Merck). MK-4180, MK-1248, and other anti-GITR antibodies are disclosed, e.g. , in US 8,709,424, WO 2011/028683, WO 2015/026684, and Mahne et al. Cancer Res 2017; 77(5):1108-1118, incorporated by reference in their entirety. In one embodiment, the anti-GITR antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of MK-4166 or MK-1248.
In one embodiment, the anti-GiTR antibody molecule is TRX518 (Leap Therapeutics). TRX518 and other anti-GITR antibodies are disclosed, e.g., in US 7,812,135, US 8,388,967, US 9,028,823, WO 2006/105021 , and Ponte J et ai. (2010) Clinical Immunology ; 135:S96, incorporated by reference in their entirety in one embodiment, the anti-GITR antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of TRX518
In one embodiment, the anti-GiTR antibody molecule is INCAGN1876 (Incyte/Agenus). INCAGN1876 and other anti-GiTR antibodies are disclosed, e.g., in US 2015/0368349 and WO 2015/184099, incorporated by reference in their entirety in one embodiment, the anti-GITR antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of IMCAGN1876.
In one embodiment, the anti-GiTR antibody molecule is AMG 228 (Amgen). AMG 228 and other anti-GITR antibodies are disclosed, e.g., in US 9,464,139 and WO 2015/031667, incorporated by reference in their entirety. In one embodiment, the anti-GITR antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of AMG 228.
In one embodiment, the anti-GiTR antibody molecule is INBRX-110 (!nhibrx). INBRX- 1 10 and other anti-GiTR antibodies are disclosed, e.g., in US 2017/0022284 and WO
2017/015623, incorporated by reference in their entirety in one embodiment, the G!TR agonist comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of INBRX-1 10.
In one embodiment, the GITR agonist (e.g., a fusion protein) is MEDI 1873
(Medimmune), also known as MEDI1873. MED! 1873 and other GITR agonists are disclosed, e.g., in US 2017/0073386, WG 2017/025610, and Ross et al. Cancer Res 2016; 76(14 Suppi): Abstract nr 561 , incorporated by reference in their entirety. In one embodiment, the GITR agonist comprises one or more of an IgG Fc domain, a functional multimerization domain, and a receptor binding domain of a glucocorticoid-induced TNF receptor iigand (G!TRL) of MED! 1873
Further known GITR agonists (e.g., anti-GiTR antibodies) include those described, e g., in WO 2016/054638, incorporated by reference in its entirety.
In one embodiment, the anti-GiTR antibody is an antibody that competes for binding with, and/or binds to the same epitope on GITR as, one of the anti-GITR antibodies described herein.
In one embodiment, the GITR agonist is a peptide that activates the GITR signaling pathway in one embodiment, the GITR agonist is an immunoadhesin binding fragment (e.g., an immunoadhesin binding fragment comprising an extracellular or GITR binding portion of GITRL) fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
Table 8: Amino acid sequence of other exemplary anti-GiTR antibody molecules
Figure imgf000433_0001
In certain embodiments, the immunomoduiafor is an inhibitor of an immune checkpoint molecule in one embodiment, the immunomodulator is an inhibitor of PD-1 , PD-L1 , PD-L2, CTLA4, TIM3, LAGS, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4 and/or TGFRbeta. in one embodiment, the inhibitor of an immune checkpoint mo!ecuie inhibits PD-1 , PD-L1 , LAG-3, TIM- 3 or CTLA4, or any combination thereof. The term“inhibition” or“inhibitor” includes a reduction in a certain parameter, e.g., an activity, of a given molecule, e.g., an immune checkpoint inhibitor. For example, Inhibition of an activity, e.g., a PD-1 or PD-L1 activity, of at least 5%,
10%, 20%, 30%, 40%, 50% or more is included by this term. Thus, inhibition need not be 100%.
Inhibition of an inhibitory molecule can be performed at the DNA, RNA or protein level in some embodiments, an inhibitory nucleic acid (e.g., a dsRNA, siRNA or shRNA), can be used to inhibit expression of an inhibitory molecule. In other embodiments, the inhibitor of an inhibitory signal is a polypeptide e.g., a soluble iigand (e.g., PD-1-lg or CTLA-4 Ig), or an antibody or antigen-binding fragment thereof, that binds to the inhibitory molecule; e.g., an antibody or fragment thereof (also referred to herein as“an antibody molecule”) that binds to PD-1 , PD-L1 , PD-L2, CTLA4, TIM3, LAGS, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4 and/or TGFR beta, or a combination thereof.
In one embodiment, the antibody molecule is a full antibody or fragment thereof (e.g., a Fab, F(ab')2, Fv, or a single chain Fv fragment (scFv)). In yet other embodiments, the antibody molecule has a heavy chain constant region (Fc) selected from, e.g., the heavy chain constant regions of lgG1 , !gG2, !gG3, lgG4, IgM, lgA1 , igA2, IgD, and igE; particularly, selected from, e.g., the heavy chain constant regions of lgG1 , !gG2, lgG3, and lgG4, more particularly, the heavy chain constant region of !gG1 or !gG4 (e.g.., human lgG1 or lgG4). In one embodiment, the heavy chain constant region is human lgG1 or human lgG4. In one embodiment, the constant region is altered, e.g., mutated, to modify the properties of the antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).
In certain embodiments, the antibody molecule is in the form of a bispecific or multispecific antibody molecuie. In one embodiment, the bispecific antibody molecule has a first binding specificity to PD-1 or PD-L1 and a second binding specifity, e.g., a second binding specificity to TIM-3, LAG-3, or PD-L2. In one embodiment, the bispeeific antibody molecule binds to PD-1 or PD-L1 and TIM-3. In another embodiment, the bispecific antibody molecule binds to PD-1 or PD-L1 and LAG-3 in another embodiment, the bispeeific antibody molecule binds to PD-1 and PD-L1 . In yet another embodiment, the bispecific antibody molecule binds to PD-1 and PD-L2. in another embodiment, the bispecific antibody molecule binds to TIM-3 and LAG-3. Any combination of the aforesaid molecules can be made in a multispecific antibody molecule, e.g., a tnspecific antibody that includes a first binding specificity to PD-1 or PD-1 , and a second and third binding specifities to two or more of: TIM-3, LAG-3, or PD-L2.
In certain embodiments, the immunomoduiator is an inhibitor of PD-1 , e.g., human PD-1. In another embodiment, the immunomoduiator is an inhibitor of PD-L1 , e.g., human PD-L1. In one embodiment, the inhibitor of PD-1 or PD-L1 is an antibody molecule to PD-1 or PD-L1. The PD-1 or PD-L1 inhibitor can be administered alone, or in combination with other
immunomodulators, e.g., in combination with an inhibitor of LAG-3, TIM-3 or CTLA4. in an exemplary embodiment, the inhibitor of PD-1 or PD-L1 , e.g., the anti-PD-1 or PD-L1 antibody molecule, is administered in combination with a LAG-3 inhibitor, e.g., an anii-LAG~3 antibody molecule. In another embodiment, the inhibitor of PD-1 or PD-L1 , e.g., the anti-PD-1 or PD-L1 antibody molecule, is administered in combination with a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody molecule. In yet other embodiments, the inhibitor of PD-1 or PD-L1 , e.g., the anti-PD-1 antibody molecule, is administered in combination with a LAG-3 inhibitor, e.g., an anti-LAG-3 antibody molecule, and a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody molecule.
Other combinations of immunomodulators with a PD-1 inhibitor (e.g., one or more of PD- L2, CTLA4, TIM3, LAGS, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4 and/or TGFR) are also within the present invention. Any of the antibody molecules known in the art or disclosed herein can be used in the aforesaid combinations of inhibitors of checkpoint molecule.
PD-1 inhibitors In some embodiments, the antibody conjugate of the present Invention is administered in combination with a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is selected from PDR001 (Novartis), Nivolumab (Bristol-Myers Squibb), Pembro!izumab (Merck & Co),
Pidilizumab (CureTech), MEDI0680 (Medimmune), REGN281 Q (Regeneron), TSR-042
(Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR121 G
(incyte), or AMP-224 (Ampiimmune).
Exemplary PD-1 Inhibitors
In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecuie as described in US
2015/0210789, published on July 30, 2015, entitled“Antibody Molecules to PD-1 and Uses Thereof,” incorporated by reference in its entirety.
In one embodiment, the anti-PD-1 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in
Table 9 (e.g. , from the heavy and light chain variable region sequences of BAP049-Clone-E or BAP049-Clone-B disclosed in Table 9), or encoded by a nucleotide sequence shown in Table 9. In some embodiments, the CDRs are according to the Kabat definition (e.g , as set out in Table 9). In some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 9). In some embodiments, the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g., as set out in Table 9). In one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GYTFTTYWMH (SEQ ID NO: 96). In one embodiment, one or more of the CDRs (or collectively ail of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 9, or encoded by a nucleotide sequence shown in Table 9.
In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 57, a VHCDR2 amino acid sequence of SEQ ID NO: 58, and a VHCDR3 amino acid sequence of SEQ ID NO: 59; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO:
66, a VLCDR2 amino acid sequence of SEQ ID NO: 67, and a VLCDR3 amino acid sequence of SEQ ID NO: 68, each disciosed in Table 9.
In one embodiment, the antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 79, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 80, and a VHGDR3 encoded by the nucleotide sequence of SEQ ID NO: 81 ; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO:
84, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 85, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 86, each disclosed in Table 9. In one embodiment, the anii-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 62. in one embodiment, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 75. In one embodiment, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 71 , or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 71 . in one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 75. In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the a ino acid sequence of SEQ ID NO: 71
In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 63, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 63. in one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 76 or 72, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 76 or 72. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 63 and a VL encoded by the nucleotide sequence of SEQ ID NO: 76 or 72.
In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 64. In one embodiment, the anti-PD- 1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 77. In one embodiment, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 73. In one embodiment, the anti-PD- 1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 and a light chain comprising the amino acid sequence of SEQ ID NO: 77. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 and a light chain comprising the amino acid sequence of SEQ ID NO: 73.
In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 65, or a nucleotide sequence at least 85%, 90%, 95%, or 99% Identical or higher to SEQ ID NO: 65. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 78 or 74, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 78 or 74. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 65 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 78 or 74.
The antibody molecules described herein can be made by vectors, host ceils, and methods described in US 2015/0210769, incorporated by reference in its entirety.
Table 9. Amino acid and nucleotide sequences of exemplary anti-PD-1 antibody molecules
Figure imgf000437_0001
Figure imgf000438_0001
Figure imgf000439_0001
Figure imgf000440_0001
Figure imgf000441_0001
Figure imgf000442_0001
Other Exemplary PD-1 inhibitors
In some embodiments, the anti-PD-1 antibody Is Nivolumab (CAS Registry Number: 946414-94-4). Alternative names for Nivolumab include MDX-1 106, MDX-1 106-04, ONO-4538, BMS-936558 or OPDIVG©. Nivolumab is a fully human igG4 monoclonal antibody which specifically blocks PD1 . Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed In US Pat No. 8,008,449 and PGT Publication No.
W02006/121 168, incorporated by reference in their entirety in one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Nivolumab, e g., as disclosed in Table 10.
In other embodiments, the anti-PD-1 antibody is Pembrolizumab. Pembrolizumab (Trade name KEYTRUDA formerly Lambrolizumab, also known as Merck 3745, MK-3475 or SCH- 900475) is a humanized igG4 monoclonal antibody that binds to PD1 . Pembrolizumab is disclosed, e.g ., in Hamid, O. et ai. (2013) New England Journal of Medicine 369 (2): 134-44, PCT Publication No. W02009/114335, and US Patent No. 8,354,509, incorporated by reference in their entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively ail of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Pembrolizumab, e.g., as disclosed in Table 10.
In some embodiments, the anti-PD-1 antibody is Pidi!izumab Pidilizumab (CT-01 1 ; Cure Tech) is a humanized lgG1 k monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in PCT Publication No.
W02009/10161 1 , incorporated by reference in their entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively ail of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Pidilizumab, e.g., as disclosed in Table 10.
Other anti-PD1 antibodies are disclosed in US Patent No. 8,609,089, US Publication No. 2010028330, and/or US Publication No. 20120114649, incorporated by reference in their entirety. Other anti-PD1 antibodies include AMP 514 (Amplimmune).
In one embodiment, the anti-PD-1 antibody molecule is MEDI0680 (Medimmune), also known as AMP-514. MEDIQ680 and other anti-PD-1 antibodies are disclosed in US 9,205,148 and WO 2012/145493, incorporated by reference in their entirety. In one embodiment, the anti- PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of MEDI0680.
In one embodiment, the anti-PD-1 antibody molecule is REGN2810 (Regeneron). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of REGN2810.
In one embodiment, the anti-PD-1 antibody molecule is PF-06801591 (Pfizer). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively ail of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of PF-06801591. In one embodiment, the anii-PD-1 antibody molecule is BGB-A317 or BGB-1 G8
(Beigene). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of BGB-A317 or BGB-108.
In one embodiment, the anti-PD-1 antibody molecule is INCSHR121 Q (Incyte), also known as INCSHR0121 G or SHR-1210. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of INCSHR1210.
In one embodiment, the anti-PD-1 antibody molecule is TSR-Q42 (Tesaro), also known as ANB011. in one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of TSR-G42.
Further known anti-PD-1 antibodies include those described, e.g., in WO 2015/1 12800, WO 2016/092419, WO 2015/085847, WO 2014/179664, WO 2014/194302, WO 2014/209804, WO 2015/200119, US 8,735,553, US 7,488,802, US 8,927,697, US 8,993,731 , and US
9,102,727, incorporated by reference in their entirety.
In one embodiment, the anti-PD-1 antibody is an antibody that competes for binding with, and/or binds to the same epitope on PD-1 as, one of the anti-PD-1 antibodies described herein.
In one embodiment, the PD-1 inhibitor is a peptide that inhibits the PD-1 signaling pathway, e.g., as described in US 8,907,053, incorporated by reference in its entirety. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224 (B7-DCig (Amplimmune), e.g., disclosed in WO 2010/027827 and WO 2011/066342, incorporated by reference in their entirety).
Table 10. Amino acid sequences of other exemplary anii-PD-1 antibody molecules
Figure imgf000444_0001
Figure imgf000445_0001
PD-L1 inhibitors
In certain embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-L1. in some embodiments, the antibody conjugate of the present invention is administered in combination with a PD-L1 inhibitor in some embodiments, the PD-L1 inhibitor is selected from FAZ053 (Novartis), Atezo!izumab (Genentech/Roche), Avelumab (Merck Serono and Pfizer), Durvalumab (Medimmune/AstraZeneca), or BMS-938559 (Bristol-Myers Squibb). Exemplary PD-L 1 1nhibitors
In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule as disclosed in US 2016/0108123, published on April 21 , 2016, entitled“Antibody Molecules to PD-L1 and Uses Thereof,” incorporated by reference in its entirety.
In one embodiment, the anti-PD-L1 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 11 (e.g., from the heavy and light chain variable region sequences of BAPQ58-Clone O or BAP058-Clone N disclosed in Table 11), or encoded by a nucleotide sequence shown in Table 11. In some embodiments, the CDRs are according to the Kabat definition (e.g., as set out in Table 1 1). in some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 11). In some embodiments, the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g., as set out in Table 1 1). In one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GYTFTSYWMY (SEQ ID NO: 142). In one embodiment, one or more of the CDRs (or collectively all of the GDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 1 1 , or encoded by a nucleotide sequence shown in Table 1 1.
In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 97, a VHCDR2 amino acid sequence of SEQ ID NO: 98, and a VHCDR3 amino acid sequence of SEQ ID NO: 99; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 105, a VLCDR2 amino acid sequence of SEQ ID NO: 106, and a VLCDR3 amino acid sequence of SEQ ID NO: 107, each disclosed in Table 11
In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 123, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 124, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 125; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 128, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 129, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 130, each disclosed in Table 11 .
In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 102, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 102. In one embodiment, the anti-PD-L1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 1 11 , or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 1 11 . In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 115, or an amino acid sequence at ieast 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 1 15. in one embodiment, the anti-PD-L1 antibody molecuie comprises a VL comprising the amino acid sequence of SEQ ID NO: 1 19, or an amino acid sequence at ieast 85%, 90%, 95%, or 99% identicai or higher to SEQ ID NO: 1 19. In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 102 and a VL comprising the amino acid sequence of SEQ ID NO:
1 11. In one embodiment, the anti-PD-LI antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 115 and a VL comprising the amino acid sequence of SEQ ID NO: 119.
In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 103. in one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 112, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 112. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 116, or a nucleotide sequence at Ieast 85%, 90%, 95%, or 99% identicai or higher to SEQ ID NO: 1 16. in one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 12Q, o a nucleotide sequence at ieast 85%, 90%, 95%, or 99% identicai or higher to SEQ ID NO: 120. in one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 103 and a VL encoded by the nucleotide sequence of SEQ ID NO: 112. in one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 116 and a VL encoded by the nucleotide sequence of SEQ ID NO: 120.
In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence at Ieast 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 104. in one embodiment, the anti- PD-LI antibody molecuie comprises a light chain comprising the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence at Ieast 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 1 13. In one embodiment, the anti-PD-L1 antibody molecuie comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 117, or an amino acid sequence at Ieast 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 117. In one embodiment, the anti-PD-LI antibody molecuie comprises a light chain comprising the amino acid sequence of SEQ ID NO: 121 , or an amino acid sequence at Ieast 85%, 90%, 95%, or 99% identicai or higher to SEQ ID NO: 121. in one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 104 and a light chain comprising the amino acid sequence of SEQ ID NO: 1 13. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 121
In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 1 10, or a nucleotide sequence at least 85%, 90%, 95%, or 99% Identical or higher to SEQ ID NO: 1 10. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 1 14, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 1 14. in one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 1 18, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 1 18. in one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 122, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 122 in one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 1 10 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 1 14 in one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 1 18 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 122.
The antibody molecules described herein can be made by vectors, host ceils, and methods described in US 2016/0108123, incorporated by reference in its entirety. Table 1 1 . Amino acid and nucleotide sequences of exemplary anti-PD-L1 antibody molecules
Figure imgf000448_0001
Figure imgf000449_0001
Figure imgf000450_0001
Figure imgf000451_0001
Figure imgf000452_0001
Figure imgf000453_0001
Figure imgf000454_0001
Other Exemplary PD-L1 inhibitors
In some embodiments, the PD-L1 inhibitor is anti-PD-U antibody. In some
embodiments, the anti-PD-L1 inhibitor is selected from YW243.55.S70, MPDL328QA, MEDi- 4738, or MDX-11 Q5MSB-0010718C (also referred to as AQ9-248-2) disclosed in, e.g., WO 2013/0179174, and having a sequence disclosed herein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified).
In one embodiment, the PD-L1 inhibitor is MDX-1105. MDX-1 105, also known as BMS- 938559, is an anti-PD-L1 antibody described in PCI Publication No. WO 2007/005874
In one embodiment, the PD-L1 inhibitor is YW243.55.S70. The YW243.55.S70 antibody is an anti-PD-L1 described in PCT Publication No. WO 2010/077834.
In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech / Roche) also known as Atezoiizumabm, RG7446, ROS541267, YW243.55.S70, or TECENTRIQ™. MDPL3280A is a human Fc optimized !gG1 monoclonal antibody that binds to PD-L1 . MDPL328GA and other human monoclonal antibodies to PD-L1 are disclosed in U.S. Patent No.: 7,943,743 and U.S Publication No.: 20120039906 incorporated by reference in its entirety in one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or coliectiveiy all of the CDR sequences), the heavy chain or Sight chain variable region sequence, or the heavy chain or light chain sequence of Aiezoiizumab, e.g., as disclosed in Table 12.
In other embodiments, the PD-L2 inhibitor is AMP-224. AMP-224 is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1 (B7-DC!g; Amplimmune; e.g., disclosed in PCT Publication Nos. WO2010/027827 and WO201 1/066342).
In one embodiment the PD-L1 inhibitor is an anti-PD-L1 antibody molecule in one embodiment, the anti-PD-L1 antibody molecule is Aveiumab (Merck Serono and Pfizer), also known as MSBQ010718C. Aveiumab and other anti-PD-L1 antibodies are disclosed in WO 2013/079174, incorporated by reference in its entirety in one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively ail of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Aveiumab, e.g., as disclosed in Table 12.
In one embodiment, the anti-PD-L1 antibody molecule is Durvalumab
(Medlmmune/AstraZeneca), also known as MEDI4736. Durvalumab and other anti-PD-L1 antibodies are disclosed in US 8,779,108, incorporated by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Durvalumab, e.g , as disclosed In Table 12
In one embodiment, the anti-PD-L1 antibody molecule is BMS-936559 (Bristol-Myers
Squibb), also known as MDX-1105 or 12A4. BMS-936559 and other anti-PD-L1 antibodies are disclosed in US 7,943,743 and WG 2015/081158, incorporated by reference in their entirety in one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of BMS-936559, e.g., as disclosed in Table 12.
Further known anii-PD-L1 antibodies include those described, e.g., in WO 2015/181342, WO 2014/100079, WO 2016/000619, WO 2014/022758, WO 2014/055897, WO 2015/061668, WO 2013/079174, WO 2012/145493, WO 2015/1 12805, WO 2015/109124, WO 2015/195163, US 8,168,179, US 8,552,154, US 8,460,927, and US 9,175,082, incorporated by reference in their entirety.
In one embodiment, the anti-PD-L1 antibody is an antibody that competes for binding with, and/or binds to the same epitope on PD-L1 as, one of the anti-PD-L1 antibodies described herein. Table 12. Amino acid sequences of other exemplary anti-PD-L1 antibody molecules
Figure imgf000455_0001
Figure imgf000456_0001
LAG-3 inhibitors
In certain embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of LAG-3. In some embodiments, the antibody conjugate of the present invention is administered in combination with a LAG-3 inhibitor. In some embodiments, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986018 (Bristol-Myers Squibb), or TSR-Q33 (Tesaro).
Exemplary LAG-3 Inhibitors
In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule in one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule as disclosed in US 2015/0259420, published on September 17, 2015, entitled“Antibody Molecules to LAG-3 and Uses Thereof,” incorporated by reference in its entirety.
In one embodiment, the anti-LAG-3 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 13 (e.g., from the heavy and light chain variable region sequences of BAPQ5Q-Glone I or BAPOSG-Clone J disclosed in Table 13), or encoded by a nucleotide sequence shown in Table 13. In some embodiments, the CDRs are according to the Kabat definition (e.g., as set out in Table 13). in some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 13). In some embodiments, the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g., as set out in Table 13). in one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the a ino acid sequence GFTLTNYGMN (SEQ ID NO: 207). In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 13, or encoded by a nucleotide sequence shown in Table 13.
In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEG ID NO: 143, a VHCDR2 amino acid sequence of SEG ID NO: 144, and a VHCDR3 amino acid sequence of SEQ ID NO: 145; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEG ID NO: 152, a VLCDR2 amino acid sequence of SEG ID NO: 153, and a VLCDR3 amino acid sequence of SEG ID NO: 154, each disclosed in Table 13.
In one embodiment, the anti-LAG-3 antibody molecuie comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 177 or 178, a VHCDR2 encoded by the nucleotide sequence of SEG ID NO: 179 or 180, and a VHCDR3 encoded by the nucleotide sequence of SEG ID NO: 181 or 182; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 187 or 188, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 189 or 190, and a VLCDR3 encoded by the nucleotide sequence of SEG ID NO: 191 or 192, each disclosed In Table 13. in one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 199 or 178, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 200 or 180, and a VHGDR3 encoded by the nucleotide sequence of SEQ ID NO: 201 or 182; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 187 or 188, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 189 or 190, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 191 or 192, each disclosed in Table 13.
In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEG ID NO: 148, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 148. In one embodiment, the anti-LAG-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 159. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 165. in one embodiment, the anti-LAG-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 171 , or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 171. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 148 and a VL comprising the amino acid sequence of SEQ ID NO:
159. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 171.
In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 149 or 150, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 149 or 150. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 160 or 161 , or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 160 or 161. in one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 166 or 167, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 166 or 167. in one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 172 or 173, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 172 or 173. In one
embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 149 or 150 and a VL encoded by the nucleotide sequence of SEQ ID NO: 160 or 161. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 166 or 167 and a VL encoded by the nucleotide sequence of SEQ ID NO: 172 or 173.
In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ iD NO: 151 , or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 151 in one embodiment, the anti- LAG-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 162, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 162. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 168, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 168. In one embodiment, the anti-LAG-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 174, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 174 In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 151 and a light chain comprising the amino acid sequence of SEQ ID NO: 162. in one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain comprising the amino acid sequence of SEQ ID NO: 174.
In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 157 or 158, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 157 or 158. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 163 or 164, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 163 or 164. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 169 or 170, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 169 or 170. in one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 175 or 176, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 175 or 176 in one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 157 or 158 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 163 or 164. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 169 or 17Q and a light chain encoded by the nucleotide sequence of SEQ ID NO: 175 or 176.
The antibody molecules described herein can be made by vectors, host ceils, and methods described in US 2015/0259420, incorporated by reference in its entirety.
Table 13. Amino acid and nucleotide sequences of exemplary anti-LAG-3 antibody molecules
Figure imgf000459_0001
Figure imgf000460_0001
Figure imgf000460_0002
Figure imgf000460_0003
Figure imgf000461_0001
Figure imgf000462_0001
Figure imgf000463_0001
Figure imgf000464_0001
Figure imgf000465_0001
Figure imgf000466_0001
Figure imgf000467_0001
Figure imgf000468_0001
Other Exemplary LAGS inhibitors
In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitor is BMS-986G18 (Bristol-Myers Squibb), also known as BMS986016. BMS-986Q16 and other anti-LAG-3 antibodies are disclosed in WO 2015/1 16539 and US 9,505,839, incorporated by reference in their entirety in one embodiment, the anti- LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively ail of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of BMS-986016, e.g., as disclosed in Table 14.
In one embodiment, the anti-LAG-3 antibody molecule is TSR-033 (Tesaro). In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the GDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of TSR-033
In one embodiment, the anti-LAG-3 antibody molecule is IMP731 or GSK2831781 (GSK and Prima BioMed). IMP731 and other anti-LAG-3 antibodies are disclosed in WO
2008/132601 and US 9,244,059, incorporated by reference in their entirety. In one
embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively ail of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of I P731 , e.g , as disclosed in Table 14. In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of GSK2831781.
in one embodiment, the anti-LAG-3 antibody molecule is IMP761 (Prima BioMed). in one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of IMP761.
Further known anti-LAG-3 antibodies include those described, e.g , in WO 2008/132601 , WO 2010/019570, WO 2014/140180, WO 2015/116539, WO 2015/2001 19, WO 2016/028672, US 9,244,059, US 9,505,839, incorporated by reference in their entirety.
In one embodiment, the anti-LAG-3 antibody is an antibody that competes for binding with, and/or binds to the same epitope on LAG-3 as, one of the anti-LAG-3 antibodies described herein.
In one embodiment, the anti-LAG-3 inhibitor is a soluble LAG-3 protein, e.g., IMP321 (Prima BioMed), e.g , as disclosed in WO 2009/044273, incorporated by reference in its entirety.
Table 14. Amino acid sequences of other exemplary anti-LAG-3 antibody molecules
Figure imgf000469_0001
Figure imgf000470_0001
T!M-3 inhibitors
In certain embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIM-3. In some embodiments, the antibody conjugate of the present invention is administered in combination with a TIM-3 inhibitor. In some embodiments, the TIM-3 inhibitor is MGB453 (Novartis) or TSR-022 (Tesaro).
Exemplary TiM-3 inhibitors
In one embodiment, the TilVS-3 inhibitor is an anti-TIM-3 antibody molecule. In one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule as disclosed in US
2015/0218274, published on August 6, 2015, entitled“Antibody Molecules to TIM-3 and Uses Thereof,” incorporated by reference in its entirety.
In one embodiment, the anti-TIM-3 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 15 (e.g., from the heavy and light chain variable region sequences of ABTIM3-hum1 1 or ABTIM3-hum03 disclosed in Table 15), or encoded by a nucleotide sequence shown in Table 15. In some embodiments, the CDRs are according to the Kabat definition (e.g., as set out in Table 15). In some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 15). in one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 15, or encoded by a nucleotide sequence shown in Table 15.
In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 208, a VHCDR2 amino acid sequence of SEQ ID NO: 209, and a VHCDR3 amino acid sequence of SEQ ID NO: 210; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 217, a VLCDR2 amino acid sequence of SEQ ID NO: 218, and a VLCDR3 amino acid sequence of SEQ ID NO: 219, each disclosed in Table 15. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 208, a VHCDR2 amino acid sequence of SEQ ID NO: 226, and a VHCDR3 amino acid sequence of SEQ ID NO: 210; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 217, a VLCDR2 amino acid sequence of SEQ ID NO: 218, and a VLCDR3 amino acid sequence of SEQ ID NO: 219, each disclosed in Table 15.
In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 213, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 213. In one embodiment, the anti-TIM-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 222, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 222. In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 228, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 228 in one embodiment, the anti-TIM-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 232, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 232. in one embodiment, the anti-TIM-3 antibody molecuie comprises a VH comprising the amino acid sequence of SEQ ID NO: 213 and a VL comprising the amino acid sequence of SEQ ID NO: 222. in one embodiment, the anti-TIM-3 antibody molecuie comprises a VH comprising the amino acid sequence of SEQ ID NO: 228 and a VL comprising the amino acid sequence of SEQ ID NO: 232.
In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 214, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 214. in one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 223, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 223 in one embodiment, the antibody molecule comprises a VH encoded by the nucieotide sequence of SEQ ID NO: 229, or a nucieotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 229. in one embodiment, the antibody molecule comprises a VL encoded by the nucieotide sequence of SEQ ID NO: 233, or a nucieotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 233. in one embodiment, the antibody molecule comprises a VH encoded by the nucieotide sequence of SEQ ID NO: 214 and a VL encoded by the nucleotide sequence of SEQ ID NO: 223. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 229 and a VL encoded by the nucleotide sequence of SEQ iD NO: 233.
In one embodiment, the anti-TiM-3 antibody molecule comprises a heavy chain comprising the a ino acid sequence of SEQ ID NO: 215, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 215. In one embodiment, the anti- TIM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 224, or an amino acid sequence at ieast 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 224. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 230, or an amino acid sequence at ieast 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 230. In one embodiment, the anti-TiM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ iD NO: 234, or an amino acid sequence at Ieast 35%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 234. in one embodiment, the anti-TiM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 215 and a light chain comprising the amino acid sequence of SEQ ID NO: 224. In one embodiment, the anii-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 230 and a light chain comprising the a ino acid sequence of SEQ ID NO: 234
In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucieotide sequence of SEQ ID NO: 216, or a nucleotide sequence at Ieast 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 216. In one embodiment, the antibody molecule comprises a light chain encoded by the nucieotide sequence of SEQ ID NO: 225, or a nucieotide sequence at Ieast 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 225. in one embodiment, the antibody molecule comprises a heavy chain encoded by the nucieotide sequence of SEQ ID NO: 231 , or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 231 . in one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 235, or a nucieotide sequence at Ieast 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 235. in one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 216 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 225. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 231 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 235.
The antibody molecules described herein can be made by vectors, host ceils, and methods described in US 2015/0218274, incorporated by reference in its entirety.
Table 15. Amino acid and nucleotide sequences of exemplary anti-TIM-3 antibody molecules
Figure imgf000473_0001
Figure imgf000474_0001
Figure imgf000475_0001
Figure imgf000476_0001
j
i i i i | | | | j j j i i i i i
Figure imgf000477_0001
Other Exemplary TIM-3 Inhibitors
In one embodiment, the anti-TIM-3 antibody molecule is TSR-022 (AnaptysBio/Tesaro). In one embodiment, the anti-TilVS-3 antibody molecule comprises one or more of the GDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of TSR-022. in one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of APE5137 or APE5121 , e.g. , as disclosed in Table 18.
APE5137, APE5121 , and other anti-T!M-3 antibodies are disclosed In WO 2016/161270, incorporated by reference in its entirety.
In one embodiment, the anti-T!M-3 antibody molecule is the antibody clone F38-2E2. in one embodiment, the anti-T!M-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of F38-2E2.
Further known anti-TIM-3 antibodies include those described, e g., in WO 2016/11 1947, WO 2016/071448, WO 2016/144803, US 8,552,156, US 8,841 ,418, and US 9,163,087, incorporated by reference in their entirety.
In one embodiment, the anti-TIM-3 antibody is an antibody that competes for binding with, and/or binds to the same epitope on TIM-3 as, one of the anti-TIM-3 antibodies described herein.
Table 16. Amino acid sequences of other exemplary anti-TIM-3 antibody molecules
Figure imgf000478_0001
Cytokines
In yet another embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with one or more cytokines, including but not limited to, interferon, !L-2, IL-15, IL-7, or IL21. In certain embodiments, antibody conjugate is administered in combination with an !L- 15/IL-15Ra complex. In some embodiments, the !L-15/IL-15Ra complex is selected from
IZ985 (Novartis), ATL-8G3 (A!tor) or CYP0150 (Cytune).
Exemplary IL-15/IL~15Ra complexes
In one embodiment, the cytokine is IL-15 co plexed with a soluble form of 1L-15 receptor alpha (IL-15Ra). The IL-15/IL-15Ra complex may comprise IL-15 covalently or noncovalently bound to a soluble form of IL-15Ra. In a particular embodiment, the human IL-15 is noncovalently bonded to a soluble form of IL-15Ra. In a particular embodiment, the human IL- 15 of the composition comprises an amino acid sequence of SEQ ID NO: 240 in Table 17 or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 240, and the soluble form of human IL-15Ra comprises an amino acid sequence of SEQ ID NO: 241 in Table 17, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEG ID NO: 241 , as described in WO 2014/066527, incorporated by reference in its entirety. The molecules described herein can be made by vectors, host ceils, and methods described in WO 2QQ7084342, incorporated by reference in its entirety. Table 17. Amino acid and nucleotide sequences of exemplary IL-15/IL-15Ra complexes
Figure imgf000479_0002
Other exemplary !L-15/!L-15Ra complexes
In one embodiment, the IL-15/IL-15Ra complex Is ALT-803, an IL-15/IL-15Ra Fc fusion protein (!L-15N72D:!L-15RaSu/Fc soluble complex) ALT-803 is described in WO 2008/143794, incorporated by reference in its entirety in one embodiment, the IL-15/IL-15Ra Fc fusion protein comprises the sequences as disclosed in Table 18.
In one embodiment, the !L-15/IL-15Ra complex comprises IL-15 fused to the sushi domain of IL-15Ra (CYP0150, Cytune). The sushi domain of IL-15Ra refers to a domain beginning at the first cysteine residue after the signal peptide of IL-i SRa, and ending at the fourth cysteine residue after said signal peptide. The complex of IL-15 fused to the sushi domain of !L-15Ra is described in WO 2007/04806 and WO 2012/175222, incorporated by reference in their entirety. In one embodiment, the IL-15/IL-15Ra sushi domain fusion comprises the sequences as disclosed in Table 18.
Table 18. Amino acid sequences of other exemplary IL-15/IL-15Ra complexes
ALT-803
Figure imgf000479_0001
Figure imgf000480_0001
In yet another embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in Gornbination with one or more agonists of toll like receptors (TLRs, e.g., TLR7, TLR8, TLR9). in some embodiments, the antibody conjugate of the present invention can be used in combination with a TLR7 agonist or a TLR7 agonist conjugate.
In another embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with one or more angiogenesis inhibitors, e.g., Bevacizumab (Avastin®), axitinib (Inlyta®); Brivanib a!aninate (BMS-582864, (S)-((ff)-1-(4-(4-Fluoro-2-methyl-1 H-indol-5-yloxy)- 5-metbyipyrroio[2,1-/][1 ,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate); Sorafenib (Nexavan®); Pazopanib (Votrient®); Sunitinib maiate (Sutent®); Cediranib (AZD2171 , CAS 288383-20-1); Vargatef (BIBF1 120, GAS 928328-83-4); Foretinib (GSK1363089); Telatinib (BAY57-9352, CAS 332012-40-5); Apatinib (YN968D1 , CAS 81 1803-05-1 ); Imatinib
(Gleevec®); Ponatinib (AP24534, CAS 943319-70-8); Tivozanib (AV951 , CAS 475108-18-0); Regorafenib (BAY73-4506, CAS 755037-Q3-7); Vatalanib dihydrochloride (PTK787, CAS 212141-51 -0) ; Brivanib (BMS-540215, CAS 649735-46-6); Vandetanib (Capreisa® or AZD6474); Motesanib diphosphate (A G7Q6, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl- 1 H-indol-6-y!)-2-[(4-pyridinylmeihyi)aminoj-3-pyridinecarboxamide, described in PCT Publication No. WO 02/066470); Dovitinib dilactic acid (TKI258, CAS 852433-84-2); Linfanib (ABT869, CAS 796967-16-3); Cabozantinib (XL184, CAS 849217-68-1); Lestaurtinib (CAS 11 1358-88- 4); N-[5-[[[5-(1 ,1-Dimethylethyl)-2-oxazo!yl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7); (3R,4R)-4-Amino-1-((4-((3-methQxyphenyi)amino)pyrrolo[2,1 - f][1 ,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); /V-(3,4-Dichloro-2-fluorophenyl)-6- methoxy-7-[[(3aa,5|3,6aa)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]- 4- quinazolsnamine (XL647, CAS 781613-23-8); 4-Methyl-3-[[1 -methyl-6--(3-pyndinyl)--1 /-/- pyrazolo[3,4-djpyrimidin-4-yl]amino]-/\/-[3-(trifluoromethyl)phenylJ-benzamide (BHG712, CAS 940310-85-0); or Aflibercept (Eylea®).
In another embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with one or more heat shock protein inhibitors, e.g., Tanespimycin (17-ai!yiamino- 17-demethoxygeidanamycin, also known as KOS-953 and 17-AAG, available from SIGMA, and described in US Patent No 4,261 ,989); Retaspimycin (IPI504), Ganetespib (STA-9Q90): [6- Chloro-9-(4-metboxy-3,5-dimethylpyridin-2-ylmethyl)-9H-purin-2-yl]amine (BHB021 or CNF2024, CAS 848695-25-0); irans-4-[[2-(Aminocarbonyl)-5-[4,5,6,7-tetrahydro-6,6-dimethyl-4-oxo-3- (trif!uorometbyl)-1 H-indazoi-1 -yijphenyi]amino]cyciohexyl glycine ester (SNX5422 or
PF049291 13, CAS 9081 15-27-5); 5-[2,4-Dibydroxy-5-(1 -metbyieibyi)phenyl]-A/-eibyi-4-[4-(4- morpbolinyimethyi)phenylj- 3-lsoxazolecarboxamide (AUY922, CAS 747412-49-3); or 17- Dimethylaminoetbyiamino-17-demethoxygeldanamycin (17-DMAG).
In another embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with one or more HDAC inhibitors or other epigenetic modifiers. Exemplary HDAC inhibitors include, but not limited to, Voninostat (Zolinza®); Romidepsin (isiodax®);
Treichostatin A (TSA); Gxamfiatin; Vorinostat (Zolinza®, Suberoylanilide hydroxamic acid); Pyroxamide (syberoyl-3-aminopyridineamide hydroxamic acid); Trapoxin A (RF-1023A);
Trapoxin B (RF-1 G238); Cyclo[(aS,2S)-a-amino-r|-oxo-2-oxiraneocianoyl-0-methyl-D-tyrosyl-L- iso!eucyi-L-prolyi] (Cyl-1); Cyclo[(aS,2S)-a-amino-r|-oxo-2-oxiraneoctanoyl-0-methyl-D-tyrosyl- L-iso!eucy!-(2S)-2-piperidinecarbonyl] (Cyl-2); Cyclic[L-alanyl·D-alanyl-(2S)-p-oxo-L-a- aminooxiraneoctanoyl-D-prolyl] (HC-toxin); Cyclo[(aS,2S)-a-amino-r|-oxo-2-oxiraneoctanoyl-D- phenylalanyi-L-ieucyl-(2S)-2-piperidinecarbonyl] (WF-3161); Chlamydocin ((S)-Cyclic(2- methylalanyi-L-pbenylalanyi-D-prolyl-n-oxo-L-a-aminooxiraneoctanoyl); Apicidin (Cyclo(8-oxo-L- 2-aminodecanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-D-2-piperidinecarbonyl); Romidepsin (Istodax®, FR-901228); 4-Phenylbutyrate; Spiruchostatin A; Mylproin (Valproic acid);
Entinostat (MS-275, N-(2-Aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)-amino-methyl]- benzamide); Depudecin (4, 5:8, 9-dianhydro-1 ,2,6,7,11 -pentadeoxy- D-/ireo-D-/do-Undeca-1 ,6- dienitoi); 4-(Acetylamino)-N-(2-aminophenyl)-benzamide (aiso known as C!-994); N1-(2- Aminophenyi)-N8-phenyi-octanediamide (also known as BML-21 G); 4-(Dimethylamino)-N-(7- (hydroxyamino)-7-oxoheptyl)benzamide (also known as M344); (E)-3-(4-(((2-(1 H-indol-3- yl)ethyl)(2-hydroxyethyl)amino)-methyl)phenyl)-N-hydroxyacrylamide; Panobinostat(Farydak®); Mocetinostat, and Belinostat (also known as PXD1 G1 , Beieodaq®, or (2£)-A-Hydroxy~3-[3- (phenyisulfamoyi)pbenyl]prop-2-enamide), or chidamide (also known as CS055 or HBI-8GG0, (E)-N-(2-amino-5-fluorophenyl)-4-((3-(pyridin-3-yl)acrylamido)methyl)benzamide). Other epigenetic modifiers include but not limited to inhibitors of EZH2 (enhancer of zeste homolog 2), EED (embryonic ectoderm development), or LSD1 (lysine-specific bistone demetbyiase 1 A or KDM1A).
In yet another embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with one or more inhibitors of indoleamine-pyrrole 2,3-dioxygenase (IDO), for example, Indoximod (aiso known as NLG-8189), a-Cyclobexyl-5H~imidazo[5,1-a]isoindole-5~ ethanol (also known as NLG919), or (4E)-4-[(3-Chloro-4-fluoroanilino)-nitrosomethylidene]- 1 ,2,5-oxadiazol-3-amine (also known as INCB024360).
In yet another embodiment, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with one or more agents that control or treat cytokine release syndrome (CRS). Therapies for CRS include but not are limited to, IL-8 inhibitor or IL-6 receptor (IL-6R) inhibitors (e.g., tociiizumab or siituximab), bazedoxifene, sgp13G blockers, vasoactive medications, corticosteroids, immunosuppressive agents, histamine H2 receptor antagonists, anti-pyretics, analgesics (e.g., acetaminophen), and mechanical ventilation. Exemplary therapies for CRS are described in International Application WO201401 1984, which is hereby incorporated by reference.
Tociiizumab is a humanized, immunoglobulin G1 kappa anti-human IL-6R monoclonal antibody. Tociiizumab blocks binding of IL-6 to soluble and membrane bound IL-6 receptors (!L- 6Rs) and thus inhibitos classical and trans-IL-6 signaling. In embodiments, tociiizumab is administered at a dose of about 4-12 mg/kg, e.g., about 4-8 mg/kg for adults and about 8-12 mg/kg for pediatric subjects, e.g., administered over the course of 1 hour.
In some embodiments, the CRS therapeutic is an inhibitor of IL-6 signalling, e.g , an inhibitor of IL-8 or IL-6 receptor in one embodiment, the inhibitor is an anti-IL-6 antibody, e.g., an anti-IL-6 chimeric monoclonal antibody such as si!tuximab. In other embodiments, the inhibitor comprises a soluble gp130 (sgp130) or a fragment thereof that is capable of blocking IL-6 signalling. In some embodiments, the sgp130 or fragment thereof is fused to a heterologous domain, e.g., an Fc domain, e.g., is a gp130-Fc fusion protein such as FE301. In embodiments, the inhibitor of IL-8 signalling comprises an antibody, e.g., an antibody to the IL-6 receptor, such as sarilumab, olokizumab (CDP8038), elsilimomab, sirukumab (CNTO 136), ALD518/BMS-945429, ARGX-1 Q9, or FM101. in some embodiments, the inhibitor of IL-6 signalling comprises a small molecule such as CPSI-2364.
Exemplary vasoactive medications include but are not limited to angiotensin-11 , endotheiin-1 , alpha adrenergic agonists, rostanoids, phosphodiesterase inhibitors, endothelin antagonists, inotropes (e.g., adrenaline, dobutamine, isoprenaline, ephedrine), vasopressors (e.g., noradrenaline, vasopressin, metaramino!, vasopressin, methylene blue), Inodi!ators (e.g., milrinone, levosimendan), and dopamine.
Exemplary vasopressors include but are not limited to norepinephrine, dopamine, phenylephrine, epinephrine, and vasopressin. In some embodiments, a high-dose vasopressor includes one or more of the following: norpepinephrine monotherapy at >20 ug/min, dopamine monotherapy at >10 ug/kg/min, phenylephrine monotherapy at >200 ug/min, and/or epinephrine monotherapy at >10 ug/min. In some embodiments, if the subject is on vasopressin, a high-dose vasopressor includes vasopressin + norepinephrine equivalent of >10 ug/min, where the norepinephrine equivalent dose = [norepinephrine (ug/min)] + [dopamine (ug/kg/min) / 2] + [epinephrine (ug/min)] + [phenylephrine (ug/min) / 10] in some embodiments, if the subject is on combination vasopressors (not vasopressin), a high-dose vasopressor includes
norepinephrine equivalent of >20 ug/min, where the norepinephrine equivalent dose =
[norepinephrine (ug/min)] + [dopamine (ug/kg/min) / 2] + [epinephrine (ug/min)] + [phenylephrine (ug/min) / 10] See e.g., Id.
In some embodiments, a low-dose vasopressor is a vasopressor administered at a dose less than one or more of the doses listed above for high-dose vasopressors.
Exemplary corticosteroids include but are not limited to dexamethasone, hydrocortisone, and methylprednisolone. in embodiments, a dose of dexamethasone of 0 5 mg/kg is used in embodiments, a maximum dose of dexamethasone of 1 Q mg/dose is used in embodiments, a dose of methylprednisolone of 2 mg/kg/day is used.
Exemplary immunosuppressive agents include but are not limited to an inhibitor of TNFa or an inhibitor of !L-1 in embodiments, an inhibitor of TNFa comprises an anti-TNFa antibody, e.g., monoclonal antibody, e.g., infliximab in embodiments, an inhibitor of TNFa comprises a soluble TNFa receptor (e.g., etanercept). In embodiments, an IL-1 or IL-1 R inhibitor comprises anakinra.
Exemplary histamine h receptor antagonists include but are not limited to cimetidine (Tagamet®), ranitidine (Zantac®), famotidine (Pepcid®) and nizatidine (Axid®).
Exemplary anti-pyretic and analgesic includes but is not limited to acetaminophen (Tylenol®), ibuprofen, and aspirin.
In some embodiments, the present invention provides a method of treating cancer by administering to a subject in need thereof antibody conjugate of the present invention in combination with two or more of any of the above described inhibitors, activators,
immunomodu!ators, agonists, or modifiers. For example, the antibody conjugate of the present invention can be used In combination with one or more checkpoint inhibitors and/or one or more immune activators.
In addition to the above therapeutic regimes, the patient may be subjected to surgical removal of cancer cells and/or radiation therapy.
Pharmaceutical Compositions
To prepare pharmaceutical or sterile compositions including one or more antibody conjugates described herein, provided antibody conjugate can be mixed with a pharmaceutically acceptable carrier or excipient.
Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g , lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman et al., Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y., 2001 ; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis, et a! (eds.), Pharmaceutical Dosage Forms:
Parenteral Medications, Marcel Dekker, NY, 1993; Lleberman, et al. (eds ), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY, 1990; Lleberman, et al. (eds.) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y., 2000).
In some embodiments, the pharmaceutical composition comprising the antibody conjugate of the present invention is a iyophilisaie preparation. In certain embodiments a pharmaceutical composition comprising the antibody conjugate is a iyophilisaie in a vial containing an antibody conjugate, histidine, sucrose, and polysorbate 20. In certain
embodiments the pharmaceutical composition comprising the antibody conjugate is a
Iyophilisaie in a vial containing an antibody conjugate, sodium succinate, and polysorbate 20 in certain embodiments the pharmaceutical composition comprising the antibody conjugate is a iyophilisaie in a vial containing an antibody conjugate, trehalose, citrate, and polysorbate 8. The iyophilisate can be reconstituted, e.g., with water, saline, for injection. In a specific embodiment, the solution comprises the antibody conjugate, histidine, sucrose, and polysorbate 20 at a pH of about 5.0. In another specific embodiment the solution comprises the antibody conjugate, sodium succinate, and polysorbate 20. in another specific embodiment, the solution comprises the antibody conjugate, trehalose dehydrate, citrate dehydrate, citric acid, and polysorbate 8 at a pH of about 6.6. For intravenous administration, the obtained solution will usually be further diluted into a carrier solution.
Selecting an administration regimen for a therapeutic depends on severai factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the
immunogenicity of the entity, and the accessibility of the target ceils in the biological matrix. In certain embodiments, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub Ltd, Oxfordshire, UK, 1996; Kresina (ed ), Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y., 1991 ; Bach (ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y., 1993; Baert et al., New Engl. J Med. 348:601-608, 2003; Milgrom et al, New Engl. J. Med. 341 :1966-1973, 1999; Slamon et a!., New Engl. J. Med. 344:783-792, 2001 ; Beniaminovitz et a!., New Engl. J. Med. 342:613-619, 2000; Ghosh et a!., New Engl. J. Med. 348:24-32, 2003; Lipsky et al., New Engi. J Med 343:1594-1602, 2000).
Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects important diagnostic measures include those of symptoms of, e.g. , the inflammation or level of inflammatory cytokines produced.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors known in the medical arts.
Compositions comprising the antibody conjugate of the invention can be provided by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week, once every other week, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once very eight weeks. Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectaliy, intramuscular, iniracerebrai!y, or by inhalation. A specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects.
For the antibody conjugates of the invention, the dosage administered to a patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body weight. The dosage may be between 0.001 mg/kg and 50 mg/kg, 0.005 mg/kg and 20 mg/kg, 0.01 mg/kg and 20 mg/kg, 0.02 g/kg and 10 mg/kg, 0 05 and 5 mg/kg, 0.1 g/kg and 10 mg/kg, 0.1 mg/kg and 8 mg/kg, 0.1 mg/kg and 5 mg/kg, 0 1 mg/kg and 2 mg/kg, 0.1 mg/kg and 1 mg/kg of the patient's body weight. The dosage of the antibody conjugate may be calculated using the patient’s weight in kilograms (kg) multiplied by the dose to be administered in mg/kg.
Doses of the antibody conjugates the invention may be repeated and the administrations may be separated by less than 1 day, at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, 4 months, 5 months, or at least 6 months. In some embodiments, an antibody conjugate of the invention is administered twice weekly, once weekly, once every two weeks, once every three weeks, once every four weeks, or less frequently. In a specific embodiment, doses of the antibody conjugates of the invention are repeated every 2 weeks.
An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route and dose of administration and the severity of side effects (see, e.g., Maynard et a! , A Handbook of SOPs for Good Clinical Practice, !nterpharm Press, Boca Raton, Fla , 1996; Dent, Good Laboratory and Good Clinical Practice, Urch PubL, London, UK, 2001).
The route of administration may be by, e.g., topical or cutaneous application, injection or infusion by subcutaneous, intravenous, intraperitoneai, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesionai administration, or by sustained release systems or an implant (see, e.g., Sidman et a!., Biopolymers 22:547-556, 1983; Langer ei a/., J. Blomed. Mater. Res. 15:167-277, 1981 ; Langer, Cbem. Tech. 12:98-105, 1982; Epstein et a!., Proc. Natl. Acad. Sci. USA 82:3888-3692, 1985; Hwang et at., Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980; U.S. Pat. Nos. 8,350,466 and 8,318,024). Where necessary, the composition may also include a solubilizing agent or a local anesthetic such as lidocaine to ease pain at the site of the injection, or both. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos.
6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entirety.
Examples of such additional ingredients are well-known in the art.
Methods for co-administration or treatment with a second therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation, are known in the art (see, e.g., Hardman et ai, (eds.) (20Q1) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10.sup.th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practiced Practical Approach, Lippincott, Williams & Wilkins, Pbiia , Pa ; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective amount of therapeutic may decrease the symptoms by at least 10%; by at least 20%; at least about 30%; at least 40%, or at least 50%.
Additional therapies (e.g., prophylactic or therapeutic agents), which can be
administered in combination with the antibody conjugates of the invention may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 1 1 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 38 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 80 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart from the antibody conjugates of the invention. The two or more therapies may be administered within one same patient visit.
In certain embodiments, the antibody conjugates of the invention can be formulated to ensure proper distribution in vivo. Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et a/.); mannosides (Umezawa et ai. , (1988) Biochem. Biophys Res. Commun. 153:1038); antibodies (Bioeman et ai. , (1995) FEBS Lett. 357:140; Owais et ai., (1995) Anti icrob. Agents Che other. 39:180); surfactant Protein A receptor (Briscoe et a!., (1995) Am. J. Physiol. 1233:134); p 120 (Schreier et al, (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 348:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.
The invention provides protocols for the administration of pharmaceutical composition comprising antibody conjugates of the invention alone or in combination with other therapies to a subject in need thereof. The therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the present invention can be administered concomitantly or sequentially to a subject. The therapy (e.g., prophylactic or therapeutic agents) of the combination therapies of the present invention can also be cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time and repeating this sequential
administration, i.e., the cycle, in order to reduce the development of resistance to one of the therapies (e.g., agents) to avoid or reduce the side effects of one of the therapies (e.g., agents), and/or to improve, the efficacy of the therapies.
The therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the invention can be administered to a subject concurrently.
The term "concurrently" is not limited to the administration of therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but. rather it is meant that a pharmaceutical composition comprising antibodies or fragments thereof the invention are administered to a subject in a sequence and within a time interval such that the antibodies or antibody conjugates of the invention can act together with the other therapy(ies) to provide an increased benefit than if they were administered otherwise. For example, each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. Each therapy can be administered to a subject separately, in any appropriate form and by any suitable route in various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered to a subject less than 5 minutes apart, less than 15 minutes apart, less than 30 minutes apart, less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 8 hours apart, at about 8 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart in other embodiments, two or more therapies (e.g., prophylactic or therapeutic agents) are administered within the same patient visit.
Prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions. The prophyiactic or therapeutic agents may be administered to a subject by the same or different routes of administration.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be inciuded within the spirit and purview of this application and scope of the appended claims.
EXAMPLES
The invention is further described in the following examples, which are not intended to limit the scope of the invention described in the claims.
Temperatures are given in degrees Celsius If not mentioned otherwise, ail evaporations are performed under reduced pressure, typically between about 15 mm Hg and 100 mm Hg (= 20-133 bar). The structure of final products, intermediates and starting materials is confirmed by standard analytical methods, e.g., microanalysis and spectroscopic characteristics, e.g., MS, IR, NMR. Abbreviations used are those conventional in the art.
All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized to synthesis the compounds of the present invention are either commercially available or can be produced by organic synthesis methods known to one of ordinary skill in the art or can be produced by organic synthesis methods as described herein.
For illustrative purposes, the general reaction schemes depicted herein provide potential routes for synthesizing the compounds of the present invention as well as key intermediates. For a more detailed description of the individual reaction steps, see the Examples section beiow. Although specific starting materials and reagents are depicted in the schemes and discussed below, other starting materials and reagents can be easily substituted to provide a variety of derivatives and/or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.
Abbreviations:
Abbreviations used are those conventional in the art or the following:
Figure imgf000489_0001
Example 1 : Synthesis of Linker Intermediates
Example 1-1 : Synthesis of 4-((S)-2-((S)-2-(3-(2,5-d!oxo-2,5-dihydro-1 H-pyrrol-1 - yl)propanamido)-3-methyibutanamido)-5-ureidopentanamido)benzyl (4- nitrophenyl) carbonate (LI-1)
Figure imgf000490_0001
Step 1 : (S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5- ureidopentanamide (valcit-pab-OH) (100 mg, 0.264 mmol) (purchased from Levena Biopharma, San Diego) was added io 2,5-dioxopyrrolidin-1-y! 3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)propanoate (77 mg, 0 29 mol) in DMF (5ml) at room temperature, followed by the addition of DIEA (70 mg, 0 54 mmol). The mixture was stirred at room temperature for 2 hrs, concentrated and then purified by ISCO using 5Gg C18 aq column, eluted with 10-25% ACN- water with 0.05%TFA Fractions containing (S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (MP- valcit-pab-OH) were combined and concentrated (79 8 mg, 0 150 mmol, 57.1 % yield). LCMS MW+1 = 531 3, tr= 0 687 min.
Step 2: A solution of MP-valcit-pab-OH (33 mg, 0.062 mmol), bis(4-nitrophenyl) carbonate (189 mg, 0.622 mmol) and DIEA (0.033 mL, 0.19 mmol) in DMF-DCM (1 :4, 5 ml) was stirred at room temperature for 1 week, then concentrated and purified by silica gel column, eluted with eOH:DCM (2% to 10%). Fractions containing the desired compound were combined and concentrated to give 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (LI-1 ) (20 mg, 0.029 mmol, 46 % yield). LCMS MW+1 =696.3, tr= 1 .039 min.
Example 2: Synthesis of cvcSic Buildiiiq Block Intermediates
Figure imgf000490_0002
Example 2-1 : Synthesis of (2R,3R,4R,5R)-2~((bis(4-met.hoxyphenyi)(pbeny!)metboxy)methyl)~4- ((tert-butyidimetby!si!yl)oxy)~5-f6~cbioro~9H-purin~9-yi)tetrahydrofuran-3~ol (2- OTBS-BB) and (2R,3R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(6- chloro-9H-purin-9-yl)tetrahydrofuran-3-ol (3-OTBS-BB) 6-C
Figure imgf000491_0001
oro- - - -r o uranosy - -pur ne
Step 1 -1): Synthesis of (2R,3S,4R,5R)-2-((bis(4-melhoxyphenyl)(phenyl)methoxy)methyl)-5-(6- ch!oro-9H-purin-9-yi)tetrahydrofuran-3,4-diol.
To a stirred solution of 6-Ghloro-9-B-D-ribofuranosyl-9H-purine (1 equiv) in pyridine (0.3M) was added 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (DMTCI, 1 .1 equiv).
The reaction mixture was stirred at room temperature for 15 hours, quenched with saturated NaHCOs (aq.) and extracted with DCM. The organic phase was washed with brine, dried over Na2S04 and concentrated under vacuum. The residue was purified by column chromatography (0-100% Ethyl acetate/Heptanes) to afford (2R,3S,4R,5R)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyi)-5-(6-chloro-9H-purin-9-y!)tetrabydrofuran-3,4-dioi (86%). Ή NMR (500 MHz, Methanol-*) 0 8.65 (s, 1 H), 8.63 (s, 1 H), 7.41 - 7.37 (m, 2H), 7.29 - 7.16 (m, 7H), 6.78 (dd, J = 9.0, 7.6 Hz, 4H), 6.14 (d, J = 4.7 Hz, 1 H), 4.96 (t, J = 4.9 Hz, 1 H), 4.53 (s, 1 H), 4.26 (q, J = 4.3 Hz, 1 H), 3.76 (d, J = 1 .5 Hz, 6H), 3.42 (dd, J = 4.Q, 2.2 Hz, 2H).
Step 1 -2: Synthesis of (2R,3R,4R,5R)-2-((bis(4-methoxypbenyi)(pbenyi)metboxy)metbyl)-4- ((iert-butyidimethylsilyl)oxy)-5-(6-chloro-9H-piirin-9-yi)tetrahydrofuran-3-ol (2-OTBS-BB) and (2R,3R,4S,5R)-5-((b!S(4-methoxyphenyl)(phenyl)meihoxy)methyl)-4-((tert- biityldimeihylS!lyl)oxy)-2-(6-chloro-9H-purin-9-yl)tetrahyd!Ofuran-3-oi (3-OTBS-BB).
To a stirred solution of (2R,3S,4R,5R)-2~((bis(4- methoxypheny!)(pheny!)methGxy)methyl)~5-(6~chloro~9H-purin~9-yi)tetrahydrofuran-3,4-diol (1 equiv) in DMF (G.3M) was added imidazole (1 .7 equiv), then TBSCi (1 .03 equiv). The reaction mixture was stirred at room temperature for 15 hours, quenched with saturated NaHC03 (aq.) and extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2S04 and concentrated under vacuum. The residue was purified by column chromatography (0-100% Ethyl acetate/Heptanes) to afford 2-OTBS-BB (26%, yellow foam, retention time=3Gmin) and 3- OTBS-BB (43%, yellow foam, retention time=36min).
(2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert- butyldimethylsilyl)oxy)-5-(6-chloro-9H-purin-9-yl)tetrahydrofuran-3-ol (2-OTBS-BB): 1 H NMR (500 MHz, Chloroform-d) d 8.67 (s, 1 H), 8.36 (s, 1 H), 7.45 - 7.41 (m, 2H), 7.35 - 7.25 (m, 7H), 6.81 (dd, J = 8.9, 2.0 Hz, 4H), 6.1 1 (d, J = 5.4 Hz, 1 H), 4.98 (t, J = 5.2 Hz, 1 H), 4.41 - 4.34 (m, 1 H), 4.29 (q, J = 3.3 Hz, 1 H), 3.79 (d, J = 1 .3 Hz, 7H), 3.52 (dd, J = 10.7, 3.0 Hz, 1 H), 3.42 (dd, J = 10.7, 3.7 Hz, 1 H), 2.68 (d, J = 4.0 Hz, 1 H), 0.84 (s, 9H), 0.00 (s, 3H), -0.15 (s, 3H).
(2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert- butyldimethylsilyl)oxy)-2-(6-chloro-9H-purin-9-yl)tetrahydrofuran-3-ol (3-OTBS-BB): 1 H NMR (500 MHz, Chloroform-d) d 8.71 (s, 1 H), 8.36 (s, 1 H), 7.41 - 7.36 (m, 2H), 7.32 - 7.18 (m, 7H), 6.79 (dd, J = 9.0, 1 .2 Hz, 4H), 6.06 (d, J = 5.0 Hz, 1 H), 4.76 (dt, J = 6.8, 5.2 Hz, 1 H), 4.59 (dd, J = 5.3, 4.0 Hz, 1 H), 4.20 (q, J = 3.7 Hz, 1 H), 3.78 (d, J = 1 .3 Hz, 7H), 3.51 (dd, J = 10.7, 3.4 Hz,
1 H), 3.28 (dd, J = 10.8, 3.9 Hz, 1 H), 3.07 (d, J = 6.9 Hz, 1 H), 0.90 (s, 9H), 0.10 (s, 3H), 0.03 (s, 3H).
2-2: Synthesis of (2R,3R,4R<5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5- (6-chioiO-9H-purin-9-yi)-4-fluorotetrahydrofuran-3-o! (3-F-BB)
Figure imgf000492_0001
Step 1 -1 : Synthesis of 9-((2R,3R,4R,5R)-4-((tert-butyldimethyisiiyl)oxy)-5-(((tert- butyidimetbyisi!yl)oxy)methyl)-3-fiuQroteirahydrofuran-2-yl)-9H-purin-6-amine.
To a solution of 2’-Deoxy-2'-fluoroadenosine (1 equiv) and imidazole (3 equiv) in DMF (0.3M) was added TBSCI (2.5 equiv). The reaction mixture was stirred at room temperature for 15 hours, quenched with saturated NaHCOs (aq.) and extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2S04 and concentrated under vacuum. The residual was purified by column chromatography (0-100% ethyl acetate/heptanes) to afford 9- ((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3- fluorotetrahydrofuran-2-yl)-9H-purin-6-amine (93%, white solid). 1 H NMR (500 MHz, Meihanol- cf4) 6 8.29 (s, 1 H), 8.18 (s, 1 H), 6.25 (dd, J = 18 2, 1 .8 Hz, 1 H), 5.60 (ddd, J = 53 1 , 4.3, 1 .7 Hz,
1 H), 4.97 (ddd, J = 20.2, 7.5, 4.3 Hz, 1 H), 4.10 - 3.96 (rn, 2H), 3.81 (dd, J = 1 1 .9, 2 6 Hz, 1 H), 0.97 (s, 9H), 0.82 (s, 9H), 0 20 (d, J = 1 .8 Hz, 6H), 0.03 (s, 3H), -0.05 (s, 3H).
Step 1 -2: Synthesis of 9~((2R,3R,4R,5R)~4~((iert-butyldimethyisiiyl)oxy)-5~(((teri- biityldimeihyisiiy!)oxy)methyi)-3~fluoroietrahydrofuran-2~y!)-6~chloro~9H-purine.
To a 0°C solution of 9-((2R,3R,4R,5R)~4-((tert-buiyldimeihyLsiiyl)oxy)~5-(i(†ert- butyidimeihyisily!)oxy)meihyi)-3-fiuorotetrahydrofuran-2-y!)-9H-purin-6-amine (1 equiv) in DCM (0.3M) was added T SCi (2 equiv) followed by tBuONO (5 equiv). The reaction mixture was stirred at 0°C for 2 hours then allowed to warm to room temperature for 1 hour. The reaction mixture was quenched with saturated NaHCOs (aq.) and extracted with DCM The organic phase was washed with brine, dried over Na2S04 and concentrated under vacuum. The residue was purified by column chromatography (0-100% Ethyl aeetate/Heptanes) to afford 9- ((2R,3R,4R,5R)-4-((tert-butyidimethyisilyl)oxy)-5-(((fert-butyldimethylsilyl)oxy)methyl)-3- fiuorofetrahydrofuran-2-yl)-8-chioro-9H-purine (40%, yellow syrup). 1H NMR (500 MHz, Methanol-*) d 8.74 (s, 1 H), 8.71 (s, 1 H), 6.37 (dd, J = 18.1 , 1 .6 Hz, 1 H), 5.70 (ddd, J = 52.8, 4.2, 1 .6 Hz, 1 H), 4.99 (ddd, J = 20.7, 7.7, 4.3 Hz, 1 H), 4.1 1 (dd, J = 7.3, 2.0 Hz, 1 H), 4.02 (dd, J = 1 1 .9, 2.5 Hz, 1 H), 3.81 (dd, J = 1 1 .9, 2.5 Hz, 1 H), Q.97 (s, 9H), 0.77 (s, 9H), 0.21 (d, J = 3.3 Hz, 6H) 0 02 (s, 3H), -0 08 (s, 3H).
Step 2-1 : Synthesis of (2R,3R,4R,5R)-5-(6-chloro-9H-purin-9-yl)-4-fluoro-2- (hydroxymethyl)tetrahydrofuran-3-ol.
To a 0°C solution of 9-((2RI3R 4R 5R)-4-((teri-buiyldimefhylsilyl)oxy)-5-(((teri- buty!dimethyisily!)oxy)methyi)-3-fiuorotetrahydrofuran-2-y!)-6-ch!oro-9H-purine (1 equiv) in
DCM/THF (1/1 , 0.2M) was added Et3N (5 equiv) foiiowed by Et3N(HF)3 (13 equiv). The reaction mixture was allowed to warm to room temperature and stirred for 15 hours. The reaction mixture concentrated under vacuum, quenched with saturated NaHCG3 (aq.). The precipitated product was collected by vacuum filtration, washed with diethyl ether and dried under high vacuum to afford (2R,3R,4R,5R)-5-(8-chioro-9H-purin-9-yi)-4-fluoro-2- (hydroxymethyl)tetrahydrofuran-3-o! (85%). 1H NMR (500 MHz, Methanoi-cf4) d 8.87 (s, 1 H),
8.76 (s, 1 H), 6.44 (dd, J = 16.4, 2.2 Hz, 1 H), 5.46 (ddd, J = 52.6, 4.4, 2.2 Hz, 1 H), 4.67 (ddd, J = 19.3, 7.2, 4.4 Hz, 1 H), 4.21 - 4.07 (m, 1 H), 3.97 (dd, J = 12.5, 2.4 Hz, 1 H), 3.79 (dd, J = 12.5, 3.3 Hz, 1 H).
Step 2-2: Synthesis of (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6- ch!oro-9H-purin-9-yl)-4-fiuorotetrahydrofuran-3-ol (3-F-BB).
To a stirred solution of (2R,3R,4R,5R)-5-(6-chloro-9H-purin-9-yl)-4-fIuoro-2- (hydroxymethyl)tetrahydrofuran-3-ol (1 equiv) in pyridine (0.3M) was added
Diisopropy!ethylamine (1 .5 equiv) foiiowed by 4,4'-
(chloro(phenyi)methylene)bis(methoxybenz.ene) (DMTCI, 1 .5 equiv). The reaction mixture was stirred at room temperature for 3 hours, quenched with saturated NaHC03 (aq.) and extracted with DCM. The organic phase was washed with brine, dried over NasSC* and concentrated under vacuum. The residual was purified by column chromatography (0-100% Ethyl a cetate/He planes) to afford (2R,3R,4R,5R)-2-((bis(4-methoxyphenyi)(pheny!)methoxy)methy!)- 5-(6-chioro-9H-purin-9-yl)-4-fiuorotetrahydrofuran-3-ol (3-F-BB) (86%, yellow foam).
Figure imgf000493_0001
NMR (500 MHz, Methanol-*) d 8.69 (s, 1 H), 8.69 (s, 1 H), 7.37 - 7.28 (m, 2H), 7.27 - 7.09 (m, 7H),
6.76 (dd, J = 9.0, 6.7 Hz, 4H), 6.41 (dd, 2 = 18.2, 1 .5 Hz, 1 H), 5.70 (ddd, J = 52.5, 4.4, 1 .4 Hz,
1 H), 4.95 (ddd, J = 21 .7, 8.1 , 4.4 Hz, 1 H), 4.24 (dt, J = 4.4, 2.8 Hz, 1 H), 3.76 (s, 3H), 3.76 (s, 3H), 3.52 - 3.37 (m, 2H).
Example 3: Synthesis of Cyclic Pimjcleotides
Figure imgf000493_0002
Example 3-1 : Synthesis of 2R,3R,3aR,5R,7aR,9R,10R,10aS,12R,14aR)-2-(6-amino-9H-purin-
9-yl)-3-fluoro-9-(6-hydrazinyl-9H-purin-9-yl)-10-hydroxy-5,12- dimercaptooctahydro-2H,7H-difuro[3,2-d:3\2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyciododeclne 5,12-dioxide (CDISI-1 ) and (2R,3R,3aR,5S,7aR,9R,1 GR,1 GaS,12R,14aR)-2-(6-amino-9H-purin-9-yl)-3-fluoro- 9-(6-hydrazinyl-9H-purin-9-yl)-1 Q-hydroxy-5,12-dimercaptooctahydro-2H,7H- difuro[3,2-d:3’,2‘-j][1 ,3,7,9]tetraoxa[2,8]diphosphacyciododecine 5, 12-dioxide (CDN-2)
Figure imgf000494_0001
Step 1 : Synthesis of (2R,3R,4R,5R)-2-((bis(4-methoxyphenyi)(phenyi)methoxy)methyi)-4-((tert- butyldimethyisiiyl)oxy)-5-(6-chloro-9H-purin-9-yl)tetrahydrofuran-3-yi hydrogen phosphonate (2) To a stirred solution of compound 2-OTBS-BB (1 equiv) in dioxane (0.3M) and pyridine (47 equiv) was added 2-chloro-4H-benzo[d][1 ,3,2]dioxaphosphinin-4-one (1 ) (1 .5 equiv, added as 2 portions, each dissolved in 5mL pyridine and immediately added) which was purchased from TCI Chemicals. The reaction was stirred at room temperature for 1 hour, quenched with sat. aq. NaHC03 and extracted with ethyl acetate. The organic layers were combined and concentrated under vacuum. The residue was azeotroped twice using toluene to obtain a crude solid that was a mixture of product (2) and unreacted starting materiai. This mixture was purified by column chromatography (0-10% (10% TEA in MeOH)/DCM) to afford (2R,3R,4R,5R)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethyisilyl)oxy)-5-(6-chloro-9H- purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate (2) (TEA salt) as a yellow foam (74%): 1H NMR (500 MHz, Chloroform-d) d 8.63 (s, 1 H), 8.34 (s, 1 H), 7.44 (d, J = 7.3 Hz, 2H), 7.34 - 7.17 (m, 7H), 6.80 (dd, J = 8.9, 1 .4 Hz, 4H), 6.21 (d, J = 5.7 Hz, 1 H), 4.97 (t, J = 5.1 Hz, 1 H), 4.86 (dt,
J = 9.9, 4.0 Hz, 1 H), 4.53 (d, J = 3.2 Hz, 1 H), 3.51 (d, J = 3.1 Hz, 2H), 0.78 (s, 9H), Q.Q8 (s, 3H), -0.15 (s, 3H).
Step 2 and Step 3: Synthesis of (N-(9-((2R,3R,3aR,7aR,9R,10R,10aR,14aR)-10-((tert- butyidimethylsilyl)Qxy)-9-(6-chlorQ-9H-purin-9-yl)-5-(2-cyanoetboxy)-3-fluoro-12-mercapio-12- oxido-5-sulfidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecin- 2-yl)~9H-purin~8-yi)benzamide (8): To a stirred solution of compound 2 (1 equiv) in DCM (0.1 M) was added water (9 equiv) followed by 6% dichloroacetic acid (DCA) in DCM (v/v) (7 equiv). The bright red reaction mixture was stirred a room temp for 10 minutes, quenched with anhydrous pyridine (14 equiv) and concentrated under vacuum. The residue was rotoevaporated from anhydrous MeCN (0.G7M, 3x). During the final concentration, the crude mixture was concentrated to -"0.1 M in MeCN. To this crude solution containing the free alcohol product was added a stir bar and used in the next step.
In a separate flask, phosphoroa idiie (3) (1 equiv., purchased from Cbemgens Corp.) was rotoevaporated from anhydrous MeCN (G.07M, 3x). During the final concentration, phosphoroamidiie (3) was concentrated to G.1 M in MeCN and used immediately. To a stirred MeCN solution of the free alcohol product from above was added phosphoroamidite (3) via syringe under argon atmosphere. The reaction mixture was stirred for 20 minutes at room temperature, quenched with (E)-N,N~dimethyl-N'-(3-tbioxo~3H-1 ,2 ,4-d ith iazol-5- yi)formimidamide (4) (DDTT, 1 .07 equiv), stirred for an additional 30 minutes at room temperature and then concentrated under vacuum to give reside (5) which was used in the next step without purification.
To a stirred solution of compound (5) in DCM (G.2M) was added water (9 equiv) followed by 6% dichloroacetic acid (DCA) in DCM (v/v) (7 equiv). The bright red reaction mixture was stirred a room temp for 10 minutes, quenched with anhydrous pyridine (32 equiv) and concentrated under vacuum. The residue was rotoevaporated from anhydrous pyridine (0.1 M, 3x). During the final concentration, the crude mixture was concentrated to ~Q.7M in pyridine.
To this stirred crude solution was added 2-chloro-5,5-dimethyl-1 ,3,2-dioxaphosphinane 2-oxide (6) (DMOCP, 3 equiv). The reaction mixture was stirred at room temperature for 15 minutes, quenched with water (3 equiv) and treated with 3H-benzo[c][1 ,2]dithiol-3-one (7) (1.3 equiv).
The reaction mixture was stirred at room temperature for 25 minutes, quenched with sat. aq. NaHCOs and extracted with ethyl acetate. The organic phase was concentrated under vacuum. The residue was suspended in DCM, adsorbed onto silica gei under vacuum and purified by column chromatography (0-10% MeOH/DCM) to afford N-(9~
(^R.SR.SaRTaR.QR.I QFTI OaR^aFO-I Q-^tert-butyldimethyisiiyDoxyl-Q-fe-ehloro-QH-purin-Q- yl)-5-(2-cyanoethoxy)-3-fluoro-12-mercapto-12-oxido-5-suifidooctahydro-2H,7H-difuro[3,2- d:3',2'-j][1 ,3,7,9]tetraoxa[2,8]diphosphacyc!ododecin-2-yi)-9H-purin-6-yl)benzamide (8) as a mixture of phophorothioaie stereoisomers.
Step 4: Synthesis of 2R,3R,3aR,5R,7aR,9R,10R,10aS,12R,14aR)-2-(6-amino-9H-purin-9-yl)-3- fluoro-9-(8-hydrazinyi-9H-purin-9-yi)-10-hydroxy-5,12-dimercaptooctahydro-2H,7H-difuro[3,2- d:3',2'-j][1 ,3,7,9]tetraoxa[2,8]diphosphacyc!ododecine 5,12-dioxide (CDN-1) and
(2R,3R,3aR,5S,7aR,9R,10R,10aS,12R,14aR)-2-(6-amino-9H-purin-9-yi)-3-fluoro-9-(6- bydrazinyi-9H-purin-9-yi)-1 Q-hydroxy-5,12-dimercaptooctabydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine 5,12-dioxide (CDN-2)
A solution of compound (8) (1 equiv) in 1 :1 mixture of MeOH:Hydrazine hydrate (0.1 M) was stirred in a sealed reaction vessel at 50 °C for 1 .5 hours. The reaction mixture was transferred to a plastic vessel and concentrated to afford a yellow solid. The residue was treated with triethylamine trihydrofluoride (120 equiv) and was stirred in a sealed plastic reaction vessel at 50°C for 2 hours. The reaction mixture was quenched by slowly pouring into a large flask containing 1 M triethylammonium bicarbonate. The reaction mixture was allowed to cool to room temperature and stirred until the foam subsided. The entire aqueous solution was then loaded onto a reverse phase ISCO column (C18, 40g) for purification. The column gradient was 0% B in A for 10 minutes followed by ramping 0-20% B in A over 30 minutes, where A=1 QmM Triethyl ammonium acetate (Et3N~HOAc) in water and B=MeCN. Compound CDN-2 (RS- isomer) was collected between 25-27minutes and Compound CD -1 (RR-isomer) was collected between 27-31 min. The column fractions were concentrated under vacuum and lyophoiized to afford bis-triethylammonium salts of CDN-1 (35%) and CDN-2 (45%).
(2R,3R,3aR,5R,7aR,9R,10R,10aS,12R,14aR)-2-(6-amino-9H-purin-9-yl)-3-fluoro-9-(6- hydrazinyl-9H-purin-9-yl)-10-hydroxy-5,12-dimercaptoociahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine 5,12-dioxide, CDISI-1 (RR-isomer, bis- triethylammonium salt): !H NMR (500 MHz, Deuterium Oxide) d 8.33 (s, 1 H), 8.30 (s, 1 H), 8 22 (s, 1 H), 8.10 (s, 1 H), 8.38 (d, J = 16.7 Hz, 1 H), 6.12 (s, 1 H), 5.59 (d, J = 51 .9 Hz, 1 H), 5.22 (d, J = 22.8 Hz, 1 H), 5.09 (d, J = 4.5 Hz, 1 H), 4.85 (d, J = 4.3 Hz, 1 H), 4.57 (dd, J = 26.9, 1 1 .2 Hz, 4H), 4.09 (dd, J = 1 1 .6, 6.0 Hz, 2H). 19F NMR (471 MHz, Deuterium Oxide) 6 -122.29. 31 P NMR (202 MHz, Deuterium Oxide) d 54.51 , 54.16.
(2R,3R,3aR,5S,7aR,9R,10R,10aS,12R,14aR)-2-(6-amino-9H-purin-9-yl)-3-fluoro-9-(6- bydrazinyi-9H-purin-9-yi)-1 G-hydroxy-5,12-dimereaptooctahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine 5,12-dioxide, CDN-2 (RS-isomer, bis- triethylammonium salt): 1H NMR (500 MHz, Deuterium Oxide) d 8.58 (s, 1 H), 8.38 (s, 1 H), 8.29 (s, 1 H), 8.22 (s, 1 H), 6.48 (d, J = 17.1 Hz, 1 H), 6.22 (s, 1 H), 5.87 (dd, J = 51 .3, 4.0 Hz, 1 H), 5.13 - 4.99 (m, 2H), 4.61 (d, J = 1 1 .6 Hz, 2H), 4.50 (dd, J = 24.1 , 10.1 Hz, 2H), 4.19 - 4.08 (m, 2H). 19F NMR (471 MHz, Deuterium Oxide) 6 -201 .67. 31P NMR (202 MHz, Deuterium Oxide) d 54.69, 54.43.
Example 3-2: Synthesis Qf (2R,3R,3aR,5R,7aR,9R,10R,10aR,12R,14aR)-2-(6-amino-9H-purin- 9-yl)-3,10-difluoro-9-(6-hydrazinyl-9H-purin-9-yl)-5,12-dimercaptooctahydro- 2H,7H-d ifuro[3,2-d:3',2'-j][1 ,3,7,9]tetraoxa[2,8]diphosphacyciododecine 5, 12- dioxide (CDM-5) and (2R,3R,3aR,5S,7aR,9R,1 GR,10aR,12R,14aR)-2-(6-amino- QH-purin-Q-y -S.I O-difiuoro-Q-iS-hydrazinyl-gH-purin-Q-y -S,^- dimercaptooctahydro-2H,7H-difuro[3,2-d:3',2’- j][1 ,3,7,9]tetraoxa[2,8]dipbesphacyciododecine 5,12-dioxide (CDiSS-6)
Figure imgf000497_0001
Compounds CDN-5 and CDN-6 were synthesized using a synthetic route similar to the synthesis of compounds CON-1 and C D-2, however CDN building block 3-F-BB was used in place of CDN building block 2-OTBS-BB.
(2R,3R,3aR,5R,7aR,9R,10R,10aR,12R,14aR)-2-(6-amino-9H-purin-9-yl)-3,10-difluoro-9-(6- hydrazinyl-9H-purin-9-yl)-5,12-dimercaptooctahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyciododecine 5,12-dioxide (CDN-5): 1H NMR (500 MHz, Deuterium Oxide) d 8.32 (s, 1 H), 8.30 (s, 1 H), 8.21 (s, 1 H), 8.12 (s, 1 H), 6.40 (dd, J = 16.4, 4.0 Hz, 2H), 5.65 (dd, J = 13.9, 3.8 Hz, 1 H), 5.59 - 5.49 (m, 1 H), 5.25 - 5.12 (m, 2H), 4.60 (d, J = 10.9 Hz, 4H), 4.1 1 (dd, J = 12.3, 5.7 Hz, 2H). 19F NMR (471 MHz, Deuterium Oxide) d -201 .18, -201 .35. 31P NMR (202 MHz, Deuterium Oxide) d 54.57, 54.48.
(2R,3R,3aR,5S,7aR,9R,1 GR,10aR,12R,14aR)-2-(6-amino-9H-purin-9-y!)-3,10-difluoro-9-(6- bydrazinyi-9H-purin-9-yi)-5,12-dimercaptoQctahydrQ-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]teiraoxa[2,8]diphospbacyclododecine 5,12-dioxide (CDM-6): !H NMR (500 MHz,
Deuterium Oxide) d 8.53 (s, 1 H), 8.38 (s, 1 H), 8.24 (s, 1 H), 8.21 (s, 1 H), 6.48 (dd, J = 16.6, 6.6 Hz, 2H), 5.91 (dd, J = 51 .3, 3.9 Hz, 1 H), 5.56 (d, J = 51 .3 Hz, 1 H), 5.24 - 4.95 (m, 2H), 4.68 - 4.45 (m, 4H), 4.14 (dd, J = 32.7, 14.2 Hz, 2H). 19F NMR (471 MHz, Deuterium Oxide) d - 201 .39, -201 .77. 31P NMR (2Q2 MHz, Deuterium Oxide) d 54.76, 54.51 .
Example 3-3: Synthesis of (2R,3R,3aR,5R,7aR,9R,10R,10aR,12R,14aR)-2-(6-amino-9H-purin- 9-yl)-3,10-difluoro-9-(6-(hydroxyamino)-9H-purin-9-yl)-5,12-dimercaptooctahydro- 2H,7H-difuro[3,2-d:3',2'-j][1 ,3,7,9]tetraoxa[2,8]diphosphacyciododecine 5,12- dioxide (CDN-9) and (2R,3R,3aR,5S,7aR,9R,10R,10aR,12R,14aR)-2-(6-amino- 9H-purin-9-yl)-3,10-difluoro-9-(6-(hydroxyamino)-9H-purin-9-yi)-5,12- dimercaptooctahydro-2H,7H-difuroi3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyciododecine 5,12-dioxide (CDN-10)
Figure imgf000497_0002
Compounds CDN-9 and CDN-10 were synthesized using a synthetic route simiiar to the synthesis of compounds CDN-5 and CDN-6, however hydroxyiamine hydrate was used in place of hydrazine monohydrate
(2R 3RI3aRI5R,7aR,9R,10R,10aR,12R,14aR)-2-(6-amino-9H-purin-9-yl)-3 10-difluoro-9-(6- (hydroxyamino)-9H-purin-9-yl)-5,12-dimercaptooctahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine 5,12-dioxide (CDN-9): 1H NMR (500 MHz, Deuterium Oxide) d 8 34 (s, 1 H), 8.18 (s, 1 H), 8.13 (d, J = 2.1 Hz, 1 H), 7 92 (s, 1 H), 6.40 (dd, J = 27.6, 17.0 Hz, 2H), 5.59 (d, J = 51 .5 Hz, 2H), 5.17 (d, J = 23.2 Hz, 2H), 4.65 - 4.52 (m, 4H), 4.Q9 (dd, J = 1 1 .0, 5.5 Hz, 2H). 19F NMR (471 MHz, Deuterium Oxide) d -200.53, -2Q1 .13. 31P NMR (202 MHz, Deuterium Oxide) d 54.74, 54.80.
(2R,3R,3aR,5S,7aR,9R,10R,10aR,12R,14aR)-2-(6-amino-9H-purin-9-yl)-3,10-difluoro-9-(6-
(hydroxyamino)-9H-purin-9-yl)-5,12-dimercaptooctahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]teiraoxa[2,8]diphosphacyclododecine 5,12-dioxide (GDN-10): 1 H NMR (50Q MHz, Deuterium Oxide) d 8.37 (s, 2H), 8.22 (s, 1 H), 8.02 (d, J = 10.6 Hz, 1 H), 6.52 - 6.32 (m, 2H), 5.93 (dd, J = 51 .1 , 4.0 Hz, 1 H), 5.56 (dd, J = 51 .8, 4.0 Hz, 1 H), 5.26 - 4.99 (m, 2H), 4.66 - 4.43 (m, 4H), 4.13 (ddd, J = 27.1 , 11.8, 4.8 Hz, 2H). 19F NMR (471 MHz, Deuterium Oxide) d - 201 .14, -201 .82. 31P NMR (202 MHz, Deuterium Oxide) d 54.66, 54.57.
Example 3-4: Synthesis of CDN-D: (Diastereomers {CDN~13), (CDN-14), (CDN-15) and (CDN-
18))
Figure imgf000498_0001
Compounds CDN-13, CDN-14, CDN-15 and GDN-16 (Diastereomers CDN-13, CDN-14, CDN- 15 and CDN-18 (bis-iriethylammonium salts) were synthesized using a route simiia to the synthesis of compounds CDN-1 and CDN-2, however 3-OTBS-BB was used in place of 2- OTBS-BB. Specific stereochemistry was not assigned to the compounds.
CDN-13: ¾H NMR (500 MHz, Deuterium Oxide) 0 8.67 (s, 1 H), 8.23 (s, 1 H), 8.19 (s, 1 H), 8.1 1 (s, 1 H), 6.37 (d, J = 15.5 Hz, 1 H), 6.20 (d, J = 8.1 Hz, 1 H), 5.85 (d, J = 50.9 Hz, 1 H), 5.50 - 5.43 (m, 1 H), 5.31 - 5.22 (m, 1 H), 4.62 (d, J = 4.0 Hz, 1 H), 4.56 - 4.52 (m, 1 H), 4.50 - 4.40 (m, 2H), 4.28 - 4.22 (m, 1 H), 4.19 - 4.07 (m, 2H). 19F NMR (471 MHz, Deuterium Oxide) d -75.55, - 202.45. 31P NMR (202 MHz, Deuterium Oxide) d 55.77, 54.60. CDI4-14: NMR (500 MHz, Deuterium Oxide) 08.71 (s, 1H), 8.32 (s, 1H), 8.19 (s, 1H), 8.08
(s, 1 H), 6.43 (dd, J= 16.1, 7.7 Hz, 1H), 6.36-6.22 (m, 1H), 5.97 (d, J= 50.9 Hz, 1H), 5.46 (d, J = 3.3 Hz, 1 H), 5.02 (d, J = 4.0 Hz, 1 H), 4.68 - 4.57 (m, 2H), 4.52 (dd, J = 21.5, 11.0 Hz, 2H), 4.41 - 4.14 (m, 3H).19F NMR (471 MHz, Deuterium Oxide) d -202.02. 31P NMR (202 MHz, Deuterium Oxide) d 53.85, 51.96.
GD -15: ¾H NMR (500 MHz, Deuterium Oxide) 08.71 (s, 1H), 8.32 (s, 1H), 8.19 (s, 1H), 8.08 (s, 1 H), 6.43 (d, J = 15.6 Hz, 1 H), 6.25 (d, J = 8.4 Hz, 1 H), 5.97 (d ,J = 50.4 Hz, 1 H), 5.45 (d, J = 3.4 Hz, 1 H), 5.16 (d, J = 20.1 Hz, 1H), 5.02 (d, J= 3.9 Hz, 1H), 4.64 (d, 2 = 8.3 Hz, 1H), 4.52 (d, J = 18.5 Hz, 2H), 4.37 (d, J= 16.3 Hz, 1H), 4.28 (d, J= 14.9 Hz, 2H). 19F NMR (471 MHz, Deuterium Oxide) d -202.01. 31P NMR (202 MHz, Deuterium Oxide) d 53.85, 51.96.
GD -16: ¾H NMR (500 MHz, Deuterium Oxide) 08.46 (s, 1H), 8.31 (s, 1H), 8.12 (s, 1H), 8.05 (s, 1 H), 6.44 (d, J= 16.0 Hz, 1H), 6.26 (d, J= 8.3 Hz, 1H), 5.66 (dd, J = 51.1 , 3.8 Hz, 1H), 5.52 (id, J = 8.5, 4.0 Hz, 1 H), 5.37 - 5.26 (m, 1 H), 4.94 (d, J = 4.1 Hz, 1 H), 4.62 (d, J = 7.7 Hz, 1 H), 4.54 (s, 1 H), 4.51 -4.43 (m, 2H), 4.35 (dd, J= 10.8, 4.7 Hz, 1H), 4.14 (dd, J= 11.5, 4.0 Hz,
1 H). 19F NMR (471 MHz, Deuterium Oxide) d -200.78. 31P NMR (202 MHz, Deuterium Oxide) d 54.89, 52.11.
Example 3-5: Synthesis of (GDN-E): (Diasiereomers (CDN-17), (CDN-18), (CDN-19) and
(CDN-20))
Figure imgf000499_0002
Compounds CDN-17, CDN-18, CDN-19 and CDN-20 (Diasiereomers CDN-17, CDN-18, CDN- 19 and CDN-2Q were synthesized using a route simiiarto the synthesis of compounds CDN-1 and C D-2, however 3-OTBS-BB was used in place of 2-OTBS-BB and commercial
(2R,3R,4R,5R)-2-(8-benzamido-9H-purin-9-yl)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yi (2- cyanoethyl) diisopropylphosphoramidite was used in place of phosphoroamidite (3).
CDN-17:
Figure imgf000499_0001
Deuterium Oxide) 6
8.51 (s, 1H), 8.38 (s, 1H), 8.16 (s, 1H), 8.05 (s, 1H), 6.26 (d. J = 8.4 Hz, 1H), 6.17 (d, J= 1.6 Hz, 1 H), 5.48 (td, J = 8 7, 4.1 Hz, 1 H), 5.19 (td, J = 8.1 , 4.6 Hz, 1 H), 4.97 (d, J = 4.0 Hz, 1 H), 4.90 (d, J = 2.9 Hz, 1 H), 4.59 (s, 1 H), 4.54 (s, 1 H), 4.42 (id, J = 7.1 , 6.4, 3.2 Hz, 2H), 4.35 (dd, J = 10.2, 5.0 Hz, 1 H), 4.14 (dd, J = 1 1 .7, 4.3 Hz, 1 H). 31P SMMR (202 MHz, Deuterium Oxide) d 54.87, 52.41 .
Example 4: Synthesis of Exemplary Compounds
Figure imgf000500_0001
Example 4-1 : Synthesis of 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl 2-(9~ ((2R,3R,3aS,5R,7aR,9R,10R,10aR,12R,14aR)-9-(6-amino-9H-purin-9-yl)-10- fluoro-3-hydroxy-5, 12-dime reapto-5,12-dioxidooctahydro-2H, 7H-difuro[3,2-d :3', 2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecin-2-yl)-9H-purin-8-yl)hydrazine-1 -
Figure imgf000500_0002
To a solution of Cyclic Dinuc!eotide (CDM-1 ) Et3N salt (5 mg, 0.0055 mmol) in DMF (1 ml ) was added MP-vc-pabc-PNP (linker-intermediate (LI-1 )) (3.8 mg, 0.0055 mmol), DIEA (3.6 mg,
0.Q27 mmol) and HOAT (0.8 mg, 0.0055 mmol). The reaction was stirred at room temperature for 18 hrs and then concentrated. The residue was purified by reverse phase ISCO using 5Qg C18 column, eluted with 5-35% acetonitrile-H20 (aqueous phase containing 10 mM Et3N HOAc). Fractions containing the desired product were concentrated to obtain 4-((S)-2-((S)-2-(3- (2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)-3-methylbutanamido)-5- ureidopentanamido)benzyl 2-(9-((2R,3R,3aS,5R,7aR,9R,10R,10aR,12R,14aR)-9-(8-amino-9H- purin~9-yi)-10-fiuoro-3~hydroxy~5,12~dimercapto-5,12-dioxidoociahydro~2H,7H-diiuro[3,2-d:3’,2·- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecin-2-yl)-9H-purin-6-yl)hydrazine-1 -carboxylate (C1 ) as Et3N salt. LCMS M+1 =1284.1 , tr= 0.670 min.
Example 4-2: Synthesis of 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl 2-(9- ((2R,3R,3aS,5S,7aR,9R,1 GR,1 QaR,12R,14aR)-9-(6-amino-9H-purin-9-yi)-1 G~ fluoro-3-hydroxy-5,12-dimercapto-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3\2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecin-2-yl)-9H-purin-6-yl)hydrazine-1 - carboxylaie (C2)
Figure imgf000500_0003
Compound (C2) was synthesized using using a route similar to the synthesis of Compound (C1), except Cyclic Dinucleotide Compound (CDISI-2) was used in place of Cyclic Dinucleotide Compound (COM-1 ) LCMS M/2+1 =632.7, tr= 0.732 min
Example 4-3: Synthesis of 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yi)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzy! 2-(9- ((2R,3R,3aR,5R,7aR,9R,1 GR,1 GaR,12R,14aR)-9-(6-amino-9H-purin-9-y!)-3,10- difluoro-5,12-dimercapto-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]dipbosphacyclododecin-2-yl)-9H-purin-6-yl)hydrazine-1- carboxylate (C3)
Figure imgf000501_0001
Compound (03) was synthesized using using a route similar to the synthesis of Compound (C1), except Cyclic Dinucleotide Compound (CDM-5) was used in place of Cyclic Dinucleotide Compound {CDM-1 ). LCMS M+1 =1266.1 , tr= 0.818 min.
Example 4-4: Synthesis of 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrroi-1 - yi)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzy! 2-(9- ((2R,3R,3aR,5R,7aR,9R,10R,10aR,12S,14aR)-9-(6-amino-9H-purin-9-yl)-3,10- difluoro-5,12-dimercapto-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecin-2-yl)-9H-pifrin-6-yl)hydrazine-1- carboxylate (C4)
Figure imgf000501_0002
Compound (C4) was synthesized using using a route similar to the synthesis of Compound (C1 ), except Cyclic Dinucleotide Compound fCD!M-S) was used in place of Cyclic Dinucleotide Compound (CDM-1 ). LCMS M+1 =1266.1 , tr= 0 619 in.
Example 4~S: Synthesis of Compound (CS)
Figure imgf000502_0001
Compound (CS) was synthesized using using a route similar to the synthesis of Compound (G1), except Cyclic Dinucleotide Compound (CDIM-17) was used in place of Cyclic Dinucleotide
Compound (CDN-1 ). LCMS M+1 =1262.1 , tr= 0.664 min.
Figure imgf000502_0002
A) Preparation of ar¾li-HER2 antibody with specific Cysteine mutations
Figure imgf000502_0003
Preparation of anti-HER2 antibodies, e.g., trastuzumab, and other antibodies with site-specific cysteine mutations has been described previously in WO 2014/124316 and WO 2015/138615, each of which was incorporated by reference herein. Briefly, DMA encoding variable regions of the heavy and light chains of an anti-HER2 antibody, e.g., trastuzumab, were chemically synthesized and cloned into two mammalian expression vectors that contain constant regions of human !gG1 heavy chain and human kappa light chain. The heavy chain vector encodes the constant region of the human IgGi antibody, includes a signal peptide (MPLLLLLPLLWAGALA) (SEG ID NO: 28), a CMV promoter to drive expression of the heavy chain, and appropriate signal and selection sequences for stable transfection into CHO ceils. The light chain vector encodes the constant region of the human kappa light chain, includes a signal peptide (MSVLTGVLALLLLWLTGTRC)
(SEG ID NO: 29), a CMV promoter to drive expression of the light chain, and appropriate signal and selection sequences for stable transfection into CHO cells. In some examples, the constant regions encoded in the vectors have been modified by site-directed
mutagenesis to introduce non-native cysteines at specific sites.
For example, cysteines were introduced at one or more of the following positions (all positions by EU numbering) in an anti~HER2 antibody: (a) positions 152, 360 and/or 375 of the antibody heavy chain, and (b) positions 107, 159, and/or 165 of the antibody light chain. For example, cysteines were introduced at positions 152 and 375 of the heavy chain resulting in anti-HER2 mAbi , which has a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 19. In some embodiments, cysteine was also introduced at position 152 of the heavy chain, resulting in anti-HER2 mAb4, which has a heavy chain sequence of SEQ ID NO: 30 and a light chain sequence of SEQ ID NO: 19. In some embodiments, cysteine was also introduced at position 159 of the light chain resulting in anti-HER2 mAb6, which has a heavy chain sequence of SEQ ID NO: 23 and a light chain sequence of SEQ ID NO: 34.
To produce antibodies, a heavy chain vector and a light chain vector were co-transfected into a CHO cell line. Cells underwent selection, and stably transfected cells were then cultured under conditions optimized for antibody production. Antibodies were purified from the ceil supernatants by standard Protein A affinity chromatography.
Alternatively, GHO stable lines can be produced by transfection of cells with a single bicistronic vector. In this case, the heavy chain and light chain were cloned into the same vector, each downstream of a CMV promoter and appropriate signal peptide coding sequence in this case, CHO ceils were transfected with the single vector and underwent selection, and stably transfected cells were then cultured under conditions optimized for antibody production. Antibodies were purified from the ceil supernatants by standard Protein A affinity
chromatography.
Alternatively, antibodies or Cys mutants of antibodies were expressed in 293
Freestyle™ cells by co-transfecting heavy chain and light chain plasmids using transient transfection methods as described previously (Meissner, ef a/., Biotechnol Bioeng. 75:197- 203 (2001)) The expressed antibodies were purified from the cel! supernatants by
standard Protein A affinity chromatography.
In some instances, antibodies may be further purified prior to conjugation. One example Is to apply the antibody to a size exclusion chromatography (SEC) column such as one with Superdex-200 resin (GE) and collect the peak corresponding to the antibody monomer.
Reduction, reoxidation and conjugation of Cvs mutant anti-HER2 antibodies to STING agonists
Some compounds described herein comprising a linker were conjugated to Cys residues engineered into an antibody similar to what is described in Junutula JR, et ai. , Nature
Biotechnology 28:925-932 (20Q8).
Because engineered Cys residues in antibodies expressed in mammalian ceils are modified by adducts (disulfides) such as glutathione (GSH) and/or cysteine during
biosynthesis (Chen et al. 2009), the modified Cys as initially expressed is unreactive to thiol reactive reagents such as aieimido or bromo-acetamide or iodo-acetamide groups.
To conjugate engineered Cys residues, glutathione or cysteine, adducts need to be
removed by reducing disulfides, which generally entails reducing all disulfides in the expressed antibody. This can be accomplished by first exposing antibody to a reducing agent such as dithiothreito! (DTT) followed by reoxidation of ail native disulfide bonds of the antibody to restore and/or stabilize the functional antibody structure. Accordingly, in order to reduce native disulfide bonds and disulfide bonds between the cysteine or GSH adducts of engineered Cys residue(s), freshly prepared DTT was added to previously purified Cys mutant antibodies to a final concentration of 10 mM or 20 mM. After antibody incubation with DTT at 37°C for 1 hour, mixtures were dialyzed against PBS for three days with daily buffer exchange to remove DTT. Alternatively, DTT can be removed by a gel filtration step. After removal of DTT, antibody solutions are allowed to reoxidize to reform native disulfide bonds. The reoxidation process was monitored by reverse-phase HPLC, which is able to separate antibody tetra er from individual heavy and light chain
molecules. Reactions were analyzed on a PRLP-S 4000A column (50 mm x 2.1 mm,
Agilent) heated to 80°C and column elution was carried out by a linear gradient of 30-60% acetonitrile in water containing 0.1 % TFA at a flow rate of 1.5 m!/min. The elution of proteins from the column was monitored at 280 nm. Incubation was allowed to continue until reoxidation was complete. After reoxidation, a maleimide-containing compound
(either (C1), (C2), (C3), (C4) or (C5)) was added to reoxidized antibodies in PBS buffer (pH 7.2) at molar ratios of typically 1 :1 , 1 5:1 , 2.5:1 , or 5:1 to engineered Cys, and
incubations were carried out for 5 to 60 minutes or longer Typically, excess free
compound was removed by purification over Protein A resin by standard methods followed by buffer exchange into PBS.
Cys mutant antibodies were alternatively reduced and reoxidized using an on-resin method. Protein A Sepharose beads (1 ml per 10 mg antibody) were equilibrated in PBS (no calcium or magnesium salts) and then added to an antibody sample in batch mode. A stock of 0.5 M cysteine was prepared by dissolving 850 mg of cysteine HGI in 10 ml of a solution prepared by adding 3.4 g of NaOH to 250 ml of 0.5 M sodium phosphate pH 8.0 and then 20 mM cysteine was added to the antibody/bead slurry, and mixed gently at room temperature for 30-60 minutes. Beads were loaded to a gravity column and washed with 50 bed volumes of PBS in less than 30 minutes, then the column was capped with beads resuspended in one bed volume of PBS. To modulate the rate of reoxidation, 50 nM to 1 mM copper chloride was optionally added. The reoxidation progress was monitored by removing a small test sample of the resin, eluting in IgG Elution buffer (Thermo), and analyzing by RP-HPLC as described above. Once reoxidation progressed to desired completeness, conjugation could be initiated immediately by addition of 1-5 molar equivalent of compound over engineered cysteines, and allowing the mixture to react for 5-1 Q minutes at room temperature before the column was washed with at least 20 column volumes of PBS. Antibody conjugates were eluted with IgG elution buffer and neutralized with 0.1 volumes Q.5 M sodium phosphate pH 8.Q and buffer exchanged to PBS. Alternatively, instead of initiating conjugation with antibody on the resin, the column was washed with at least 20 column volumes of PBS, and antibody was eluted with IgG elution buffer and neutralized with buffer pH 8.0. Antibodies were then either used for conjugation reactions or flash frozen for future use. For example, conjugation of compound (C4) is illustrated below:
Figure imgf000505_0001
Properties of the an÷.i-HER2-STlNG agonist conjugates
Antibody-STING agonist conjugates were analyzed to determine extent of
conjugation. A compound-to-antibody ratio was extrapolated from LC-MS data for reduced and deglycosylated samples. LC-MS allows quantitation of the average number of molecules of linker-payload (compound) attached to an antibody in a conjugate sample. HPLC separates antibody into light and heavy chains, and separates heavy chain (HC) and light chain (LC) according to the number of linker-payload groups per chain. Mass spectral data enables identification of the component species in the mixture, e.g., LC, LC+1 , LC÷2, HC, HC+1 , HC+2, etc. From the average loading on the LC and HC chains, the average compound to antibody ratio can be calculated for an antibody conjugate. A compound-to- antibody ratio for a given conjugate sample represents the average number of compound (linker-payload) molecules attached to a tetrameric antibody containing two light chains and two heavy chains.
Conjugates were profiled using analytical size-exclusion chromatography (AnSEC) on Zenix C-30Q 3 urn 7.8x150mm column (Sepax Technologies); aggregation was analyzed based on analytical size exclusion chromatography.
Pharmacokinetics of the conjugates were studied following injection of 1 or 4 mg/kg into CD1 mice. Serum samples were taken at various time points and stored frozen for analysis. The level of the human antibodies or antibody conjugates in serum samples was measured by immunoassays on a Gyros instrument. In both cases, capture was performed with an anti-human Fc reagent. Detection was performed with an anti-hlgG antibody to determine concentration of the antibody, and with a payload-specific antibody to determine levels of payload remaining on the conjugates. Payload retention was also studied by LC- MS. Briefly, samples were mixed 1 :1 with PBS pH 7.2, 5 mM EDTA, clarified by
centrifugation, and then loaded on IgG Select sepharose 6 fast flow resin (GE Healthcare) in 96-well plate format. Resin was washed with PBS and moved to a fresh plate to avoid background due to serum protein binding to the plates. Samples were then treated with
PNGaseF at 37 °C for 2 hours. Samples were washed with PBS again and then eluted in 1 % formic acid. Samples were reduced with 133 mM TCEP, Q.67 M ammonium acetate pH 5.Q for 30 minutes at room temperature. Samples were then measured by LC-MS and analyzed for compound-to-antibody ratio as described above.
Most conjugates achieved high compound-to-antibody ratio and were mainly monomeric. Conjugation through this method results in conjugation efficiencies of greater than 90% for most compounds (Table 19, below). The majority of the conjugates contain less than 10% dimeric and oligomeric material (Table 19).
B¾ Generation of aiiti-HER2-STS G agonist conjugates through partial reduction of native disuSfide bonds of nors-engineered anti-HER2 antibodies
Some compounds of the invention can also be conjugated to native cysteine residues of non-engineered antibodies using a procedure that involves partial reduction of the antibodies (Doronina, S. O. et al. , Nat Biotechnol. 21, 778-784, 2003).
Inter- and intra-chain disulfides bonds of anti-HER2 mAb3 (at a concentration of 10 mg/ml) can be first partially reduced in PBS pH 8.Q containing 2 mM EDTA by adding TCEP to a final concentration of 10 mM and incubating the mixture at 37°C for 1 hour. After desalting and addition of 1 % w/v PS-20 detergent, the partially reduced antibody samples (1 mg/ml) can be reacted overnight at 4°C with 5:1 or 10:1 molar ratio of compound (C1), (C2), (C3), (C4) or (C5). Resulting conjugates can be purified by Protein A chromatography by standard methods and buffer exchanged to PBS, and profiled by MS and AnSEC as described above.
D) Generation of arsti- agonist conjugates by conjugation to native lysine
Figure imgf000506_0001
Native antibodies can be functionalized with certain compounds of the invention through established methods. For example, anti-HER2 mAb3 with a heavy chain of SEQ ID NO: 23 and a light chain of SEQ ID NO: 19, in PBS pH 8.0 at 2 mg/mi can be mixed with 20-fold molar excess of a compound of the invention comprising an NHS ester, a penfafluorophenyl ester or a tetrafluorophenyl ester. The reaction can be incubated at room temperature overnight, and then buffer exchanged by standard methods to PBS pH 7.2. Table 19. Properties of anti-HER2-STMMG agonist conjugates
Figure imgf000507_0002
Example S: Anti-HER2-ST!NG agonist conjugates induce IP-10 secretion from HER2+ HCC1 S54 breast cancer cel!s in a target dependent manner.
HCC1954 cells were suspended into 384 well plates and allowed to attach overnight. Ceils were treated with anti-HER2-STING agonist conjugates or unconjugated payloads the next day and incubated for approximately 3 days. Geli culture supernatants were harvested and human !P-10 levels in the media were measured using a Human IP-10 Tissue Culture Kit from Mesoscale Devices. The EC5o values (triplicates) were calculated by performing logistic regression on measured dose-response curves. Data were curve fitted with the following formula to obtain ECso values:
Figure imgf000507_0001
where Y is the observed value, Bottom is the lowest observed value, Top is the highest observed value, and the Hill coefficient gives the largest absolute value of the slope of the curve. ECso value characterizes the concentration of the compound for a 50% activation, i.e., = (Top+Bottom)/2. The curve fitting is carried out by a curve fitting program using
Matlab.
The maximum activity was compared to 2’,3’-cGAMP (100 mM) treated ceils to determine %Efficacy. Values reported are the average of independent experiments when the conjugates or drugs were tested multiple times. Representative data are summarized in the Table 20.
Table 20. IP-10 secretion from HER2+ HCC1954 breast cancer cells
Figure imgf000508_0001
Example 6: In vivo testing of anti~HER2 mAb1~C4 irs 87 gastric tumor xenograft mode! Materials and Methods
For in vivo testing of the anii-Her2 mAb1-C4 conjugate in the N87 gastric carcinoma xenograft mouse model, female SCID-beige mice at 6-8 weeks of age (purchased from Harlan Laboratories) were used for implantation. N87 cells (obtained from ATCC, Catalog#CRL-5822, Vendor !ot#7686255) were grown in sterile conditions in a 37°C incubator with 5% C02 for two weeks. Ceils were grown in RPM! medium with 10% fetal bovine serum. Cells were passaged every 3-4 days with 0.05% Trypsin/EDTA. On the day of implantation, N87 cells were lifted (passage x17) and re-suspended in RPMI1640 serum-free media at a concentration of 5 x 10s cells and 50% matrigel/100 p!. Cells were Radii tested to assure that they are free of mycoplasma and murine viruses.
N87 cells were implanted with a subcutaneous injection into the lower flank using a 28 ½ G needle (5 x 1 G6 ceils /100 pi injection volume per mouse). After cell implantation, tumors were measured by caliper and mice were weighed three times per week once tu ors were palpable. Tumors were measured in two dimensions. Caliper measurements were calculated using (L x \IR){2. Mice were fed with normal diet and housed in a SPF animal facility in accordance with the Guide for Care and Use of Laboratory Animals and regulations of the Institutional Animal Care and Use Committee.
When xenograft tumors reached about 190 rnm3, mice were administered by intravenous route 1 g/kg or 5 g/kg of anti-HER2 mAb1-C4. Tumors continued to be measured three times a week. Average tumor volumes were plotted using Prism 5 (GraphPad) software. An endpoint for efficacy studies was achieved when tumor size reached a volume of 2000 mm3. Following injection, mice were also closely monitored for signs of clinical deterioration if for any reason mice showed any signs of morbidity, including respiratory distress, hunched posture, decreased activity, hind leg paralysis, tachypnea as a sign for pleural effusions, weight loss approaching 20% or 15% plus other signs, or if their ability to cany on normal activities (feeding, mobility), was impaired, mice were euthanized.
Results
N87 gastric tumor xenograft mice were treated intravenously with a single dose of anti- HER2 mAb1-C4 1 g/kg or 5 mg/kg (9 mice per group). Anti-HER2 mAb1 -C4 showed dose- dependent efficacy on N87 model in SCID mice inhibition of tumor growth was observed in mice treated with anti-HER2 mAb1-C4 at 1 g/kg or 5 mg/kg compared to the untreated mice (FIG. 1). All treatments were well tolerated except moderate (<-4%) but transient body weight loss was observed immediately after dosing mice with 3 mg/kg of Anti-HER2 mAb1 -C4 (FIG. 2).
If is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Claims

1 . An immunoconjugate of having a structure selected from the following:
Figure imgf000510_0001
Formula (AA-c) Formula (AA-d)
Figure imgf000510_0002
Formula (BB-c) Formula (BB-d)
Figure imgf000511_0001
Formula (CC-c) Formula (CC-d)
Figure imgf000511_0002
Formula (DD-c) Formula (DD-d)
Figure imgf000512_0001
Formula (EE-c) Formula (EE-d)
Figure imgf000512_0002
Formula (EE-e) Formula (FF-a)
Figure imgf000513_0001
Formula (FFd) Formula (FF-e)
Figure imgf000514_0001
Formula (FF-f) Formula (FF-g) wherein:
each Gi is independent^ selected from
Figure imgf000514_0002
the * of Gi indicates the point of attachment to -CR8RS-;
XA is C(=0)-, -C(=S)- or -~C(=NR11)- and each Zi is NR12;
XB is C, and each Z2 is N;
G2
Figure imgf000514_0003
, where the * of G2 indicates the point of attachment to --GRS3R9a-;
Xc is C(=0)-, -C(=S)- or C(=NR11)- and each Z3 is NR12:
XD is Cs and each Z4 is N;
Yi is -0-, -NH-, -S-, -S(=0)-, -SO2-, -CHr, or -CF2-;
Y2 is -0-, -NH-, -S-, -S(=0)-, -SOr, -CHr, or -CF2-;
Y3 is OH, O , OR10, N(R1C)2, SR10, SeH, Se , BH3, SH or S ;
Y,. is OH, O , OR10, N(R1C)2, SR10, SeH, Se , BH3 SH or S ;
Ys is -CH2", -NH-, -O- or -S;
Ys is -CH2", -NH-, -O- or -S;
Y-/ is O or S;
Ys is O or S; Yg is -GH2-, -NH-, -0- or -S;
Yio is -CH2-, -NH-, -O- or -S;
Yu is -O-, -NH-, -S-, -S(=0)-, -SOr, -CH2-, or -CF2-;
q is 1 , 2 or 3:
each R1 is independently a partially saturated or aromatic monocyclic heterocyclyl or
partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1 is substituted with Q, 1 , 2, 3 or 4 substituents independently selected from ~NH2, ~NHNH2, -NHOH, F, Cl, Br, Ci-C3alkyl and a Ci-C6alkyi substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3, or wherein when Ab is attached to R1 then R1 is substituted with -NHLiR115, -NHNHLiR1 15, -NHOLiR115- or -NHN=CR12iCH2),iL,R1 15- and Ab is attached to the R115 moiety;
each R1a is independently a partially saturated or aromatic monocyclic heterocyclyl or
partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -NHGH, F, Cl, Br, Ci-Csalkyl and a Ci-G6alkyi substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3, or wherein when Ab is attached to R1a then R1a is substituted with -NHLiR115, -NHNHLiR115, -NHOL1R115- or - NHN=CR (CH2)r.LiR115- and Ab is attached to the R115 moiety;
each R1b is independently a partially saturated or aromatic monocyclic heterocyclyl or
partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R,b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -NHOH, F, Cl, Br, CrC6a!kyi and a GrGsalkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3, or wherein when Ab is attached to R1b then R1b is substituted with -NHLiR115, -NHNHLiR115, -NHOL1R115- or - attached to the R115 moiety;
Figure imgf000515_0001
each R2 is independently selected from H and Ci-C6alkyl;
each R3 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-G6a!kyi substituted with a NHOH, NHNH2 or NH2; or when Ab is attached to R3, then R3 is -NHL1R115, -NHNHLiR115, -NHOL1R115- or -NHN=CR12(CH2)nLiR115- and Ab is attached to the R115 moiety; each R4 is independently selected from H, CrC6alkyl and Ci-C6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R5 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-Geaikyi substituted with a NHOH, NHNH2 or NH2; or when Ab is attached to R5, then R5 is -NHL1R115, -NHNHL1R115, -NHOL1R115- or -NHN=CR12(CH2)nLiR1 15- and Ab is attached to the R115 moiety;
each R6 is independently selected from H, CrC6alkyl and C i-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7 is independently selected from H, CrC6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R8 is independently selected from H, CrC6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R9 is independently selected from H, CrC6alkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R2a is independently selected from H and CrC6aikyl;
each R3a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Ci-CBalkyi substituted with a NHOH, NHNH2 or NH2: or when Ab is attached to R3a, then R3a is -NHL1R115, -NHNHL1R115, -NHOL1R115- or -NHN=CR12(CH2)nLiR115- and Ab is attached to the R115 moiety;
each R4a is independently selected from H, Ci-C5alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R5a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a CrC6alkyi substituted with a NHOH, NHNH2 or NH2; or when Ab is attached to R5a, then R5a is -NHL1R115, -NHNHL1R115, -NHOL1R115- or -NHN=CR (CH2)nLiR115- and Ab is attached to the R115 moiety;
each R6a is independently selected from H, C -C3alkyi and CrCgalkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7a is independently selected from H, C -C3alkyi and CrCgalkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each RSa is independently selected from H, Ci-C3alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R9a is independently selected from H, Ci-C6alkyi and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3; eac yl, Ci
Figure imgf000517_0001
wherein the Cr
Cl2alkyl and Ci-Csheteroalkyi of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, Ci-G |2aikoxy, -S-C(=G)Ci-G6alkyi, halo, -CN, Gr
Ci2aikyl, -O-aryl, -Q-heteroaryl, -O-cycloalkyi, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, -OC(Q)QCrCsaikyland C(0)0Ci-C3alkyl, wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from C -C alkyl, 0-CrCi2alkyl, Ci~Ci2heieroaikyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heferoaryl, -C(=0)Ci-Ci2alkyl, -0C(=0)Ci-Ci2alkyl, -C(=0)0C Ci2alkyi, -0C(=0)0Ci-Ci2aikyl, -C(=0)N(R, 1)-Ci-Ci2alkyl, ~N(R11)C(=G)~C Ci2aikyl; - OC(=0)N(Rn)-CrCi2aikyl, -C(=0)-aryi, -C(=G)-heteroaryl, -OC(=G)-aryl, -C(=0)0-aryl, - GC(=0)-heteroaryl, -C(=0)0-heteroaryl, -C(=0)0-aryl, -C(=Q)G-heteroaryl, - C(=0)N(Ri 1)-aryl, -C(=Q)N(Ri 1)-heteroaryl, -IM(R11)C(G)-aryl, -N(R11)2C(G)-aryi, - N(R11)C(Q)-heteroaryl, and S(0)2N(R11)-aryl;
each R11 is independently selected from H and CrC6aikyl;
each R12 is independently selected from H and CrC6aikyl;
optionally R3 and R6 are connected to form -Q-G i-C6aikylene, such that when R3 and Rs are connected, the O is bound at the R3 position
optionally R3a and R6a, are connected to form -0-Ci-C6alkylene, such that when R3a and RSa are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form -0-CrC6alkyiene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form -G~CrC6a!kylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form ~0-CrC6aikylene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -G-CrCsa!kylene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form -0-CrC6aikylene, such that when R5 and R6 are connected, the O is bound at the R5 position;
optionally R5a and R6a, are connected to form -0-Ci-G6alkylene, such that when R5a and RSa are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form -Q-CrC6aikylene, such that when R5 and R7 are connected, the O is bound at the R5 position; optionally R5a and R7a, are connected to form -0-CrCBalkyiene, such that when R5a and R7a are connected, the O is bound at the R5a position:
Li is a linker;
each R115 is independently
Figure imgf000518_0001
Figure imgf000518_0002
Figure imgf000519_0001
of R1 indicates the point of attachment to Ab;
R13 is H or methyl;
R14 is H, -CHs or phenyl;
R110 is independently selected from H, Gi-C6aikyl, F, Cl, and -OH;
R111 is independently selected from H, Ci-C6alkyl, F, Cl, -NH2, -OCHs, -OCH2CH3,
N(CH3)2, -CN, -NO2 and -OH;
each R112 is independently selected from H, Ci-Salkyl, fluoro, benzyloxy substituted with - C(=0)0H, benzyl substituted with -C(=0)0H, G^alkoxy substituted with -~C(=Q)OH and Ci-4alkyl substituted with -C(=0)0H ;
each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 and 1 8;
Ab is an antibody or fragment thereof; and
y is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
2. The immunoconjugate of claim 1 , having a structure selected from the following :
Figure imgf000519_0002
Formula (AA-3a) Formula (AA-3b)
Figure imgf000520_0001
Formula (CC-3c) Formula (CC-3d)
Figure imgf000521_0001
Figure imgf000521_0002
Formula (EE-3a) Formula (EE-3b)
Figure imgf000521_0003
Formula (EE-3c) Formula (EE-3d)
Figure imgf000522_0001
Formula (FF-3b) Formula (FF-3c)
Figure imgf000523_0001
Formula (FF-3f) Formula (FF-3g)
wherein: Ab, y, R1 , R1a, R2, R2a, R3, R3a, R4, R4a, R5, R5a, R6, R6a, R7, R7a, Yi , Y2> Y3, Y4, Y7, Ys and Yu are as defined in claim 1 .
3. The immunoconjugate of claim 1 or claim 2, having a structure selected from the following :
Figure imgf000523_0002
Formiila (AA-4a) Formula (AA-4a)
Figure imgf000524_0001
Formula (AA-4k) Formula (AA-41)
Figure imgf000525_0001
Formula (AA-5a) Formula (AA-5b)
Figure imgf000526_0002
Formula (AA-5e) Formula (AA-5f)
Figure imgf000526_0001
Formula (AA-5Ϊ) Formula (AA-5j)
Figure imgf000527_0001
Formula (AA-5q) Formula (AA-5r)
Figure imgf000528_0001
Formula (AA-5aa) Formula (AA-5bb)
Figure imgf000529_0001
Figure imgf000529_0002
Formula (BB-4a) Formula (BB-4b)
Figure imgf000529_0003
Formula (BB-4c) Formula (BB-4d)
Figure imgf000530_0001
Formula (BB-4g) Formula (BB-4h)
Figure imgf000530_0002
Formula (BB-4k) Formula (BB-4I)
Figure imgf000531_0001
Formula (BB-4o) Formula (BB-4p)
Figure imgf000531_0002
Formula (BB-4s) Formula (BB-4t)
Figure imgf000532_0001
Formula (BB-5c) Formula (BB-5d)
Figure imgf000532_0002
Formula (BB-5g) Formula (BB-5h)
Figure imgf000533_0001
Formula (CC-4e) Formula (CC-41)
Figure imgf000534_0001
Formula (CC-4o) Formula (CC~4p)
Figure imgf000535_0001
Formula (CC-5e) Formula (CC-5!)
Figure imgf000536_0001
Formula (DD-4c) Formula (DD-4d)
Figure imgf000537_0001
Formula (DD-4I) Formula (DD-4j)
Figure imgf000538_0001
Formula (DD-4o) Formula (DD-4p) wherein: Ab, y, R1 , R1a, R3, R3a, R5, R5a, R6, RSa, Y3 and Y4 are as defined in claim 1 .
4. The immunoconjugate of any one of claims 1 to 3, wherein
Figure imgf000538_0002
Figure imgf000539_0001
Figure imgf000540_0001
Figure imgf000541_0001
Figure imgf000542_0001
Figure imgf000543_0001
Figure imgf000544_0001
, , , stituents independently selected from F, Ci, Br, NHQH, NH2, CrCsalky! and Ci-C6alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N3, and each R200 is independently selected from H and LiR115;
R1a and R1b are each independently
Figure imgf000544_0002
Figure imgf000544_0003
Figure imgf000545_0001
Figure imgf000546_0001
Figure imgf000547_0001
Figure imgf000548_0001
Figure imgf000549_0001
Figure imgf000550_0001
, wherein: R1 a is substituted with Q, 1 , 2 or 3 substituents independently selected from F, Cl, Br, NHQH, NH2, Ci-C3alkyl and Cr Csalkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, i, OH, CN, and Na; and each R21C is independently selected from H and LiR115, and n, L-, and R115 are as defined in claim 1
5. The immunoconjugate of any one of claims 1 to 4, wherein
Figure imgf000550_0002
115;
Figure imgf000550_0003
, ,
and n. Li and R115 are as defined in claim 1 .
6. The immunoconjugate of any one of claims 1 to 5, wherein the Ab specifically binds a target antigen.
7. The immunoconjugate of any one of claims 1 to 6, wherein the Ab is human or humanized.
8. The immunoconjugate of any one of claims 1 to 7, wherein the Ab is a monoclonal antibody.
9. The immunoconjugate of any one of claims 1 to 8, wherein the Ab comprises a modified Fc region.
10. The immunconjugate of of any one of claims 1 to 9, wherein the Ab comprises cysteine at one or more of the following positions, which are numbered according to EU numbering:
(a) positions 152, 360 and 375 of the antibody heavy chain, and
(b) positions 107, 159, and 165 of the antibody light chain.
1 1. The immunoconjugate of any one of claims 1 to 10, wherein the LiR115 group Is attached to the Ab via conjugation to one or more modified cysteine residues in the Ab.
12. The immunoconjugate of claim 10, wherein the UR115 group is conjugated to the Ab via modified cysteine residues at positions 152 and 375 of the heavy chain of the Ab, wherein the positions are determined according to EU numbering.
13. The immunoconjugate of any one of claims 1 to 12, wherein the target antigen is a tumor antigen.
14. The immunoconjugate of any one of claims 1 to 13, wherein the immunoconjugate is
parenteraily administered.
15. The immunoconjugate of any one of claims 1 to 14, wherein the immunoconjugate has in vivo anti-tumor activity.
16. The immunoconjugate of any one of claims 1 to 15 comprising a structure selected from:
Figure imgf000551_0001
Figure imgf000552_0001
17. A pharmaceutical composition comprising the immunconjugate of any one of claims 1 to 16 and a pharmaceutically acceptable excipient.
18. A composition comprising the immunoconjugate of any one of claims 1 to 16 in combination and one or more additional therapeutic agents.
19. The composition of claim 18, wherein the additional therapeutic agent is selected from the group consisting of an inhibitor of a co-inhibitory molecule, an activator of a co-stimulatory molecule, a cytokine, an agent that reduces cytokine release syndrome (CRS), a chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a vaccine, or a ceil therapy.
20. The composition of claim 18, wherein the additional therapeutic agent is an inhibitor of a co- inhibitory molecule, an activator of a co-stimulatory molecule, or a cytokine, wherein: (i) the co-inhibiiory molecule is selected from Programmed death-1 (PD-1), Programmed death-ligand 1 (PD-L1), Lymphocyte activation gene-3 (LAG-3), or T-celi immunoglobulin domain and mucin domain 3 (TIM-3),
(ii) the co-stimulatory molecule is Glucocorticoid-induced TNFR-re!ated protein (GITR), and
(iii) the cytokine is IL-15 compiexed with a soluble form of IL-15 receptor alpha (IL- 15Ra).
21. A method of treating cancer comprising administering to a patient in need thereof a
therapeutically effective amount of the immunconjugate of any one of claims 1 to 18, the pharmaceutical composition of claim 17, or the composition of any one of claims 18 to 20.
22. Use of the immunconjugate of any one of claims 1 to 16, the pharmaceutical composition of claim 17, or the composition of any one of claims 18 to 20 for treatment of a cancer in a subject in need thereof.
23. The immunconjugate of of any one of claims 1 to 16, the pharmaceutical composition of claim 17, or the composition of any one of claims 18 to 20, for use in the treatment of cancer.
24. Use of the immunconjugate of any one of claims 1 to 16, the pharmaceutical composition of claim 17, or the composition of any one of claims 18 to 20 in the manufacture of a medicament for treatment of a cancer in a subject in need thereof.
25. The method of claim 21 , the use of claim 22 or 24, or the immunoconjugate of claim 23, wherein the cancer is selected from sarcomas, adenocarcinomas, blastemas, carcinomas, liver cancer, lung cancer, non-small ceil lung cancer, small ceil lung cancer, breast cancer, lymphoid cancer, colon cancer, renal cancer, urothelial cancer, prostate cancer, cancer of the pharynx, rectal cancer, renal cell carcinoma, cancer of the small intestine, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, colorectal cancer, cancer of the anal region, cancer of the peritoneum, stomach or gastric cancer, esophageal cancer, salivary gland carcinoma, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, penile carcinoma, glioblastoma, neuroblastoma, cervical cancer , Hodgkin lymphoma , non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi’s sarcoma, neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet ceil cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, epidermoid cancer, squamous ceil cancer, T-ceil lymphoma, environmentally induced cancers including those induced by asbestos, leukemia, lymphoma, acute myelogenous leukemia (AML), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphoid leukemia (CLL), myelodysplastic syndromes, B-celi acute lymphoid leukemia (“BALL”), T-cel! acute lymphoid leukemia (“TALL”), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, Follicular lymphoma, Hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant iymphoproiiferative conditions,
MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, p!asmabiastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.
26. The method of claim 21 , the use of claim 20 or 22, or the immunoconjugate of claim 21 , wherein the immunoconjugate is administered to the subject intravenously, intratumora!!y, or subcutaneously.
27. The immunconjugate of any one of claims 1 to 16, the pharmaceutical composition of claim 17, or the composition of any one of claims 18 to 20, for use as a medicament.
28. A compound having a structure selected from Formula (A), Formula (B), Formula (C),
Formula (D), Formula (E), or Formula (F) or stereoisomers or pharmaceutically acceptable salts thereof,
Figure imgf000554_0001
Formula (A) Formula (i
Figure imgf000555_0001
Formula (E) Formula (F) wherein:
each Gi Is Independently selected from
Figure imgf000555_0002
the * of Gi Indicates the point of attachment to -CR8RS-;
XA Is C(=0)-, -C(=S)- or -C(=NR11)- and each
Figure imgf000555_0003
XB Is C, and each Z2 is N;
Figure imgf000555_0004
Xc is G(=0)-, -C(=S)- or -C(=NR11)- and each Z3 is NR12;
XD is C, and each Z& is N;
Yi is -0-, -NH-, -S-, -S(=0)-, -SO2-, -CHr, or -CF2-; Y2 is -O, -NH-, -S-, -S(=0)-, -SO2-, -CHr, or -CF2-;
Figure imgf000556_0001
Y6 is -CH2-, -NH-, -G- or S;
Y7 is O or S;
Y8 is O or S;
Y9 is -CHz-. -NH-, -O- or -S;
Y10 is -CH2-, -NH-, -O- or -S;
Y11 is -O-, -NH-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2~;
q is 1 , 2 or 3;
R1 is a partially saturated or aromatic monocyclic heterocyclyl or partially saturated or aromatic fused bicyciic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatorns, and each heteroatorns is independently selected from O, N or S, or a tautomer thereof, wherein R1 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHLR15, -NHNHL,R15, -NHOLiR15, -NHN=CR12(CH2)nL,R15, NH2 -NHNH2, - NHOH, F, Cl, Br, CrC6alkyl and CrCBalkyi substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N3;
R18 is a partially saturated or aromatic monocyclic heterocyclyl or partially saturated or aromatic fused bicyciic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatorns, and each heteroatorns is independently selected from O, N or S, or a tautomer thereof, wherein R18 is substituted with Q, 1 , 2, 3 or 4 substituents independently selected from -NHL R15, -NHNHL1R15, -NHOLiR15, -NHN=CR12(CH2),iLiR15, ~NH2, -NHNH2, - NHOH, F, Cl, Br, CrC6alkyl and Ci-C6alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N3;
R1b is a partially saturated or aromatic monocyclic heterocyclyl or partially saturated or aromatic fused bicyciic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatorns, and each heteroatorns is independently selected from O, N or S, or a tautomer thereof, wherein R1b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHL1R15, -NHNHL1R15, -NHOL,R15, -NHN=CR12(CH2)nLiR15, -NH2, -NHNH2, - NHOH, F, Cl, Br, CrC6aikyl and CrCsaikyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R2 is independently selected from H and Ci-C6alkyi;
each R3 is independently selected from -NHL1R15, -NHNHL1R15, -NHOL1R15, -
NHN=CR12(CH2)r.LiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, -NHNH2, NH2 and a CrC6alkyi substituted with a NHOH, NHNH2 or NH2; each R4 is independently selected from H, CrC6alkyl and Ci-C6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R5 is independently selected from -NHL,R15, -NHNHLR15, -NHOLiR15, -NHN=CR12(CH2)nLiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, -NHNH2 NH2 and a Ci-C3aikyi substituted with a NHQH, NHNH2 or NH2;
each R6 is independently selected from H, CrCsalky! and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, !, OH, CN, and N3;
each R7 is independently selected from H, Ci-C6alkyl and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R8 is independently selected from H, Ci-C6alkyl and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each Rs is independently selected from H, Ci-C6alkyl and CrC6aikyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R2a is independently selected from H and CrC6a!kyi;
R3a is independently selected from -NHL,R15, -NHNHL1R15, -NHGL,Ri5, -NHN=CR12(CH2)nL1R15, H, -OH, -SH, F, Cl, Br, i, NHOH, -NHNH2, NH2 and a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH2;
R4a is independently selected from H, CrCsalkyl and CrCsalkyl substituted by 1 , 2 or 3
substituents independently selected from F, Cl, Br, I, OH, GN, and N3;
R5a is independently selected from -NHL1R15, -NHNHL1R15, -NHOL1R15, -NHN=CR12(CH2)nLiR15, H, -OH, -SH, F, Cl, Br, I, NHOH, -NHNH2, NH2 and a Ci-C6alkyl substituted with a NHOH, NHNH2 or NH2;
R68 is independently selected from H, CrCsalky! and CrC6alkyi substituted by 1 , 2 or 3
substituents independently selected from F, Cl, Br, !, OH, CN, and N3;
R78 is independently selected from H, CrCsalky! and CrC6aikyl substituted by 1 , 2 or 3
substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R8a is independently selected from H, CrCsalky! and CrC6aikyl substituted by 1 , 2 or 3
substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R9a is independently selected from H, CrCsalky! and CrC6aikyl substituted by 1 , 2 or 3
substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R10 is independently selected from the group consisting of H, CrCsalkyl, Cr
Csheteroalkyl,
Figure imgf000557_0001
wherein the C
Ci2alkyi and Ci-C6heteroaikyi of R10 is substituted by Q, 1 , 2 or 3 substituents independently selected from -OH, Ci-Ci2alkoxy, -S-C(=0)Ci-C6alkyl, halo, -CN, CrCi2alkyl, -O-aryi, -O- heieroaryl, -O-cycloalkyl, oxo, cycioaiky!, heterocydyi, aryl, or heteroaryl, -0C(0)0Cr Csa!kyland C(G)OCi-C3alkyl, wherein each alkyl, cycloalkyi, heterocydyi, aryl, and heieroaryl Is substituted by 0,1 , 2 or 3 substituents independently selected from C1-C12 alkyl, 0-Ci-Ci2alkyl, Ci-C^heteroalkyl, halo, GN, OH, oxo, aryl, heieroaryl, O-aryl, O-heteroaryl, -
Figure imgf000558_0001
-aryl, -C(=:0)-heteroa!yl, -0G(=0)-aryi, -C(=:0)0-aryl -Oe(=Q)-heteroaryi, -C^G^-heteroaryi, - C(=0)0-aryi, -C(=0)0-heteroaryl, -C(=0)N(R1 1)-aryl, -C(=0)fM(R1 1)-heteroaryi, ~N(R11)C(Q)~ aryl, -N(R11)2C(0)-aryl, -M(R1 1)C(0)-heteroaryl, and S(0)2N(R11)-aryi;
R1 1 is independently selected from H and Ci-C6alkyi;
R12 is independently selected from H and Ci-C6alkyi;
optionally R3 and R6 are connected to form -G-Ci-C6aikylene, such that when R3 and R6 are connected, the O Is bound at the R3 position
optionally R3a and R6a, are connected to form -O-CrGsalkylene, such that when R3a and R6a are connected, the O is bound at the R3a position;
optionaliy R2 and R3 are connected to form -0-CrC6alkyiene, such that when R2 and R3 are connected, the O is bound at the R3 position:
optionally R2a and R3a, are connected to form -O-Ci-Cealkylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form -0-Ci-C6alkyiene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -0-Ci-C6alkylene, such that when R48 and R38 are connected, the O is bound at the R38 position;
optionally R5 and R6 are connected to form -0-Ci~C6alkylene, such that when R5 and R6 are connected, the O is bound at the R5 position;
optionally R5a and R6a, are connected to form -0-CrC6aikyiene, such that when R£,a and R6a are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form ~0-CrC6alkylene, such that when R5 and R7 are connected, the O is bound at the R5 position;
optionaliy R5a and R7a, are connected to form -0-CrCBalkylene, such that when R5a and R7a are connected, the O is bound at the R5a position;
Li is a linker;
Figure imgf000559_0001
Figure imgf000560_0001
each R110 is independently selected from H, CrC6alkyi, F, Cl, and -OH;
each R111 is independently selected from H, Ci-C6aikyl, F, Cl, -NH2, ~OCH3, -GCH2CH3, - N(CH3)2I -CN , -NO2 and -OH;
each Ri 12 is independently selected from H, Chalky!, fiuoro, benzyioxy substituted with - C(=0)0H, benzyl substituted with -C(=0)0H, Ci-4aikoxy substituted with -C(=0)0H and Ci- 4alkyl substituted with -C(=0)0H;
each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16,17 and 18,
and provided at least one of R1 , R1a or R1b is substituted with -NHL1R15, -NHNHL1R15, - least one of R3, R5, R33 or R53 is -NHL1R15, -
Figure imgf000560_0002
2)nLiR15.
29. The compound of claim 28, having the structure selected from Formula (A-3), Formula (B-3), Formula (C-3), Formula (D-3), Formula (E-3), or Formula (F-3) or stereoisomers or pharmaceutically acceptable salts thereof,
Figure imgf000560_0003
Formula (C-3) Formula (D-3)
Figure imgf000561_0001
Formula (E-3) Formula (F-3)
wherein: R1, Ria, R2, R2a, R3, R3a, R4, R4a, R5, R£'a, R6, RSa, R7, R7a, Yi, Y2, Y3, Y«, Y7, Ys and Yu are as defined in claim 28.
30. The compound of claim 28 or claim 29, wherein
Figure imgf000561_0002
Figure imgf000562_0001
Figure imgf000563_0001
Figure imgf000564_0001
Figure imgf000565_0001
Figure imgf000566_0001
wherein: R1 is substituted with 0, 1 , 2 or 3 substituents independently selected from F, Cl, Br, CrC6alkyi and Ci-C3alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CM, and N3, and each R20 is independently selected from H and UR15; R11 and R1D are independently
Figure imgf000567_0001
Figure imgf000567_0002
Figure imgf000568_0001
Figure imgf000569_0001
Figure imgf000570_0001
Figure imgf000571_0001
Figure imgf000572_0001
wherein: R18 is substituted with 0, 1 , 2 or 3 substituents independently selected from F, Cl, Br, NHOH, NH2, C -Cgalkyi and CrCgalkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3, and each R21 is independently selected from H and LiR15;
and n, Li and R15 are as defined in claim 28.
31. The compound of any one of claims 29 to 30, wherein
Figure imgf000572_0002
Figure imgf000573_0002
and n, Li and R15 are as defined in claim 28.
32. The immunoconjugate of any one of claims 1 to 18 or the compound of any one of claims 28 to 31 , wherein
Figure imgf000573_0001
Figure imgf000574_0001
Figure imgf000575_0001
Figure imgf000576_0001
C(=0)XiC(=0)(CH2)mNHC(=0)(CH2)m-, where the ** of indicates the point of attachment to R115 or R15;
each R12 is independently selected from H and Ci-C3alkyl;
Xi s
Figure imgf000576_0002
, where the * of Xi indicates the point of attachment to X2;
Figure imgf000576_0003
the * of X2 indicates the point of attachment to Xi , -NH-, -NHNH-, -NHO- or -NHN=GR12(CH2)n-
Figure imgf000577_0001
the ** of X5 indicates orientation toward R115 or R15 ;
each m is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10;
and
each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 and 18.
33. The immunoconjugate of any one of claims 1 to 16 or the compound of any one of claims 28 to 32, wherein
Figure imgf000577_0002
C(=0)((CH2)m0)„(CH2)mNHC(=0)X5((CH2)m0)„(CH2)m-**; - C(=0)((CH2)m0)n(CH2)mNHC(=0)X5((CH2)m0)„(CH2)mNHC(=0)(CH2)m-**; - C(=0)((CH2)mO)n(CH2)mNHC(=0)X5((CH2)mO)n(CH2)mNHC(=0)(CH2)mX3(CH2)m-**;
C(-0)((CH2)r!,0)n(CH2)mNHC(=0)X5((CH2)m0)n(CH2)mX3(CH2)m-*i
C(=0)((CH2)mO)n(CH2)mNHC(=0)X5(CH2)mNH((CH2)mO)n(CH2)nr**; - C(=0)((CH2)mO)n(CH2)mNHC(=0)X5C(=0)(CH2)mNH((CH2)mO)„(CH2)mX3(CH2)m-**; - C(=0)((CH2)mO)n(CH2)mNHC(=0)X5(CH2)m-**; or -C(=0)(CH2)mC(=0)NH(CH2)m-M, where the ** of Li indicates the point of attachment to R115 or R15 and where R12, Xi , X2, X3, Xs, m and n are as defined in claim 32.
34. The compound of any one of claims 28 to 33 selected from
Figure imgf000578_0001
and
Figure imgf000579_0001
35. A compound having a structure selected from Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or Formula (F) or a stereoisomer or pharmaceutically acceptable salt thereof,
Figure imgf000579_0002
Formula (C) Formula (D)
Figure imgf000580_0001
Formula (E) Formula (F) wherein:
eacb Gi is independently selected from
Figure imgf000580_0002
where the 4 of Gi indicates the point of attachment to -CR8R9-;
XA is C(=0)-, -C(=
XB is C, and each
Figure imgf000580_0003
Ys
Figure imgf000580_0004
Yi is -0-, -NH-, -S-, -S(=0)-, -SO2-, -CHr, or-CF2-;
Y2 is -0-, -NH-, -S-, -S(=0)-, -S02-, -CHr, or -CF2-;
Y3 is OH, O , OR10, N(R10)2I SR1C, SeH, Se, BH3, SH or S;
Y4 is OH, O, OR10, N(R10)2, SR1C, SeH, Se, BH3, SH or S;
Y5 is -CH2-, -NH-, -O- or-S;
Y6 is -CH2-, -NH-, -O- or-S;
Y7 is O or S;
Y8 is O or S;
Y9 is -CH2-, -NH-, -O- or-S; Yio is -CH2-, -NH-, -O- or -S:
Yu is -O-, -NH-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2-;
q is 1 , 2 or 3;
R1 is a partially saturated or aromatic monocyclic heierocyclyl or partially saturated or aromatic fused bicyclic heierocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1 is substituted with Q, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -fsSHOH, F, Cl, Br, CrC6alkyl and a Ci-C3a!ky! substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R18 is a partially saturated or aromatic monocyclic heierocyclyl or partially saturated or aromatic fused bicyclic heierocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -NHGH, F, Cl, Br, CrC6alkyl and a CrCsalkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R1 b is a partially saturated or aromatic monocyclic heterocyclyl or partially saturated or aromatic fused bicyclic heterocyclyl containing from 5-10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S, or a tautomer thereof, wherein R1 b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NH2, -NHNH2, -NHOH, F, Cl, Br, CrC6alky! and CrC6alkyl substituted with 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R2 is independently selected from H and CrC6a!kyi;
each R3 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a CrCsalkyl substituted with a NHOH, NHNH2 or NH2;
each R4 is independently selected from H, CrCsalkyl and CrCsalkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R5 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a CrCsalkyl substituted with a NHOH, NHNH2 or NH2;
each R6 is independently selected from H, Ci-C6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R7 is independently selected from H, Ci-C6alkyl and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R8 is independently selected from H, CrC6alky! and CrC6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3; each R9 is independently selected from H, CrC6alkyl and CrC6aikyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R23 is independently selected from H and CrCsaikyi
R3a is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a C1- Cgalkyi substituted with a NHOH, NHNH2 or NH2;
R43 is independently selected from H, Ci-C6alkyl and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Gi, Br, I, OH, CN, and N3;
R58 is independently selected from H, -OH, -SH, F, Cl, Br, I, NHOH, NHNH2, NH2 and a Cr Csaikyi substituted with a NHOH, NHNH2 or NH2;
R68 is independently selected from H, CrC6alky! and CrC6alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R7a is independently selected from H, Ci-C6aikyi and Ci-C6alkyl substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R8a is independently selected from H, CrC6alkyl and CrC6alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
R9a is independently selected from H, CrC6alkyl and CrC6alkyi substituted by 1 , 2 or 3 substituents independently selected from F, Cl, Br, I, OH, CN, and N3;
each R10 is independently selected from the group consisting of H, Ci-Ci2alkyl, C1-
Cgheteroalkyi, -(CH2CH20)nCH2CH2C(=0)0Ci-C6al , wherein the C
Ci2alkyi and Ci-C3heteroa!kyi of R1C is substituted b ents independently selected from -OH, Ci-Ci2alkoxy, -S-
Figure imgf000582_0001
o, -CN, Cr Ci2alkyi, -O-aryl, -O-heteroaryl, -O-cycloalkyl, oxo, cycloalkyl, heterocyciyi, aryl, or heteroaryl, -QC(0)OCi-C6a!kyiand C(0)0Ci-C6alkyl, wherein each alkyl, cycloalkyl, heterocyciyi, aryl, and heteroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from C1-C12 alkyl, 0-Ci-Ci2alkyl, Ci-Ci2heteroalkyl, halo, CM, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroaryl, -C(=0)Ci-Ci2alkyl, -0C(=0)CrCi2alkyl, -C(=0)0Ci- Cisa!kyi, -0C(=0)0Ci-Ci2alkyl, -C(=0)N(R11)-CrCi2alkyl, -N(R11)C(=0)-Ci-Ci2alkyl; - OC(=0)N(R11)-Ci-Ci2alkyl, -C(=0)-aryl, -C(=0)-heteroaryl, -0C(=0)-aryl, -C(=0)0-aryl, - QC(=G)-heteroaryi, -C(=0)0-heteroaryl, -C(=0)0-aryl, -C(=0)0-heteroaryl, - C(=0)N(R11)-aryl, -C(=0)N(R11)-heteroaryl, -N(R11)C(0)-aryl, -N(R11)2C(Q)-aryi, - N(R11)C(0)-heteroai l, and S(G)2N(R11)-aryl;
R11 is independently selected from H and CrC6aikyi;
R12 is independently selected from H and Ci-C6alkyi; optionally R3 and R6 are connected to form -Q-Ci-C6alky!ene, such that when R3 and R6 are connected, the O is bound at the R3 position
optionally R3a and R6a, are connected to form -0-CrCBalkyiene, such that when R3a and R6a are connected, the O is bound at the R3a position:
optionally R2 and R3 are connected to form -Q-G i-C6aikylene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form -0-CrC6alkylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form -0-CrC6aikyiene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form -0-Ci-C6alkylene, such that when R48 and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form ~0-Ci-C6aikylene, such that when R5 and Rs are connected, the O is bound at the R5 position;
optionally R5a and R6a, are connected to form -G-CrCsa!kyiene, such that when R£,a and RSa are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form -0-CrC6aikylene, such that when R5 and R7 are connected, the O is bound at the R5 position,
and
optionally R5a and R7a, are connected to form -0-GrC6alkyiene, such that when R5a and R7a are connected, the O is bound at the R5a position.
36. The compound of claim 35, having the structure selected from Formula (A-3), Formula (B-3), Formula (G-3), Formula (D-3), Formula (E-3), or Formula (F-3) or a stereoisomer or a pharmaceutically acceptable salt thereof,
Figure imgf000583_0001
Formula (A- 3) Formula (B-3)
Figure imgf000584_0001
Formula (E-3) Formula (F-3)
wherein: R1, R1a, R2, R2a, R3, R3a, R4, R4a, R5, R5a R6, R63, R7, R7a, Yi, Y2, Y3, Y4, Y-/, Ye and Y, , are as defined in claim 35.
37. The compound of claim 35 or claim 38, wherein
R1 , R1a and R1 b are each independently selected from
Figure imgf000584_0002
Figure imgf000584_0003
Figure imgf000585_0001
Figure imgf000586_0001
Figure imgf000587_0001
substituted with 0, 1 , 2 or 3 substituents independently selected from halogen, Ci-C6a!kyi and Ci-C3a!kyi substituted with 1 , 2 or 3 substituents independently selected from F, Ci, Br, !, OH, CN, and N3.
38. The compound of any one of claims 35 to 37, wherein
R1 , R1a and R1b are each independently selected from
Figure imgf000587_0002
Figure imgf000587_0003
39. The compound of any one of claims 35 to 38 selected from:
Figure imgf000587_0004
Figure imgf000588_0001
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