EP3615080A1 - Antibody conjugates comprising sting agonist - Google Patents

Antibody conjugates comprising sting agonist

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Publication number
EP3615080A1
EP3615080A1 EP18724677.2A EP18724677A EP3615080A1 EP 3615080 A1 EP3615080 A1 EP 3615080A1 EP 18724677 A EP18724677 A EP 18724677A EP 3615080 A1 EP3615080 A1 EP 3615080A1
Authority
EP
European Patent Office
Prior art keywords
alkyl
alkynyl
alkenyl
crc
independently selected
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP18724677.2A
Other languages
German (de)
French (fr)
Inventor
Thomas W. Dubensky Jr.
Jacob Robert BRUML
Stephen CANHAM
Charles Y. CHO
Kelsey GAUTHIER
Laura Hix GLICKMAN
Xueshi Hao
David Kanne
Shailaja Kasibhatla
George Edwin KATIBAH
Thanh Ngoc Lan LE
Justin Leong
Jeffrey Mckenna
Sarah McWHIRTER
Chudi Obioma Ndubaku
Weijia Ou
Leonard Sung
George Scott Tria
Tetsuo Uno
Tom Yao-Hsiang Wu
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
Original Assignee
Novartis AG
Aduro Biotech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novartis AG, Aduro Biotech Inc filed Critical Novartis AG
Publication of EP3615080A1 publication Critical patent/EP3615080A1/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
    • C07K9/003—Peptides being substituted by heterocyclic radicals, e.g. bleomycin, phleomycin

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 cells and/or CD8+ cytotoxic T cells, to promote cellular and humoral immunity.
  • Antigen presentation cells of the innate immune system such as dendritic cells 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 al., 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., IFN-a and IFN- ⁇ ) and exert a potent anti-viral state following expression
  • type I interferon e.g., IFN-a and IFN- ⁇
  • 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 immunostimmulatory 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-(L-(D)m)n (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; 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.
  • 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 cell, 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 cell 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-Dual 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-Dual 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, IFN- ⁇ , type 3 interferon, IFN , IP10, TNF, IL-6, CXCL9, CCL4, CXCL1 1 , CCL5, CCL3, or CCL8.
  • 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-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.
  • D, or the cleavage product thereof has STING agonist activity if it binds to STING and is able to stimulate expression of a STING-dependent transcript selected from any one of the transcripts listed in Fig. 1A - Fig. 10 and Fig. 2A - Fig. 2L in a STING-expressing cell at least 5-fold or greater than the expression level in an untreated STING-expressing cell.
  • expression of the STING-dependent transcript is increased 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 700-fold or greater.
  • D or the cleavage product thereof, has STING agonist activity if it binds to STING and is able to stimulate expression of a luciferase reporter gene controlled by interferon (IFN)-stimulated response elements in a STING-expressing cell at an EC50 of 20 micromolar ⁇ M), 15 ⁇ , 10 ⁇ , 9 ⁇ , 8 ⁇ , 7 ⁇ , 6 ⁇ , 5 ⁇ , 4 ⁇ , 3 ⁇ , 2 ⁇ , 1 ⁇ , 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 luciferase reporter gene controlled by interferon (IFN)-stimulated response elements in a STING-expressing cell to a level equal to or greater than the level of stimulation of 50 ⁇ of 2'3'-cGAMP.
  • IFN interferon
  • the STING-expressing cell is THP1 -Dual cell
  • the luciferase reporter gene is the IRF-Lucia reporter gene in THP1 -Dual cell
  • the STING agonist activity is determined by the THP1 -Dual assay described for Table 7.
  • the luciferase reporter gene is the 5xlSRE-mlFNb-GL4 reporter gene and the STING-expressing cell is a cell expressing wild-type human STING protein, and optionally the STING agonist activity is determined by the hSTING wt assay described in Table 7.
  • the immunoconjugate stimulates IP-10 secretion from a STING-expressing cell targeted by the Ab at an EC50 of 5 nanomolar (nM) or less in an IP-10 secretion assay.
  • the immunoconjugate is parenterally 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.
  • the Ab comprises a modified Fc region.
  • 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 maleimide linkage to the cysteine.
  • D is a dinudeotide.
  • D is a cyclic dinudeotide (CDN).
  • CDN cyclic dinudeotide
  • D is a compound selected from any one of the compounds of Table 1 , Table 2, Table 3, or Table 4.
  • 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-immolative 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.
  • L 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 55 to 69;
  • 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.
  • L has a structure selected from the following, or L comprises a structural
  • the immunoconjugate is selected from the
  • each Gi is independently
  • XD is C, and each Z 4 is N;
  • Y 3 is OH, 0-, OR 10 , N(R 10 ) 2 , SR 10 , SeH, Se , BH 3 , SH or S
  • Y 4 is OH, 0 " , OR 10 , N(R 10 ) 2 , SR 10 , SeH, Se " , BH 3 , SH or S
  • Ys is -CHr, -NH-, -0- or -S;
  • Y 6 is -CHr, -NH-, -0- or -S;
  • Y 8 is 0 or S
  • Y 9 is -CHr, -NH-, -0- or -S;
  • Yio is -CHr, -NH-, -0- or -S;
  • q is 1 , 2 or 3;
  • R 1 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 0, N or S, or a tautomer thereof, wherein R 1 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHL ⁇ R 115 , F, CI, Br, OH, SH, NH 2 , D, CD 3 , d-C e alkyl, Ci-Cealkoxyalkyl, d- C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(C r C 6 alkyl), -0(C 3 -C 8 cycloalkyl), -S(C r C 6 alkyl), - S(C r C 6
  • R 1a 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 0, N or S, or a tautomer thereof, wherein R 1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHUR 115 , F, CI, Br, OH, SH, NH 2 , D, CD 3 , C r C 6 alkyl, C r C 6 alkoxyalkyl, C r C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(C r C 6 alkyl), -0(C 3 -C 8 cycloalkyl), -S(C r C 6 alkyl), - S(C r C 6
  • R 1 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 0, N or S, or a tautomer thereof, wherein R 1b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHUR 115 , F, CI, Br, OH, SH, NH 2 , D, CD 3 , d-C 6 alkyl, C r C 6 alkoxyalkyl, d- C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(C r C 6 alkyl), -0(C 3 -C 8 cycloalkyl), -S(C r C 6 alkyl), - S(C r C 6 amino
  • OC(0)C 2 -C 6 alkynyl of R 5 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • each R 11 is independently selected from H and CrCsalkyl
  • each R 12 is independently selected from H and CrCealkyl
  • R 3 and R 6 are connected to form CrCsalkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, - 0-CrC 6 alkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 3 and R 6 are connected, the 0 is bound at the R 3 position
  • R 3a and R 6a are connected to form CrCsalkylene, C 2 -C 5 alkenylene, C 2 -C 6 alkynylene, -O-CrCsalkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 3a and R 6a are connected, the 0 is bound at the R 3a position;
  • R 2 and R 3 are connected to form CrCgalkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, - O-CrCsalkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 2 and R 3 are connected, the 0 is bound at the R 3 position;
  • R 2a and R 3a are connected to form C r C 3 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, -O-CrCealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 2a and R 3a are connected, the 0 is bound at the R 3a position;
  • R 4 and R 3 are connected to form CrCsalkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, - O-CrCsalkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 4 and R 3 are connected, the 0 is bound at the R 3 position;
  • R and R are connected to form C ⁇ Csalkylene, C 2 -C 3 alkenylene, C 2 -C 6 alkynylene, -O-CrCealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 4a and R 3a are connected, the 0 is bound at the R 3a position;
  • R 5 and R 6 are connected to form CrCealkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, - ⁇ -C ! -Cealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 5 and R 6 are connected, the 0 is bound at the R 5 position;
  • R 5a and R 6a are connected to form CrCgalkylene, C 2 -C 8 alkenylene, C 2 -C 6 alkynylene, -O-CrCealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 5a and R 6a are connected, the 0 is bound at the R 5a position;
  • R 5 and R 7 are connected to form CrCealkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, - ⁇ -C ! -Cealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 5 and R 7 are connected, the 0 is bound at the R 5 position;
  • R 5a and R 7a are connected to form C ⁇ Csalkylene, C 2 -C 3 alkenylene, C 2 -C 6 alkynylene, -O-CrCealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 5a and R 7a are connected, the 0 is bound at the R 5a position;
  • R 8 and R 9 are connected to form a CrCealkylene, C 2 -C 6 alkenylene, C 2 -C 3 alkynylene, and
  • R 8a and R 9a are connected to form a Ci-C 6 alkylene, C 2 -C 6 alkenylene, C 2 - C 6 alkynylene,
  • L- ! is a linker
  • R 1J is H or methyl
  • R 14 is H, -CH 3 or phenyl
  • each R 110 is independently selected from H, d-Cealkyl, F, CI, and -OH;
  • each R 111 is independently selected from H, C r C 6 alkyl, F, CI, -NH 2 , -OCH 3 , -OCH 2 CH 3 , -
  • Ab is an antibody or a functional fragment thereof
  • y is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the immunoconjugate has in vivo anti-tumor activity.
  • the present application also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising an immunconjugate as disclosed herein and a pharmaceutically acceptable excipient.
  • the present application also discloses an immunoconjugate 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 drug, a cytotoxic agent, an immune-based therapy, a vaccine, or a cell therapy.
  • 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-cell 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-cell immunoglobulin domain and mucin domain 3
  • the co-stimulatory molecule is Glucocorticoid-induced TNFR-related protein (GITR), and
  • the cytokine is IL-15 complexed with a soluble form of IL-15 receptor alpha (IL-15Ra).
  • 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, blastomas, 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 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 tumor
  • myelogenous leukemia A L
  • 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 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 lymphoproliferative conditions MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom
  • 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 also discloses a method of manufacturing any of the immunoconjugates as disclosed herein comprising the steps of:
  • this application discloses a compound having a structure selected from Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or Formula (F)
  • each Gi is independently selected from * indicates the point of attachment to -CR 1
  • X B is Z 2 is N; * indicates the point of attachment to -
  • X D is C, and each Z 4 is N;
  • Y 3 is OH, 0 " , OR 10 , N(R 10 ) 2 , SR 10 , SeH, Se , BH 3 , SH or S
  • Y4 is OH, 0-, OR 10 , N(R 10 ) 2 , SR 10 , SeH, Se , BH 3 , SH or S
  • Y 5 is -CH 2 -, -NH-, -0- or -S;
  • Ye is -CH ri -NH-, -0- or -S;
  • Ys is 0 or S
  • Y 9 is -CHr, -NH-, -0- or -S;
  • Y 10 is -CH 2 -, -NH-, -0- or -S;
  • q is 1 , 2 or 3;
  • R 1 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 0, N or S, or a tautomer thereof, wherein R 1 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHUR 15 , F, CI, Br, OH, SH, NH 2 , D, CD 3 , Ci-C e alkyl, C r C 6 alkoxyalkyl, C r C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(C r C 6 alkyl), -0(C 3 -C 8 cycloalkyl), -S(C r C 6 alkyl), - S(C r C 6 aminoalky
  • R 1a 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 0, N or S, or a tautomer thereof, wherein R 1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHL ⁇ R 15 , F, CI, Br, OH, SH, NH 2 , D, CD 3 , CrCealkyl, C r C 6 alkoxyalkyl, C r C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2
  • R 1 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 0, N or S, or a tautomer thereof, wherein R 1b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHUR 15 , F, CI, Br, OH, SH, NH 2 , D, CD 3 , d-Cealkyl, C r C 6 alkoxyalkyl, C r C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(C r C 6 alkyl), -0(C 3 -C 8 cycloalkyl), -S(C r C 6 alkyl), - S(C r C 6 aminoal
  • each R 11 is independently selected from H and CrCsalkyl
  • each R 12 is independently selected from H and CrCsalkyl
  • R 3 and R 6 are connected to form CrC 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, - 0-C r C 6 alkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 3 and R 6 are connected, the 0 is bound at the R 3 position
  • R 3a and R 6a are connected to form Ci-C 3 alkylene, C 2 -C 3 alkenylene, C 2 -C 6 alkynylene, -0-CrC 6 alkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 3a and R 6a are connected, the 0 is bound at the R 3a position;
  • R 2 and R 3 are connected to form CrC 6 alkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, - 0-CrC 6 alkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 2 and R 3 are connected, the 0 is bound at the R 3 position;
  • R and R are connected to form C ⁇ Csalkylene, C 2 -C 3 alkenylene, C 2 -C 6 alkynylene, -O-CrCealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 2a and R 3a are connected, the 0 is bound at the R 3a position;
  • R 4 and R 3 are connected to form CrCealkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, - ⁇ -C ! -Cealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 4 and R 3 are connected, the 0 is bound at the R 3 position;
  • R a and R 3a are connected to form CrCgalkylene, C 2 -C 8 alkenylene, C 2 -C 6 alkynylene, -O-CrCealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 4a and R 3a are connected, the 0 is bound at the R 3a position;
  • R 5 and R 6 are connected to form CrCealkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, - ⁇ -C ! -Cealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 5 and R 6 are connected, the 0 is bound at the R 5 position;
  • R 5a and R 6a are connected to form C ⁇ Csalkylene, C 2 -C 3 alkenylene, C 2 -C 6 alkynylene, -O-CrCealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 5a and R 6a are connected, the 0 is bound at the R 5a position;
  • R 5 and R 7 are connected to form CrCealkylene, C 2 -C 6 alkenylene, C 2 -C 6 alkynylene, - 0-C r C 6 alkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 5 and R 7 are connected, the 0 is bound at the R 5 position;
  • R 5a and R 7a are connected to form C ⁇ Csalkylene, C 2 -C 3 alkenylene, C 2 -C 6 alkynylene, -0-C r C 6 alkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 5a and R 7a are connected, the 0 is bound at the R 5a position;
  • R 8 and R s are connected to form a CrCealkylene, C 2 -C 6 alkenylene, C 2 -C 3 alkynylene, and
  • R 8a and R 9a are connected to form a C Cealkylene, C 2 -C 6 alkenylene, C 2 - C 6 alkynylene,
  • Xi is chment to X 2 ; X 2 is
  • X 4 is -0(CH 2 ) n SSC(R 12 ) 2 (CH 2 ) n - or -(CH 2 ) n C(R 12 ) 2 SS(CH 2 ) n O-;
  • X 5 is where the ** indicates orientation toward the Drug moiety;
  • X 6 is or, where the ** indicates orientation toward the Drug moiety
  • R 17 is 2-pyridyl or 4-pyridyl
  • each R 11 is independently selected from H and CrCealkyl
  • each R 12 is independently selected from H and d-Cealkyl
  • 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, CrCealkyl, F, CI, and -OH;
  • each R 111 is independently selected from H, C r C 6 alkyl, F, CI, -NH 2 , -OCH 3 , -OCH 2 CH 3 , -
  • R 1 , R 1a or R 1b is substituted with -NHL1R 15 , or at least one of R 3 , R 4 ,
  • R 5 , R 7 , R 3a , R a , R 5a or R 7a is -OLiR 15 .
  • FIG. 1A-10 are a series of a tables listing various transcripts in HCC1954 cells whose expression increased by at least 5-fold upon exposure to either cGAMP or compound T1 -1.
  • FIG. 2A-2L are a series of tables listing various transcripts in THP1 dual cells whose expression increased by at least 5-fold upon exposure to compound T1 -2.
  • FIG. 3 is a line graph showing STING agonist payload retention as determined by IPMS (intact protein mass spectrometry).
  • FIG. 4 is a line graph showing the anti-HER2-STING agonist conjugates induce IP-10 secretion from HER2+ HCC1954 breast cancer cells.
  • FIG. 5 is a line graph showing the anti-HER2 mAb1 -C1 conjugate inhibits N87 gastric tumor growth in mice.
  • FIG. 6 is a line graph showing the anti-HER2 mAb1 -C1 conjugate is well tolerated in the N87 gastric tumor xenograft mice.
  • FIG. 7 is a line graph showing the anti-HER2 mAb1 -C1 conjugate inhibits HCC1954 breast tumor growth in mice.
  • FIG. 8 is a line graph showing the anti-HER2 mAb1 -C1 conjugate is well tolerated in the HCC1954 breast tumor xenograft mice.
  • FIG. 9A is a line graph showing the anti-HER2 mAb1-C1 conjugate inhibits SKOV3 ovarian carcinoma growth in mice.
  • FIG. 9B is a line graph showing the anti-HER2 mAb1-C1 conjugate is well tolerated in the SKOV3 ovarian carcinoma xenograft mice.
  • FIG. 10 illustrates certain compounds which can be used as a Drug moiety.
  • FIG. 1 1 illustrates certain compounds which can be used as a Drug moiety.
  • FIG. 12 illustrates certain compounds which can be used as a Drug moiety.
  • FIG. 13A is a graph depicting efficacy of P-Cad mAb1 -C1 conjugate in an C38 murine colon adenocarcinoma model in mice.
  • FIG. 13B is a graph showing that P-Cad mAb1 -C1 conjugate is well tolerated in the MC38 murine colon adenocarcinoma model in mice.
  • FIG. 14 is a graph depicting efficacy of Target B mAb1 -C1 in a breast cancer xenograft model in mice.
  • FIG. 15 is a graph depicting efficacy of Target C mAb1-C1 conjugate in a lung cancer xenograft model in mice.
  • C r C 6 alkyl 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 molecule by a single bond.
  • Non-limiting examples of “CrCealkyl” groups include methyl, ethyl, 1 -methylethyl , n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl and hexyl.
  • C 2 -C 6 alkenyr refers to a straight or branched hydrocarbon chain radical group consisting solely 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 molecule by a single bond.
  • Non-limiting examples of "C 2 -C 6 alkenyl” groups include ethenyl, prop-1 -enyl, but-1 -enyl, pent-1 -enyl, pent-4-enyl and penta-1 ,4-dienyl.
  • C 2 -C 6 alkynyl refers to a straight or branched hydrocarbon chain radical group consisting solely 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 molecule by a single bond.
  • Non-limiting examples of "C 2 -C 6 alkynyl” groups include ethynyl, prop-1-ynyl, but-1 -ynyl, pent-1 -ynyl, pent-4-ynyl and penta-1 , 4-diynyl.
  • CrCealkylene 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.
  • C 2 -C 6 alkenyl refers to a bivalent straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to six carbon atoms.
  • C 2 -C 6 alkynyl refers to a bivalent straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having from two to six carbon atoms.
  • C ⁇ alkoxyalkyl refers to a radical of the formula -Ra-O-Ra, where each Ra is independently a Ci -6 alkyl radical as defined above.
  • the oxygen atom may be bonded to any carbon atom in either alkyl radical. Examples of include, but are not limited to, methoxy-methyl, methoxy-ethyl, ethoxy-ethyl, 1 -ethoxy-propyl and 2-methoxy-butyl.
  • C r C 6 hydroxyalkyl refers to a C 1-6 alkyl radical as defined above, wherein one of the hydrogen atoms of the C 1-6 alkyl radical is replaced by OH.
  • hydroxyC ⁇ alkyl include, but are not limited to, hydroxy-methyl, 2-hydroxy-ethyl, 2-hydroxy- propyl, 3-hydroxy-propyl and 5-hydroxy-pentyl
  • C 3 -C 8 cycloalkyl refers to a saturated, monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic ring system.
  • Non-limiting examples of fused bicyclic or bridged polycyclic ring systems include bicyclo[1 .1 .1 ]pentane, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[3.2.1 ]octane, bicyclo[2.2.2]octane and adamantanyl.
  • Non-limiting examples monocyclic C 3 -C 8 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups.
  • C r C 6 haloalkyl refer to the respective "CrC 6 alkyl", as defined herein, wherein at least one of the hydrogen atoms of the "CrC 6 alkyl” is replaced by a halo atom.
  • the CrC 6 haloalkyl groups can be monoCrC 6 haloalkyl, wherein such C r C s haloalkyl groups have one iodo, one bromo, one chloro or one fluoro.
  • the C r C 6 haloalkyl groups can be diCrC 6 haloalkyl wherein such CrCehaloalkyl groups can have two halo atoms independently selected from iodo, bromo, chloro or fluoro.
  • the CrCshaloalkyl groups can be polyC r C 6 haloalkyl wherein such C r C 6 haloalkyl groups can have two or more of the same halo atoms or a combination of two or more different halo atoms.
  • Such polyCr C 6 haloalkyl can be perhaloCrC 6 haloalkyl where all the hydrogen atoms of the respective d- C 6 alkyl have been replaced with halo atoms and the halo atoms can be the same or a combination of different halo atoms.
  • Non-limiting examples of CrC 6 haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, trifluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl.
  • C 2 -C 6 haloalkenyl refer to the respective "C r C 6 alkenyl", as defined herein, wherein at least one of the hydrogen atoms of the "C r C 6 alkenyl” is replaced by a halo atom.
  • the C 2 -C 6 haloalkenyl groups can be monoC r C 6 haloalkenyl, wherein such C r C 6 haloalkenyl groups have one iodo, one bromo, one chloro or one fluoro.
  • the C 2 - C 6 haloalkenyl groups can be diC 2 -C 6 haloalkenyl wherein such C 2 -C 6 haloalkenyl groups can have two halo atoms independently selected from iodo, bromo, chloro or fluoro.
  • the C 2 -C 6 haloalkenyl groups can be polyC 2 -C 6 haloalkenyl wherein such C 2 -C 6 haloalkenyl groups can have two or more of the same halo atoms or a combination of two or more different halo atoms.
  • C 2 -C 6 haloalkynyl refer to the respective “CrC 6 alkynyl", as defined herein, wherein at least one of the hydrogen atoms of the "C r C 6 alkynyl” is replaced by a halo atom.
  • the C 2 -C 6 haloalkynyl groups can be monoC r C 6 haloalkynyl, wherein such C r C 6 haloalkynyl groups have one iodo, one bromo, one chloro or one fluoro.
  • the C 2 - C 6 haloalkynyl groups can be diC 2 -C 6 haloalkynyl wherein such C 2 -C 6 haloalkynyl groups can have two halo atoms independently selected from iodo, bromo, chloro or fluoro.
  • the C 2 -C 6 haloalkynyl groups can be polyC 2 -C 6 haloalkynyl wherein such C 2 -C 6 haloalkenyl groups can have two or more of the same halo atoms or a combination of two or more different halo atoms.
  • heteroalkyl refers to an "alkyl” moiety wherein at least one of the carbon atoms has been replaced with a heteroatom such as 0 S, or N.
  • 3 to 6 membered heterocycloalkyl refers to a monocyclic ring structure having 3 to 6 ring members, wherein one to two of the ring members are
  • Non-limiting examples of 3-6 membered heterocycloalkyl groups include aziridin-1 -yl, 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-yl, thietanyl, thietan-2-yl, thietan-3-yl, thietan-4-yl, pyrrolidinyl, pyrrolidin-1 -yl, pyrrol id in-2-yl, pyrrolidin-3-yl, pyrrolidin-4-yl, pyrrolidinyl, pyrrolidin-1 -yl, pyrrol id in-2-yl, pyrrolidin-3-y
  • 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, C 1-6 alkyl, C ⁇ alkoxy, amino, C ⁇ alkylamino, di-C ⁇ alkylamino.
  • 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-b]pyridinyl, pyrrolo[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, pyrazolo[4,3-c]pyridinyl, pyrazolo[3,4- c]pyridinyl, pyrazolo[3,4-d]pyridinyl, pyrazolo[3,4-b]pyridinyl, imidazo[1 ,2-a]pyridinyl, pyrazolo[1 ,5-a]pyridinyl, pyrrolo[1 ,2-b]pyridazinyl, imidazo[1 ,2-c]pyrimidinyl, pyri
  • bicyclic heterocyclyl ring systems include heterocyclyl ring systems wherein one of the fused rings is aromatic but the other is non-aromatic.
  • the heterocyclyl is said to be partially saturated.
  • partially saturated bicyclic 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-yl-6-one.
  • Other examples of partially saturated bicyclic system are examples of partially saturated bicyclic system.
  • Heterocyclyl also includes a 5- or 6- membered ring aromatic heterocyclyl having 2 to 3 heteroatom (preferably nitrogen) (also referred to as 5- to 6-membered heteroaryl).
  • monocyclic heteroaryl are: imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, 1 , 2, 3-oxadiazolyl, 1 ,2,4- oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,5- thiadiazolyl, 1 ,3,4-thiadiazolyl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl, oxazol-2-yl, oxazol- 4-yl, oxazol-5-yl, isoxazol-3-yl
  • Heterocyclyl also includes 6-membered monocyclic partially saturated ring having 1-3 heteroatoms (preferably nitrogen).
  • Examples of partially saturated monocyclic heterocyclyl are pyrimidine-one and pyrimidine-dione, specifically pyrimidin-2(1 H)-one and pyrimidin-1 -yl-2,4(1 H, 3H)-dione.
  • Heterocyclyl can exist in various tautomeric forms.
  • a heterocyclyl 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 follow:
  • 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.
  • 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.
  • phosporothioic acid and phosphoric acid moieties can exist in the respective equilibrium as shown below.
  • 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, alkenes, alkynes and azides.
  • such functional groups include reactive groups of Table 5 provided herein.
  • Tvw wavy line
  • 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 17q12, and the genomic sequence of HER2/NEU gene can be found in GenBank at NG_007503.1.
  • GenBank GenBank at NG_007503.1.
  • a human HER2 protein also encompasses proteins that have over its full length at least about 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 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.
  • HER2 isoform A The mRNA and protein sequences for human HER2 isoform A, the longest isoform, are: Homo sapiens erb-b2 receptor tyrosine kinase 2 (ERBB2), transcript variant 1 , mRNA [NM_004448.3]
  • Receptor tyrosine-protein kinase erbB-2 isoform a precursor [Homo sapiens] [NP_004439.2]
  • HER2 isoform B NM_001005862.2 (mRNA) ⁇ NP_001005862.1 (protein);
  • HER2 isoform C NM_001289936.1 (mRNA) ⁇ NP_001276865.1 (protein);
  • HER2 isoform D NM_001289937.1 (mRNA) ⁇ NP_001276866.1 (protein);
  • HER2 isoform E NM_001289938.1 (mRNA) ⁇ NP_001276867.1 (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 CH3.
  • 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 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant 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., IgG 1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass.
  • 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 CH 1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid 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, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1 126-1 136, 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).
  • Fn3 fibronectin type III
  • 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-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-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 "hypervariable loops.”
  • VH heavy chain variable domain
  • HCDR1 e.g., insertion(s) after position 35
  • HCDR2 HCDR2
  • HCDR3 CDR amino acid residues in the light chain variable domain
  • VL CDR amino acid residues in the light chain variable domain
  • LCDR1 e.g., insertion(s) after position 27
  • 50-56 LCDR2
  • LCDR3 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)
  • the amino acid residues in VL are numbered 26-32 (LCDR1) (e.g., insertion(s) after position 30), 50-52 (LCDR2), and 91 -96 (LCDR3).
  • 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 amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.
  • 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 (CDR3) (numbering according to "Kabat").
  • 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 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, bispecific 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 germline 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 al. (2000. J Mol Biol 296, 57-86).
  • immunoglobulin variable domains e.g., CDRs
  • CDRs immunoglobulin variable domains
  • the structures and locations of immunoglobulin variable domains may be defined using well 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.; Al Lazikani et al., (1997) J. Mol. Bio. 273:927 948); Kabat et al., (1991) Sequences of Proteins of
  • 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 germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • recombinant human antibody includes all 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 DNA sequences.
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • 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, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • Fc region refers to a polypeptide comprising the CH3, 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.
  • the invention comprises an Fc region, a CH1 region and a Ckappa/lambda region from the constant domain of an antibody.
  • a binding molecule of the invention comprises a constant region, e.g., a heavy chain constant region.
  • 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 CH3) 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-covalent 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, isoleucine, proline, phenylalanine, methionine
  • beta- branched side chains e.g., threonine, valine, isoleucine
  • 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.
  • homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • two nucleic acid molecules such as, two DNA molecules or two RNA molecules
  • polypeptide molecules between two polypeptide molecules.
  • 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 10), 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 result 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.
  • 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.com), using either a Blossum 62 matrix or a PAM250 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.com), 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 Blossum 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 algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 1 1 -17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 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.ncbi.nlm.nih.gov.
  • cancer refers 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, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial 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.
  • solid tumors and hematological cancers including carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma),
  • 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.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, aden
  • 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 cell, 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 cell.
  • a tumor antigen is a marker expressed by both normal cells and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells.
  • a tumor antigen is a cell surface molecule that is
  • a tumor antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell.
  • 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 cell receptors
  • the MHC class I complexes are constitutively expressed by all nucleated cells.
  • virus-specific and/or tumor-specific peptide/MHC complexes represent a unique class of cell surface targets for immunotherapy.
  • 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 cell 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 cells.
  • a "HER2-positive cancer” or "HER2-expressing cancer” is a cancer comprising cells that have HER2 protein present at their cell surface. Many methods are known in the art for detecting or determining the presence of HER2 on a cancer cell. 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
  • ком ⁇ онент 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.
  • 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.
  • 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 least 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 superimposable 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- superimposable 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 (dextro- 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)-.
  • P-cadherin (also known as P-Cadherin, P-cad, P-Cad, Pcad, PCad, Cadherin 3, Cadherin-3, Cad3, Cad-3, CAD3, CAD-3, CDH3 or CDH-3) refers to the nucleic acid and amino acid sequences of P-cadherin, which have been published in GenBank Accession Nos. NP_ 001784, NP_001784.2 (amino acid sequence), and N _001793.4, GenBank Accession Nos. AA14462, NG_009096, and NG_009096.1 (nucleotide sequences). Sequence information for human P-cadherin domains 1 -5 are extracellular and are published in GenBank Acession Nos. NM_001793.4 and NP_001784.
  • P-cadherin also refers to proteins and amino acid sequences that over their full length have at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of the above GenBank accession Nos.
  • a P-cadherin nucleic acid sequence has over its extracellular domain at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence of GenBank accession numbers NM_001793.4, GenBank Accession Nos. AA14462, NG_009096, and NG_009096.1 .
  • 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
  • STING and “STING receptor” are used interchangeably, and include different isoforms and variants of STING.
  • the mRNA and protein sequences for human STING isoform 1 are:
  • TME 173 Homo sapiens transmembrane protein 173 (TME 173), transcript variant 1 , mRNA
  • gagccccagc agaagaatgg agaggaggag gaggctgagt ttggggtatt gaatcccccg
  • the mRNA and protein sequences for human STING isoform 2, a shorter isoform, are:
  • TAE 173 Homo sapiens transmembrane protein 173 (TME 173), transcript variant 2, mRNA
  • Homo sapiens stimulator of interferon genes protein isoform 2 [NP_001288667.1 ]
  • polymorphisms include the following and those described in Yi, PLoS One. 2013 Oct 21 ;
  • hSTING wt wild type: Reference SNP (refSNP) Cluster Report: rs1 131769
  • 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- ⁇ , type 3 interferons, e.g., IFN , IP10, TNF, IL-6, CXCL9, CCL4, CXCL11 , CCL5, CCL3, or CCL8.
  • interferons such as type 1 interferons, including IFN-a, IFN- ⁇ , type 3 interferons, e.g., IFN , IP10, TNF, IL-6, 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.
  • TK 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 [3H]cGAMP 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).
  • “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • “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
  • terapéuticaally 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).
  • a therapeutically effective amount does not induce or cause undesirable side effects.
  • 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 “prophylactically effective dose” or a “prophylactically effect amount”, of the molecules 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 molecules 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®).
  • 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 (
  • X B is C, and each Z 2 is N;
  • G 2 is * indicates the point of attachment to -CR 8a R 9a -;
  • X D is C, and each Z 4 is N;
  • Y 3 is OH, 0 " , OR 10 , N(R 10 ) 2 , SR 10 , SeH, Se " , BH 3 , SH or S " ;
  • Y 4 is OH, 0 " , OR 10 , N(R 10 ) 2 , SR 10 , SeH, Se " , BH 3 , SH or S " ;
  • Y 5 is -CH 2 -, -NH-, -0- or -S;
  • Y 6 is -CH 2 -, -NH-, -0- or -S;
  • Y 7 is 0 or S
  • Y 8 is 0 or S
  • Yg is -CHr, -NH-, -O- or -S;
  • Y 10 is -CH 2 -, -NH-, -0- or -S;
  • q is 1 , 2 or 3;
  • R 1 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 R 1 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from F, CI, Br, OH, SH, NH 2 , D, CD 3 , Ci-C 6 alkyl, C r C s alkoxyalkyl, C r C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, - ⁇ (C Cealkyl), -
  • R 1a 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 R 1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from F, CI, Br, OH, SH, NH 2 , D, CD 3 , d-Csalkyl,
  • C r C s alkoxyalkyl, C r C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -Oid-Cealkyl), - 0(C 3 -C 8 cycloalkyl), -S(C r Csalkyl), -StCrCsaminoalkyl), -S(C r C 6 hydroxyalkyl), -S(C 3 - Cgcycloalkyl), -NH(C r C 6 alkyl), -NH(C 3 -C 8 cycloalkyl), -N(C r C 6 alkyl) 2 , -NCd-Cealkyl) (C 3 - C 8 cycloalkyl), -CN, -P( 0)(OH) 2 , -O(CH 2 ) 1 . 10 C(
  • R 1b 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 R 1b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from F, CI, Br, OH, SH, NH 2 , D, CD 3 , CrC s alkyl,
  • C r C s alkoxyalkyl, C r C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC 6 alkyl), - 0(C 3 -C 8 cycloalkyl), -S(C r C s alkyl), -S(C r C 6 aminoalkyl), -S(C r C 6 hydroxyalkyl), -S(C 3 - C 8 cycloalkyl), -NH(C r C 6 alkyl), -NH(C 3 -C 8 cycloalkyl), -N(CrC 6 alkyl) 2 , -N(CrC 6 alkyl) (C 3 - Cscycloalkyl), -CN, -P( 0)(OH) 2 , -O(CH 2 ) 1 . 10 C(
  • -OC(0)OCrC 6 alkyl, -OC(0)OC 2 -C 6 alkenyl, -OC(0)OC 2 -C 6 alkynyl, - OC(0)C r C 6 alkyl, -OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 2 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • each R 3 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD 3 , CN, N 3 , CrCsalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, CrC 6 haloalkyl, C 2 -C 3 haloalkenyl, C 2 -
  • CrCshaloalkyl C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0(C r C 6 alkyl), -0(C 2 -C 3 alkenyl), - 0(C 2 -C 6 alkynyl), -OC(0)OCrC 6 alkyl, -OC(0)OC 2 -C 6 alkenyl, -OC(0)OC 2 -C 6 alkynyl, - OC(0)C r C 6 alkyl, -OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 3 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • -OC(0)OCrC 6 alkyl, -OC(0)OC 2 -C 6 alkenyl, -OC(0)OC 2 -C 6 alkynyl, - OC(0)C r C 6 alkyl, -OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 4 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • each R 5 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD 3 , CN, N 3 , CrCsalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, CrC 6 haloalkyl, C 2 -C 3 haloalkenyl, C 2 -
  • CrCshaloalkyl C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0(C r C 6 alkyl), -0(C 2 -C 3 alkenyl), - 0(C 2 -C 6 alkynyl), -OC(0)OCrC 6 alkyl, -OC(0)OC 2 -C 6 alkenyl, -OC(0)OC 2 -C 6 alkynyl, - OC(0)CrC 6 alkyl, -OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 5 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • C r C s alkyl, C 2 -C 3 alkenyl and C 2 -C 6 alkynyl of the C r C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, CrCshaloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0(CrC 6 alkyl), -0(C 2 -C 3 alkenyl), - 0(C 2 -C 6 alkynyl), -OC(0)OCi-C 6 alkyl, -0C(0)0C 2 -C 6 alkenyl, -0C(0)0C 2 -C 6 alkynyl, - OC(0)C r C 6 alkyl, -OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R s are substituted by 0,1 , 2 or
  • OC(0)C 2 -C 6 alkynyl wherein the -OC(0)Ophenyl of R 3a and the C r C 6 alkyl, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl of the CrC 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, CrCghaloalkyl, C 2 - C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0(C r C 6 alkyl), -0(C 2 -C 6 alkenyl), -0(C 2 -C 6 alkynyl), - OC(0)OC r C 6 alkyl, -OC(0)OC 2 -C 6 alkenyl, -OC(0)OC 2 -C 6 alkynyl, -OC(0)C r C 6 alkyl, -OC(0)OC 2 -C 6 alkenyl, -OC(0)OC
  • OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 4a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • OC(0)OC r C 6 alkyl, -OC(0)OC 2 -C 6 alkenyl, -OC(0)OC 2 -C 6 alkynyl, -OC(0)C r C 6 alkyl, - OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 5a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • R 6a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD 3 , CN, N 3 , CrCealkyl, C 2 -C 3 alkenyl, C 2 -C 6 alkynyl, C r C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0(C r
  • OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 6a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • OC(0)OC r C 6 alkyl, -OC(0)OC 2 -C 6 alkenyl, -OC(0)OC 2 -C 6 alkynyl, -OC(0)C r C 6 alkyl, - OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 7a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • R 8a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD 3 , CN, N 3 , CrCealkyl, C 2 -C 3 alkenyl, C 2 -C 6 alkynyl, C r C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0(C r
  • C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0(C r C 6 alkyl), -0(C 2 -C 6 alkenyl), -0(C 2 -C 6 alkynyl), - OC(0)OC r C 6 alkyl, -OC(0)OC 2 -C 6 alkenyl, -OC(0)OC 2 -C 6 alkynyl, -OC(0)C r C 6 alkyl, - OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 8 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • each R 10 is independently selected from the group consisting of H, d-C ⁇ alkyl, C r
  • each R 11 is independently selected from H and Chalky!
  • each R 12 is independently selected from H and CrCsalkyl
  • R 3 and R 6 are connected to form d-Calkylene, C 2 -C 6 alkenylene, C 2 -
  • R 3a and R 6a are connected to form Crdalkylene, C 2 -C 6 alkenylene, C 2 - C 6 alkynylene, -0-C r C 6 alkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, 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 d-Calkylene, C 2 -C 6 alkenylene, C 2 -
  • R 2a and R 3a are connected to form Crdalkylene, C 2 -C 6 alkenylene, C 2 - C 6 alkynylene, -O-CrCealkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, 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 Crdalkylene, C 2 -C 6 alkenylene, C 2 -
  • R 5 and R 6 are connected to form C ⁇ Cealkylene, C 2 -C 6 alkenylene, C 2 - C 6 alkynylene, -0-C r C 6 alkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 5 and R 6 are connected, the 0 is bound at the R 5 position;
  • R 5a and R 6a are connected to form CrQjalkylene, C 2 -C 6 alkenylene, C 2 - C 6 alkynylene, -0-C r C 6 alkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, 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 C ⁇ Cealkylene, C 2 -C 6 alkenylene, C 2 -
  • R 5a and R 7a are connected to form CrCsalkylene, C 2 -C 6 alkenylene, C 2 - C 6 alkynylene, -0-C r C 6 alkylene, -0-C 2 -C 6 alkenylene, -0-C 2 -C 6 alkynylene, such that when R 5a and R 7a are connected, the O is bound at the R 5a position;
  • R 8 and R 9 are connected to form a d-Calkylene, C 2 -C 6 alkenylene, C 2 - C 6 alkynylene, and
  • R 8a and R 9a are connected to form a CrC 6 alkylene, C 2 -C 6 alkenylene, C 2 - C 6 alkynylene.
  • Embodiment 1 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
  • Formula (E-1) Formula (F-1) wherein 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 8a , R 9 , Y t l Y 2 , Y 3 , Y 4 , Y5, ⁇ , Y7, Ye, Y9, Y10 and Yn are as defined above for compounds of Formula (A) , Formula (B), Formula (C), Formula (D), Formula (E) and Formula (F).
  • Embodiment 2 A compound of Formula( 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 nucleic acid base or analogue thereof, and R 1 is a pyrimidine or purine nucleic acid base or analogue thereof, each of which is substituted as described in R 1 , R 1a or R 1 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).
  • Embodiment 3 A compound of Formula (A-2), Formula (B-2), Formula (C-2), Formula (D- 2), Formula (E-2) or Formula (F-2):
  • Formula (E-2) Formula (F-2) wherein R 1 , R 1a , R 1 , 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 8a , R 9 , Y, , Y 2 , Y 3 , Y 4 , Y5, Ye, Y7, Ys, Y9, Y1 0 and Y are as defined above for compounds of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) and Formula (F).
  • Embodiment 4 A compound of Formula (A), Formula (A-1) or Formula (A-2) of
  • Embodiment 1 , 2 or 3 wherein:
  • C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 3 or R 4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • R 7 and R 7a are H
  • R 6 and R 6a are H
  • R 8 , R 9 , R 8a and R 9a are independently H or C r C 6 alkyl
  • C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 3a or R 4a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 .
  • Embodiment 5 A compound of Formula (A), Formula (A-1) or Formula (A-2) of
  • Embodiment 1 2, 3 or 4 wherein:
  • Y 3 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ;
  • Y 4 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ;
  • Y 5 and Y 6 are 0 or S;
  • Y 7 and Y 8 are 0 or S;
  • Y 9 and Y 10 are 0 or S;
  • R 2 , R 2a , R 6 , R 6a , R 7 and R 7a are H;
  • R 3a and R a is H and the other is H, OH or F
  • one of R 3 and R 4 is H and the other is H, OH or F
  • R 8a , R 9a , R 8 and R 9 are independently selected from H or CrCealk l.
  • Embodiment 6 A compound of Formula (B), Formula (B-1) or Formula (B-2) of
  • Embodiment 1 , 2 or 3 wherein:
  • R 2 and R 2a are H
  • C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 3a or R a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • R 7a and R 6a are H
  • R s and R 4 are H
  • R 8 , R 9 , R 8a and R 9a are independently H or C r C 6 alkyl
  • C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 5 or R 7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 .
  • Embodiment 7 A compound of Formula (B), Formula (B-1) or Formula (B-2) of
  • Embodiment 1 2, 3 or 6 wherein:
  • ⁇ ⁇ and Y 2 are O, CH 2 or S;
  • Y 3 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ;
  • Y 4 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ; Y 5 and Y 6 are 0 or S;
  • Y 7 and Y 8 are 0 or S;
  • Y g and Y 10 are 0 or S;
  • R 2 , R 2a , R 7a , R Sa , R 6 and R 4 are H;
  • R 3a and R 4a are H and the other is H, OH or F;
  • R 5 and R 7 are H and the other is H, OH or F, and
  • R 8a , R 9a , R 8 and R 9 are independently selected from H or C 1 -C 3 alkyl.
  • Embodiment 8 A compound of Formula (C), Formula (C-1) or Formula (C-2) of
  • Embodiment 1 , 2 or 3 wherein:
  • R 2 and R 2a are H
  • R 4a and R 6a are H;
  • R 6 and R 7 are H
  • R 8 , R 9 , R 8a and R 9a are independently H or C r C 6 alkyl
  • Embodiment 1 , 2, 3 or 8 wherein:
  • Y 3 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ;
  • Y 4 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ;
  • Y 5 and Y 6 are 0 or S;
  • Y 7 and Y 8 are 0 or S;
  • Y 9 and Yio are 0 or S;
  • R 2 , R 2a , R 4a , R 8a , R 6 and R 7 are H;
  • R 3 and R 4 is H and the other is H, OH or F;
  • R 5a and R 7a are H and the other is H, OH or F, and
  • Embodiment 10 A compound of Formula (D), Formula (D-1) or Formula (D-2) of
  • Embodiment 1 , 2 or 3 wherein:
  • R 2 and R 2a are H
  • C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 5a or R 7a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • R a and R 6a are H
  • R 6 and R 4 are H
  • R 8 , R 9 , R 8a and R 9a are independently H or C r C 6 alkyl
  • Embodiment 11 A compound of Formula (D), Formula (D-1) or Formula (D-2) of
  • Embodiment 1 1, 2, 3 or 10 wherein:
  • Y 1 and Y 2 are 0, CH 2 or S;
  • Y 3 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ;
  • Y 4 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ;
  • Y 5 and Y 6 are 0 or S;
  • Y 7 and Y 8 are 0 or S;
  • Y 9 and Y 10 are 0 or S;
  • R 2 , R 2a , R 4a , R Sa , R 6 and R 4 are H;
  • R 5a , R 7a is H and the other is H, OH or F;
  • R 5 and R 7 are H and the other is H, OH or F, and
  • R 8 , R 9 , R 8a and R 9a are independently H or C r C 6 alkyl.
  • Embodiment 12 A compound of Formula (E), Formula (E-1) or Formula (E-2) of
  • Embodiment 1 , 2 or 3 wherein:
  • R 2 and R 2a are H
  • R 6 and R 6a are H
  • R 7a is H
  • R 8 , R 9 , R 8a and R 9a are independently H or C r C 6 alkyl
  • R 3a and R 4a is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD 3 , CN, N 3 , C r C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, CrCehaloalkyl, C 2 -
  • C 6 alkyl, -OC(0)OC 2 -C 6 alkenyl, -OC(0)OC 2 -C 6 alkynyl, -OC ⁇ CrCealkyl, -OC(0)C 2 - C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 3 or R 4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 , and one of R 5 and R 7 is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD 3 , CN, N 3 , C r C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C r C 6 haloalkyl, C 2 - C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0(C r Csal
  • C 6 alkenyl and -0C(0)C 2 -C 6 alkynyl of R 5 or R 7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 .
  • Embodiment 13 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 O, CH 2 or S;
  • Y 3 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ;
  • Y 5 is O or S
  • Y 7 is O or S
  • Y 9 is O or S
  • R 2 , R 2a , R 5a , R 8a , R 6 and R 7a are H;
  • R 3a , R a is H and the other is H, OH, OCH 3 or F;
  • R 3 , R 4 is H and the other is H, OH, OCH 3 or F;
  • R 5 and R 7 are H and the other is H, OH, OCH 3 or F, and
  • R 8 , R 9 , R 8a and R 9a are independently H or C r C 6 alkyl.
  • Embodiment 14 A compound of Formula (F), Formula (F-1) or Formula (F-2) of
  • Embodiment 1 , 2 or 3 wherein:
  • R 2 and R 2a are H; each R e and R ea are H;
  • each R 7a and R 7 are H;
  • R 8 , R 9 , R 8a and R 9a are independently H or C r C 6 alkyl
  • R 3a and R a is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD 3 , CN, N 3 , C r C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C r C 6 haloalkyl, C 2 -
  • C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 3 or R 4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 , and
  • R 5 is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD 3 , CN, N 3 , C r
  • Embodiment 15 A compound of Formula (F), Formula (F-1) or Formula (F-2) of
  • Embodiment 1 1, 2, 3 or 12 wherein:
  • Y 1 and Y 2 are 0, CH 2 or S;
  • each Y 3 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ;
  • each Y 5 is O or S
  • each Y 7 is independently 0 or S;
  • each Y g is independently 0 or S;
  • Y 11 is O, CH 2 or S
  • R 2 , R 2a , R 6 , R 6a , R 6 , R 7 and R 7a are H;
  • R 3a , R 4a is H and the other is H, OH, OCH 3 or F;
  • R 3 , R 4 is H and the other is H, OH, OCH 3 or F;
  • R 5 is H, OH, OCH 3 or F
  • R 8 , R 9 , R 8a and R 9a are independently H or C r C 6 alkyl.
  • Embodiment 16 A compound of any one of Embodiments 1 to 15 wherein:
  • R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH 2 , D, CD 3 , C r C s alkyl, C r
  • C 6 alkoxyalkyl, Ci-C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -CHCrCealkyl), - 0(C 3 -C 8 cycloalkyl), -S(C r C s alkyl), -S(C r C 6 aminoalkyl), -S(C r C 6 hydroxyalkyl), -S(C 3 - Cgcycloalkyl), -NH(C r C 6 alkyl), -NH(C 3 -C 8 cycloalkyl), -N(C r C 6 alkyl)2, -NCd-Csalkyl) (C 3 - Cgcycloalkyl), -CN, -P( 0)(OH) 2 , -O(CH 2 ) 1 .
  • R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH 2 , D, CD 3 , C r C 6 alkyl, C r
  • R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH 2 , D, CD 3 , C r C s alkyl, C r
  • C 6 alkoxyalkyl, C r C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -C CrCealkyl), - 0(C 3 -C 8 cycloalkyl), -S(C r C 3 alkyl), -S(C r C 6 aminoalkyl), -S(C r C 6 hydroxyalkyl), -S(C 3 - Cgcycloalkyl), -NH(C r C 6 alkyl), -NH(C 3 -C 8 cycloalkyl), -N(C r C 6 alkyl) 2 , -N d-Cealkyl) (C 3 - Cgcycloalkyl), -CN, -P( 0)(OH) 2 , -O(CH 2 ) 1 .
  • Embodiment 17 A compound of Formula (A-3), Formula (B-3), Formula (C-3) , Formula -3), Formula (E-3) or Formula (F-3):
  • Y 3 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ;
  • Y 4 is OH, 0 " , OR 10 , N(R 10 ) 2 , SH or S " ;
  • Y 7 is 0 or S
  • R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH 2 , D, CD 3 , C r C 6 alkyl, C r C s alkoxyalkyl, C r C 6 hydroxyalkyl, C 3 -C 8 cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -C ⁇ CrCsalkyl), - 0(C 3 -C 8 cycloalkyl), -S(C r C 3 alkyl), -S(C r C 6 aminoalkyl), -S(C r C 6 hydroxyalkyl), -S(C 3 - Cgcycloalkyl), -NH(C r C 6 alkyl), -NH(C 3 -C 8 cycloalkyl), -N(C r C 6 alkyl) 2 , -
  • -OC(0)OCrC 6 alkyl, -OC(0)OC 2 -C 6 alkenyl, -OC(0)OC 2 -C 6 alkynyl, - OC(0)C r C 6 alkyl, -OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R 5 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;
  • each R 6 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD 3 , CN, N 3 , C r C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, CrCehaloalkyl, C 2 -C 3 haloalkenyl, C 2 -
  • CrCshaloalkyl C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0(C r C 6 alkyl), -0(C 2 -C 3 alkenyl), - 0(C 2 -C 6 alkynyl), -OC(0)OCrC 6 alkyl, -0C(0)0C 2 -C 6 alkenyl, -0C(0)0C 2 -C 6 alkynyl, - OC(0)C r C 6 alkyl, -OC(0)C 2 -C 6 alkenyl and -OC(0)C 2 -C 6 alkynyl of R s are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N 3 ;

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Abstract

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

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 cells and/or CD8+ cytotoxic T cells, to promote cellular and humoral immunity. Antigen presentation cells of the innate immune system, such as dendritic cells 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 al., 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., IFN-a and IFN-β) and exert a potent anti-viral state following expression
(Ishikawa and Barber, Nature 2008, 455(7213):674-678; Chen et al., Cell (201 1) 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 immunostimmulatory 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)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 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 cell, 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 cell 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-Dual 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, IFN-β, type 3 interferon, IFN , IP10, TNF, IL-6, CXCL9, CCL4, CXCL1 1 , CCL5, CCL3, 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-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 further embodiments, D, or the cleavage product thereof, has STING agonist activity if it binds to STING and is able to stimulate expression of a STING-dependent transcript selected from any one of the transcripts listed in Fig. 1A - Fig. 10 and Fig. 2A - Fig. 2L in a STING-expressing cell at least 5-fold or greater than the expression level in an untreated STING-expressing cell. In some
embodiments, expression of the STING-dependent transcript is increased 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 700-fold or greater. 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 luciferase reporter gene controlled by interferon (IFN)-stimulated response elements in a STING-expressing cell at an EC50 of 20 micromolar ^M), 15 μΜ, 10 μΜ, 9 μΜ, 8 μΜ, 7 μΜ, 6 μΜ, 5 μΜ, 4 μΜ, 3 μΜ, 2 μΜ, 1 μ , 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 luciferase reporter gene controlled by interferon (IFN)-stimulated response elements in a STING-expressing cell to a level equal to or greater than the level of stimulation of 50 μΜ of 2'3'-cGAMP. In some embodiments, the STING-expressing cell is THP1 -Dual cell, and the luciferase reporter gene is the IRF-Lucia reporter gene in THP1 -Dual cell, and optionally the STING agonist activity is determined by the THP1 -Dual assay described for Table 7. In another embodiment, the luciferase reporter gene is the 5xlSRE-mlFNb-GL4 reporter gene and the STING-expressing cell is a cell expressing wild-type human STING protein, and optionally the STING agonist activity is determined by the hSTING wt assay described in Table 7. In other embodiments, the immunoconjugate stimulates IP-10 secretion from a STING-expressing cell targeted by the Ab at an EC50 of 5 nanomolar (nM) or less in an IP-10 secretion assay.
In some embodiments disclosed herein, the immunoconjugate is parenterally 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 maleimide linkage to the cysteine.
In one embodiment of the immunoconjugates disclosed herein, D is a dinudeotide. In some cases, D is a cyclic dinudeotide (CDN). In other embodiments, D is a compound selected from any one of the compounds of Table 1 , Table 2, Table 3, or Table 4.
In some embodiments disclosed herein, D is a compound selected from
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-immolative 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, L has a structure selected from:
-(Lc)x-CE-(Lc)y-CE-D gnd
-H(Lc)x-CE)p-(Lc)y-CE-D
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 55 to 69;
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 some embodiments, L has a structure selected from the following, or L comprises a structural
component selected from the
In some embodiments disclosed herein, the immunoconjugate is selected from the
Formula (AA-a) Formula (AA-b)
Formula (BB-c) Formula (BB-d)
Formula (BB-e) Formula (BB-f)
Formula (CC-e) Formula (CC-f)
Formula (EE-a) Formula (EE-b)
14
Formula (FFc) Formula (FF-d)
Formula (FF-g) Formula (FF-h)
-i) Formula (FF-j)
Formula (FF-k);
wherein:
each Gi is independently
indicates the point of attachment to -CR8R9-;
XA is C(=0)-, -C(=S)- or -C(=NR11)- and each Z is NR XB is C, and each Z2 is N; , where * indicates the point of attachment to R8aR9a
Xc is C(=0)-, -C(=S)- or -C(=NR11)- and each Z3 is NR12;
XD is C, and each Z4 is N;
YI iS -O-, -S-, -S(=0)-, -SOr, -CHr, or -CF2-;
Y2 is -0-, -S-, -S(=0)-, -SOr, -CHr, or -CFr;
Y3 is OH, 0-, OR10, N(R10)2, SR10, SeH, Se , BH3, SH or S
Y4 is OH, 0", OR10, N(R10)2, SR10, SeH, Se", BH3, SH or S
Ys is -CHr, -NH-, -0- or -S;
Y6 is -CHr, -NH-, -0- or -S;
Y8 is 0 or S;
Y9 is -CHr, -NH-, -0- or -S;
Yio is -CHr, -NH-, -0- or -S;
Yl 1 is -0-, -S-, -S(=0)-, -SOr, -CHr, or -CFr;
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 0, N or S, or a tautomer thereof, wherein R1 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHL^R115, F, CI, Br, OH, SH, NH2, D, CD3, d-Cealkyl, Ci-Cealkoxyalkyl, d- C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), - S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3- Cecycloalkyl), -N(Ci-Cealkyl)2, -N(Ci-Cealkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -0(CH2)i.
-(CH2)1.10C(=O)OH,-CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3- C8cycloalkyl), -NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
R1a 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 0, N or S, or a tautomer thereof, wherein R1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHUR115, F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, CrC6alkoxyalkyl, Cr C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), - S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3- Cgcycloalkyl), -N(CrC6alkyl)2, -N(CrC6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -0{C 2)^. 10C(=O)OH, -(CH2)1.1oC(=0)OH,-CH=CH(CH2)i.ioC(=0)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3- C8cycloalkyl), -NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
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 0, N or S, or a tautomer thereof, wherein R1b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHUR115, F, CI, Br, OH, SH, NH2, D, CD3, d-C6alkyl, CrC6alkoxyalkyl, d- C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), - S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3- Cgcycloalkyl), -N(CrC6alkyl)2, -N(CrC6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -0{C 2 - 10C(=O)OH, -(CH2)1.10C(=O)OH,-CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3- Cscycloalkyl), -NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
each R2 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(Ci-C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl, wherein the -OC(0)Ophenyl of R2 and the CrCsalkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC^CrCealkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R2 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R3 is independently selected from the group consisting of -OL^R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -0(CH2)i. -OC(0)Ophenyl, -OC(0)OCrCsalkyl, -OC(0)OC2-
C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)Ci-C6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and the CrCealkyl, C2-C6alkenyl and C2- C6alkynyl of the Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- Cehaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - 0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -
OC(0)C2-C6alkynyl of R3 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; each R4 is independently selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -0(CH2)i.
-O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrCsalkyl, -OC(0)OC2- C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R4 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R5 is independently selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -0(CH2)i.
-0(CH2)i-ioP(=0)(OH)2, -OC(0)Ophenyl, -OC(0)OCrCsalkyl, -OC(0)OC2-
C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrCealkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -
OC(0)C2-C6alkynyl of R5 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R6 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl, wherein the -OC(0)Ophenyl of R6 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- Cehaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R6 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R7 is independently selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -0(CH2)i. 10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrCsalkyl, -OC(0)OC2- C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R7 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R8 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl, wherein the -OC(0)Ophenyl of R8 and the CrCsalkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2- Cehaloalkynyl, -0(CrC6alkyl), -0(C2-Cealkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - 0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R8 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R9 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl, wherein the -OC(0)Ophenyl of R9 and the CrCsalkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R9 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R2a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2- C6alkenyl, C2-C6alkynyl, CrCghaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R2a and the CrC3alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2- C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3 R3a is selected from the group consisting of -OL^R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -0{C 2)^. 10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R4a is selected from the group consisting of -OL^R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Ci- C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -0{C 2)^. 10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R5a is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C^Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -0{C 2)^. -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, - 0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R6a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2- C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -OiCrCealkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of RSa and the CrCsalkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2- C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R6aare substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R7a is selected from the group consisting of -OL^R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -0{CH2)^. 10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Ci- C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R8a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2- C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R8a and the CrCsalkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2- C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R9a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2- C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R9a and the CrC3alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2- C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; each R10 is independently selected from the group consistin of H, CrC12alkyl, Cr
Csheteroalkyl, -(CH2CH20)nCH2CH2C(=0)OC1-C3alkyl, and wherein the Cr C12alkyl and CrCeheteroalkyl of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, CrC12alkoxy, -S-C(=0)CrC6alkyl, halo, -CN, CrC12alkyl, -O-aryl, _0- heteroaryl, -O-cycloalkyl, oxo, cycloalkyi, heterocyclyl, aryl, or heteroaryl, -OC(0)OCr
C6alkyland C(0)OCrC6alkyl, wherein each alkyl, cycloalkyi, heterocyclyl, aryl, and heteroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from CrCi2 alkyl, O-CrC^alkyl, CrC12heteroalkyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroaryl, -C(=0)C1-C12alkyl,
-C(=0)-aryl, -C(=0)-heteroaryl, - OC(=0)-aryl, -C(=0)0-aryl, -OC(=0)-heteroaryl, -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(0)-aryl, - N(R11)C(0)-heteroaryl, and S(0)2N(R11)-aryl;
each R11 is independently selected from H and CrCsalkyl;
each R12 is independently selected from H and CrCealkyl;
optionally R3 and R6 are connected to form CrCsalkylene, C2-C6alkenylene, C2-C6alkynylene, - 0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3 and R6 are connected, the 0 is bound at the R3 position
optionally R3a and R6a, are connected to form CrCsalkylene, C2-C5alkenylene, C2-C6alkynylene, -O-CrCsalkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3a and R6a are connected, the 0 is bound at the R3a position;
optionally R2 and R3 are connected to form CrCgalkylene, C2-C6alkenylene, C2-C6alkynylene, - O-CrCsalkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2 and R3 are connected, the 0 is bound at the R3 position;
optionally R2a and R3a, are connected to form CrC3alkylene, C2-C6alkenylene, C2-C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2a and R3a are connected, the 0 is bound at the R3a position;
optionally R4 and R3 are connected to form CrCsalkylene, C2-C6alkenylene, C2-C6alkynylene, - O-CrCsalkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4 and R3 are connected, the 0 is bound at the R3 position; optionally R and R , are connected to form C^Csalkylene, C2-C3alkenylene, C2-C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4a and R3a are connected, the 0 is bound at the R3a position;
optionally R5 and R6 are connected to form CrCealkylene, C2-C6alkenylene, C2-C6alkynylene, - Ο-C!-Cealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R6 are connected, the 0 is bound at the R5 position;
optionally R5a and R6a, are connected to form CrCgalkylene, C2-C8alkenylene, C2-C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R6a are connected, the 0 is bound at the R5a position;
optionally R5 and R7 are connected to form CrCealkylene, C2-C6alkenylene, C2-C6alkynylene, - Ο-C!-Cealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R7 are connected, the 0 is bound at the R5 position;
optionally R5a and R7a, are connected to form C^Csalkylene, C2-C3alkenylene, C2-C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R7a are connected, the 0 is bound at the R5a position;
optionally R8 and R9 are connected to form a CrCealkylene, C2-C6alkenylene, C2-C3alkynylene, and
optionally R8a and R9a are connected to form a Ci-C6alkylene, C2-C6alkenylene, C2- C6alkynylene,
L-! is a linker;
-C(=0)-, -ON=***, -S-, -NHC(=0)CH2-***, -S(=0)2CH2CH2-***, - (CH2)2S(=0)2CH2CH2-***, -NHS(=0)2CH2CH2.***,, -NHC(=0)CH2CH2-***, -CH2NHCH2CH2-
R1J is H or methyl;
R14 is H, -CH3 or phenyl;
each R110 is independently selected from H, d-Cealkyl, F, CI, and -OH;
each R111 is independently selected from H, CrC6alkyl, F, CI, -NH2, -OCH3, -OCH2CH3, -
N(CH3)2, -CN, -N02 and -OH;
each R112 is independently selected from H, C^alkyl, fluoro, benzyloxy substituted with - C(=0)OH, benzyl substituted with -C(=0)OH, CMalkoxy substituted with -C(=0)OH and C^. 4alkyl substituted with -C(=0)OH;
Ab is an antibody or a functional fragment thereof; and
y is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
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In some embodiments, the immunoconjugate has in vivo anti-tumor activity.
The present application also discloses a pharmaceutical composition comprising an immunconjugate as disclosed herein and a pharmaceutically acceptable excipient.
The present application also discloses an immunoconjugate 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 drug, a cytotoxic agent, an immune-based therapy, a vaccine, or a cell 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-cell immunoglobulin domain and mucin domain 3 (TIM-3),
(ii) the co-stimulatory molecule is Glucocorticoid-induced TNFR-related protein (GITR), and
(iii) 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, blastomas, 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 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 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 (A L), 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, 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 lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.
In some embodiments, the immunoconjugate is administered to the subject
intravenously, intratu morally, 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)m with Ab to form the immunoconjugate Ab— (L— (D)m)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)
Formula (C) Formula (D)
Formula (E) Formula (F)
wherein:
each Gi is independently selected from * indicates the point of attachment to -CR1
XA is C(=0)-, -C(=S)- or -C(=NR11)- and each is NR
XB is Z2 is N; * indicates the point of attachment to -
CR8aR9a-;
Xc is C(=0)-, -C(=S)- or -C(=NR11)- and each Z3 is NR12;
XD is C, and each Z4 is N;
Yi is -O-, -S-, -S(=0)-, -SO2-, -CH2-, or -CF2-;
Y2 is -O-, -S-, -S(=0)-, -SO2-, -CHr, or -CF2-;
Y3 is OH, 0", OR10, N(R10)2, SR10, SeH, Se , BH3, SH or S
Y4 is OH, 0-, OR10, N(R10)2, SR10, SeH, Se , BH3, SH or S
Y5 is -CH2-, -NH-, -0- or -S;
Ye is -CHri -NH-, -0- or -S;
Ys is 0 or S;
Y9 is -CHr, -NH-, -0- or -S;
Y10 is -CH2-, -NH-, -0- or -S;
Y11 is -0-, -S-, -S(=0)-, -SOr, -CHr, or -CFr;
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 0, N or S, or a tautomer thereof, wherein R1 is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHUR15, F, CI, Br, OH, SH, NH2, D, CD3, Ci-Cealkyl, CrC6alkoxyalkyl, Cr C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), - S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3- Cgcycloalkyl), -N(CrC6alkyl)2, -N(CrC6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -0{C 2)^. -NHC(0)(Ci-C6alkyl), -NHC(0)(C3- C8cycloalkyl), -NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2; R1a 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 0, N or S, or a tautomer thereof, wherein R1a is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHL^R15, F, CI, Br, OH, SH, NH2, D, CD3, CrCealkyl, CrC6alkoxyalkyl, Cr C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), - S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3- C8cycloalkyl), -N(CrC6alkyl)2, -N(CrC6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(0H)2, -0(CH2)1. 10C(=O)OH, -(CH2)1.10C(=O)OH,-CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3- C8cycloalkyl), -NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
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 0, N or S, or a tautomer thereof, wherein R1b is substituted with 0, 1 , 2, 3 or 4 substituents independently selected from -NHUR15, F, CI, Br, OH, SH, NH2, D, CD3, d-Cealkyl, CrC6alkoxyalkyl, Cr C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), - S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3- C8cycloalkyl), -N(CrC6alkyl)2, -N(CrC6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(0H)2, -0{C 2 1oC(=0)OH, -(CH2)1.10C(=0)OH,-CH=CH(CH2)1.10C(=0)OH,-NHC(0)(C1-C6alkyl), -NHC(0)(C3- C8cycloalkyl), -NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
each R2 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC^CrCealkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl, wherein the -OC(0)Ophenyl of R2 and the Ci-C8alkyl, C2-C8alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2- Cehaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R2 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R3 is independently selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -0(CH2)i. 10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC8alkyl, -OC(0)OC2- C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R3 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R4 is independently selected from the group consisting of -OL^R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- Cehaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -0(CH2) . 10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrCsalkyl, -OC(0)OC2- C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4 and the CrCealkyl, C2-C6alkenyl and C2- C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- Cehaloalkynyl, -0(CrC6alkyl), -0(C2-Cealkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - 0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R5 is independently selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -0(CH2)i. 10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrCsalkyl, -OC(0)OC2- C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R5 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R6 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl, wherein the -OC(0)Ophenyl of R6 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R6 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R7 is independently selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -0(CH2)i. 10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrCsalkyl, -OC(0)OC2- C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R7 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R8 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl, wherein the -OC(0)Ophenyl of R8 and the CrCsalkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R8 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R9 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl, wherein the -OC(0)Ophenyl of R9 and the CrC8alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl of R9 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; R2a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, Chalky!, C2- C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -OtCrCealkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH,
-OC(0)Ophenyl, -OC^OCrCealkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R2a and the CrCsalkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2- C6alkenyl, C2-C6alkynyl, CrCghaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -OiCrCealkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3 R3a is selected from the group consisting of -OL^R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -0{C 2)^. 10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C8alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R a is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C^Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -0{C 2)^. 10P(=O)(OH)2, -OC(0)Ophenyl, -OC^Od-Cealkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Ci- C6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R5a is selected from the group consisting of -OL^R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -0{C 2)^. 10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and the d-Cealkyl, C2-C6alkenyl and C2-C6alkynyl of the Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C^Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R6a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2- C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -OtCrCfjalkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -
OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R8a and the CrCsalkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2- C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)Ci-C6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R6aare substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R7a is selected from the group consisting of -OL^R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Ci- C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -0{C 2)^. 10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, - 0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R8a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, d-C3alkyl, C2- C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R8a and the CrCsalkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2- C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; R9a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, Chalky!, C2- C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -O(CrCsalkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH,
-OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R9a and the CrCsalkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2- C6alkenyl, C2-C6alkynyl, CrCghaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), - 0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -OC(0)OC2- C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; each R10 is independently selected from the group consisting of H, CrC12alkyl, Cr
C6heteroalkyl, , wherein the d-
C12alkyl and C C6heteroalkyl of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, CrC12alkoxy, -S-C(=0)CrC6alkyl, halo, -CN, CrC12alkyl, -O-aryl, _0- heteroaryl, -O-cycloalkyl, oxo, cycloalkyi, heterocyclyl, aryl, or heteroaryl, -OC(0)OCr
C6alkyland C(0)OCrC6alkyl, wherein each alkyl, cycloalkyi, heterocyclyl, aryl, and heteroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from CrCi2 alkyl, 0-CrCi2alkyl, CrCi2heteroalkyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroaryl, -C(=0)CrCi2alkyl, -OC(=0)C1-C12alkyl, -C(=0)OCrC12alkyl, -OC(=0)OCrC12alkyl, -C(=0)N(R11)-CrC12alkyl, - N(R11)C(=0)-CrC12alkyl; -OC(=0)N(R11)-CrC12alkyl, -C(=0)-aryl, -C(=0)-heteroaryl, -
OC(=0)-aryl, -C(=0)0-aryl, -OC(=0)-heteroaryl, -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(0)-aryl, - N(R11)C(0)-heteroaryl, and S(O)2N(R11)-aryl;
each R11 is independently selected from H and CrCsalkyl;
each R12 is independently selected from H and CrCsalkyl;
optionally R3 and R6 are connected to form CrC6alkylene, C2-C6alkenylene, C2-C6alkynylene, - 0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3 and R6 are connected, the 0 is bound at the R3 position
optionally R3a and R6a, are connected to form Ci-C3alkylene, C2-C3alkenylene, C2-C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3a and R6a are connected, the 0 is bound at the R3a position;
optionally R2 and R3 are connected to form CrC6alkylene, C2-C6alkenylene, C2-C6alkynylene, - 0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2 and R3 are connected, the 0 is bound at the R3 position; optionally R and R , are connected to form C^Csalkylene, C2-C3alkenylene, C2-C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2a and R3a are connected, the 0 is bound at the R3a position;
optionally R4 and R3 are connected to form CrCealkylene, C2-C6alkenylene, C2-C6alkynylene, - Ο-C!-Cealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4 and R3 are connected, the 0 is bound at the R3 position;
optionally R a and R3a, are connected to form CrCgalkylene, C2-C8alkenylene, C2-C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4a and R3a are connected, the 0 is bound at the R3a position;
optionally R5 and R6 are connected to form CrCealkylene, C2-C6alkenylene, C2-C6alkynylene, - Ο-C!-Cealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R6 are connected, the 0 is bound at the R5 position;
optionally R5a and R6a, are connected to form C^Csalkylene, C2-C3alkenylene, C2-C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R6a are connected, the 0 is bound at the R5a position;
optionally R5 and R7 are connected to form CrCealkylene, C2-C6alkenylene, C2-C6alkynylene, - 0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R7 are connected, the 0 is bound at the R5 position;
optionally R5a and R7a, are connected to form C^Csalkylene, C2-C3alkenylene, C2-C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R7a are connected, the 0 is bound at the R5a position;
optionally R8 and Rs are connected to form a CrCealkylene, C2-C6alkenylene, C2-C3alkynylene, and
optionally R8a and R9a are connected to form a C Cealkylene, C2-C6alkenylene, C2- C6alkynylene,
Li is -C(=0)0(CH2)mNR11C(=0)(CH2)m-**; -C(=0)0(CH2)mNR11C(=0)(CH2)mO(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;
-C(=0)OC(R12)2(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-**;
-C(=0)0(CH2)mNR11C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**;
-C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -C(=0)(CH2) J\IR1 C(=0)(CH2)m-**;
-C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-ii,
-C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**,
-C(=0)0(CH2)mX6C(=0)(CH2)m-**, -C(=0)0(CH2)mX6C(=0)(CH2)mO(CH2)m-**,
-C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**,
-C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)mO(CH2)m-**,
-C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)- **,
-C(=0)0(CH2)mX6C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-",
-C(=0)X4C(=0)X6(CH2)mNR11C(=0)(CH2)mO(CH2)m-**,
-C(=0)(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**,
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0))X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-";
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)m-
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR1 1 C(=0)X5((CH2)mO)n(CH2)mNR1 1 C(=0)(CH2)m-**; -C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mNR11((CH2)mO)n(CH2)m-**; -
C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)„(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**; -C(=0)0(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)m-**; -C(=0)0(CH2)mNR11 (CH2)m-**;
-C(=0)0(CH2)mNR11 (CH2)mC(=0)X2X1C(=0)-**;
-C(=0)0(CH2)mX3(CH2)m-**; -C(=0)0(CH2)mX6C(=0)XiX2C(=0)((CH2)mO)n(CH2)m-**; - C(=0)0((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)nX3(CH2)m-**; -C(=0)0((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mC(=0)NR1 1(CH2)m-**; -C(=0)0(CH2)mC(R12)2-**;
-C(=0)OCH2)mC(R12)2SS(CH2)mNR1 1C(=0)(CH2)m-**;
-C(=0)0(CH2)mC(=0)NR11(CH2)m-**; -C(=0)(CH2)m-**; -C(=0)((CH2)mO)n(CH2)m-**; -C(=0)(CH2)mNR11(CH2)m-**; -C(=0)(CH2)mNR11(CH2)mC(=0)X2X1C(=0)-**;
-C(=0)(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)(CH2)mNR11C(=0)(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**; -
C(=0)(CH2)mNR11C(=0(CH2)mX3(CH2)m-**; -(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;
-(CH2)m(CHOH)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m **;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)nX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mC(=0)NR11(CH2)m-**; -
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-*i;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-
-C(=0)((CH2)mO)n(CH2)mNR11C(=0))X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-";
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)m-
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-*i;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mNR11((CH2)mO)n(CH2)m-**; -
C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)m-**; - C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**; -
C(=0)(CH2)mC(R12)2SS(CH2)mNR11C(=0)(CH2)m-**; -C(=0)(CH2)mC(=0)NR11(CH2)m-**;
-C(=0)X1X2C(=0)(CH2)m-**; -C(=0)X1X2C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X1X2C(=0)(CH2)mX3(CH2)m-"; -C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mX3(CH2)m-*i;
-C(=0)X1X2C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)X1X2C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)X1X2(CH2)mX3(CH2)m-*i; -C(=0)X1X2((CH2)mO)n(CH2)m-ii;
-C(=0)X1X2((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X1X2((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**; -C(=0)X1X2((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)X1X2(CH2)mNRl 1((CH2)mO)n(CH2)m-**; -
C(=0)X1X2C(=0)(CH2)mNR11 ((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)NR1 1(CH2)m-**; -C(=0)NR11 (CH2)mX3(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)0(CH2)m-**; -C(=0)NR11 (CH2)mNR11C(=0)X,X2-**; -
C(=0)NR11 (CH2)mNR11C(=0)X5-; -C(=0)NR11(CH2)mNR11C(=0)(CH2)mX5(CH2)m-**; -
C(=0)X1C(=0)NR11(CH2)mX5(CH2)m-**; -C(=0)X6C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-
-C(=0)NR1 1(CH2)mNR11C(=0)(CH2)m-**; -C(=0)NR11 (CH2)mNR11C(=0)(CH2)mO(CH2)m-**; -C(=0)NR1 1(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-**;
-C(=0)NR11(CH2)mNR11C(=0)X4C(=0)NR1 1(CH2)mNR11C(=0)(CH2)mO(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -
C(=0)NR11 (CH2)mNR11C(=0)X5C(=0)(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)NRl 1(CH2)mNR11C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR1 lC(=0)(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-*i; -C(=0)NR1 1(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)m-*i;
-C(=0)NR1 1(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-";
-C(=0)NR1 1(CH2)mNR11C(=0)X5(CH2)mNR11 ((CH2)mO)n(CH2)m-**; -
C(=0)NR11 (CH2)mNR11C(=0)X5C(=0)(CH2)mNR11 ((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)NRl 1(CH2)mNR11C(=0)X5(CH2)m-**; -
C(=0)NR11 (CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**; -
C(=0)X1C(=0)NR11(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X1C(=0)NR11 (CH2)mX3(CH2)m-**; -C(=0)NR11 (CH2)mNR11C(=0)(CH2)m-**;
-C(=0)NR1 1(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**; -C(=0)NR11 (CH2)mNR11C(=0)-**;
-C(=0)X1X2(CH2)m-**; -C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**; -C(=0)X1X2(CH2)mX3(CH2)m-**; -C(=0)NR11(CH2)mX3(CH2)m-**; - C(=0)NR11((CH2)mO)n(CH2)mX3(CH2)m-**; -C(=0)X1X2C(=0)((CH2)mO)n(CH2)m- -C(=0)X1X2C(=0)(CH2)m-**; -C(=0)X,C(=0)(CH2)mNR11C(=0)(CH2)m-**; and -C(=0)X1C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
Xi is chment to X2; X2 is
* indicates the point of attachment to Xi or to NR11 ;
X4 is -0(CH2)nSSC(R12)2(CH2)n- or -(CH2)nC(R12)2SS(CH2)nO-; X5 is where the ** indicates orientation toward the Drug moiety;
X6 is or, where the ** indicates orientation toward the Drug moiety;
R17 is 2-pyridyl or 4-pyridyl;
each R11 is independently selected from H and CrCealkyl;
each R12 is independently selected from H and d-Cealkyl;
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, CrCealkyl, F, CI, and -OH;
each R111 is independently selected from H, CrC6alkyl, F, CI, -NH2, -OCH3, -OCH2CH3, -
N(CH3)2, -CN, -N02 and -OH;
each R112 is independently selected from H, C1-6alkyl, fluoro, benzyloxy substituted with - C(=0)OH, benzyl substituted with -C(=0)OH, CMalkoxy substituted with -C(=0)OH and C^. 4alkyl substituted with -C(=0)OH;
and provided at least one of R1 , R1a or R1b is substituted with -NHL1R15, or at least one of R3, R4,
R5, R7, R3a, R a, R5a or R7a is -OLiR15.
In some embodiments l_i is -C(=0)0(CH2)mNR11C(=0)(CH2)m-**; - C(=0)0(CH2)mNR11C(=0)(CH2)mO(CH2)m-**; -C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -
C(=0)0C(R12)2(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -
C(=0)0(CH2)mNR6C(=0)X1X2C(=0)(CH2)mO(CH2)m-**; -
C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=O *; -
C(=0)0(CH2)mNR11C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0)XsC(=0)(CH2)mNR11C(=0)(CH2)m-**; -
C(=0)0(CH2)mX6C(=0)XiX2C(=0)((CH2)mO)n(CH2)m-**; -(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-
**; -(CH2)m(CH0H)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m**; -
C(=0)X6C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -
C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**; - C(=0)(CH2)mNR1 1C(=0)X1X2C(=0)(CH2)m-**; -C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**, or -
C(=0)(CH2)mNR1 1C(=0)((CH2)mO)n(CH2)m-**, where the ** indicates the point of attachment to R15.
68
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75 BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-10 are a series of a tables listing various transcripts in HCC1954 cells whose expression increased by at least 5-fold upon exposure to either cGAMP or compound T1 -1.
FIG. 2A-2L are a series of tables listing various transcripts in THP1 dual cells whose expression increased by at least 5-fold upon exposure to compound T1 -2.
FIG. 3 is a line graph showing STING agonist payload retention as determined by IPMS (intact protein mass spectrometry).
FIG. 4 is a line graph showing the anti-HER2-STING agonist conjugates induce IP-10 secretion from HER2+ HCC1954 breast cancer cells.
FIG. 5 is a line graph showing the anti-HER2 mAb1 -C1 conjugate inhibits N87 gastric tumor growth in mice.
FIG. 6 is a line graph showing the anti-HER2 mAb1 -C1 conjugate is well tolerated in the N87 gastric tumor xenograft mice.
FIG. 7 is a line graph showing the anti-HER2 mAb1 -C1 conjugate inhibits HCC1954 breast tumor growth in mice.
FIG. 8 is a line graph showing the anti-HER2 mAb1 -C1 conjugate is well tolerated in the HCC1954 breast tumor xenograft mice.
FIG. 9A is a line graph showing the anti-HER2 mAb1-C1 conjugate inhibits SKOV3 ovarian carcinoma growth in mice.
FIG. 9B is a line graph showing the anti-HER2 mAb1-C1 conjugate is well tolerated in the SKOV3 ovarian carcinoma xenograft mice.
FIG. 10 illustrates certain compounds which can be used as a Drug moiety.
FIG. 1 1 illustrates certain compounds which can be used as a Drug moiety.
FIG. 12 illustrates certain compounds which can be used as a Drug moiety.
FIG. 13A is a graph depicting efficacy of P-Cad mAb1 -C1 conjugate in an C38 murine colon adenocarcinoma model in mice.
FIG. 13B is a graph showing that P-Cad mAb1 -C1 conjugate is well tolerated in the MC38 murine colon adenocarcinoma model in mice.
FIG. 14 is a graph depicting efficacy of Target B mAb1 -C1 in a breast cancer xenograft model in mice.
FIG. 15 is a graph depicting efficacy of Target C mAb1-C1 conjugate in a lung cancer xenograft model in 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 "CrC6alkyl", 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 molecule by a single bond. Non-limiting examples of "CrCealkyl" groups include methyl, ethyl, 1 -methylethyl , n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl and hexyl.
The term "C2-C6alkenyr, as used herein, refers to a straight or branched hydrocarbon chain radical group consisting solely 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 molecule by a single bond. Non-limiting examples of "C2-C6alkenyl" groups include ethenyl, prop-1 -enyl, but-1 -enyl, pent-1 -enyl, pent-4-enyl and penta-1 ,4-dienyl.
The term "C2-C6alkynyl", as used herein, refers to a straight or branched hydrocarbon chain radical group consisting solely 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 molecule by a single bond. Non-limiting examples of "C2-C6alkynyl" groups include ethynyl, prop-1-ynyl, but-1 -ynyl, pent-1 -ynyl, pent-4-ynyl and penta-1 , 4-diynyl.
The term "CrCealkylene", 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 "C2-C6alkenyl", as used herein, refers to a bivalent straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to six carbon atoms.
The term "C2-C6alkynyl", as used herein, refers to a bivalent straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having from two to six carbon atoms.
The term "C^alkoxyalkyl ", as used herein, refers to a radical of the formula -Ra-O-Ra, where each Ra is independently a Ci-6alkyl radical as defined above. The oxygen atom may be bonded to any carbon atom in either alkyl radical. Examples of include, but are not limited to, methoxy-methyl, methoxy-ethyl, ethoxy-ethyl, 1 -ethoxy-propyl and 2-methoxy-butyl.
The term "CrC6hydroxyalkyl", as used herein, refers to a C1-6alkyl radical as defined above, wherein one of the hydrogen atoms of the C1-6alkyl radical is replaced by OH. Examples of hydroxyC^alkyl include, but are not limited to, hydroxy-methyl, 2-hydroxy-ethyl, 2-hydroxy- propyl, 3-hydroxy-propyl and 5-hydroxy-pentyl The term "C3-C8cycloalkyl," as used herein, refers to a saturated, monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic ring system. Non-limiting examples of fused bicyclic or bridged polycyclic ring systems include bicyclo[1 .1 .1 ]pentane, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[3.2.1 ]octane, bicyclo[2.2.2]octane and adamantanyl. Non-limiting examples monocyclic C3-C8cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups.
The term "CrC6haloalkyl", as used herein, refer to the respective "CrC6alkyl", as defined herein, wherein at least one of the hydrogen atoms of the "CrC6alkyl" is replaced by a halo atom. The CrC6haloalkyl groups can be monoCrC6haloalkyl, wherein such CrCshaloalkyl groups have one iodo, one bromo, one chloro or one fluoro. Additionally, the CrC6haloalkyl groups can be diCrC6haloalkyl wherein such CrCehaloalkyl groups can have two halo atoms independently selected from iodo, bromo, chloro or fluoro. Furthermore, the CrCshaloalkyl groups can be polyCrC6haloalkyl wherein such CrC6haloalkyl groups can have two or more of the same halo atoms or a combination of two or more different halo atoms. Such polyCr C6haloalkyl can be perhaloCrC6haloalkyl where all the hydrogen atoms of the respective d- C6alkyl have been replaced with halo atoms and the halo atoms can be the same or a combination of different halo atoms. Non-limiting examples of CrC6haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, trifluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl.
The term "C2-C6haloalkenyl", as used herein, refer to the respective "CrC6alkenyl", as defined herein, wherein at least one of the hydrogen atoms of the "CrC6alkenyl" is replaced by a halo atom. The C2-C6haloalkenyl groups can be monoCrC6haloalkenyl, wherein such Cr C6haloalkenyl groups have one iodo, one bromo, one chloro or one fluoro. Additionally, the C2- C6haloalkenyl groups can be diC2-C6haloalkenyl wherein such C2-C6haloalkenyl groups can have two halo atoms independently selected from iodo, bromo, chloro or fluoro. Furthermore, the C2-C6haloalkenyl groups can be polyC2-C6haloalkenyl wherein such C2-C6haloalkenyl groups can have two or more of the same halo atoms or a combination of two or more different halo atoms.
The term "C2-C6haloalkynyl", as used herein, refer to the respective "CrC6alkynyl", as defined herein, wherein at least one of the hydrogen atoms of the "CrC6alkynyl" is replaced by a halo atom. The C2-C6haloalkynyl groups can be monoCrC6haloalkynyl, wherein such Cr C6haloalkynyl groups have one iodo, one bromo, one chloro or one fluoro. Additionally, the C2- C6haloalkynyl groups can be diC2-C6haloalkynyl wherein such C2-C6haloalkynyl groups can have two halo atoms independently selected from iodo, bromo, chloro or fluoro. Furthermore, the C2-C6haloalkynyl groups can be polyC2-C6haloalkynyl wherein such C2-C6haloalkenyl groups can have two or more of the same halo atoms or a combination of two or more different halo atoms.
The term "heteroalkyl", as used herein, refers to an "alkyl" moiety wherein at least one of the carbon atoms has been replaced with a heteroatom such as 0 S, or N.
The term "3 to 6 membered 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, 0 or -S-, wherein R16 is Ci-Cealkyl. Non-limiting examples of 3-6 membered heterocycloalkyl groups, as used herein, include aziridin-1 -yl, 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-yl, thietanyl, thietan-2-yl, thietan-3-yl, thietan-4-yl, pyrrolidinyl, pyrrolidin-1 -yl, pyrrol id in-2-yl, pyrrolidin-3-yl, pyrrolidin-4-yl, pyrrolidin-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-yl, tetrahydropyran-3-yl, tetrahydropyran-4- yl, tetrahydropyran-5-yl, tetrahydropyran-6-yl, tetrahydrothiopyranyl, tetrahydrothiopyran-2-yl, tetrahydrothiopyran-3-yl, tetrahydrothiopyran-4-yl, tetrahydrothiopyran-5-yl, tetrahydrothiopyran-
6- yl, piperazinyl, piperazin-1 -yl, piperazin-2-yl, piperazin-3-yl, piperazin-4-yl, piperazin-5-yl, piperazin-6-yl, morpholinyl, morpholin-2-yl, morpholin-3-yl, morpholin-4-yl, morpholin-5-yl, morpholin-6-yl, thiomorpholinyl, thiomorpholin-2-yl, thiomorpholin-3-yl, thiomorpholin-4-yl, thiomorpholin-5-yl, thiomorpholin-6-yl, oxathianyl, oxathian-2-yl, oxathian-3-yl, oxathian-5-yl, oxathian-6-yl, dithianyl, dithian-2-yl, dithian-3-yl, dithian-5-yl, dithian-6-yl, dioxolanyl, dioxolan-2- yl, dioxolan-4-yl, dioxolan-5-yl, thioxanyl, thioxan-2-yl, thioxan-3-yl, thioxan-4-yl, thioxan-5-yl, dithiolanyl, dithiolan-2-yl, dithiolan-4-yl, dithiolan-5-yl, pyrazolidinyl, pyrazolidin-1 -yl, pyrazolidin- 2-yl, pyrazolidin-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, C1-6alkyl, C^alkoxy, amino, C^alkylamino, di-C^alkylamino. 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-b]pyridinyl, pyrrolo[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, pyrazolo[4,3-c]pyridinyl, pyrazolo[3,4- c]pyridinyl, pyrazolo[3,4-d]pyridinyl, pyrazolo[3,4-b]pyridinyl, imidazo[1 ,2-a]pyridinyl, pyrazolo[1 ,5-a]pyridinyl, pyrrolo[1 ,2-b]pyridazinyl, imidazo[1 ,2-c]pyrimidinyl, pyrido[3,2- d]pyrimidinyl, pyrido[4,3-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrido[2,3-d]pyrimidinyl, pyrido[2,3-b]pyrazinyl, pyrido[3,4-b]pyrazinyl, pyrimido[5,4-d]pyrimidinyl, pyrazino[2,3-
Additionally, bicyclic heterocyclyl ring systems include heterocyclyl ring systems wherein one of the fused rings is aromatic but the other is non-aromatic. For such systems, the heterocyclyl is said to be partially saturated. Examples of partially saturated bicyclic 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-yl-6-one. Other examples of partially saturated bicyclic system are
Heterocyclyl also includes a 5- or 6- membered ring aromatic heterocyclyl having 2 to 3 heteroatom (preferably nitrogen) (also referred to as 5- to 6-membered heteroaryl). Examples of monocyclic heteroaryl are: imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, 1 , 2, 3-oxadiazolyl, 1 ,2,4- oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,5- thiadiazolyl, 1 ,3,4-thiadiazolyl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl, oxazol-2-yl, oxazol- 4-yl, oxazol-5-yl, isoxazol-3-yl, isoxazol-4-yl, isoxazol-5-yl, 1 ,2,4-triazol-3-yl, 1 ,2,4-triazol-5-yl, 1 ,2, 3-triazol-4-yl, 1 ,2, 3-triazol-5-yl, tetrazolyl, pyrid-2-yl, pyrid-3-yl, 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.
Heterocyclyl also includes 6-membered monocyclic partially saturated ring having 1-3 heteroatoms (preferably nitrogen). Examples of partially saturated monocyclic heterocyclyl are pyrimidine-one and pyrimidine-dione, specifically pyrimidin-2(1 H)-one and pyrimidin-1 -yl-2,4(1 H, 3H)-dione.
Heterocyclyl can exist in various tautomeric forms. For example, when a heterocyclyl 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 follow:
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.
Similarly, phosphoric acid exists as 2 tautomeric forms which interconvert in an equilibrium.
Additional examples of tautomers are phosporothioic acid which can exist in an equilibrium as shown below.
Similarly, phosphoric acid exists as 2 tautomeric forms which interconvert in an equilibrium.
In addition the phosporothioic acid and phosphoric acid moieties can exist in the respective equilibrium as shown below.
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, alkenes, alkynes and azides. In certain embodiments, such functional groups include reactive groups of Table 5 provided herein.
The term "sugar moiety", as used herein, refers to the following ring structures of the compounds of the invention , wherein Y Y2 and Y3 are each independently selected from -0-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2-.
As used herein, when partial structures of the compounds are illustrated a wavy line ( Tvw ) 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 17q12, 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%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 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 erb-b2 receptor tyrosine kinase 2 (ERBB2), transcript variant 1 , mRNA [NM_004448.3]
1 gcttgctccc aatcacagga gaaggaggag gtggaggagg agggctgctt gaggaagtat 61 aagaatgaag ttgtgaagct gagattcccc tccattggga ccggagaaac caggggagcc 121 ccccgggcag ccgcgcgccc cttcccacgg ggccctttac tgcgccgcgc gcccggcccc 181 cacccctcgc agcaccccgc gccccgcgcc ctcccagccg ggtccagccg gagccatggg
241 gccggagccg cagtgagcac catggagctg gcggccttgt gccgctgggg gctcctcctc 301 gccctcttgc cccccggagc cgcgagcacc caagtgtgca ccggcacaga catgaagctg 361 cggctccctg ccagtcccga gacccacctg gacatgctcc gccacctcta ccagggctgc 421 caggtggtgc agggaaacct ggaactcacc tacctgccca ccaatgccag cctgtccttc 481 ctgcaggata tccaggaggt gcagggctac gtgctcatcg ctcacaacca agtgaggcag
541 gtcccactgc agaggctgcg gattgtgcga ggcacccagc tctttgagga caactatgcc 601 ctggccgtgc tagacaatgg agacccgctg aacaatacca cccctgtcac aggggcctcc 661 ccaggaggcc tgcgggagct gcagcttcga agcctcacag agatcttgaa aggaggggtc 721 ttgatccagc ggaaccccca gctctgctac caggacacga ttttgtggaa 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 tgctgccggc tgcacgggcc ccaagcactc tgactgcctg 1021 gcctgcctcc acttcaacca cagtggcatc tgtgagctgc actgcccagc cctggtcacc 1081 tacaacacag acacgtttga gtccatgccc aatcccgagg gccggtatac attcggcgcc 1 141 agctgtgtga ctgcctgtcc ctacaactac ctttctacgg acgtgggatc ctgcaccctc 1201 gtctgccccc tgcacaacca agaggtgaca gcagaggatg gaacacagcg gtgtgagaag 1261 tgcagcaagc cctgtgcccg agtgtgctat ggtctgggca tggagcactt gcgagaggtg 1321 agggcagtta ccagtgccaa tatccaggag tttgctggct gcaagaagat ctttgggagc 1381 ctggcatttc tgccggagag ctttgatggg gacccagcct ccaacactgc cccgctccag 1441 ccagagcagc tccaagtgtt tgagactctg gaagagatca caggttacct atacatctca 1501 gcatggccgg acagcctgcc tgacctcagc gtcttccaga acctgcaagt aatccgggga 1561 cgaattctgc acaatggcgc ctactcgctg accctgcaag ggctgggcat cagctggctg 1621 gggctgcgct cactgaggga actgggcagt ggactggccc tcatccacca taacacccac 1681 ctctgcttcg tgcacacggt gccctgggac cagctctttc ggaacccgca ccaagctctg 1741 ctccacactg ccaaccggcc agaggacgag tgtgtgggcg agggcctggc ctgccaccag 1801 ctgtgcgccc gagggcactg ctggggtcca gggcccaccc agtgtgtcaa ctgcagccag 1861 ttccttcggg gccaggagtg cgtggaggaa tgccgagtac tgcaggggct ccccagggag 1921 tatgtgaatg ccaggcactg tttgccgtgc caccctgagt gtcagcccca gaatggctca 1981 gtgacctgtt ttggaccgga ggctgaccag tgtgtggcct gtgcccacta taaggaccct 2041 cccttctgcg tggcccgctg ccccagcggt gtgaaacctg acctctccta catgcccatc 2101 tggaagtttc cagatgagga gggcgcatgc cagccttgcc ccatcaactg cacccactcc 2161 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 2461 gtctacaagg gcatctggat ccctgatggg gagaatgtga aaattccagt ggccatcaaa 2521 gtgttgaggg aaaacacatc ccccaaagcc aacaaagaaa tcttagacga agcatacgtg 2581 atggctggtg tgggctcccc atatgtctcc cgccttctgg gcatctgcct gacatccacg 2641 gtgcagctgg tgacacagct tatgccctat ggctgcctct tagaccatgt ccgggaaaac 2701 cgcggacgcc tgggctccca ggacctgctg aactggtgta tgcagattgc caaggggatg 2761 agctacctgg aggatgtgcg gctcgtacac agggacttgg ccgctcggaa cgtgctggtc 2821 aagagtccca accatgtcaa aattacagac ttcgggctgg ctcggctgct ggacattgac 2881 gagacagagt accatgcaga tgggggcaag gtgcccatca agtggatggc 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 actgatggct acgttgcccc 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 gggaggagct gcccctcagc cccaccctcc tcctgccttc 3901 agcccagcct tcgacaacct ctattactgg gaccaggacc caccagagcg gggggctcca 3961 cccagcacct tcaaagggac acctacggca gagaacccag agtacctggg tctggacgtg 4021 ccagtgtgaa ccagaaggcc aagtccgcag aagccctgat gtgtcctcag ggagcaggga 4081 aggcctgact tctgctggca tcaagaggtg ggagggccct ccgaccactt ccaggggaac 4141 ctgccatgcc aggaacctgt cctaaggaac cttccttcct gcttgagttc ccagatggct 4201 ggaaggggtc cagcctcgtt ggaagaggaa cagcactggg gagtctttgt ggattctgag 4261 gccctgccca atgagactct agggtccagt ggatgccaca gcccagcttg gccctttcct 4321 tccagatcct gggtactgaa agccttaggg aagctggcct gagaggggaa gcggccctaa 4381 gggagtgtct aagaacaaaa gcgacccatt cagagactgt ccctgaaacc tagtactgcc
4441 ccccatgagg aaggaacagc aatggtgtca gtatccaggc tttgtacaga gtgcttttct
4501 gtttagtttt tacttttttt gttttgtttt tttaaagatg aaataaagac ccagggggag
4561 aatgggtgtt gtatggggag gcaagtgtgg ggggtccttc tccacaccca ctttgtccat
4621 ttgcaaatat attttggaaa acagctaaaa aaaaaaaaaa aaaa (SEQ ID NO: 25)
Receptor tyrosine-protein kinase erbB-2 isoform a precursor [Homo sapiens] [NP_004439.2]
MELAALCRWG LLLALLPPGA ASTQVCTGTD MKLRLPASPE THLDMLRHLY
QGCQWQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC AAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP
YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL
PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAWG ILLWVLGW FGILIKRRQQ KIRKYTMRRL LQETELVEPL TPSGAMPNQA QMRILKETEL RKVKVLGSGA FGTVYKGIWI PDGENVKIPV AIKVLRENTS PKANKEILDE AYVMAGVGSP YVSRLLGICL TSTVQLVTQL
MPYGCLLDHV RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR LVHRDLAARN VLVKSPNHVK ITDFGLARLL DIDETEYHAD GGKVPIKWMA LESILRRRFT HQSDVWSYGV TVWELMTFGA KPYDGIPARE IPDLLEKGER LPQPPICTID VYMIMVKCWM IDSECRPRFR ELVSEFSRMA RDPQRFWIQ NEDLGPASPL DSTFYRSLLE DDDMGDLVDA EEYLVPQQGF FCPDPAPGAG GMVHHRHRSS
STRSGGGDLT LGLEPSEEEA PRSPLAPSEG AGSDVFDGDL GMGAAKGLQS LPTHDPSPLQ RYSEDPTVPL PSETDGYVAP LTCSPQPEYV NQPDVRPQPP SPREGPLPAA RPAGATLERP KTLSPGKNGV VKDVFAFGGA VENPEYLTPQ GGAAPQPHPP PAFSPAFDNL YYWDQDPPER GAPPSTFKGT PTAENPEYLG LDVPV
(SEQ ID NO: 26)
The mRNA and protein sequences of the other human HER2 isoforms can be found in
GeneBank with the following Accession Nos:
HER2 isoform B: NM_001005862.2 (mRNA)→ NP_001005862.1 (protein);
HER2 isoform C: NM_001289936.1 (mRNA)→ NP_001276865.1 (protein);
HER2 isoform D: NM_001289937.1 (mRNA)→ NP_001276866.1 (protein);
HER2 isoform E: NM_001289938.1 (mRNA)→ NP_001276867.1 (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 CH3. 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 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant 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., IgG 1 , lgG2, lgG3, lgG4, lgA1 and lgA2) 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 CH 1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid 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, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1 126-1 136, 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-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-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), Al-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, 132-136 (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 CDR3, 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 "hypervariable 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
(HCDR2), 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., insertion(s) after position 30), 50-52 (LCDR2), and 91 -96 (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 amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Under IMGT, 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 (CDR3) (numbering according to "Kabat"). Under IMGT, 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, bispecific 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 germline 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 al. (2000. J Mol Biol 296, 57-86). The structures and locations of immunoglobulin variable domains, e.g., CDRs, may be defined using well 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.; Al Lazikani et al., (1997) J. Mol. Bio. 273:927 948); Kabat et al., (1991) Sequences of Proteins of
Immunological Interest, 5th edit., NIH 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 al., (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 germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The phrase "recombinant human antibody" as used herein, includes all 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 DNA 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, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
The term "Fc region" as used herein refers to a polypeptide comprising the CH3, 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 CH3) 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-covalent 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, isoleucine, proline, phenylalanine, methionine), beta- branched side chains (e.g., threonine, valine, isoleucine) 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 DNA molecules or two RNA molecules, or between two polypeptide molecules. When 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 10), 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 result 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.com), using either a Blossum 62 matrix or a PAM250 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.com), 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 Blossum 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 algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 1 1 -17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 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.ncbi.nlm.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, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial 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 cell, 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 cell. In some embodiments, a tumor antigen is a marker expressed by both normal cells and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cell 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 cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. 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 cell receptors (TCRs) on CD8 + 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 cell 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 cells.
A "HER2-positive cancer" or "HER2-expressing cancer" is a cancer comprising cells that have HER2 protein present at their cell surface. Many methods are known in the art for detecting or determining the presence of HER2 on a cancer cell. 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 least 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 superimposable 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- superimposable 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 (dextro- 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 "P-cadherin" (also known as P-Cadherin, P-cad, P-Cad, Pcad, PCad, Cadherin 3, Cadherin-3, Cad3, Cad-3, CAD3, CAD-3, CDH3 or CDH-3) refers to the nucleic acid and amino acid sequences of P-cadherin, which have been published in GenBank Accession Nos. NP_ 001784, NP_001784.2 (amino acid sequence), and N _001793.4, GenBank Accession Nos. AA14462, NG_009096, and NG_009096.1 (nucleotide sequences). Sequence information for human P-cadherin domains 1 -5 are extracellular and are published in GenBank Acession Nos. NM_001793.4 and NP_001784.
"P-cadherin" also refers to proteins and amino acid sequences that over their full length have at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of the above GenBank accession Nos.
NP_001784, NP_ 001784.2.
Structurally, a P-cadherin nucleic acid sequence has over its extracellular domain at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence of GenBank accession numbers NM_001793.4, GenBank Accession Nos. AA14462, NG_009096, and NG_009096.1 .
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 mRNA and protein sequences for human STING isoform 1 , the longest isoform, are:
Homo sapiens transmembrane protein 173 (TME 173), transcript variant 1 , mRNA
[NM_198282.3]
1 tataaaaata gctcttgtta ccggaaataa ctgttcattt ttcactcctc cctcctaggt 61 cacacttttc agaaaaagaa tctgcatcct ggaaaccaga agaaaaatat gagacgggga
121 atcatcgtgt gatgtgtgtg ctgcctttgg ctgagtgtgt ggagtcctgc tcaggtgtta
181 ggtacagtgt gtttgatcgt ggtggcttga ggggaacccg 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 cccaaatgcg 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 tgataacctg agtatggctg accccaacat tcgcttcctg gataaactgc cccagcagac
1021 cggtgaccat gctggcatca aggatcgggt ttacagcaac agcatctatg agcttctgga
1081 gaacgggcag cgggcgggca cctgtgtcct ggagtacgcc acccccttgc agactttgtt
1 141 tgccatgtca caatacagtc aagctggctt tagccgggag gataggcttg agcaggccaa
1201 actcttctgc cggacacttg aggacatcct ggcagatgcc 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 ccagtggtct ccaagcctct ggactggggg ctctcttcag tggctgaatg tccagcagag
1561 ctatttcctt 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 ttatactcca
2161 cccctttctc aacggtcctt ttttaaagca catctcagat tacccaaaaa aaaaaaaaaa
2221 aaa [SEQ ID NO: 180]
Homo sapiens stimulator of interferon genes protein isoform 1 [NP_938023.1]
MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVC SLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQ ALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAV SQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCV LEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSS FSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTST SQEPELLISGMEKPLPLRTDFS [SEQ ID NO: 181]
The mRNA and protein sequences for human STING isoform 2, a shorter isoform, are:
Homo sapiens transmembrane protein 173 (TME 173), transcript variant 2, mRNA
[NM_001301738.1]
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 cacctagcct ccctgcagct gggactgctg ttaaacgggg tctgcagcct ggctgaggag 301 ctgcgccaca tccactccag gtaccggggc agctactgga ggactgtgcg ggcctgcctg
361 ggctgccccc 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 ggaacctgca gatgacagca gcttctcgct gtcccaggag gttctccggc
901 acctgcggca ggaggaaaag gaagaggtta ctgtgggcag cttgaagacc tcagcggtgc
961 ccagtacctc cacgatgtcc caagagcctg agctcctcat cagtggaatg gaaaagcccc
1021 tccctctccg cacggatttc tcttgagacc cagggtcacc aggccagagc ctccagtggt
1081 ctccaagcct ctggactggg ggctctcttc agtggctgaa tgtccagcag agctatttcc
1 141 ttccacaggg ggccttgcag ggaagggtcc aggacttgac atcttaagat gcgtcttgtc
1201 cccttgggcc agtcatttcc cctctctgag cctcggtgtc ttcaacctgt gaaatgggat
1261 cataatcact gccttacctc cctcacggtt gttgtgagga ctgagtgtgt ggaagttttt
1321 cataaacttt ggatgctagt gtacttaggg ggtgtgccag gtgtctttca tggggccttc
1381 cagacccact ccccaccctt ctccccttcc tttgcccggg gacgccgaac tctctcaatg
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 acccccaccc cactagagta ctccctctca cttgcggttt ccttatactc cacccctttc
1741 tcaacggtcc ttttttaaag cacatctcag attacccaaa aaaaaaaaaa aaaaa [SEQ ID NO: 182]
Homo sapiens stimulator of interferon genes protein isoform 2 [NP_001288667.1 ]
MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVC SLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQ ALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAV SQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRNLQMT AASRCPRRFSGTCGRRKRKRLLWAA [SEQ ID NO: 183]
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.
hSTING wt (wild type): Reference SNP (refSNP) Cluster Report: rs1 131769
atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcc tggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgct gttaaacggggtctgcagcctggctgaggagctgcgccacatccactccaggtaccggggcagctactggaggactgtgcgggcct gcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcact tggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccccagctgagatctctgcagtgtgt gaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggccc ggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtatattctcctcccattggactgtggg gtgcctgataacctgagtatggctgaccccaacattcgcttcctggataaactgccccagcagaccggtgaccgtgctggcatcaagg atcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgc agactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggataggcttgagcaggccaaactcttctgccggacacttg aggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgct gtcccaggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagt acctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga [SEQ ID NO: 184] hSTING R293Q: Reference SNP (refSNP) Cluster Report: rs1131769 rs7380824
atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcc tggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgct gttaaacggggtctgcagcctggctgaggagctgcgccacatccactccaggtaccggggcagctactggaggactgtgcgggcct gcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcact tggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccccagctgagatctctgcagtgtgt gaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggccc ggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtatattctcctcccattggactgtggg gtgcctgataacctgagtatggctgaccccaacattcgcttcctggataaactgccccagcagaccggtgaccgtgctggcatcaagg atcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgc agactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggataggcttgagcaggccaaactcttctgccagacacttg aggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgct gtcccaggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagt acctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga [SEQ ID NO: 185] hSTING G230A/R293Q: Reference SNP (refSNP) Cluster Report: rs1131769 rs7380824 rs78233829 atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcc tggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgct gttaaacggggtctgcagcctggctgaggagctgcgccacatccactccaggtaccggggcagctactggaggactgtgcgggcct gcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcact tggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccccagctgagatctctgcagtgtgt gaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggccc ggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtatattctcctcccattggactgtggg gtgcctgataacctgagtatggctgaccccaacattcgcttcctggataaactgccccagcagaccgctgaccgtgctggcatcaagg atcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgc agactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggataggcttgagcaggccaaactcttctgccagacacttg aggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgct gtcccaggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagt acctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga [SEQ ID NO: 186] hSTING R71 H/G230A/R293Q: Reference SNP (refSNP) Cluster Report:
rs1131769 rs7380824 rs78233829 rs11554776
atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcc tggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgct gttaaacggggtctgcagcctggctgaggagctgcaccacatccactccaggtaccggggcagctactggaggactgtgcgggcct gcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcact tggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaagggcctggccccagctgagatctctgcagtgtgt gaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggccc ggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtatattctcctcccattggactgtggg gtgcctgataacctgagtatggctgaccccaacattcgcttcctggataaactgccccagcagaccgctgaccgtgctggcatcaagg atcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgc agactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggataggcttgagcaggccaaactcttctgccagacacttg aggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgct gtcccaggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagt acctccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga [SEQ ID NO: 187]
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-β, type 3 interferons, e.g., IFN , IP10, TNF, IL-6, 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 [3H]cGAMP 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 "prophylactically effective dose" or a "prophylactically effect amount", of the molecules 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 molecules 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. Immunostimulatorv Compounds of the Invention
Drug Moiety (D)
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 (
Formula (A) Formula (B)
Formula (C) Formula (D)
Formula (E) Formula (F) wherein:
each G
XA is C(=0)-, -C(=S)- or -C(=NR11)- and each Z^ is NR12;
XB is C, and each Z2 is N;
G2 is * indicates the point of attachment to -CR8aR9a-;
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-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2-;
Y2 is -0-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2-;
Y3 is OH, 0", OR10, N(R10)2, SR10, SeH, Se", BH3, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SR10, SeH, Se", BH3, SH or S";
Y5 is -CH2-, -NH-, -0- or -S;
Y6 is -CH2-, -NH-, -0- or -S;
Y7 is 0 or S;
Y8 is 0 or S;
Yg is -CHr, -NH-, -O- or -S;
Y10 is -CH2-, -NH-, -0- or -S;
Yii is -0-, -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 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 0, 1 , 2, 3 or 4 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, Ci-C6alkyl, CrCsalkoxyalkyl, CrC6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -©(C Cealkyl), -
0(C3-C8cycloalkyl), -S(CrCsalkyl), -StCrCsaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- Cgcycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -NCd-Cealkyl) (C3- Cgcycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
R1a 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 F, CI, Br, OH, SH, NH2, D, CD3, d-Csalkyl,
CrCsalkoxyalkyl, CrC6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -Oid-Cealkyl), - 0(C3-C8cycloalkyl), -S(CrCsalkyl), -StCrCsaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- Cgcycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -NCd-Cealkyl) (C3- C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,-
CH=CH(CH2)1.10C(=O)OH,-NHC(O)(C1-C3alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2; R1b 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 F, CI, Br, OH, SH, NH2, D, CD3, CrCsalkyl,
CrCsalkoxyalkyl, CrC6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), - 0(C3-C8cycloalkyl), -S(CrCsalkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- C8cycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(CrC6alkyl) (C3- Cscycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH,
CH=CH(CH2)1.10C(=O)OH,-NHC(O)(C1-C3alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
each R2 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C3haloalkenyl, C2- Cshaloalkynyl, -0(CrC8alkyl), -0(C2-C8alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, -
O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), -
0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R3 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C3haloalkenyl, C2-
C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)Ci-C6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and the CrC3alkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl,
CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R4 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C3haloalkenyl, C2- Cshaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), -
0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R5 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C3haloalkenyl, C2-
C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl,
CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R6 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6 and the
CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCi-C6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of Rs are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R7 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R8 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R8 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R9 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R9 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R2a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Ci- C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- Cshaloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3 R3a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC^Od-Cealkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -
OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCghaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R4a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, -
OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C3haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R5a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -
O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R6a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr
C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC^Od-Cealkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R6aare substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R7a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -
O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R8a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr
C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - 0C(0)0C2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R8a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2-
C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R9a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R9a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R10 is independently selected from the group consisting of H, d-C^alkyl, Cr
C6heteroalkyl, -(CH2CH20)nCH2CH2C(=0)0C1-C6alkyl, and , wherein the d C12alkyl and CrCeheteroalkyl of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, CrC12alkoxy, -S-C(=0)CrC6alkyl, halo, -CN, Cr C12alkyl, -O-aryl, _0-heteroaryl, -O-cycloalkyl, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, -OC^Od-Cealkyland C(0)OCrC6alkyl, wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is substituted by 0,1 , 2 or 3 substituents independently selected from CrC12 alkyl, 0-CrC12alkyl, d-C^heteroalkyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroaryl, -C(=0)C1-C12alkyl, -OC(=0)C C12alkyl, -C(=0)OCi- C12alkyl, -OC(=0)OCrC12alkyl, -C(=0)N(R11)-CrC12alkyl, -N(R11)C(=0)-CrC12alkyl; - OC(=0)N(R11)-CrC12alkyl, -C(=0)-aryl, -C(=0)-heteroaryl, -OC(=0)-aryl, -C(=0)0-aryl, OC(=0)-heteroaryl, -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(0)-aryl, - N(R11)C(0)-heteroaryl, and S(0)2N(R11)-aryl;
each R11 is independently selected from H and Chalky!;
each R12 is independently selected from H and CrCsalkyl;
optionally R3 and R6 are connected to form d-Calkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3 and R6 are connected, the 0 is bound at the R3 position
optionally R3a and R6a, are connected to form Crdalkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3a and R6a are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form d-Calkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2 and R3 are connected, the 0 is bound at the R3 position;
optionally R2a and R3a, are connected to form Crdalkylene, C2-C6alkenylene, C2- C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form Crdalkylene, C2-C6alkenylene, C2-
C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4 and R3 are connected, the 0 is bound at the R3 position; optionally R and R , are connected to form CrCsalkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form C^Cealkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R6 are connected, the 0 is bound at the R5 position;
optionally R5a and R6a, are connected to form CrQjalkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R6a are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form C^Cealkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R7 are connected, the 0 is bound at the R5 position;
optionally R5a and R7a, are connected to form CrCsalkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R7a are connected, the O is bound at the R5a position;
optionally R8 and R9 are connected to form a d-Calkylene, C2-C6alkenylene, C2- C6alkynylene, and
optionally R8a and R9a are connected to form a CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene.
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. 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
Formula (A-1) Formula (B-1)
Formula (E-1) Formula (F-1) wherein R1 , R1a, R1 b, R2, R2a, R3, R3a, R4, R4a, R5, R5a, R6, R6a, R7, R7a, R8, R8a, R9, Yt l Y2, Y3, Y4, Y5, Υβ, Y7, Ye, Y9, Y10 and Yn are as defined above for compounds of Formula (A) , Formula (B), Formula (C), Formula (D), Formula (E) and Formula (F).
Embodiment 2. A compound of Formula( 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 nucleic acid base or analogue thereof, and R1 is a pyrimidine or purine nucleic acid base or analogue thereof, each of which is substituted as described in R1 , R1a or R1 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).
Embodiment 3. A compound of Formula (A-2), Formula (B-2), Formula (C-2), Formula (D- 2), Formula (E-2) or Formula (F-2):
Formula (E-2) Formula (F-2) wherein R1 , R1a, R1 , R2, R2a, R3, R3a, R4, R4a, R5, R5a, R6, R6a, R7, R7a, R8, R8a, R9, Y, , Y2, Y3, Y4, Y5, Ye, Y7, Ys, Y9, Y10 and Y are as defined above for compounds of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) and Formula (F).
Embodiment 4. A compound of Formula (A), Formula (A-1) or Formula (A-2) of
Embodiment 1 , 2 or 3 wherein:
R2 and R2a are H; one of R3 and R4 is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R3 or R4 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the C^Cealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr C6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC^d-Cealkyl, -OC(0)C2-
C6alkenyl and -OC(0)C2-C6alkynyl of R3 or R4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R7 and R7a are H;
R6 and R6a are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R3a and R a is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(Ci-C3alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R3a and R a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC^d-Cealkyl, -OC(0)C2-
C6alkenyl and -OC(0)C2-C6alkynyl of R3a or R4a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3.
Embodiment 5. A compound of Formula (A), Formula (A-1) or Formula (A-2) of
Embodiment 1 , 2, 3 or 4 wherein:
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S;
Y9 and Y10 are 0 or S;
R2, R2a, R6, R6a, R7 and R7a are H;
one of R3a and R a is H and the other is H, OH or F; one of R3 and R4 is H and the other is H, OH or F; and
R8a, R9a, R8 and R9 are independently selected from H or CrCealk l.
Embodiment 6. A compound of Formula (B), Formula (B-1) or Formula (B-2) of
Embodiment 1 , 2 or 3 wherein:
R2 and R2a are H;
one of R3a and R4a is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -
OC(0)CrC6alkyl, -0C(0)C2-C6alkenyl and -0C(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R3a and R4a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr CsalkyI, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-
C6alkenyl and -OC(0)C2-C6alkynyl of R3a or R a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R7a and R6a are H;
Rs and R4 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R5 and R7 is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -
OC(0)CrC6alkyl, -0C(0)C2-C6alkenyl and -0C(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R5 and R7 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC8alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-
C6alkenyl and -OC(0)C2-C6alkynyl of R5 or R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3.
Embodiment 7. A compound of Formula (B), Formula (B-1) or Formula (B-2) of
Embodiment 1 , 2, 3 or 6 wherein:
ΥΪ and Y2 are O, CH2 or S;
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S"; Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S;
Yg and Y10 are 0 or S;
R2, R2a, R7a, RSa, R6 and R4 are H;
one of R3a and R4a is H and the other is H, OH or F;
one of R5 and R7 is H and the other is H, OH or F, and
R8a, R9a, R8 and R9 are independently selected from H or C1-C3alkyl.
Embodiment 8. A compound of Formula (C), Formula (C-1) or Formula (C-2) of
Embodiment 1 , 2 or 3 wherein:
R2 and R2a are H;
one of R3 and R4 is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R3 and R4 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC^CrCealkyl, -OC(0)C2- C6alkenyl and -0C(0)C2-C6alkynyl of R3 or R4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R4a and R6a are H;
R6 and R7 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl;
one of R5a and R7a is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(Ci-C3alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R5a and R7a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC^CrCealkyl, -OC(0)C2- C6alkenyl and -0C(0)C2-C6alkynyl of R5a or R7a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3. Embodiment 9. A compound of Formula (C), Formula (C-1) or Formula (C-2) of
Embodiment 1 , 2, 3 or 8 wherein:
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S;
Y9 and Yio are 0 or S;
R2, R2a, R4a, R8a, R6 and R7 are H;
one of R3 and R4 is H and the other is H, OH or F;
one of R5a and R7a is H and the other is H, OH or F, and
R8a Rga R8 a nd R9 gre jncjepencjentiy selected from H or C1-C6alkyl.
Embodiment 10. A compound of Formula (D), Formula (D-1) or Formula (D-2) of
Embodiment 1 , 2 or 3 wherein:
R2 and R2a are H;
one of R5a and R7a is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(Ci-C3alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R5a and R7a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC^d-Cealkyl, -OC(0)C2-
C6alkenyl and -OC(0)C2-C6alkynyl of R5a or R7a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R a and R6a are H;
R6 and R4 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R5 and R7 is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R5 and R7 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)Ci-Cealkyl, -OC(0)C2- C6alkenyl and -OC(0)C2-C6alkynyl of R5 or R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3.
Embodiment 11. A compound of Formula (D), Formula (D-1) or Formula (D-2) of
Embodiment 1 , 2, 3 or 10 wherein:
Y1 and Y2 are 0, CH2 or S;
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S;
Y9 and Y10 are 0 or S;
R2, R2a, R4a, RSa, R6 and R4 are H;
one of R5a, R7a is H and the other is H, OH or F;
one of R5 and R7 is H and the other is H, OH or F, and
R8, R9, R8a and R9a are independently H or CrC6alkyl.
Embodiment 12. A compound of Formula (E), Formula (E-1) or Formula (E-2) of
Embodiment 1 , 2 or 3 wherein:
R2 and R2a are H;
R6 and R6a are H;
R7a is H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R3a and R4a is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-
C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -0C(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R3a and R4a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC^CrCealkyl, -OC(0)C2- C6alkenyl and -OC(0)C2-C6alkynyl of R3a or R a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
one of R3 and R4 is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(Ci-Cealkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R3 and R4 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the
CrCsalkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr
C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC^CrCealkyl, -OC(0)C2- C6alkenyl and -OC(0)C2-C6alkynyl of R3 or R4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and one of R5 and R7 is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R5 and R7 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC^CrCealkyl, -OC(0)C2-
C6alkenyl and -0C(0)C2-C6alkynyl of R5 or R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3.
Embodiment 13. A compound of Formula (E), Formula (E-1) or Formula (E-2) of
Embodiment 1 , 2, 3 or 12 wherein:
Y1 and Y2 are O, CH2 or S;
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y5 is O or S;
Y7 is O or S;
Y9 is O or S;
R2, R2a, R5a, R8a, R6 and R7a are H;
one of R3a, R a is H and the other is H, OH, OCH3 or F;
one of R3, R4 is H and the other is H, OH, OCH3 or F;
one of R5 and R7 is H and the other is H, OH, OCH3 or F, and
R8, R9, R8a and R9a are independently H or CrC6alkyl.
Embodiment 14. A compound of Formula (F), Formula (F-1) or Formula (F-2) of
Embodiment 1 , 2 or 3 wherein:
R2 and R2a are H; each Re and Rea are H;
each R7a and R7 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R3a and R a is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-
C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R3a and R a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC^CrCealkyl, -OC(0)C2- C6alkenyl and -OC(0)C2-C6alkynyl of R3a or R4a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
one of R3 and R4 is H and the other is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(Ci-C3alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the - OC(0)Ophenyl of R3 and R4 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the C^Cealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCr C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC^d-Cealkyl, -OC(0)C2-
C6alkenyl and -OC(0)C2-C6alkynyl of R3 or R4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and
R5 is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr
C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C3haloalkenyl, C2- Cshaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrCealkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2- C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-
C6alkenyl), -0(C2-C6alkynyl), -OC^OCrCealkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl of R5 is substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3.
Embodiment 15. A compound of Formula (F), Formula (F-1) or Formula (F-2) of
Embodiment 1 , 2, 3 or 12 wherein:
Y1 and Y2 are 0, CH2 or S;
each Y3 is OH, 0", OR10, N(R10)2, SH or S";
each Y5 is O or S;
each Y7 is independently 0 or S;
each Yg is independently 0 or S;
Y11 is O, CH2 or S;
R2, R2a, R6, R6a, R6, R7 and R7a are H;
one of R3a, R4a is H and the other is H, OH, OCH3 or F;
one of R3, R4 is H and the other is H, OH, OCH3 or F;
R5 is H, OH, OCH3 or F, and
R8, R9, R8a and R9a are independently H or CrC6alkyl.
Embodiment 16. A compound of any one of Embodiments 1 to 15 wherein:
or wherein R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrCsalkyl, Cr
C6alkoxyalkyl, Ci-C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -CHCrCealkyl), - 0(C3-C8cycloalkyl), -S(CrCsalkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- Cgcycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -NCd-Csalkyl) (C3- Cgcycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- CH=CH(CH2)i-ioC(=0)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - -N(C3-C8cycloalkyl)2;
wherein: R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, Cr
C6alkoxyalkyl, CrCehydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -CKCrCealkyl), - 0(C3-C8cycloalkyl), -S(CrC3alkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-
Cgcycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -NCd-Csalkyl) (C3- Cgcycloalkyl), -CN, -P(=0)(0H)2, -(CH2)1.10C(=O)OH,- CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
and
, wherein R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrCsalkyl, Cr
C6alkoxyalkyl, CrC6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -C CrCealkyl), - 0(C3-C8cycloalkyl), -S(CrC3alkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- Cgcycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N d-Cealkyl) (C3- Cgcycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2.
Embodiment 17. A compound of Formula (A-3), Formula (B-3), Formula (C-3) , Formula -3), Formula (E-3) or Formula (F-3):
Formula (E-3) Formula (F-3)
ΥΪ is -0-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2-; Y2 is -0-, -S-, -S(=0)-, -SOr, -CH2-, or -CF2-;
Y is -0-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2-;
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
Y7 is 0 or S;
d from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, CrC6alkoxyalkyl, CrCehydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), -0(C3-C8cycloalkyl), -S(Cr C6alkyl), -S(CrC6aminoalkyl), -StCrCehydroxyalkyl), -S(C3-C8cycloalkyl), -NH(Cr C6alkyl), -NH(C3-C8cycloalkyl), -NCC Cealky^, -N(CrC6alkyl) (C3-C8cycloalkyl), -CN, - P(=0)(OH)2, -0(CH2)i-ioC(=0)OH, -(CH2)1.10C(=O)OH,-CH=CH(CH2)1.10C(=O)OH, - NHC(0)(d-C6alkyl), -NHC(0)(C3-C8cycloalkyl), -NHC(0)(phenyl), and -N(C3- C8cycloalkyl)2;
, wherein: R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, CrCsalkoxyalkyl, CrC6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -C^CrCsalkyl), - 0(C3-C8cycloalkyl), -S(CrC3alkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- Cgcycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -NCd-Cealkyl) (C3- Cgcycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
and
selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, d-Cealkoxyalkyl, Cr C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), -0(C3- Cgcycloalkyl), -S(CrC6alkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- C8cycloalkyl), -NH(d-C6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(Ci-Cealkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH,
CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
each R2 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R3 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R4 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C3haloalkenyl, C2- Cshaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R5 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, -
O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), -
0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R6 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C3haloalkenyl, C2-
C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)Ci-C6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of Rs and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl,
CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of Rs are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R7 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2- C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2- C6alkenyl), -0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl of R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; R2a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC^Od-Cealkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -
OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCghaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3
R3a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, -
OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C3haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R4a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -
O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R5a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr
C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC^Od-Cealkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R6a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - 0C(0)C2-C6alkenyl and -0C(0)C2-C6alkynyl of R6aare substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R7a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC^Od-Cealkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R10 is independently selected from the group consisting of H, CrCealkyl, -
, wherein the CrC12alkyl of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, Cr C12alkoxy, -S-C(=0)Ci-C6alkyl and C(0)OCrC6alkyl; optionally R3 and Rs are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3 and R6 are connected, the 0 is bound at the R3 position
optionally R3a and R6a, are connected to form CrCsalkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3a and R6a are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form d-Calkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2 and R3 are connected, the 0 is bound at the R3 position;
optionally R2a and R3a, are connected to form CrCsalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4 and R3 are connected, the 0 is bound at the R3 position;
optionally R a and R3a, are connected to form CrCsalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and Rs are connected to form CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R6 are connected, the O is bound at the R5 position;
optionally R5a and RSa, are connected to form CrCsalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R6a are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R7 are connected, the 0 is bound at the R5 position, and
optionally R5a and R7a, are connected to form CrCsalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R7a are connected, the O is bound at the R5a position.
Embodiment 18. The compound Formula (A-3) , or a pharmaceutically acceptable salt thereof, having the structure of Formula (A-4), or a pharmaceutically acceptable salt thereof:
Formula (A-4)
wherein: R , R , R , R , Rb, R , Y3 and Y4 are as defined in Embodiment 17.
Embodiment 19. 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:
Formula (A-4a) Formula (A-4b)
Formula (A-4c) Formula (A-4d) wherein: R1 , R1a, R3, R3a, R6 and R6a are as defined in Embodiment 17;
Y3 is OR10, N(R10)2, SH or S", and
Y4 is OR10, N(R10)2, SH or S".
Embodiment 20. 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-4I), Formula (A-4m), Formula (A-4n), Formula (A-4o) or Formula (A-4p), or a pharmaceutically acceptable salt thereof:
-4e) Formula (A-4f) Formula (A-4g)
-4h) Formula (A-4i) Formula (A-4j)
Formula (A-4n) Formula (A-4o) Formula (A-4p) wherein: R1 , R1a, R3, R3a, R6 and R6a are as defined in Embodiment 17;
Y3 is OR10, N(R10)2, SH or S", and
Y4 is OR10, N(R10)2, SH or S\
Embodiment 21. The compound of Formula (B-3) having the structure of Formula (B-4), a pharmaceutically acceptable salt thereof:
Formula (B-4)
wherein: R1 , R1a, R3, R3a, R5, R6a, Y3 and Y4 are as defined in Embodiment 17.
Embodiment 22. 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:
-4a) Formula (B-4b) Formula (B-4c)
Formula (B-4d)
wherein: R1 , R1a, R3a, R5 and R6a are as defined in Embodiment 13;
Y3 is OR10, N(R10)2, SH or S", and
Y4 is OR10, N(R10)2, SH or S".
Embodiment 23. 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:
Formula (B-4e) Formula (B-4f) Formula (B-4g)
Formula (B-4h)
wherein: R1 , R1a and R5 are as defined in Embodiment 17;
Y3 is OR10, N(R10)2, SH or S", and
Y4 is OR10, N(R10)2, SH or S".
Embodiment 24. The compound of Formula (C-3) having the structure of Formula (C-4), or a pharmaceutically acceptable salt thereof:
Formula (C-4)
wherein: R1 , R1a, R3, R5a, Rs, RSa, Y3 and Y4 are as defined in Embodiment 17.
Embodiment 25. 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 -4d), or a pharmaceutically acceptable salt thereof:
Formula (C-4a) Formula (C-4b) Formula (C-4c)
Formula (C-4d)
wherein: R1 , R1a, R3, R5a and R6 are as defined in Embodiment 17;
Y3 is OR10, N(R10)2, SH or S", and
Y4 is OR10, N(R10)2, SH or S".
Embodiment 26. 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 -4h), or a pharmaceutically acceptable salt thereof:
Formula (C-4h)
wherein: R1 , R1a and R5a are as defined in Embodiment 17;
Y3 is OR10, N(R10)2, SH or S", and
Y4 is OR10, N(R10)2, SH or S".
Embodiment 27. 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:
Formula (D-4)
wherein: R1 , R1a, R5, R5a, Y3 and Y4 are as defined in Embodiment 17.
Embodiment 28. 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 Formula
(D-4d), or a pharmaceutically acceptable salt thereof:
Formula (D-4a) Formula (D-4b) Formula (D-4c)
Formula (D-4d)
wherein: R1 , R1a, R5 and R5a are as defined in Embodiment 17;
Y3 is OR10, N(R10)2, SH or S", and
Y4 is OR10, N(R10)2, SH or S".
Embodiment 29. The compound of Formula (E-3), or a pharmaceutically acceptable salt thereof, having the structure eutically acceptable salt thereof:
Formula (E-4)
wherein: R1 , R1a, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 17.
Embodiment 30. 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:
Formula (E-4a) Formula (E-4b)
wherein: R1 , R1a, R3, R3a, R4, R a, R5 and R7 are as defined in Embodiment 17;
and
OR , N(R10)2, SH or S-
Embodiment 31. 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:
Formula (F-4)
wherein: R1 , R1a, R1 b, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 17.
Embodiment 32. 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:
-4a) -4b)
Formula (F-4c) Formula (F-4d) wherein: R1 , R1a, R1 b, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 17; and
each Y3 is independenly selected from OR10, N(R10)2, SH and S".
Embodiment 33. The compound of any one of Embodiments 1 to 32, wherein R
Embodiment 34. The compound of any one of Embodiments 1 to 32, wherein R1a is
Embodiment 35. The compound of any one of Embodiments 1 to 32, wherein R1 is
Embodiment 36. The compound of any one of Embodiments 1 to 32, wherein R1
Embodiment 37. The compound of any one of Embodiments 1 to 32, wherein R1a is
Em mpound of any one of Embodiments 1 to 32, wherein R1 is
Em und of any one of Embodiments 1 to 32, wherein R
Embodiment 40. The compound of any one of Embodiments 1 to 32 wherein R1
and R1a is τ Em of any one of Embodiments 1 to 32, wherein R1 is
■""T and R a is ι
Em of any one of Embodiments 1 to 32, wherein R1 is
and R1a is
Embodiment 43. The compound of any one of Embodiments 1 to 32, wherein R
and R a is i
Embodiment 44. The compound of any one of Embodiments 1 to 32, wherein R1 is
I , R is and R a is ι
Embodiment 45. The compound of any one of Embodiments 1 to 44, wherein:
Y3 is OH, 0", SH or S", and
Y4 is OH, 0", SH or S\
Embodiment 46. The compound of any one of Embodiments 1 to 44, wherein:
Y3 is OH or O", and
Y4 is OH or O"
Embodiment 47. The compound of any one of Embodiments 1 to 44, wherein:
Y3 is SH or S", and
Y4 is OH or 0"
Embodiment 48. The compound of any one of Embodiments 1 to 44, wherein:
Y3 is OH or 0", and
Y4 is SH or S"
Embodiment 49. The compound of any one of Embodiments 1 to 44, wherein:
Y3 is SH or S" , and
Y4 is SH or S"
Embodiment 50. The compound of any one of Embodiments 1 to 49 wherein: R3 is -OH or F;
R3a is -OH or F;
R5 is -OH or F;
R5a is -OH or F;
R6 is H, and
R6a is H.
Embodiment 51. The compound of any one of Embodiments 1 to 49 wherein
R3 is H, -OH or F;
R3a is H, -OCH3, -OH or F;
R5 is -OH or F;
R4, R4a, R6, R6a, R7, R7a are H, and
R6a is H.
Embodiment 52. A Drug moiety (D) is a compound of Table 1 :
Table 1
Embodiment 53. A Drug moiety (D) is a compound of Table 2:
Table 2
Embodiment 54. A Drug moiety (D) is a compound of Table 3: Table 3
Embodiment 55. The Drug moiety (D) is
Embodiment 56. The Drug moiety (D) is
Embodiment 57. The Drug moiety (D) is
5 Embodiment 58. The Drug moiety (D) is
Embodiment 59. The Drug moiety (D) is Em
Em
Embodiment 62. The Drug moiety (D) is Embodiment 63. The Drug moiety (D) is
Embodiment 64. The Drug moiety (D) is
Embodiment 65. The Drug moiety (D) is
Embodiment 66. The Drug moiety (D) is Embodiment 67. The Drug moiety (D) is
Embodiment 68. The Drug moiety (D) is
Embodiment 69. The Drug moiety (D) is
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Aduro (WO2016/145102).
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Aduro Biotech (WO2014/093936).
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Aduro and Novartis unpublished US Provisional application
USSN:62/362907 filed July 15, 2016 .
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Aduro and Novartis unpublished PCT application PCT/US2016/059506 filed 28 October 2016..
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Memorial Sloan Kettering et al (WO2014/179335). Such compounds are listed in Table 4.
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 4.
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Merck & Co (WO2017/027645). Such compounds are listed in Table 4.
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in GlaxoSmithKline (WO2015/185565). Such compounds are listed in Table 4.
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Brock University (WO2015/074145). Such compounds are listed in Table 4. In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Rutgers (US9315523). Such compounds are listed in Table 4.
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Spring Bank (WO2007070598, WO2017004499 and WO201701 1622). Such compounds are listed in Table 4.
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Invivogen (WO2016/096174. Such compounds are listed in Table 4.
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Regents of Univ. California and Aduro Biotech (WO2014/189805). Such compounds are disclosed herein in FIG. 10, FIG. 1 1 , and FIG. 12.
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Sperovie (WO2018009648).
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Sperovie (WO2018009652).
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Sperovie (WO2018013887).
In another aspect the Drug moiety (D) of the immunoconjugates of the invention are the compounds disclosed in Sperovie (WO2018013908). Each of the preceding applications are incorporated by reference in their entirety.
Table 4
as RR, RS, SR and SS diastereomers
172
174
175
177
181
182
Example synthesis of compounds of Formula (A)
Compounds of Formula (A) were made according to the synthetic description in
WO2016145102.
Specifically, (2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12R, 14aR)-2,9-bis(6-amino-9H-purin-9-yl)-3, 10- difluorooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine-5, 12- bis(thiolate) 5,12-dioxide (T1 -1 ), and (2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12S, 14aR)-2,9-bis(6- amino-9H-purin-9-yl)-3, 10-difluorooctahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine-5, 12-bis(thiolate) 5, 12-dioxide (T1-6) were synthesized according to the scheme below:
(T1-1) (T1-6)
Step 1 : Preparation of (2R,3R,4R,5R)-5-(6-benzamido-9 H-purin-9-yl)-4-fluoro-2- (hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate (2): To a solution of N-(9- ((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4- hydroxytetrahydrofuran-2-yl)-9-/-/-purin-6-yl)benzamide (1 , 2.0 g, 3.0 mmol, ChemGenes) in 1 ,4- dioxane (25 mL) and pyridine (8 mL) was added a solution of 2-Chloro-1 ,3,2- benzodioxaphosphorin-4-one (SalPCI) (0.84 g, 4.1 mmol) in 1 ,4-dioxane (12 mL). After 30 min, to the stirred reaction mixture at room temperature was introduced water (4 mL), and the resulting mixture was poured into a 1 N aqueous NaHC03 solution (100 mL). This aqueous mixture was extracted with EtOAc (3 x 100 mL) and the layers were partitioned. The EtOAc extracts were combined and concentrated to dryness in vacuo as a colorless foam. The colorless foam was dissolved in CH2CI2 (30 mL) to give a colorless solution. To this solution was added water (0.5 mL) and a 6% (v/v) solution of dichloroacetic acid (DCA) in CH2CI2 (30 mL). After ten min of stirring at room temperature, to the red solution was charged pyridine (3.5 mL). The resulting white mixture was concentrated in vacuo and water was removed as an azeotrope after concentration with MeCN (30 mL). This azeotrope process was repeated two more times with MeCN (30 mL). On the last evaporation, the resulting white slurry of compound 2 was left in MeCN (15 mL).
Step 2: Preparation of (2R,3R,4R,5R)-5-(6-benzamido-9 H-purin-9-yl)-2-((((((2R,3R,4R,5R)-5- (6-benzamido-9 H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4- fluorotetrahydrofuran-3-yl hydrogenphosphonate (4): To a solution of (2R,3R,4R,5R)-5-(6- benzamido-9 H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (3, 2.5 g, 2.9 mmol, ChemGenes) in MeCN (20 mL) was dried through concentration in vacuo. This process was repeated two more times to remove water as an azeotrope. On the last azeotrope, to the solution of compound 3 in MeCN (7 mL) was introduced ten 3A molecular sieves and the solution was stored under an atmosphere of nitrogen. To a stirred mixture of compound 2 with residual pyridin-1-ium dichloroacetate in MeCN (15 mL) was added the solution of compound 3 in MeCN (7 mL). After five min, to the stirred mixture was added 3-((dimethylamino- methylidene)amino)-3H-1 ,2,4-dithiazole-3-thione (DDTT) (650 mg, 3.2 mmol). After 30 min, the yellow mixture was concentrated in vacuo to give compound 4 as a yellow oil.
Step 3: Preparation of A/,/V'-(((2R,3R,3aR,7aR,9R,10R,10aR,12R,14aR)-5-(2-cyanoethoxy)- 3,10-difluoro-12-mercapto-12-oxido-5-sulfidooctahydro-2 H,7H-difuro[3,2-cf:3',2'- y][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine-2,9-diyl)bis(9 H-purine-9,6-diyl))dibenzamide( 5): To a solution of compound 4 in CH2CI2 (60 mL) were added water (0.35 mL) and a 6% (v/v) solution of dichloroacetic acid (DCA) in CH2CI2 (60 mL). After ten min at room temperature, to the red solution was introduced pyridine (20 mL). The resulting yellow mixture was concentrated in vacuo until approximately 20 mL of the yellow mixture remained. To the yellow mixture was introduced pyridine (20 mL) and the mixture was concentrated in vacuo until approximately 20 mL of the yellow mixture remained. To the yellow mixture was added pyridine (30 mL) and the mixture was concentrated in vacuo until approximately 30 mL of the yellow mixture remained. To the stirred yellow mixture in pyridine (30 mL) was added 2-chloro-5,5-dimethyl-1 ,3,2- dioxaphosphorinane-2-oxide (DMOCP) (1.6 g, 8.4 mmol). After seven min, to the dark orange solution was added water (1.4 mL), followed immediately by the introduction of 3H-1 ,2- benzodithiol-3-one (0.71 mg, 4.2 mmol). After five min, the dark orange solution was poured into a 1 N aqueous NaHC03 solution (400 mL). After ten min, the biphasic
mixture was extracted with EtOAc (200 mL) and diethyl ether (200 mL). After separation, the aqueous layer was back extracted with EtOAc (200 mL) and diethyl ether (200 mL). The organic extracts were combined and concentrated in vacuo. To the concentrated yellow oil was added toluene (75 mL) and the mixture was evaporated in vacuo to remove residual pyridine. This procedure was repeated twice with toluene (75 mL). The resulting oil was purified by silica gel chromatography (0% to 10% MeOH in CH2CI2) to provide compound 5 (67 mg, 2.5% yield) as an orange oil.
Step 4: Preparation of Compound (T1 -1 ): To a stirred solution of compound 5 (65 mg, 0.07 mmol) in MeOH (0.9 mL) was added aqueous ammonium hydroxide (0.9 mL) and the orange slurry was heated at 50 °C. After two hours, the orange solution was allowed to cool and concentrated in vacuo. The orange residue was purified by reverse phase silica gel chromatography (0% to 30% MeCN in 10 mM aqueous Triethylammonium acetate (TEAA) to obtain Compound (T1-1) (18 mg, 38% yield) as a white mono-triethylammonium salt after lyophilization. LCMS-ESI: 693.25 [M-H] - (calculated for C2oH22F2N1008P2S2 : 694.305); Rt: 16.698' min by HPLC conditions (10 mM TEAA, 2% to 20%); Rt : 20.026'. min by LCMS conditions (20 mM NH4OAc, 2% to 20%). 1H NMR (400 MHz, 45 °C, D20) δ 8.44 (s, 2H), 8.24 (s, 2H), 6.52 (d, J = 16.4 Hz, 2H), 5.80 (d, J = 3.6 Hz, 1 H), 5.67 (d, J = 4.0 Hz, 1 H), 5.37-5.26 (m, 2H), 4.77-4.65 (m, 4H), 4.22 (dd, J = 1 1 .4 Hz, 6.0 Hz, 2H), 3.34 (q, J = 7.0 Hz, 6H), 1.43 (t, J = 7.0 Hz, 9H). 19F NMR (400 MHz, 45 °C, D20) δ -200.74 to -200.98 (m). 31P NMR (45 °C, D20) δ 54.46.
The stereochemistry of this compound, as depicted was confirmed by the co-crystal structure bound to wild type STING protein.
The Rp.Sp isomer was also isolated after purification in the reverse phase chromatography step, to provide Compound (T1 -6) as the bistriethylammonium salt after lyophilization. LCMS- ESI: 693.30 [M-H]" (calculated for C2oH22F2N1008P2S2 : 694.05); Rt 13.830 min by HPLC conditions (10 mM TEAA, 2% to 20%). Rt 15.032 min by LCMS conditions (20 mM NH4OAc, 2% to 20%). 1H NMR. (400 MHz, 45 °C, D20) δ 8.65 (s, 1 H), 8.50 (s, 1 H), 8.34 (s, 1 H), 8.26 (s, 1 H), 6.58 (dd, J = 16.4, 2.8 Hz, 2H), 6.00 (dd, J = 51 .2, 3.6 Hz, 1 H), 5.69 (dd, J = 51.2, 3.8 Hz, 1 H), 5.32-5.15 (m, 2H), 4.77-4.67 (m, 3H), 4.61 (d, J = 12.4 Hz, 1 H), 4.25 (dd, J = 1 1 .8, 4.2 Hz, 2H), 3.33 (q, J = 7.2 Hz, 12H), 1 .43 (t, J = 7.2 Hz, 18H). 19F NMR (400 MHz, 45 °C, D20) δ -200.75 to -201 .31 (m). 31 P NMR (45 °C, D20) δ 54.69, 54.64.
Example synthesis of compounds of Formula (B)
Compounds of Formula (B) were made according to the synthetic description in WO2014189805. Specifically, Compound (T1-2), was synthesized according to the scheme below:
To a solution of 5 g (5.15 mmol) N -benzoyl-5'-0-(4, 4'-dimethoxytrityl)-2'-0-tert- butyldimethylsilyl-3'-0-[(2-cyanoethyl)-N, N-diisopropylaminophinyl]adenosine (1 ) in 25ml acetonitrile was added 0.18ml (10 mmole) water and 1.20 g (6.2 mmole) pyridinium
trifluoroacetate. After 5 minutes stirring at room temperature 25 ml tertbutylamine was added and the reaction stirred for 15 minutes at room temperature. The solvents were removed under reduced pressure to give (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl hydrogen phosphonate as a foam which was then coevaporated with acetonitrile (2x50 ml), then dissolved in 60 ml dichloromethane. To this solution was added water (0.9 ml, 50 mmole) and 60 ml of 6% (v/v) dichloroacetic acid (44 mmol) in dichloromethane. After 10 minutes at room temperature the reaction was quenched by the addition of pyridine (7.0 ml, 87 mmol), and concentrated to an oil which was dried by three co-evaporations with 40 ml anhydrous acetonitrile giving (2) in a volume of 12 ml.
N -benzoyl-5'-0-(4, 4'-dimethoxytrityl)-3'-0-tert-butyldimethylsilyl-2'-0-[(2-cyanoethyl)-N, N-diisopropylaminophinyl]adenosine ((3), 6.4 g, 6.6 mmole) was dissolved in 40 ml anhydrous acetonitrile and dried by three co-evaporations with 40 ml anhydrous acetonitrile, the last time leaving 20 ml. 3A molecular sieves were added and the solution stored under argon until used. Azeo dried (3) (6.4 g, 6.6 mmole) in 20 ml acetonitrile was added via syringe to a solution of (2) (5.15 mmol) in 12 ml of anhydrous acetonitrile. After 5 minutes stirring at room temperature, 1.14g (5.6 mmol) of 3-((N,N-dimethylaminomethylidene) amino)-3H-1 , 2,4-dithiazole-5-thione (DDTT) was added and the reaction stirred for 30 minutes at room temperature. The reaction was concentrated and the residual oil dissolved in 80 ml dichloromethane. Water (0.9 ml, 50 mmol) and 80 ml of 6% (v/v) dichloroacetic acid (58 mmol) in dichloromethane was added, and the reaction stirred for 10 minutes at room temperature. 50 ml pyridine was added to quench the dichloroacetic acid. The solvents were removed under reduced pressure to give crude
(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin- 9-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2- cyanoethoxy)phosphorothioyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl hydrogen phosphonate as a solid, which was then dissolved in 150 ml dry pyridine and concentrated down to a volume of approximately 100 ml. 2-chloro-5, 5-dimethyl-1 , 3,2- dioxaphosphorinane-2-oxide (DMOCP, 3.44 g, 18 mmole) was then added and the reaction stirred for 5 minutes at room temperature. 3.2 ml water was added immediately followed by addition of 3-H-1 ,2-benzodithio1 -3-one (1.3 g, 7.7 mmole), and the reaction stirred for 5 minutes at room temperature. The reaction mix was then poured into 700 ml water containing 20 g NaHC03 and stirred for 5 minutes at room temperature, then poured into a separatory funnel and extracted with 800 ml 1 : 1 ethyl acetate:diethyl ether. The aqueous layer was extracted again with 600 ml 1 : 1 ethyl acetate:diethyl ether. The organic layers were combined and concentrated under reduced pressure to yield approximately 1 1 g of an oil containing diastereoisomers (5a) and (5b). The crude mixture above was dissolved in dichloromethane and applied to a 250 g silica column. The desired diastereoisomers were eluted from the column using a gradient of ethanol in dichloromethane (0-10%). Fractions containing the desired diastereoisomers (5a) and (5b) were combined and concentrated, giving 2.26 g of approximately 50% (5a) and 50% (5b).
2.26g of crude (5a) and (5b) from the silica gel column was transferred to a thick-walled glass pressure tube. 60 ml methanol and 60 ml concentrated aqueous ammonia was added and the tube was heated with stirring in an oil bath at 50°C for 16 h. The reaction mixture was cooled to near ambient temperature, sparged with a stream of nitrogen gas for 30 minutes, and then transferred to a large round bottom flask. Most of the volatiles were removed under reduced pressure with caution so as to avoid foaming and bumping. If water was still present the residue was frozen and lyophilized to dryness. The lyophilized crude mixture was taken up in approximately 50ml of CH3CN/10 mM aqueous triethylammonium acetate (60/40). After 0.45 micron PTFE filtration, 4-5ml sample portions were applied to a C-18 Dynamax column
(40x250mm). Elution was performed with a gradient of acetonitrile and 10 mM aqueous triethylammonium acetate (30% to 50% CH3CN over 20 minutes at 50 ml/min flow). Fractions from the preparative HPLC runs containing pure (6) were pooled, evaporated to remove CH3CN and lyophilized to give 360mg of pure (6) (the RpRp diastereoisomer) as the bis- triethylammonium salt.
To 270 mg (0.24 mmol) of (6) was added 5.0 ml of neat trimethylamine trihydrofluoride. The mixture was stirred at room temperature for approximately 40 h. After confirming completion of reaction by analytical HPLC, the sample was neutralized by dropwise addition into 45 ml of chilled, stirred 1 M triethylammonium bicarbonate. The neutralized solution was desalted on a Waters C-18 Sep-Pak and the product eluted with CH3CN/10 mM aqueous triethylammonium acetate (5: 1).The CH3CN was evaporated under reduced pressure and the remaining aqueous solution was frozen and lyophilized. Multiple rounds of lyophilization from water gave 122 mg (57%) of (T1 -2) as the bis-triethylammonium salt. 1H NMR (500 MHz, 45 °C, (CD3)2SO-15 L D20) δ 8.58 (s, 1 H), 8.41 (s, 1 H), 8.18 (s, 1 H), 8.15 (s, 1 H), 6.12 (d, J= 8.0, 1 H), 5.92 (d, J = 7.0, 1 H), 5.30 (td, J= 8.5, 4.0, 1 H), 5.24-5.21 (m, 1 H), 5.03 (dd, J= 7.5, 4.5, 1 H), 4.39 (d, J= 4, 1 H), 4.23 (dd, J= 10.5, 4.0, 1 H), 4.18 (s, 1 H), 4.14-4.08 (m, 2H), 3.85-3.83 (m, 1 H), 3.73 (d, J= 12.0, 1 H), 3.06 (q, J= 7.5.12H), 1 .15 (t, J= 7.5, 1 H); 31 P NMR (200 MHz, 45 °C, (CD3)ISO- 15pL D20) 6 58.81 , 52.54; HRMS (FT-ICR) l/z calcd for C20H24O10N10P2S2 (M— H) 689.0521 , found 689.0514.
Example synthesis of compounds of Formula (A)
Synthesis of (2R,3R,3aS,5R,7aR,9S, 10R, 10aS, 12R, 14aR)-2,9-bis(6-amino-9/-/-purin-9-yl)-5, 12- dimercaptotetrahydro-2/-/,7H,9H, 14H-3, 14a: 10,7a-bis(epoxymethano)difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine 5, 12-dioxide (T2-45) and
(2R,3R,3aS,5R,7aR,9S, 10R, 10aS, 12S, 14aR)-2,9-bis(6-amino-9H-purin-9-yl)-5, 12- dimercaptotetrahydro-2/-/,7H,9H, 14H-3, 14a: 10,7a-bis(epoxymethano)difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine 5, 12-dioxide (T2-44), were prepared according to the following Scheme:
Step 1 : Preparation of (1 S,3R,4R,7S)-3-(6-benzamido-9/-/-purin-9-yl)-1 -(hydroxymethyl)-2,5- dioxabicyclo[2.2.1 ]heptan-7-yl hydrogen phosphonate (2): To a solution of (1 R,3R,4R,7S)-3-(6- benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5- dioxabicyclo[2.2.1 ]heptan-7-yl (2-cyanoethyl) diisopropylphosphoramidite (1 , 1 .0 g, 1 .2 mmol, Exiqon, Woburn, MA) in MeCN (10 mL) and H20 ( 0.05 mL) was added pyridinium
trifluoroacetate (270 g, 1 .5 mmol). After 25 min, to the stirring reaction mixture at room temperature was added ferf-butyl amine (5.0 mL). After 15 min, the reaction solution was concentrated in vacuo and water was removed as an azeotrope after concentration with MeCN (3 x 15 mL) to obtain a white foam. To a solution of the white foam in 1 ,4-dioxane (13mL) was added a solution of SalPCI (226 mg, 1 .0 mmol), in 1 ,4-dioxane (5 mL). After 7 min, to the cloudy white mixture was added pyridine (3 mL). After 1 h, to the cloudy reaction mixture was introduced water (2 mL). After 5 min, the mixture was poured into a 1 N NaHC03 solution (100 mL). The solution was extracted with EtOAc (3 x 100 mL) and the organic layer was condensed to dryness in vacuo. The residue was dissolved in CH2CI2 (10 mL) to give a white mixture. To this solution was added water (150 μΙ_) and 9 % (v/v) solution of DCA in CH2CI2 (10 ml_). After 10 min of stirring at room temperature, to the orange solution was charged pyridine (1.5 ml_). The resulting clear solution was concentrated in vacuo and water was removed as an azeotrope after concentration with eCN (3 x 20 ml_). On the last evaporation, the resulting cloudy slurry of compound 2 was left in MeCN (20 ml_).
Step 2: Preparation of (1 R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((((((1 R,3R,4R,7S)-3-(6- benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5- dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-2,5- dioxabicyclo[2.2.1]heptan-7-yl hydrogen phosphonate (3): A solution of compound 1 (1.0 g, 1.2 mmol, Exiqon) in MeCN (10 mL) was dried through concentration in vacuo. This process was repeated two more times to remove water as an azeotrope. On the last azeotrope, to the solution of compound 1 in MeCN (10 mL) was introduced ten 3A molecular sieves and the solution was stored under an atmosphere of nitrogen. To a stirred mixture of compound 2 with residual pyridinium dichloroacetate in MeCN (20 mL) was added the solution of compound 1 in MeCN (10 mL). After 40 min, to the stirred mixture was added DDTT (263 mg, 1.3 mmol). After 70 min, the yellow solution was concentrated in vacuo to give compound 3 as a yellow paste. Step 3: Preparation of N,N'-(((2S,3R,3aS,7aR,9R,10R,10aS,12R,14aR)-5-(2-cyanoethoxy)-12- mercapto-12-oxido-5-sulfidotetrahydro-2H,7H,9H, 14H-3, 14a: 10,7a- bis(epoxymethano)difuro[3,2-cf:3',2'-v][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine-2,9- diyl)bis(9H-purine-9,6-diyl))dibenzamide (4): To a solution of compound 3 in CH2CI2 (30 mL) were added water (180 μΐ) and 8.5 % (v/v) solution of DCA in CH2CI2 (20 mL). After stirring for 15 min at room temperature, to the red-orange solution was introduced pyridine (10 mL). The resulting yellow solution was concentrated in vacuo until approximately 10 mL of the yellow mixture remained. To the yellow mixture was introduced pyridine (30 mL) and the mixture was concentrated in vacuo until approximately 10 mL of the yellow mixture remained. To the yellow mixture was added pyridine (30 mL) and the mixture was concentrated in vacuo until approximately 10 mL of the yellow mixture remained. To the stirred yellow mixture in pyridine (50 mL) was added DMOCP (631 mg, 3.4 mmol). After 15 min, to the brownish yellow solution was added water (750 pL), followed immediately by the introduction of 3H-1 ,2-benzodithiol-3- one (304 mg, 1.8 mmol). After 30 min, the brownish yellow solution was poured into a 1 N aqueous NaHC03 solution (250 mL). After 15 min, the biphasic mixture was extracted with EtOAc (200 mL). After separation, the aqueous layer was back extracted with EtOAc (2 x 150 mL). The organic extracts were combined and concentrated in vacuo. To the concentrated yellow oil was added toluene (20 mL) and the mixture was evaporated in vacuo to remove residual pyridine. This procedure was repeated again with toluene (30 mL). The resulting oil was purified by silica gel chromatography (0% to 50% MeOH in CH2CI2) to provide a mixture of compound 4 (604 mg, 52% yield) as beige solid. Step 4: Preparation of (T2-45) and (T2-44): To a stirred solution of compound 4 (472 mg, 0.5 mmol) in EtOH (5.0 mL) was added AMA (ammonium hydroxide/40% methylamine solution in water )(6.5 mL) and the yellow solution was heated at 50 °C. After 2 h, the yellow solution was allowed to cool and concentrated in vacuo. The yellow residue in 10mM TEAA (3 mL) was purified by reverse phase silica gel chromatography (0% to 25% MeCN in 10 mM aqueous TEAA) to obtain compound (T2-45) (92 mg, 27% yield) as a white triethylammonium salt after lyophilization. LCMS-ESI: 712.95 [M-H]" (calculated for C22H24N1o01oP2S2: 714.56); Rt: 1 .06 min by UPLC (20 mM NH4OAc, 2% to 80% MeCN). 1H NMR (400 MHz, 45 °C, D20) 0 8.45 (d, J =4.4 Hz, 2H), 8.30 (d, J = 5.6 Hz, 2H), 6.36 (d, J = 4.4 Hz, 2H), 5.12 (s, 4H), 4.63 (d, J = 12.4 Hz, 2H), 4.34-4.24 (m, 6H), 3.33 (q, J = 7.2 Hz, 12H), 2.09 (m, 1 H), 1 .40 (t, J = 5.2 Hz, 18H). 31P NMR (45 °C, D20) δ 54.57.
The Rp.Sp isomer was also isolated after purification in the reverse phase chromatography step, to provide compound (T2-44) (35 mg, 10% yield) as the triethylammonium salt after lyophilization. LCMS-ESI: 712.95 [M-H]" (calculated for C22H24N1o01oP2S2: 714.56); Rt: 1 .01 min by UPLC (20 mM NH4OAc, 2% to 80% MeCN). 1H NMR (400 MHz, 45 °C, D20) δ 8.58 (s, 1 H), 8.46 (s, 1 H), 8.31 (s, 1 H), 8.27 (s, 1 H), 6.38 (s, 2H), 5.32 (s, 1 H), 5.1 1 (s, 1 H), 5.07 (d, J = 10.4 Hz, 2H), 4.62 (d, J = 1 1 .2 Hz, 1 H), 4.53 (d, J = 1 1 .2 Hz, 1 H), 4.41 - 4.31 (m, 4H), 4.24 (t, J = 16.4 Hz, 1 H), 3.33 (q, J = 7.2 Hz, 10H), 1.41 (t, J = 7.2 Hz, 15H). 31P NMR (45 °C, D20) δ 55.33, 54.48. Example synthesis of compounds of Formula (B)
Certain compounds of Formula (B) were made enzymatically. Specificall compound T1 -25 was prepared enzymatically according to the following synthetic scheme:
The reaction was carried out in duplicate in parallel: to 100 mM aqueous (((2S,3R,4R,5R)-5-(6- amino-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)phosphonic diphosphoric anhydride (a) (250 pL, 0.025 mmol; N-1007, TriLink Biotechnologies, San Diego, CA, USA), 100 mM aqueous (((2S,3S,4R,5R)-5-(2-amino-6-oxo-1 ,6-dihydro-9H-purin-9-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methyl)phosphonic diphosphoric anhydride (b) (250 μΙ_, 0.025 mmol, Sigma Cat. No 51 120), Herring Sperm DNA solution (250 μΙ_, 10 mg/mL aq.; #9605-5-D, Trevigen Inc., Gaithersburg, MD, USA) and human cGAS (1500 μΙ_, 2.1 mg/mL, prepared as described in the next paragraph) was added reaction buffer (50 mM TRIS, 2.5 mM magnesium acetate, 10 mM KCI, pH adjusted to 8.2 with aq. NaOH 5 M; 25 mL). The reaction was incubated for 16 hours at 37 °C and 150 rpm on an orbital shaker. Completion of the reaction was confirmed through analysis of an aliquot (100 μί) of the reaction mixture, diluted with acetonitrile (100 μί), centrifuged and the desired compound formation determined by UV analysis. The reactions were mixed with acetonitrile (20 mL), incubated at room temperature on an orbital shaker for 10 minutes and after subsequent centrifugation (7000 g for 5 min) the supernatant was filtrated through a paper filter. The filtrate was mixed with acetic acid (100 μί) and directly loaded onto a 20 x 250 mm Inertsil Amide 5 μηι column (flow rate 30 mL/min; solvent A: aqueous 10 mM ammonium acetate, 2 mM acetic acid, solvent B: acetonitrile; using an isocratic elution using 26% phase A / 74% phase B, fraction size 50 mL). The fractions containing the desired compound (T1-25) were combined and the solvents were evaporated in vacuo to a final volume of about 10 mL. The concentrated compound (T1 -25) solution from the first chromatography was re-purified by direct injection onto 1 x 50 cm Sephadex G10 HPLC column (flow rate 1 .0 mL/min; mobile phase containing 0.25 mM ammonium hydroxide and 25% acetonitrile) with UV detection at 250 nm. All fractions containing the desired compound (T1 -25) were combined and dried by lyophilisation to give 4.5 mg of compound (T1-25) as the bis- ammonium salt; 1H NMR (600.1 MHz, D20) δ 8.35 (br s, 1 H), 8.06 (br s, 1 H), 7.77 (s, 1 H), 6.31 (d, J = 12.8 Hz, 1 H), 5.86 (s, 1 H), 5.62 (s, 1 H), 5.35 (d, J = 50.8 Hz, 1 H), 4.97 (d, J = 19.0 Hz, 1 H), 4.46 (s, 1 H), 4.42 (s, 1 H), 4.33 (s, 1 H), 4.24 (s, 1 H), 4.21 (s, 2H), 3.97 (s, 1 H); MS m/z 677.2 [M+H]+. The cGAS used in this example and the following example were prepared by cloning and expression of human and mouse cGAS. The coding region of human or mouse cGAS comprising amino acid 155-522 (human) and amino acid 147-507 (mouse) was cloned into a pET based expression vector. The resulting expression construct contained an N-terminal 6x- His-tag (SEQ ID NO: 930) followed by a ZZ-tag and an engineered HRV3C protease cleavage side allowing generation of human cGAS 155-522 and mouse cGas 147-507 with an N-terminal extension of a Gly-Pro. Both plasmids were transformed in the E.coli strain · BL21 (DE3) phage resistant cells (C2527H, New England BioLabs, Ipswich, MA) for bacterial expression. The phage resistant E. coli cells BL21 (DE3) harboring the cGas expression plasmids were expressed at a 1 .5 L scale in Infors bioreactors. Precultures were grown in LB medium. 1.5 L auto-induction media (Studier, Protein Expr Purif. 2005 May; 41 (1):207-34) containing
Kanamycin (50 | g/mL) were inoculated with 100 mL preculture and cultivated to an OD of approximately 10 under the following conditions: temperature 37 °C; stirrer (cascade regulation via p02) 500; pH 7.0; p02 (cascade regulation on) 5%; flow 2.5 L/min; and gas mix (cascade regulation via p02) 0. The temperature was then reduced to 18 °C and expression was run over night. Cells were harvested by centrifugation and lysed by using an Avestin EmulsiFlex French press. Purification was done according the published protocol by Kato et al. (PLoS One, 2013, 8(10) e76983) using Ni-affinity chromatography, a heparin purification step to remove DNA and a final size exclusion chromatography. cGAS eluted as a homogenous fraction and was concentrated to at least 5 mg/mL.
Human cGAS: GPDAAPGASK LRAVLEKLKL SRDDISTAAG VKGWDHLL LRLKCDSAFR GVGLLNTGSY YEHVKISAPN EFDVMFKLEV PRIQLEEYSN TRAYYFVKFK RNPKENPLSQ FLEGEILSAS KMLSKFRKII KEEINDIKDT DVIMKRKRGG SPAVTLLISE KISVDITLAL
ESKSSWPAST QEGLRIQNWL SAKVRKQLRL KPFYLVPKHA KEGNGFQEET WRLSF-SHIEK EILNNHGKSK TCCENKEEKC CRKDCLKLMK YLLEQLKERF KDKKHLDKFS SYHVKTAFFH VCTQNPQDSQ WDRKDLGLCF DNCVTYFLQC LRTEKLENYF IPEFNLFSSN LIDKRSKEFL TKQIEYERNN EFPVFDEF (SEQ ID NO: 178. Example synthesis of compounds of Formula (B)
Certain compounds of Formula (B) were made enzymatically. Specificall compound T1 -28 was prepared enzymatically according to the following synthetic scheme:
The reaction was performed four times in parallel, each on a 26 ml. scale: to 100 mM aqueous (((2S,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2- yl)methyl)phosphonic diphosphoric anhydride (a) (250 μΙ_, 0.025 mmol), 100 mM aqueous (((2S,3S,4S,5R)-5-(2-amino-6-oxo-1 ,6-dihydro-9H-purin-9-yl)-3-fluoro-4-hydroxytetrahydrofuran- 2-yl)methyl)phosphonic diphosphoric anhydride (c) (250 μΙ_, 0.025 mmol; N-3002, TriLink Biotechnologies), Herring Sperm DNA solution (800 μΙ_, 10 mg/mL aq.; #9605-5-D, Trevigen Inc.) and mouse cGAS preparation (250 μΐ, 6.5 mg/mL, prepared as described for human cGAS above) was added reaction buffer (50 mM TRIS, 2.5 mM magnesium acetate, pH adjusted to 8.2 with aq. NaOH 5 M; 25 mL). The reaction was incubated for 16 hours at 37 °C and 150 rpm on an orbital shaker. The reactions were mixed with acetonitrile (20 mL) and incubated at room temperature on an orbital shaker for 10 min. After subsequent centrifugation (7000 g for 5 min) the supernatant of all four reactions was combined and filtrated through a paper filter. The filtrate was evaporated in vacuo to a residual volume of approximately 20 mL and mixed with 0.5 mL acetic acid (0.5 mL) and 1.0M aqueous triethylammonium acetate (5 mL). The crude material was directly injected onto the Chromolith RP18e 2.1 x 10 cm column. Chromatography (flowrate 80 mL/min; isocratic mobile 10 mM triethylammonium acetate and 1 vol% acetonitrile) yielded the desired compound (T1 -28) fractions which were combined, mixed with aqueous 25 % ammonia solution (20 pL) and dried by lyophilisation. The compound (T1 -28) was obtained as bis-triethylammonium salt; 39.8 mg; 1H NMR (600.1 MHz, D20) δ 8.16 (s, 1 H), 8.13 (s, 1 H), 7.73 (s, 1 H), 6.33 (d, J = 13.9 Hz, 1 H), 5.91 (d, J = 8.6 Hz, 1 H), 5.61 (m, 1 H), 5.40 (dd, J = 51 .5, 2.6 Hz, 1 H), 5.30 (dd, J = 53.3, 3.2 Hz, 1 H), 4.98 (m, 1 H), 4.56 (d, J = 25.8 Hz, 1 H), 4.44 (d, J = 9.0 Hz, 1 H), 4.39 (d, J = 1 1 .8 Hz, 1 H), 4.20 (m, 1 H), 4.08 (d, J = 12.4 Hz, 1 H), 4.04 (d, J = 1 1.8 Hz, 1 H), 3.06 (q, J = 7.3 Hz, 12H), 1 .13 (t, J = 7.3 Hz, 18H); 31 P NMR (376.4 MHz, D20) δ - 1.68, -2.77; 19F NMR (376.4 MHz, D20) δ -199.72, -203.23; MS 677.2 [M-1 ]-.
Mouse cGAS: GPDKLKKVLD KLRLKRKDIS EAAETVNKW ERLLRRMQKR ESEFKGVEQL NTGSYYEHVK ISAPNEFDVM FKLEVPRIEL QEYYETGAFY LVKFKRIPRG NPL-SHFLEGE VLSATKMLSK FRKIIKEEVK EIKDIDVSVE KEKPGSPAVT LLIRNPEEIS VDIILALESK GSWPISTKEG LPIQGWLGTK VRTNLRREPF YLVPKNAKDG NSFQGETWRL SF-SHTEKYIL NNHGIEKTCC ESSGAKCCRK ECLKLMKYLL EQLKKEFQEL DAFCSYHVKT AIFHMWTQDP QDSQWDPRNL SSCFDKLLAF FLECLRTEKL DHYFIPKFNL FSQELIDRKS KEFLSKKIEY ERNNGFPIFD KL (SEQ ID NO: 179).
Example synthesis of compounds of Formula (D) Specifically, (1 S,3R,6R,8R,9S, 1 1 R, 14R, 16R, 17R, 18R)-8, 16-bis(6-amino-9H-purin-9-yl)-17, 18- difluoro-2,4,7, 10, 12,15-hexaoxa-3, 1 1 -diphosphatricyclo[12.2.1.16,9]octadecane-3, 1 1 - bis(thiolate) 3,1 1 -dioxide (8) (which corresponds to compound (T2-46)) was synthesized according to the scheme below:
isomers
Step 1 : Preparation of (2R,3S,4R,5R)-2-(6-benzamido-9/-/-purin-9-yl)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (2): To a solution of Compound i6 (1 , 1 g, 1.5 mmol, 1 eq) (dried via co-evaporation in vacuo with anhydrous MeCN (3 x 3 mL)) in anhydrous THF (6 mL) was added DMAP (18 mg, 0.15 mmol, 0.1 eq) and DIPEA (0.98 mL, 5.9 mmol, 4 eq). 2-cyanoethyl N,N- diisopropyl chlorophosphoramidite (360 pL, 1 .6 mmol, 1 .1 eq, ChemGenes) was added and the reaction was stirred overnight. The mixture was diluted with 100 mL of EtOAc (prewashed with 5 % NaHC03) and washed with brine (5 x 50 mL). The EtOAc layer dried over Na2S04, filtered and concentrated in vacuo. Flash chromatography (40 g silica gel, isocratic gradient - 50:44:4 DCM:Hexanes:TEA) gave 1.08 g of the compound 2.
Step 2: Preparation of (2R,3S,4R,5R)-2-(6-benzamido-9/-/-purin-9-yl)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogen phosphonate (4): To a solution of Compound i6 (1.5 g, 2.7 mmol, 1 eq) in anhydrous dioxane (17 mL) was added anhydrous pyridine (4.7 mL, 69 mmol, 26 eq) followed by a solution of 2-chloro-1 ,3,2- benzodioxaphosphorin-4-one (3, 540 mg, 3.2 mmol, 1 .2 eq, Sigma Aldrich) in 1 ,4-dioxane (8.3 mL). The reaction mixture was stirred for 1 h then diluted with 10 mL water and NaHC03 (3.72 g in 100 mL of water). The suspension was extracted with EtOAc (3 x 100 mL), the organic layers were combined, dried with Na2S04, filtered and concentrated. Chromatography (80 g of Si02, 0-50% MeOH (with 0.5% pyridine) and DCM) gave compound 4.
Step 3: Preparation of (2R,3S,4R,5R)-2-(6-benzamido-9/-/-purin-9-yl)-4-fluoro-5- (hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate (5): To a solution of compound 4 (0.78 g, 1 .1 mmol, 1 eq) in DCM (13 mL) was added water (190 pL, 1 1 mmol, 10 eq) and a solution of DCA (760 μΐ 9.2 mmol, 8.7 eq) in DCM (13 mL). The mixture was stirred for 10 min and quenched with pyridine (1 .5 mL, 18 mmol, 17 eq). The mixture was concentrated in vacuo and co-evaporated with anhydrous MeCN (3 x 10 mL) to provide compound 5 in 4 mL of MeCN.
Step 4: Preparation of (2R,3S,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((((((2R,3S,4R,5R)-2-(6- benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4- fluorotetrahydrofuran-3-yl hydrogen phosphonate (6): Compound 2 (1 .1 g, 1 .2 mmol, 1.1 eq) was dried via co-evaporation in vacuo with anhydrous MeCN (3 x 10 mL leaving 8 mL). This solution was added to the solution of compound 5 from Step 3 and stirred for 5 min. DDTT (240 mg, 1 .2 mmol, 1.1 eq) was added and the mixture was stirred for 30 min then concentrated in vacuo to provide compound 6.
Step 5: Preparation of /V,A/'-(((1 S,3R,6R,8R,9S, 1 1 R,14R, 16R,17R, 18R)-3-(2-cyanoethoxy)- 17, 18-difluoro-1 1 -mercapto-1 1 -oxido-3-sulfido-2,4,7, 10, 12, 15-hexaoxa-3, 1 1 - diphosphatricyclo[12.2.1 .16,9]octadecane-8, 16-diyl)bis(9H-purine-9,6-diyl))dibenzamide (7A): To a solution of compound 6 in DCM (25 mL) was added water (190 pL, 1 1 mmol, 10 eq) and a solution of DCA (1.5 mL, 18 mmol, 17 eq) in DCM (25 mL). The mixture was stirred for 10 min, then quenched with pyridine (1 1 mL, 130 mmol, 120 eq), then concentrated in vacuo to approximately 13 mL. An additional 30 mL of anhydrous pyridine was added. The solution was treated with DMOCP (580 mg, 3.2 mmol, 3 eq) and stirred for 3 min, after which water (570 pL, 32 mmol, 30 eq) was added followed immediately by 3/-/-1 ,2-benzodithiol-3-one (260 mg, 1.6 mmol, 1 .5 eq). After 5 min the solution was poured into saturated NaHC03 (100 mL) and extracted with EtOAc (2 x 100 mL). The organic layers were combined and concentrated to give -2.5 g of crude mixture of isomers 7A/B. Chromatography (80 g Si02, MeOH:DCM 0-15% over 54 min) gave 128 mg of compound 7A. Step 6: Preparation of (1 S,3R,6R,8R,9S,1 1 R, 14R,16R, 17R,18R)-8,16-bis(6-amino-9/-/-purin-9- yl)-17, 18-difluoro-3, 1 1 -dimercapto-2,4,7, 10, 12,15-hexaoxa-3, 1 1 - diphosphatricyclo[12.2.1 .16 9]octadecane 3, 1 1 -dioxide (8) (which corresponds to compound (T2- 46)): To a solution of 7A (70 mg) in MeOH (1 .5 mL) was added NH4OH (1 .5 mL). The reaction mixture was heated to 50 °C for 2.5 h then cooled, sparged with N2 and concentrated in vacuo. Purification (RP MPLC - 5.5 g C18 - 0-20% MeCN/TEAA (10 mM) over 90 column volumes) to give after lyophilization 10 mg of Compound 8. LCMS-ESI: 693.70 [M-H]" (calculated for CsoH FsNioOgPsSs: 694.05); Rt: 8.174 min by LCMS conditions (20 mM NH4OAc, 2% to 50%). 1H NMR. (400 MHz, 45 °C, D20) δ 8.08 (s, 1 H), 7.99 (s, 1 H), 6.17 (d, J = 8.4, 1 H), 5.84 (dd, J = 52.4, 3.6 1 H), 5.19 - 5.1 1 (m, 1 H), 4.77 (m, 1 H), 4.46-4.2 (m, 1 H), 4.10 - 4.09 (m, 1 H), 3.09 (q, J = 7.2, 6H), 1.17 (t, = 7.6 Hz, 9H).
Intermediate i6 (used above) was prepared according to the following scheme
i3 i4
Step 1 : Preparation of (2R,3R,4R,5R)-5-(6-benzamido-9/-/-purin-9-yl)-2-((bis(4-methoxyphenyl) (phenyl)methoxy)methyl)-4-((fe/if-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl trifluoromethane- sulfonate (i2): A mixture of /V-(9-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-3-((ferf-butyldimethylsilyl)oxy)-4- hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (i1 , 5.6 g, 7.11 mmol, ChemGenes) and DMAP (0.174 g, 1.42 mmol) was suspended in anhydrous THF (35 mL), addition of DIPEA (6.21 mL, 35.5 mmol) created a solution to which N-phenyltriflamide (5.08 g, 14.21 mmol), was added. The mixture was stirred for 3.5 h at rt, at which point it was poured into 5% brine (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic phases were dried (Na2S04) the drying agent filtered-off and concentrated on silica gel (10g) in vacuo. The crude material was purified by chromatography on silica gel (gradient elution 25-100% EtOAc/heptane) to give the desired compound i2 as a tan solid; 5.53 g; 1H NMR (400 MHz, CDCI3) δ 9.05 (s, 1 H), 8.68 (s, 1 H), 8.18 (s, 1 H), 8.06 (d, J = 7.5 Hz, 2H), 7.66 (t, J = 7.4 Hz, 1 H), 7.61 - 7.48 (m, 4H), 7.48 - 7.25 (m, 7H), 6.88 (d, J = 8.8 Hz, 4H), 6.04 (d, J = 7.6 Hz, 1 H), 5.50 (dd, J = 7.5, 4.7 Hz, 1 H), 5.32 (d, J = 4.5 Hz, 1 H), 4.50 (t, J = 4.1 Hz, 1 H), 3.82 (s, 6H), 3.77 (dt, J = 10.8, 5.2 Hz, 1 H), 3.41 (dd, J = 10.8, 3.7 Hz, 1 H), 0.77 (s, 9H), -0.01 (s, 3H), -0.46 (s, 3H); LCMS (Method A) R, = 1.65 min; m/z 920.5 [M+H]+.
Step 2: Preparation of (2R,3S,4R,5R)-5-(6-benzamido-9/-/-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((ferf-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl acetate (i3): A mixture of compound i2 (5.5 g, 5.98 mmol), KOAc (2.93 g, 29.9 mmol), and 18- crown-6 (1 ,4,7, 10,13, 16-hexaoxacyclooctadecane, 0.79 g, 2.99 mmol) in toluene (40 mL) was heated at 1 10 °C for 4 h. The reaction mixture was then cooled to rt and silica gel (10g) added and the solvent was removed in vacuo. The crude material was purified by chromatography on silica gel (gradient elution 25-100% EtOAc/heptane) to give the desired compound i3 as a tan solid: 3.3g; 1H NMR (400 MHz, CDCI3) δ 8.70 (s, 1 H), 8.58 (s, 1 H), 7.93 (s, 1 H), 7.84 (d, J = 7.5 Hz, 2H), 7.44 (t, J = 7.4 Hz, 1 H), 7.35 (t, J = 7.6 Hz, 2H), 7.28 (d, J = 7.2 Hz, 2H), 7.21 - 7.02 (m, 7H), 6.67 (dd, J = 8.9, 2.1 Hz, 4H), 5.98 (s, 1H), 4.97 (dd, = 3.6, 1.4 Hz, 1 H), 4.61 - 4.52 (m, 1 H), 4.35 (s, 1 H), 3.62 (s, 6H), 3.41 (dd, J = 9.8, 6.2 Hz, 1 H), 3.18 (dd, J = 9.8, 5.6 Hz, 1 H), 1.53 (s, 3H), 0.77 (s, 9H), 0.03 (s, 3H), 0.0 (s, 3H). LCMS (Method A) R, 1.68 min; m/z 830.2 [M+H]+. Step 3: Preparation of A/-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)- 3-((ferf-butyldimethylsilyl)oxy)-4-hydro (i4): Compound i3 (6.78 g, 8.17 mmol) was dissolved in MeOH (120 mL) and a 2.0 M dimethylamine solution in MeOH (20.4 mL, 40.8 mmol) was added. The reaction mixture was stirred for 17 h at rt. Silica gel (12g) was added and the solvent was removed in vacuo. The crude material was purified by chromatography on silica gel (gradient elution 25-75% EtOAc/heptane) to give the desired compound i4 as a tan solid: 3.9 g; 1 H NMR (400 MHz, CDCI3) δ 8.94 (s, 1 H), 8.65 (s, 1 H), 8.16 (s, 1 H), 7.97 - 7.90 (m, 2H), 7.58 - 7.38 (m, 3H), 7.38 - 7.32 (m, 2H), 7.32 - 7.00 (m, 7H), 6.80 - 6.65 (m, 4H), 5.83 (d, J = 1.2 Hz, 1 H), 5.38 (d, J = 8.0 Hz, 1 H), 4.42 (s, 1 H), 4.29 (t, J = 4.6 Hz, 1 H), 4.02 - 3.95 (m, 1 H), 3.75 - 3.61 (m, 6H), 3.53 (d, J = 5.0 Hz, 2H), 0.81 (s, 9H), 0.0 (s, 6H). LCMS (Method A) R, 1.57 min; m/z 788.2 [M+H]+.
Step 4: Preparation of /V-(9-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-
3- ((ierf-butyldimethylsilyl)oxy)-4-fluorotetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (i5a) and A/-(9-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((fe/if- butyldimethylsilyl)oxy)-4-fluorotetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (i5b): Compound i4 (750 mg, 0.952 mmol) was dissolved in anhydrous DCM (7 mL) under an inert nitrogen atmosphere and the solution was cooled to 0 °C. A 1.0 M solution of DAST (1.90 mL, 1.90 mmol) was added and the reaction subsequently stirred at -5 °C for 17 h using a cryo-cool to control the reaction temperature. The vessel was warmed to 0 °C and saturated NaHC03 (2 mL) was added. After 30 min of stirring the mixture was diluted with 5% brine (20 mL) and extracted with EtOAc (2 x 20 mL). The combined organics were dried (Na2S04) with the drying agent filtered off, silica gel (2g) added to the filtrate and the solvent removed in vacuo. The crude material was purified by chromatography on silica gel (gradient elution 10-75% EtOAc/heptane) to give a mixture of diastereoisomers i5a and i5b as a tan solid:193 mg; Major (2R,3S,4S,5R) diastereoisomer LCMS (Method A) R, 1.53 min; m/z 790.4 (M+H)+; Minor (2R,3S,4R,5R) diastereoisomer R, 1 .58 min; m/z 790.4 [M+H]+.
Step 5: Preparation of A/-(9-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-
4- fluoro-3-hydroxytetrahydrofuran-2-yl)-9/-/-purin-6-yl)benzamide (i6): The diastereomeric mixture of i5a and i5b (2.0 g, 2.53 mmol) was dissolved in anhydrous THF (100 mL) and cooled to -42°C under an inert nitrogen atmosphere before 1 .0 M TBAF (3.80 mL, 3.80 mmol) was added. The reaction was stirred for 2.5 h, then quenched with saturated NaHC03 (20 mL). The cold bath was removed, and the slurry was stirred for 10 min before the mixture was diluted with 5% brine (150 mL) and extracted with DCM (2 x 100 mL). The combined organic phases were dried (Na2S04), with the drying agent filtered off, silica gel (4g) added to the filtrate and the solvent removed in vacuo. The crude material was purified by chromatography on silica gel (gradient elution 25-100% EtOAc/heptane) to give the desired compound i6 as a white solid: 355 mg; 1H NMR (400 MHz, CDCI3) δ 9.16 (s, 1 H), 8.64 (s, 1 H), 8.23 (s, 1 H), 7.99 (d, J = 7.5 Hz, 2H), 7.59 (t, J = 7.4 Hz, 1 H), 7.48 (t, J = 7.6 Hz, 2H), 7.41 - 7.31 (m, 3H), 7.31 - 7.1 1 (m, 7H), 6.79 (d, J = 8.9 Hz, 4H), 6.16 (d, J = 7.3 Hz, 1 H), 5.77 (br s, 1 H), 5.27 - 5.10 (m, 2H), 4.53 (dt, J = 28.0 Hz, 3.4 Hz, 1 H), 3.77 (s, 6H), 3.51 (dd, J = 10.7, 3.7 Hz, 1 H), 3.34 (dd, J = 10.7, 3.3 Hz, 1 H); 19F NMR (376.4 MHz, CDCI3) δ -197.5; 13C NMR (101 MHz, CDCI3) δ 164.66, 158.64, 158.62, 152.60, 151.43, 149.34, 144.22, 141.66, 135.29, 135.13, 133.40, 132.93, 129.96, 128.87, 127.99, 127.93, 127.86, 127.07, 122.65, 1 13.26, 93.85, 92.02, 87.56 (d, J = 144 Hz), 83.56 (d, J = 23 Hz), 77.30, 74.63 (d, J = 16 Hz), 62.82 (d, J = 1 1 Hz), 55.26; LCMS (Method A) R, 0.89 min; m/z 676.3 [M+H]+. heme 1 A'
Step 1 : Preparation of (2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-2-(hydroxymethyl)- 4-((4- methoxybenzyl) oxy)tetrahydrofuran-3-ol (i8): To a suspension of adenosine (i7, 100g, 374 mmol) in DMF (2.64 L) at 4 °C under nitrogen was added 60% sodium hydride (19.46 g, 486 mmol) in one portion and the reaction mixture stirred under nitrogen for 60 min. 4-
Methoxybenzyl chloride (60.9 ml, 449 mmol) was added dropwise over a 10 min period and the suspension stirred and warmed to rt for 16 h. The reaction was quenched with water (50 mL), a short path condenser then fitted and the pale yellow mixture was heated (1 15°C) in vacuo to remove the DMF (60-90°C). The reaction volume was reduced to -300 mL and then partitioned between water (2.5 L) and EtOAc (2 x 500 mL) with the pH of the aqueous phase ~8. The aqueous phase was separated and then extracted with 4: 1 DCM-IPA (8x 500 mL). The combined DCM-IPA phase was dried (Na2S04), the drying agent filtered off and the filtrate concentrated in vacuo to yield a semi-solid residue. The crude residue was stirred in EtOH (130 mL) at 55 °C for 1 h, filtered off, the solid washed with EtOH and dried in vacuo to afford a white solid (55.7 g, 38%, regioisomer ratio 86: 14). This material was re-subjected to a hot slurry in EtOH (100 mL at 55 °C), hot filtered, the solid washed with cold EtOH to give the desired compound i8 as a white crystalline solid (47.22 g): 1H NMR (400 MHz, DMSO-d6) δ 8.30 (s, 1 H), 8.08 (s, 1 H), 7.33 (br s, 2H), 7.06 (d, J = 8.6 Hz, 2H), 6.73 (d, J = 8.6 Hz, 2H), 6.03 (d, J = 6.3 Hz, 1 H), 5.46 (dd, J = 7.3, 4.4 Hz, 1 H), 5.28 (d, J = 5.1 Hz, 1 H), 4.57 (d, J = 1 1.6 Hz, 1 H), 4.53 (dd, J = 6.4, 5.0 Hz, 1 H), 4.37 (d, J = 1 1 .6 Hz, 1 H), 4.33 (dd, J = 5.0, 2.9 Hz, 1 H), 4.02 (q, J = 3.3 Hz, 1 H), 3.69 (s, 3H), 3.67 (m, 1 H), 3.56 (m, 1 H); LCMS (Method B) Rt 1 .86 mins; m/z 388.0 (M+H+).
Step 2: Preparation of (2R,3R,4R,5R)-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purin-9- yl)-2-((trityloxy)methyl)tetrahydrofuran-3-ol (i9): To compound i8 (45.5g, 1 17 mmol) in DMF (310 mL) was added 2,6-lutidine (68.4 mL, 587 mmol), DMAP (3.59 g, 29.4 mmol) and trityl chloride (82 g, 294 mmol). The reaction mixture was slowly heated to 80 °C. The reaction mixture was stirred for 15 h at 80°C and then cooled to rt. The reaction was poured into aq. sat. NH4CI (1500 mL) and extracted with EtOAc (3x 1 L). The combined organic phases were dried (Na2S04), the drying agent filtered off and the filtrate concentrated in vacuo. The crude product was purified by chromatography on silica gel (gradient elution EtOAc-Heptane 0-100%) to yield the desired compound i9 as an off white solid (85.79g): 1H NMR (400 MHz, CDCI3) δ 8.01 (s, 1 H), 7.87 (s, 1 H), 7.41 (m, 12H), 7.28 (m, 18H), 7.18 (d, J = 8.6 Hz, 2H), 6.95 (s, 1 H), 6.80 (d, J = 8.6 Hz, 2H), 6.1 1 (d, J = 4.4 Hz, 1 H), 4.77-4.67 (m, 2H), 4.62 (d, J = 1 1 .6 Hz, 1 H), 4.32 (q, J = 5.3 Hz, 1 H), 4.21 (m, 1 H), 3.79 (s, 3H), 3.49 (dd, J = 10.5, 3.3 Hz, 1 H), 3.36 (dd, J = 10.5, 4.5 Hz, 1 H), 2.66 (d, J = 5.7 Hz, 1 H); LCMS (Method G) Rt 1 .53 mins; m/z 872.0 (M+H+). Step 3: Preparation of (2R,4S,5R)-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purin-9-yl)-2- ((trityloxy) methyl)dihydrofuran-3(2H)-one (MO): To a solution of Dess-Martin Periodinane (DMP, 3.04 g, 7.17 mmol) in DCM (72 mL) at rt was added fe f-butanol (0.713 mL, 7.45 mmol) and sodium carbonate (0.134 g, 1 .261 mmol), followed by a dropwise addition over 1 h of a solution of compound i9 (5.00 g, 5.73 mmol) in DCM (72 mL). The resulting reaction mixture was stirred at rt for 4 h before additional DCM (1 10 mL) was added. After a further 3 h additional DMP (0.63 g) and DCM (50 mL) were added. The reaction stirred for 13 h and then quenched by addition of sat. Na2S205 (40 mL), sat. NaHC03 (150 mL) and brine (50 mL). The organic phase was separated and the aqueous phase then re-extracted with DCM (2 x 150 mL). The combined DCM was dried (Na2S04), the drying agent filtered off and the filtrate concentrated in vacuo. The crude material was purified by chromatography on silica gel (gradient elution EtOAc / heptane (0-80%) to afford compound i10 as a white foam (4.36 g): 1H NMR (400 MHz, CDCI3) δ 7.95 (s, 1 H), 7.78 (s, 1 H), 7.46-7.15 (m, 30H), 7.05 (d, J = 8.6 Hz, 2H), 6.98 (s, 1 H), 6.73 (d, J = 8.6 Hz, 2H), 6.13 (d, J = 7.8 Hz, 1 H), 5.23 (dd, J = 7.9, 0.8 Hz, 1 H), 4.80 (d, = 1 1.8 Hz, 1 H), 4.72 (d, J = 1 1.8 Hz, 1 H), 4.35 (ddd, J = 4.0, 2.4, 0.8 Hz, 1 H), 3.76 (s, 3H), 3.52 (dd, J = 10.5, 4.0 Hz, 1 H), 3.43 (dd, J = 10.5, 2.4 Hz, 1 H); LCMS (Method C) Rt 1 .53 mins; m/z 870.0 (M+H+).
Step 4: Preparation of (2R,3S,4R,5R)-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9/-/-purin-9- yl)-2-((trityloxy)methyl)tetrahydrofuran-3-ol (i11): To a solution of compound i10 (98 mg, 0.1 13 mmol) in DCM (3 mL) at -20°C was added glacial AcOH (0.15 mL) followed by NaBH (13 mg, 0.34 mmol). After 1 h the reaction mixture was quenched with 5% brine (20 mL) and extracted with EtOAc (25 mL). The organic phase was separated and dried (Na2S04), the drying agent filtered off and the filtrate concentrated in vacuo to a white solid. The crude solid (3S:3R ratio 7: 1) was slurried in hot MeOH (3 mL, warmed to 50 °C) with DCM (-0.5 mL) added dropwise and the suspension cooled. The mother liquor was decanted off and the solid was dried in vacuo (63 mg, 3S:3R ratio 13: 1). Recrystallization from MeOH:DCM (4 mL, v/v 5:1) gave compound i11 as a single diastereomer (ratio 50: 1): 1H NMR (400 MHz, CDCI3) δ 7.90 (s, 1 H), 7.74 (s, 1 H), 7.48 - 7.13 (m, 32H), 6.95 - 6.84 (m, 2H), 5.80 (s, 1 H), 4.68 (d, J 1 1 .3 Hz, 1 H), 4.49 (d, J 1 1.3 Hz, 1 H), 4.36 (s, 1 H), 4.33 - 4.27 (m, 1 H), 4.23 (d, J 3 Hz, 1 H), 3.83 (s, 3H), 3.59 - 3.52 (m, 2H); LCMS (Method H) Rt 1 .76 mins; m/z 872.2 (M+H)+.
Step 5: Preparation of 9-((2R,3S,4R,5R)-4-fluoro-3-((4-methoxybenzyl)oxy)-5- ((trityloxy)methyl)tetrahydro-furan-2-yl)-N-trityl-9H-purin-6-amine (i12): To a solution of compound i11 (240 mg, 0.275 mmol) in anhydrous DCM (15 mL) at 0 °C was added anhydrous pyridine (0.223 mL, 2.75 mmol). After 5 min, diethylaminosulfur trifluoride (DAST, 0.182 mL, 1.38 mmol) was added dropwise. After 5 min, the cooling bath was removed and the reaction stirred for 4.5 h. The reaction mixture was diluted with chloroform (20 mL), dry silica gel was added, and the mixture concentrated in vacuo before adding toluene (20 mL) and concentrating to dryness in vacuo. The crude material was purified by chromatography on silica gel (gradient elution 10-50% EtOAc / heptane) to give the desired compound i12 as a white solid (121 mg): 1H NMR (400 MHz, CDCI3) δ 7.93 (s, 1 H), 7.82 (s, 1 H), 7.42 - 7.20 (m, 30H), 7.13-7.05 (m, 3H), 6.74 (d, J 8.3 Hz, 2H), 6.09 - 6.05 (m, 1 H), 5.15- 5.06 (m, 1 H), 5.00 (dd, J 54.4, and 4.4 Hz, 1 H), 4.60-4.50 (m, 2H), 4.49-4.39 (m, 1 H), 3.77 (s, 3H), 3.51-3.38 (m, 1 H), 3.32 (dd, J = 10.6, 4.0 Hz, 1 H); 19F NMR (376.4 MHz, CDCI3) δ -198.09; LCMS (Method I) Rt 1.27 mins; m/z 874.5 (M+H)+.
Step 6: Preparation of (2R,3S,4S,5R)-2-(6-amino-9H-purin-9-yl)-4-fluoro-5- (hydroxymethyl)tetrahydrofuran-3-ol (M3): To a solution of compound i12 (70 mg, 0.080 mmol) in DCM (1 mL) was added TFA (0.5 mL, 6.49 mmol). After 45 min the reaction mixture was diluted with MeOH (10 mL) and concentrated in vacuo. The crude material was dissolved in MeOH (10 mL) and TEA (0.1 mL) was added before silica gel was added and the suspension concentrated in vacuo. The crude material was purified by chromatography on silica gel (gradient elution 0-10% MeOH / DCM) to give the desired compound i13 as a white solid (21 mg) containing TEA. TFA salt and used as is: 1H NMR (400 MHz, Methanol-c/ ) δ 8.33 (s, 1 H), 8.21 (s, 1 H), 6.02 (d, 7.9 Hz, 1 H), 5.12 (dd, J 54.5, 4.3 Hz, 1 H), 4.96 (ddd, J 25.1 , 8.0, 4.3 Hz, 1 H), 4.44 (dt, J 27.6, 2.5 Hz, 1 H), 3.94 - 3.69 (m, 2H); 19F NMR (376.4 MHz, Methanol-cf4) δ - 200.02; LCMS (Method G) Rt 0.51 mins; m/z 270.1 (M+H)+.
Step 7: Preparation of A/-(9-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-9/-/-purin-6-yl)benzamide (i14): To compound i13 (3.88 g, 14.41 mmol) in pyridine (65 mL) at 0 °C was added benzoyl chloride (8.36 mL, 72.1 mmol) slowly followed by TMSCI (9.21 mL, 72.1 mmol). The reaction mixture was stirred while warming to rt for 4 h. After another 1 h the solution was quenched with water (35 mL), followed by cone. NH4OH (17 mL) after 5 min resulting in a pale tan solid. The mixture was diluted with water (100 mL) and extracted with MeTHF (3 x 75 mL). The combined organic phases were dried (Na2S04), the drying agent filtered off and the filtrate concentrated in vacuo to a tan semi-solid crude material, which was purified by chromatography on silica gel (gradient elution 0-20% MeOH / DCM) to give the desired compound i14 (2.75g): 1H NMR (400 MHz, CDCI3) δ 8.78 (s, 1 H), 8.09 (s, 1 H), 8.08 - 8.01 (m, 2H), 7.66 (t, J = 7.4 Hz, 1 H), 7.57 (t, J = 7.5 Hz, 2H), 6.13 (br s, 1 H), 5.92 (d, J = 7.9 Hz, 1 H), 5.41 - 5.1 1 (m, 2H), 4.60 (d, J = 28.4 Hz, 1 H), 4.13 - 3.98 (m, 2H), 3.86 (d, J = 13.0 Hz, 1 H).19F N R (376.4 MHz, CDCI3) δ -199.36; LCMS (Method G) Rt 0.72 mins; m/z 374.2 (M+H)+.
Step 8: Preparation of A/-(9-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)- 4-fluoro-3-hydroxytetrahydrofuran-2-yl)-9/-/-purin-6-yl)benzamide (i6): To compound i14 (2.73 g, 10.14 mmol) in pyridine (55 mL) was added DMTCI (4.12 g, 12.17 mmol) in one portion. The reaction was stirred at rt for 72 h before the yellowish solution was quenched by addition of MeOH (20 mL) and then concentrated in vacuo to a semi-solid following addition of toluene (2 x 50 mL) to azeotrope residual pyridine. The resulting material was dissolved in DCM (100 mL), washed with sat. NaHC03 (100 mL), brine then dried (Na2S04). The drying agent was filtered off and the filtrate evaporated in vacuo. The resulting residue was purified by chromatography on silica gel (gradient elution 0-10% MeOH / DCM with 0.04% TEA) to give compound i6 as a white solid (3.70g): 1H NMR (400 MHz, CDCI3) δ 9.16 (s, 1 H), 8.64 (s, 1 H), 8.23 (s, 1 H), 7.99 (d, J 7.5 Hz, 2H), 7.59 (t, J 7.4 Hz, 1 H), 7.48 (t, J 7.6 Hz, 2H), 7.41 - 7.31 (m, 3H), 7.31 - 7.1 1 (m, 7H), 6.79 (d, J 8.9 Hz, 4H), 6.16 (d, J 7.3 Hz, 1 H), 5.77 (br s, 1 H), 5.27 - 5.10 (m, 2H), 4.53 (dt, J 28.0 Hz, 3.4 Hz, 1 H), 3.77 (s, 6H), 3.51 (dd, J 10.7, 3.7 Hz, 1 H), 3.34 (dd, J 10.7, 3.3 Hz, 1 H); 19F NMR (376.4 MHz, CDCI3) δ -197.5; 13C NMR (101 MHz, CDCI3) δ 164.66, 158.64, 158.62, 152.60, 151.43, 149.34, 144.22, 141.66, 135.29, 135.13, 133.40, 132.93, 129.96, 128.87, 127.99, 127.93, 127.86, 127.07, 122.65, 1 13.26, 93.85, 92.02, 87.56 (d, J 144 Hz), 83.56 (d, J 23 Hz), 77.30, 74.63 (d, J 16 Hz), 62.82 (d, J 11 Hz), 55.26; LCMS (Method C) Rt 2.72 mins; m/z 676.3 (M+H)+.
Note: The LCMS or HRMS data in this example, and where indicated in the following examples, were recorded using the indicated methods as follows. In all instances, masses reported are those of the protonated parent ions unless indicated otherwise.
Method A: LCMS data were recorded using a Waters System: Micromass ZQ mass
spectrometer; Column: Sunfire C18 3.5 micron, 3.0 x 30 mm; gradient: 40-98% MeCN in water with 0.05% TFA over a 2.0 min period; flow rate 2 mL/min; column temperature 40 °C).
Method B: LCMS were recorded using a Waters System: Micromass SQ mass spectrometer;
Column: Acquity UPLC BEH C18 1.7 micron, 2.1 x 30 mm; gradient 1 % to 30% MeCN to 3.20 min then gradient: 30-98% MeCN in water with 5mM NH4OH over a 1.55 min period before returning to 1 % MeCN at 5.19 min - total run time 5.2 min; flow rate 1 mL/min; column temperature 50 °C.
Method C: LCMS were recorded using a Waters System: Micromass SQ mass spectrometer; Column: Acquity UPLC BEH C18 1.7 micron, 2.1 x 50 mm; gradient: 2-98% MeCN in water + 5mM NH4OH over a 4.40 min period isocratic for 0.65 min before returning to 2% MeCN at 5.19 min - total run time 5.2 min; flow rate 1 mL/min; column temperature 50 °C.
Method E: HRMS data were recorded using a Waters System: Acquity G2 Xevo QTof mass spectrometer; Column: Acquity BEH 1 .7 micron, 2.1 x 50 mm; gradient: 40-98% MeCN in water with 0.1 % Formic acid over a 3.4 min period, isocratic 98% MeCN for 1.75 mins returning to 40% at 5.2 mins; flow rate 1 mL/min; column temperature 50°C.
Method G: LCMS data were recorded using a Waters System: Micromass SQ mass spectrometer; Column: Acquity UPLC BEH C18 1.7 micron, 2.1 x 30 mm; gradient 1 % to 30% MeCN to 1.20 mins then gradient: 30-98% MeCN in water with 5mM NH4OAc over a 0.55 min period before returning to 1 % MeCN at 2.19 mins - total run time 2.2 mins; flow rate 1 mL/min; column temperature 50 °C.
Method H: LCMS data were recorded using a Waters System: Micromass SQ mass spectrometer; Column: Acquity UPLC BEH C18 1.7 micron, 2.1 x 30 mm; gradient 2% to 98% MeCN to 1.76 mins then isocratic to 2.00 mins and then returning to 2% MeCN using gradient to 2.20 mins in water with 0.1 % Formic acid; flow rate 1 mL/min; column temperature 50 °C. Method I: LCMS data were recorded using a Waters System: Micromass SQ mass spectrometer; Column: Acquity UPLC BEH C18 1.7 micron, 2.1 x 30 mm; gradient 40% to 98% MeCN to 1.40 mins then isocratic to 2.05 mins and then returning to 40% MeCN using gradient to 2.20 mins in water with 0.1 % Formic acid; flow rate 1 mL/min; column temperature 50 °C.
Given the synthetic methods described above, and the synthetic methods described in
WO2016/145102, WO2014/093936, WO2017/027646, WO2017/027645, WO2015/185565, WO2016/096174, WO2014/189805, US2015158886, WO201701 1622, WO2017004499 and WO2007070598 the compounds listed in Tables 1 -4 can be readily made.
Linker-Drug Moiety (L-fD)^)
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 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 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 some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid based linker.
Acid-labile linkers are linkers cleavable 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 cleavable linkers. Only certain peptides are readily cleaved inside or outside cells, see e.g. , Trout et al., 79 Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umemoto et al. 43 Int. J. Cancer, 677-684 (1989). Furthermore, peptides are composed of a-amino acids and peptidic bonds, which chemically 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 s-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. Val-Cit), are well known in the art, and can be used. See, e.g., Ducry, et al., Bioconjuqate Chem.. vol. 21 , 5-13 (2010).
In addition, cleavable linkers containing a glucuronidase-cleavable moiety, are well known in the art, and can be used. See, e.g., Ducry, et al. , Bioconjuqate Chem., vol. 21 , 5-13 (2010).
For the immunoconjugates of the invention comprising a cleavable linker, the linker is substantially stable in vivo until the immunoconjugate binds to or enters a cell, 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 immunoconjugates 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 immunoconjugates of the invention can be trivalent 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 immunoconjugates 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 immunoconjugates 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 immunoconjugates of the invention can be, for example,
a) an alkylene group: -(CH2)n- (where in this instance is n is 1-18);
b) an alkenyl group;
c) an alkynyl group;
d) an ethylene glycol unit: -CH2CH20-;
e) an polyethylene glycol unit: (-CH2CH20-)x (where x in this instance is 2-20);
f) -0-;
9) -S-;
h) a carbonyl: -C(=0)-;
i) an ester: -C(=0)-0- or -0-C(=0)-;
j) a carbonate: -OC(=0)0-;
k) an amine: -NH-;
I) an amides: -C(=0)-NH-, -NH-C(=0)- or -C(=0)N(C1.6alkyl)-;
m) a carbamate: -OC(=0)NH- or -NHC(=0)0-;
n) a urea: -NHC(=0)NH-;
o) an alkylene substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide, phosphate and phosphonate); p) an CrCioalkylene in which one or more methylene groups is replace by one or more - S-, -NH- or -O- moieties;
q) 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-C6 heteroaryl, a C3-C8 cycloalkyl (including 1 , 1 -disubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and 1 ,4-disubstituted cyclohexyl), and a C4-C8 heterocycloalkyl;
r) a residue of an amino acid selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (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), citrulline (Cit), norvaline (Nva), norleucune (Nle), selenocysteine (Sec), pyrrolysine (Pyl), 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), isoleucine (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), citrulline (Cit), norvaline (Nva), norleucune (Nle), selenocysteine (Sec), pyrrolysine (Pyl), homoserine, homocysteine, and desmethyl pyrrolysine, for example Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala; Phe-Cit; Cit- Phe; Leu-Cit; Cit-Leu; lle-Cit; Cit-lle; Phe-Arg; Arg-Phe; Cit-Trp; and Trp-Cit; a self-immolative spacer, wherein the self-immolative spacer comprises i. one or more protecting (triggering) groups which are 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
and
ii. one or more groups which can undergo 1 ,4-elimination, 1 ,6-elimination, 1 ,8- elimination, 1 ,6-cyclization elimination, 1 ,5-cyclization elimination, 1 ,3- cyclization elimination, intramolecular 5-exo-trig or 6-exo-trig cyclization,
Non-limiting examples of such self-immolative spacer include:
PG is a protecting (triggering) group;
Xa is 0, NH or S;
XJs O or NH;
Ya is CH2, 0 or NH;
Y 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. Chem. Int. Ed. 2015, 54, 7492 - 7509.
By way of example only, certain self-immolative spacers used in the
immunoconjugates of the invention are
In 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 5.
Table 5
analogue Reactive Group Reactive Group
Chemical Moiety
1 2
-l o
i \
H H OH HCT ¾
-
H OH HCT ¾
H OH 0
H μ OH 0
0 0
H H 0H °
O 0
pyridyldithiol thiol disulfide where: R in Table 5 is H, d-4 alkyl, phenyl, pyrimidine or pyridine; R in Table 5 is H, d- 6alkyl, phenyl or C1-4alkyl substituted with 1 to 3 -OH groups; each R36 in Table 5 is independently selected from H, C -6alkyl, fluoro, benzyloxy substituted with -C(=0)OH, benzyl substituted with -C(=0)OH, d.4alkoxy substituted with -C(=0)OH and C1-4alkyl substituted with -C(=0)OH; R37 in Table 5 is independently selected from H, phenyl and pyridine; n in Table 5 is 0, 1 , 2 or 3; R13in Table 5 is H or methyl; R50 in Table 5 is H or nitro; and R14 in Table 5 is H, -CH3 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-naturally 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 (201 1).
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, . A linker components formed by reaction with the amine of a lysine residue of the antibody, antigen binding fragment or their
functional equivalent include, but are not limited to, t each R is independently H or Ci-4 alkyl (preferably methyl).
A linker component formed by reaction with a pyrrolysine residue or desmethyl pyrrolysine
are not limited to, R14 OR wherein R is H or methyl, and R is H, methyl or phenyl.
In some embodiments, a linker component of linker, L, of immunoconjugates of the invention
is , which is formed upon reaction of a hydroxylamine and a X . moiety,
the moiety is formed by reduction of an interchain disulfide bridge of the
re-bridging using a 1 ,3-dihaloacetone (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 , which is
upon reaction of a hydrazine and a moiety, where the moiety is formed by reduction of an interchain disulfide bridge of the antibody and re-bridging using a 1 ,3- dihaloacetone (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 6 below:
R is independently selected from H, CM alkyl, phenyl, pyrimidine and pyridine;
R is independently selected from
R is independently selected from H, C1-4 alkyl, and d.6 haloalkyl.
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 of the immunoconjugates of the invention is designed to have one of the following structures:
-HLC)X-CE-D - (LC)X-CE-(LC)Y-CE-D or -h(LC)X-CE)P-(LC)Y-CE-D 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).
The presence of a non-cleavable linker fragment attached directly to a Drug moiety described herein is observed to decrease the activity of the Drug moiety as tested in a hSTING wt assay and THP1 -dual assay (see below for description of assays and Table 7 for results), therefore such linker designs allow for the release of active Drug moieties. hSTING wt assay:
HEK-293T cells were reverse transfected with a mixture of human STING (accession BC047779 with Arg mutation introduced at position 232 to make the clone into human STING wild type) and a 5xlSRE-mlFNb-GL4 plasmid (five interferon stimulated response elements and a minimal mouse interferon beta promoter driving expression of the firefly luciferase GL4). Cells were transfected using FuGENE transfection reagent (3:1 FuGENE:DNA ratio) by adding the FuGENE:DNA mix to HEK-293T cells in suspension and plating into 384 well plates. Cells were incubated overnight and treated with compounds. After 9-14 hours, plates were read by adding BrightGlo reagent (Promega) and 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 u . Plates were run in triplicate. EC50 values were calculated as described for the IP-10 secretion assay.
THP1 -Dual assay:
THP1 -Dual cells were purchased from Invivogen. THP1 -Dual cells were plated in 384 well plates in 20 uL of tissue culture media and incubated overnight. Compounds were added the next day and incubated 16-24 hours. Lucia reporter signal was read out by adding Quantiluc reagent (Invivogen) 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. EC50 values were calculated as described for the IP-10 secretion assay.
THP1 -Dual/STING-KO assay
Guide RNA (gRNA) oligo (TCCATCCATCCCGTGTCCCA (SEQ ID NO: 931)) for human STING was cloned into Lentivirus vector pNGx_LV_g003 and transduced into THP1 -Dual_Cas9 cells. FACS sorted single clones were then cultured in 96 well cell culture plate. Each single well also contains 500 THP1 -Dual parental cells as supporting cells. After 30 days 1 ug/ml puromycin was added to each well to eliminate supporting cells. 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, T1 -1 , T1 -2, using the methods described in the THP1 -Dual assay above.
Table 7
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 70. A linker component of linker, L, or combinations thereof, of
Embodiment 71. A linker, L selected from:
-**C(=0)0(CH2)mNR11C(=0)(CH2)m-; -**C(=0)0(CH2)mNR11C(=0)(CH2)mO(CH2)m-;
-**C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-;
-**C(=0)OC(R12)2(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-;
-**C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)m-;
-**C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-;
-**C(=0)0(CH2)mNR11C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-;
-**C(=0)0(CH2)mNR11C(=0)XsC(=0)(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-;
-**C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-;
-**C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-;
-**C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-
-**C(=0)0(CH2)mX6C(=0)(CH2)m-; -**C(=0)0(CH2)mX6C(=0)(CH2)mO(CH2)m-;
-**C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-;
-**C(=0)0(CH2)mX6C(=0)XiX2C(=0)(CH2)mO(CH2)m-;
-**C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-;
-**C(=0)0(CH2)mX6C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-;
-**C(=0)X4C(=0)X6(CH2)mNR11C(=0)(CH2)mO(CH2)m-;
-**C(=0)(CH2)mX6C(=0)X1X2C(=0)(CH2)m-; - **C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0))X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mNR11((CH2)mO)n(CH2)m-; - **C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)m-; -
**C(=0)0((CH2)mO)„(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-; -**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-; -**C(=0)0(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)m-; -**C(=0)0(CH2)mNR11(CH2)m-;
-**C(=0)0(CH2)mNR11(CH2)mC(=0)X2X1C(=0)-;
-**C(=0)0(CH2)mX3(CH2)m-; -**C(=0)0((CH2)mO)n(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-; -**C(=0)0(CH2)mNR11C(=0(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)nX3(CH2)m-; -**C(=0)0((CH2)mO)n(CH2)mX3(CH2)m-;
-**C(=0)0((CH2)mO)n(CH2)mC(=0)NR11(CH2)m-; -**C(=0)0(CH2)mC(R12)2-;
-**C(=0)OCH2)mC(R12)2SS(CH2)mNR11C(=0)(CH2)m-, and
-**C(=0)0(CH2)mC(=0)NR11(CH2)m-, where: ** indicates point of attachment to the drug moiety (D);
wherein: ent to X2;
* indicates the point of attachment to
-0(CH2)nSSC(R12)2(CH2)n- or -(CH2)nC(Ri;i)2SS(CH2)nO-;
where the ** indicates orientation toward the Drug moiety; or, where the ** indicates orientation toward the Drug moiety;
each R11 is independently selected from H and C^-C^alky!;
each R12 is independently selected from H and C Cealkyl;
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.
Embodiment 72. A linker, L selected from :
-**C(=0)(CH2)m-; -**C(=0)((CH2)mO)n(CH2)m-; -**C(=0)(CH2)mNR11(CH2)m-;
--C(=0)(CH2)mNR11(CH2)mC(=0)X2XiC(=0)-; -**C(=0)(CH2)mX3(CH2)m-;-
**C(=0)((CH2)mO)n(CH2)mX3(CH2)m-; -**C(=0)(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-;-**C(=0)(CH2)mNR11C(=0(CH2)mX3(CH2)m-;
-**C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-; -**C(=0)((CH2)mO)nX3(CH2)m-;
-**C(=0)((CH2)mO)n(CH2)mX3(CH2)m-; -**C(=0)((CH2)mO)n(CH2)mC(=0)NR11(CH2)m-;-
**C(=0)(CH2)mC(R12)2-; -**C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)m-; C(=0) (CH2)mO)n(CH2)mNR C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-;
C(=0) (CH2)mO)n(CH2)mNR C(=0)X5C(=0)(CH2)mX3(CH2)m-;
*C(=0) (CH2)mO)n(CH2)mNR C(=0)X5C(=0)((CH2)mO)n(CH2)m-;
*C(=0) (CH2)mO)n(CH2)mNR C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)n *C(=0) (CH2)mO)n(CH2)mNR C(=0))X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-;
C(=0) (CH2)mO)n(CH2)mNR C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)n
**C(=0) (CH2)mO)n(CH2)mNR C(=0)X5(CH2)mX3(CH2)m-;
**C(=0) (CH2)mO)n(CH2)mNR C(=0)X5((CH2)mO)n(CH2)m-;
k*C(=0) (CH2)mO)n(CH2)mNR C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-;
**C(=0) (CH2)mO)n(CH2)mNR C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-; **C(=0) (CH2)mO)n(CH2)mNR C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-;
k*C(=0) (CH2)mO)n(CH2)mNR C(=0)X5(CH2)mNR11((CH2)mO)n(CH2)m-;- *C(=0)( CH2)mO)n(CH2)mNR1 C(=0)X5C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-; **C(=0) (CH2)mO)n(CH2)mNR C(=0)X5(CH2)m-;- *C(=0)( CH2)mO)n(CH2)mNR1 C(=0)X5C(=0)((CH2)mO)n(CH2)m-;
**C(=0) ((CH2)mO)n(CH2)mNR C(=0)X5(CH2)mX3(CH2)m-;- **C(=0)(CH2)mC(R12)2SS(CH2)mNR11C(=0)(CH2)m-, and
-**C(=0)(CH2)mC(=0)NR11(CH2)m-,
where: ** indicates point of attachment to the drug moiety (D), and
Xi, X2, X3, X4, X5, R11, R12, n and m are as defined in Embodiment 63. Embodiment 73. A linker, L selected from :
-**C(=0)X1X2C(=0)(CH2)m-; -**C(=0)X1X2C(=0)(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)X1X2C(=0)(CH2)mX3(CH2)m-; -**C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-;
-**C(=0)X1X2C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-; - **C(=0)X1X2C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-; - **C(=0)X1X2C(=0)((CH2)mO)p(CH2)mX3(CH2)m-; - **C(=0)X1X2C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-; - **C(=0)X1X2C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)m-; - **C(=0)X1X2(CH2)mX3(CH2)m-; -**C(=0)X1X2((CH2)mO)n(CH2)m-;
-**C(=0)X1X2((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)X1X2((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-;
-**C(=0)X1X2((CH2)mO)n(CH2)mX3(CH2)m-; -**C(=0)X1X2(CH2)mNR11((CH2)mO)„(CH2) **C(=0)X1X2C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-; -**C(=0)NR11(CH2)m-;
-**C(=0)NR11(CH2)mX3(CH2)m-; -**C(=0)NR11(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-; -**C(=0)NR11(CH2)mNR11C(=0)0(CH2)m-; -**C(=0)NR11(CH2)mNR11C(=0)X1X ; -
**C(=0)NR11 (CH2)mNR11 C(=0)Xs-; -**C(=0)NR11(CH2)mNR11C(=0)(CH2)mX5(CH2)m-; -
**C(=0)X C( =0)NR11(CH2)mX5(CH2)m-; -**C(=0)NR11(CH2)mNR11C(=0)(CH2)m-; -
**C(=0)NR11 (CH2)mNR11 C(=0)(CH2)mO(CH2)m-; -
**C(=0)NR11 (CH2)mNR11 C(=0)X1X2C(=0)(CH2)mO(CH2)m-;
-**C(=0)NR' 1(CH2)mNR1 1C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X1X2C(=0)(CH2)m-; -
**C(=0)NR11 (CH2)mNR11 C(=0)X5C(=0)(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5C(=0)(CH2)mX3(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5C(=0)((CH2)mO)n(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)m-
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5(CH2)mX3(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5((CH2)mO)n(CH2)m-;
-**C(=0)NR1 VCH mNR1 1C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5(CH2)mNR1,((CH2)mO)n(CH2)m-;-
**C(=0)NR11 (CH2)mNR11 C(=0)X5C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-;
-**C(=0)NR1 1(CH2)mNR1 1C(=0)X5(CH2)m-; -
**C(=0)NR1 (CH2)mNR1 C(=0)X5C(=0)((CH2)mO)n(CH2)m-;
-**C(=0)NR 1(CH2)mNR 1C(=0)X5(CH2)mX3(CH2)m-; -
**C(=0)XiC(=0)NR11(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)X1C(=0)NRll(CH2)mX3(CH2)m-;
-**C(=0)NR11(CH2)mNR11C(=0)(CH2)m-;
-**C(=0)NR,1(CH2)mNR11C(=0)(CH2)mX3(CH2)m-;-**C(=0)NR11(CH2)mNR11C(=0)-;
-**C(=0)X1X2(CH2)m-;-**C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-;
-**C(=0)X1X2(CH2)mX3(CH2)m-; -**C(=0)NR11(CH2)mX3(CH2)m-; -
**C(=0)NR11((CH2)mO)n(CH2)mX3(CH2)m-;
--C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-;-**C(=0)X1X2C(=0)(CH2)m-;
-**C(=0)X1C(=0)(CH2)mNR11C(=0)(CH2)m-, and
-**C(=0)X1C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-, where: ** indicates point of attachment to the drug moiety (D), and Xi , X2, X3, X4, X5, R11 ,
R12, n and m are as defined in Embodiment 63.
Embodiment 74. A linker, L selected from :
-**C(=0)0(CH2)mNR11C(=0)(CH2)m-; -**C(=0)0(CH2)mNR11C(=0)(CH2)mO(CH2)m-,
-**C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-; - **C(=0)OC(R12)2(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-; - **C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)m-; - **C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-; - **C(=0)0(CH2)mNR11C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-; - **C(=0)0(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-; - **C(=0)0(CH2)mX6C(=0)XiX2C(=0)((CH2)mO)n(CH2)mC(=0)-; **- (CH2)mNR11C(=0)X1X2C(=0)(CH2)m-, -**(CH2)m(CHOH)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m; - **C(=0)X6C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-; - **C(=0)X4C(=0)NR11 (CH2)mNR11 C(=0)(CH2)mO(CH2)m- ; -
**C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-; -**(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-; - C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**, or -C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-** where: ** indicates point of attachment to the drug moiety (D), and X2, , X4, R11 , R12, n and m are as defined in Embodiment 63.
Embodiment 75. A linker, L selected from:
, where ** 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 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 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 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
one or more linkers is attached to one or more R1 , R1a and R1 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 groups of Formula (A), Formula (B), Formula
(C), Formula (D), Formula (E) or Formula (F).
Certain 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 76. 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
* indicates the point of attachment to -CR R -; XA is C(=0)-, -C(=S)- or -C(=NR11)- and each Ζ is NR12;
XB is C, and each Z2 is N;
G2 is * indicates the point of attachment to -CR8aR9a-;
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-, -S-, -S(=0)-, -SOr, -CH2-, or -CF2-;
Y2 is -0-, -S-, -S(=0)-, -SOr, -CH2-, or -CF2-;
Y3 is OH, 0", OR10, N(R10)2, SeH, Se", BH3, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SeH, Se", BH3, SH or S";
Y5 is -CH2-, -NH-, -0- or -S;
Y6 is -CH2-, -NH-, -0- or -S;
Y7 is 0 or S;
Y8 is 0 or S;
Yg is -CHr, -NH-, -O- or -S;
Y10 is -CH2-, -NH-, -0- or -S;
Y is -0-, -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 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 0, 1 , 2, 3 or 4 substituents independently selected from -NHL^ R15, F, CI, Br, OH, SH, NH2, D, CD3, Cr C6alkyl, CrC6alkoxyalkyl, CrCshydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(Cr
C6alkyl), -0(C3-C8cycloalkyl), -S^-Cealkyl), -SiCrCsaminoalkyl), -SiCrCshydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(C1-Cealkyl)2, -N(d- C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
R1a 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 -NHL^ R15, F, CI, Br, OH, SH, NH2, D, CD3, Cr C6alkyl, CrC6alkoxyalkyl, CrCehydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(Cr C6alkyl), -0(C3-C8cycloalkyl), -StCrCealkyl), -S(CrCsaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -Nid-Cealkyl^, -N(Cr C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
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 0, 1 , 2, 3 or 4 substituents independently selected from -NHUR15, F, CI, Br, OH, SH, NH2, D, CD3, Cr C6alkyl, CrC6alkoxyalkyl, CrCehydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(Cr C6alkyl), -0(C3-C8cycloalkyl), -S(Ci-C6alkyl), -S(CrCsaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -Nid-Cealkyl^, -N(Cr C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- CH=CH(CH2)i.i0C(=O)OH,-NHC(O)(Ci-C3alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
each R2 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC^d-Cealkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R3 is independently selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -Otd-Cealkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC^d-Cealkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrCealkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R4 is independently selected from the group consisting of -OL^R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R5 is independently selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R6 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; each R7 is independently selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7 are substituted by
0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R8 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -
OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R8 and the Ci-C3alkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R9 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C3haloalkenyl, C2- Cshaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, -
O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R9 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), -
0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R2a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr
C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3
R3a is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -
O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - 0C(0)C2-C6alkenyl and -0C(0)C2-C6alkynyl of R3a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R a is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -
0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-
C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R5a is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - 0C(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- Cshaloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R6a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr
C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-
C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R6aare substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R7a is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, d- C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- Cshaloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - 0C(0)0CrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R8a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -
OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R8a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; R9a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, d-Cealkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC^Od-Cealkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R9a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, d-Cshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
eac 10 is independently selected from the group consisting of H, d-C^alkyl, -
, wherein the CrC12alkyl of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, Cr
C12alkoxy, -S-C(=0)CrC6alkyl and C(0)OCrCsalkyl;
each R11 is independently selected from H and Ci-C6alkyl;
each R12 is independently selected from H and d-Cealkyl;
optionally R3 and R6 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3 and R6 are connected, the 0 is bound at the R3 position
optionally R3a and R6a, are connected to form d-Csalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3a and R6a are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2 and R3 are connected, the 0 is bound at the R3 position;
optionally R2a and R3a, are connected to form d-Csalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form d-Cealkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4 and R3 are connected, the 0 is bound at the R3 position; optionally R and R , are connected to form CrCsalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form C^Cealkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R6 are connected, the 0 is bound at the R5 position;
optionally R5a and R6a, are connected to form CrQjalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R6a are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form C^Cealkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R7 are connected, the 0 is bound at the R5 position;
optionally R5a and R7a, are connected to form CrCsalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R7a are connected, the O is bound at the R5a position;
optionally R8 and R9 are connected to form a d-Calkylene, C2-C6alkenylene, C2- C6alkynylene, and
optionally R8a and R9a are connected to form a CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene,
is a linker;
R15 is a reactive group selected from any one of the groups RG1 inTable 5;
and provided at least one of R1 , R1a or R1b is substituted with -NHUR15, or at least one of R3, R4, R5, R7, R3a, R a, R5a or R7a is -OL^R15.
Embodiment 77. A compound of Embodiment 76, wherein l_i is a linker comprising one or more cleavage elements.
Embodiment 78. 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, wherein R1 , R1 a, R1 b, R2, R2a, R3, R3a, R4, R a, R5, R5a, Rs, R6a, R7, R7a, R8, R8a, R9, Yi > Y2, Y3, Y4, Y5, Υβ, Y7, Ye, Y9, Y10 and Y are as described in Embodiment 76, and provided at least one of R1 , R1a or R1 is substituted with -NHU R15, or at least one of R3, R4, R5, R7, R3a, R a, R5a or R7a is -OL^R15.
Embodiment 79. 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 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 76. Embodiment 80. A compound of Formula (A-2), Formula (B-2), Formula (C-2), Formula (D- 2), Formula (E-2) or Formula (F-2), wherein R1 , R1a, R1 , R2, R2a, R3, R3a, R4, R4a, R5, R5a, R6, R6a, R7, R7a, R8, R8a, R9, R9a, Y1 f Y2, Y3, Y4, Y5, Y6, Y7, Ye, Yg, Yio and Y are as defined in Embodiment 76, and provided at least one of R1, R1a or R1 b is substituted with -NHUR15, or at least one of R3, R4, R5, R7, R3a, R4a, R5a or R7a is -OL^R15.
Embodiment 81. A compound of Formula (A), Formula (A-1) or Formula (A-2) of any one of Embodiments 76 to 80, wherein:
R2 and R2a are H;
one of R3 and R4 is H and the other is selected from the group consisting of -OUR15, H, - OH, F, CI, Br, I, D, CD3, CN, N3, Ci-C8alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr
C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 or R4 and the CrC6alkyl, C2-C6alkenyl and C2-
C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC3alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3 or R4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R7 and R7a are H;
Rs and RSa are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R3a and R4a is H and the other is selected from the group consisting of -OL^R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr
C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and R a and the CrCsalkyl, C2-C6alkenyl and C2-
C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3a or R a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and provided at least one of R1 or R1a is substituted with -NHUR15, or at least one of R3, R4, R3a or R a is -OUR15. Embodiment 82. A compound of Formula (A), Formula (A-1) or Formula (A-2) of any one of Embodiments 76 to 81 , wherein:
Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S;
Yg and Y10 are 0 or S;
R2, R2a, R6, R6a, R7 and R7a are H
one of R3a and R4a is H and the other is -OUR15, H, OH or F;
one of R3 and R4 is H and the other is -OU R15, H, OH or F; and
R8a, R9a, R8 and R9 are independently selected from H or Ci-Cealkyl,
and provided at least one of R1 or R1a is substituted with -NHUR15, or at least one of R3, R4, R3a or R4a is -OUR15.
Embodiment 83. A compound of Formula (B), Formula (B-1) or Formula (B-2) of any one of Embodiments 76 to 80, wherein:
R2 and R2a are H;
one of R3a and R a is H and the other is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr Cehaloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -
OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and R a and the CrCsalkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl,
-OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3a or R4a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; R7a and R6a are H;
R6 and R4 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R5 and R7 is H and the other is selected from the group consisting of -OUR15, H, - OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C6alkenyl, C2-C6alkynyl, Cr Cehaloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -
OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and R7 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the d-Cealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R5 or R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and provided at least one of R1 or R1a is substituted with -NHUR15, or at least one of R5, R7, R3a or R a is -OUR15.
Embodiment 84. A compound of Formula (B), Formula (B-1) or Formula (B-2) of any one of Embodiments 76 to 80 or 83, wherein:
Y-\ and Y2 are O, CH2 or S;
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S;
R2, R2a, R7a, RSa, R6 and R4 are H;
one of R3a and R4a is H and the other is -OUR15, H, OH or F;
one of R5 and R7 is H and the other is -OU R15, H, OH or F, and
Rsa R9a R8 a nd R9 gre jncjepencjentiy selected from H or CrCsalkyl,
and provided at least one of R1 or R1a is substituted with -NHUR15, or at least one of R5,
R7, R3a or R4a is -OUR15.
Embodiment 85. A compound of Formula (C), Formula (C-1) or Formula (C-2) of any one of Embodiments 76 to 80, wherein:
R2 and R2a are H;
one of R3 and R4 is H and the other is selected from the group consisting of -OUR15, H, -
OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C6alkenyl, C2-C6alkynyl, Cr Cehaloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and R4 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -OCrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3 or R4 are substituted by 0, 1 , 2 or
3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; R a and R6a are H; Rs and R7 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl;
one of R5a and R7a is H and the other is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -
0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R a and R7a and the CrCsalkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-
C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R5a or R7a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and provided at least one of R1 or R1a is substituted with -NHL1R15, or at least one of R5a,
R7a, R3 or R4 is -OUR15.
Embodiment 86. A compound of Formula (C), Formula (C-1) or Formula (C-2) of any one of Embodiments 76 to 80 or 85, wherein:
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S;
Yg and Y10 are 0 or S;
R2, R2a, R a, RSa, R6 and R7 are H;
one of R3 and R4 is H and the other is -OU 15, H, OH or F;
one of R5a and R7a is H and the other is -OUR15, H, OH or F, and
R8a R9a R8 a nd R9 gre jncjepencjentiy selected from H or d-C3alkyl,
and provided at least one of R1 or R1a is substituted with -NHUR15, or at least one of R5a, R7a, R3 or R4 is -OUR15.
Embodiment 87. A compound of Formula (D), Formula (D-1) or Formula (D-2) of any one of Embodiments 76 to 80, wherein:
R2 and R2a are H;
one of R5a and R7a is H and the other is selected from the group consisting of -OU R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Chalky!, C2-C6alkenyl, C2-C6alkynyl, Cr
C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R a and R7a and the CrCsalkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R5a or R7a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R4a and R6a are H;
R6 and R4 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R5 and R7 is H and the other is selected from the group consisting of -OL^R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -
OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and R7 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(CrC3alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R5 or R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and provided at least one of R1 or R1a is substituted with -NHUR15, or at least one of R5a, R7a, R5 or R7 is -OUR15.
Embodiment 88. A compound of Formula (D), Formula (D-1) or Formula (D-2) of any one of Embodiments 76 to 80 or 87, wherein:
Y1 and Y2 are O, CH2 or S;
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S;
Yg and Y10 are 0 or S;
R2, R2a, R a, RSa, R6 and R are H;
one of R5a, R7a is H and the other is -OL^R15,, OH or F;
one of R5 and R7 is H and the other is -OU R15, H, OH or F, and
R8, R9, R8a and R9a are independently H or CrC6alkyl, and provided at least one of R1 or R1a is substituted with -NHUR15, or at least one of R5a, R7a, R5 or R7 is -OL^R15.
Embodiment 89. A compound of Formula (E), Formula (E-1) or Formula (E-2) of any one of Embodiments 76 to 80, wherein:
R2 and R2a are H;
R6 and R6a are H;
R7a is H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R3a and R4a is H and the other is selected from the group consisting of -OL^R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr
C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and R a and the Ci-C3alkyl, C2-C6alkenyl and C2-
C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC3alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3a or R a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; one of R3 and R4 is H and the other is selected from the group consisting of -OUR15, H, - OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -
OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and R4 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C8haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl,
-OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3 or R4 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and one of R5 and R7 is H and the other is selected from the group consisting of -OUR15, H, - OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -
0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and R7 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl,
-OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R5 or R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and provided at least one of R1 or R1a is substituted with -NHUR15, or at least one of R3a, R4a, R3, R4, R5 or R7 is -OU R15.
Embodiment 90. A compound of Formula (E), Formula (E-1) or Formula (E-2) of any one of Embodiments 76 to 80 or 89, wherein:
Y1 and Y2 are O, CH2 or S;
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y5 is O or S;
Y7 and Y8 are 0 or S;
Y9 and Y10 are 0 or S;
R2, R2a, R5a, RSa, R6 and R7a are H;
one of R3a, R4a is H and the other is -OUR15, H, OH, OCH3 or F;
one of R3, R4 is H and the other is -OUR15, H, OH, OCH3 or F;
one of R5 and R7 is H and the other is -OU R15, -OUR15, H, OH, OCH3 or F, and
R8, R9, R8a and R9a are independently H or CrC6alkyl,
and provided at least one of R1 or R1a is substituted with -NHUR15, or at least one of R3a, R4a, R3, R4, R5 or R7 is -OU R15.
Embodiment 91. A compound of Formula (F), Formula (F-1) or Formula (F-2) of any one of Embodiments 76 to 80, wherein:
R2 and R2a are H;
each R6 and R6a are H;
each R7a and R7 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R3a and R4a is H and the other is selected from the group consisting of -OUR15, H,
-OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and R a and the CrCsalkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(Ci-Cealkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3a or R4a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; one of R3 and R4 is H and the other is selected from the group consisting of -OL^15, H, - OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and R4 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3 or R4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and
R5 is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrCealkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2- C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2- C6alkenyl), -0(C2-C6alkynyl), -OC^OCrCealkyl, -0C(0)0C2-C6alkenyl, - 0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl of R5 is substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3,
and provided at least one of R1 , R1a or R1 is substituted with -NHL^R15, or at least one of R3a, R4a, R3, R4, R5 or R7 is -OL^R15.
Embodiment 92. A compound of Formula (F), Formula (F-1) or Formula (F-2) of any one of Embodiments 76 to 80 or 91 , wherein:
Y1 and Y2 are O, CH2 or S;
each Y3 is OH, 0", OR10, N(R10)2, SH or S";
each Y5 is O or S;
each Y7 is independently are 0 or S;
each Y9 is independently 0 or S;
R2, R2a, R6, R6a, R6, R7 and R7a are H;
one of R3a, R4a is H and the other is -OUR15, H, OH, OCH3 or F;
one of R3, R4 is H and the other is -OUR15, H, OH, OCH3 or F;
R5 is -OUR15, H, OH, OCH3 or F, and
R8, R9, R8a and R9a are independently H or CrC6alkyl,
and provided at least one of R1 , R1a or R1b is substituted with -NHUR15, or at least one of R3a, R4a, R3, R4, R5 or R7 is -OUR15.
Embodiment 93. A compound of any one of Embodiments 76 to 92 wherein:
wherein: R1 is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, CrC6alkoxyalkyl, CrC6hydroxyalkyl, C3- C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms
independently selected from O, N and S, -0(CrC6alkyl), -0(C3-C8cycloalkyl), -S(Cr C6alkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(Cr C6alkyl), -NH(C3-C8cycloalkyl), -N(Ci-C6alkyl)2, -N(CrC6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,-CH=CH(CH2)1.10C(=O)OH, - NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), -NHC(0)(phenyl), and -N(C3- C8cycloalkyl)2,
and
20 is independently selected from H and UR15;
, wherein: R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, CrC6alkoxyalkyl, Cr C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -O^-Cealkyl), -0(C3- C8cycloalkyl), -S(CrC6alkyl), -StCrCeaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- Cgcycloalkyl), -NHCd-Cealkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -Nid-Cealkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- CH=CH(CH2)i-ioC(=0)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2,
and
each R21 is independently selected from H and UR15; , wherein: R1 b is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, CrC6alkoxyalkyl, Cr C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -C CrCealkyl), -0(C3- C8cycloalkyl), -S(CrC6alkyl), -S C Ceaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- Cgcycloalkyl), -NH CrCsalkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(CrC6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2,
CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2,
and
each R21 is independently selected from H and L^R15.
Embodiment 94. A compound of Formula (A-3), Formula (B-3), Formula (C-3) , Formula (D-3), Formula (E-3) or Formula (F-3), wherein:
ΥΪ is -0-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2
Y2 is -0-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2
Y is -0-, -S-, -S(=0)-, -S02-, -CH2-, or -CF
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, CrC6alkoxyalkyl, Cr C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocydyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), -0(C3- C8cycloalkyl), -S(CrC3alkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- C8cycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -NCd-Csalky s, -NCd-Csalkyl) (C3- Cscycloalkyl), -CN, -P(=0)(OH)2,
CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2,
and
each R20 is independently selected from H and UR15;
, or , wherein: R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, Cr
C6alkoxyalkyl, CrCehydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from O, N and S, -C^CrCsalkyl), - 0(C3-C8cycloalkyl), -S(CrC3alkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- Cgcycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(C Cgcycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2,
and
21 is independently selected from H and UR15;
, or , wherein: R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, Cr
C6alkoxyalkyl, Ci-C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), - 0(C3-C8cycloalkyl), -S(CrCsalkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3- Cgcycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(CrC6alkyl) (C3- Cscycloalkyl), -CN, -P(=0)(OH)2,
CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2,
and
each R21 is independently selected from H and UR15;
each R2 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)Ci-C6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R3 is independently selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; each R4 is independently selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4 are substituted by
0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R5 is independently selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -
OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the Ci-C3alkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R6 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C3haloalkenyl, C2- Cehaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, -
O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), -
0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of Rs are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R7 is independently selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl,
CrCshaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R2a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -
O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3
R3a is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -
0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2-
C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R4a is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R5a is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C^Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -
0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-
C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R6a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, Ci-C6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R6aare substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R7a is selected from the group consisting of -OUR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(Ci-C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -
OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; each R10 is independently selected from the group consisting of H, d-C^alkyl, -
, wherein the d-Ci2alkyl of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, d- C12alkoxy, -S-C(=0)CrC6alkyl and C(0)OCrC6alkyl;
optionally R3 and R6 are connected to form d-C6alkylene, d-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3 and R6 are connected, the 0 is bound at the R3 position
optionally R3a and R6a, are connected to form d-C8alkylene, d-dalkenylene, C2-
C6alkynylene, -O-d-Cealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3a and R6a are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form d-C6alkylene, C2-dalkenylene, C2-
C6alkynylene, -O-d-Cealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2 and R3 are connected, the 0 is bound at the R3 position;
optionally R2a and R3a, are connected to form d-C3alkylene, d-dalkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form d-C6alkylene, d-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4 and R3 are connected, the 0 is bound at the R3 position;
optionally R a and R3a, are connected to form d-C8alkylene, d-dalkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form d-C6alkylene, d-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R6 are connected, the 0 is bound at the R5 position;
optionally R5a and R6a, are connected to form d-C8alkylene, d-dalkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R6a are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form d-C6alkylene, C2-dalkenylene, C2-
C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R7 are connected, the 0 is bound at the R5 position, and
optionally R5a and R7a, are connected to form d-C3alkylene, d-dalkenylene, C2-
C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R7a are connected, the O is bound at the R5a position;
-C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;
(=0)OC(R12)2(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;
(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)m-**;
(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-**;
(=0)0(CH2)mNR11C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)n
(=0)0(CH2)mNR11C(=0)XsC(=0)(CH2)mNR11C(=0)(CH2)m-**;
=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-*
=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -C(=0)(CH2)mNR11C(=0)X2C(=0)(CH2)n =0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**,
=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**,
C(=0)0(CH2)mXsC(=0)(CH2)m-**,
0)0(CH2)mX6C(=0)(CH2)mO(CH2)m-**,
0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-",
0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)mO(CH2)m-**,
0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)- **,
0)0(CH2)mXeC(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**,
0)X4C(=0)X6(CH2)mNR11C(=0)(CH2)mO(CH2)m-**,
0)(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**,
0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)m-**;
0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m- =0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**; =0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)n
=0)0((CH2)mO)n(CH2)mNR11C(=0))X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR"C(=0)((CH2)mO)n(CH2)mX3(CH2)
-C =0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**;
-C =0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)m-**;
-C =0)0((CH2)mO)n(CH2)mNR11 C(=0)X5((CH2)mO)n(CH2)mNR1 1C(=0)(CH2)m-**; -C =0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**; -C =0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-";
-C =0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mNR11((CH2)mO)n(CH2)m-**; - C(: 0)0((CH2)mO)„(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-**; =0)0((CH2)mO)n(CH2)mNRMC(=0)X5(CH2)
=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)n
(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**; -C(=0)0(CH2)m-**;
(=0)0((CH2)mO)n(CH2)m-**; -C(=0)0(CH2)mNR11(CH2)m-**;
(=0)0(CH2)mNR11(CH2)mC(=0)X2X1C(=0)-**;
(=0)0(CH2)mX3(CH2)m-**; -C(=0)0(CH2)mXeC(=0)XiX2C(=0)((CH2)mO)n(CH2)m- C(=0)0((CH2)mO)n(CH2)mX3(CH2)m-**;
(=0)0((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
(=0)0(CH2)mNR11C(=0(CH2)mX3(CH2)m-**;
(=0)0((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
(=0)0((CH2)mO)nX3(CH2)m-**; -C(=0)0((CH2)mO)n(CH2)mX3(CH2)m-**;
(=0)0((CH2)mO)n(CH2)mC(=0)NR11(CH2)m-**; -C(=0)0(CH2)mC(R12)2-**;
(=0)OCH2)mC(R12)2SS(CH2)mNR11C(=0)(CH2)m-**;
(=0)0(CH2)mC(=0)NR11(CH2)m-**; -C(=0)(CH2)m-**; -C(=0)((CH2)mO)n(CH2)m-**; (=0)(CH2)mNR11(CH2)m-**; -C(=0)(CH2)mNR11(CH2)mC(=0)X2X1C(=0)-**;
(=0)(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
(=0)(CH2)mNR11C(=0)(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**; - C(=0)(CH2)mNR11C(=0(CH2)mX3(CH2)m-**; -(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -(CH2)m(CHOH)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)nX3(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mC(=0)NR11(CH2)m-**; -
=0)((CH2)mO)n(CH2)mNR C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
=0)((CH2)mO)n(CH2)mNR C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
=0)((CH2)mO)n(CH2)mNR C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
=0)((CH2)mO)n(CH2)mNR C(=0)X5C(=0)((CH2)mO)„(CH2)mNR11C(=0)(CH2)m-**; =0)((CH2)mO)n(CH2)mNR C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)n =0)((CH2)mO)n(CH2)mNR C(=0))X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
=0)((CH2)mO)n(CH2)mNR C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**; =0)((CH2)mO)n(CH2)mNR C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)n =0)((CH2)mO)n(CH2)mNR C(=0)X5(CH2)mX3(CH2)m-**;
=0)((CH2)mO)n(CH2)mNR C(=0)X5((CH2)mO)n(CH2)m-**;
=0)((CH2)mO)n(CH2)mNR C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
=0)((CH2)mO)n(CH2)mNR C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**; =0)((CH2)mO)n(CH2)mNR C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-**;
=0)((CH2)mO)n(CH2)mNR C(=0)X5(CH2)mNR11((CH2)mO)n(CH2)m-**; -
C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)n -C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)m-**; -
C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**; -
C(=0)(CH2)mC(R12)2SS(CH2)mNR11C(=0)(CH2)m-**; -C(=0)(CH2)mC(=0)NR11(CH2)m-**; -C(=0)X1X2C(=0)(CH2)m-**; -C(=0)X1X2C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X1X2C(=0)(CH2)mX3(CH2)m-**; -C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)X1X2C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)X1X2C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)X1X2(CH2)mX3(CH2)m-**; -C(=0)X1X2((CH2)mO)n(CH2)m-**;
-C(=0)X1X2((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X1X2((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-*i;
-C(=0)X1X2((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)X1X2(CH2)mNR11((CH2)mO)n(CH2)m-**; -
C(=0)X1X2C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)NR11(CH2)m-**; -C(=0)NR11(CH2)mX3(CH2)m-**;
-C(=0) NR11 (CH2)mNR11 C(=0)X1X2C(=0) (CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)0(CH2)m-**; -C(=0)NR11(CH2)mNR11C(=0)X1X2-**; -
C(=0)NR11(CH2)mNR11C(=0)Xs-; -C(=0)NR11(CH2)mNR11C(=0)(CH2)mX5(CH2)m-**; -
C(=0)X1C(=0)NR11(CH2)mXs(CH2)m-**; -C(=0)X6C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-
**; -C(=0)NR11(CH2)mNR11C(=0)(CH2)m-**; -C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)m-*i;
-C(=0)NR11(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-**;
-C(=0)NR11(CH2)mNR11C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**;
-C(=0)NR11 (CH2)mNR11 C(=0)X1X2C(=0) (CH2)m-**; -
C(=0)NR11(CH2)mNR11C(=0)X5C(=0)(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**; -C(=0)NR 1 (CH2)mNR 1 C(=0)X5((CH2)mO)n(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0) NR11 (CH2)mNR11 C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5(CH2)mNR11((CH2)mO)n(CH2)m-**; -
C(=0)NR11(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0) NR11 (CH2)mNR11 C(=0)X5(CH2)m-**; -
C(=0)NR11(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**; -
C(=0)X1C(=0)NR11(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X1C(=0)NR11(CH2)mX3(CH2)m-*i; -C(=0)NR11(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**; -C(=0)NR11(CH2)mNR11C(=0)- -C(=0)X1X2(CH2)m-**; -C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)X1X2(CH2)mX3(CH2)m-ii; -C(=0)NR11(CH2)mX3(CH2)m-ii; - C(=0)NR11((CH2)mO)n(CH2)mX3(CH2)m-**; -C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)X1X2C(=0)(CH2)m-**; -C(=0)X1C(=0)(CH2)mNR11C(=0)(CH2)m-**; and
-C(=0)X1C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
=0)2(CH=CH2), -(CH2)2S(=0)2(CH=CH2), -NHS(=0)2(CH=CH2), -NHC(=0)CH2Br, -
265
* indicates the point of attachment to
1 )2(CH2)n- or -(CH2)nC(R1 )2SS(CH2)nO-;
where the
indicates orientation toward the Drug moiety;
X6 is or, where the ** indicates orientation toward the Drug moiety;
R is 2-pyridyl or 4-pyridyl;
each R11 is independently selected from H and d-C6alkyl;
each R12 is independently selected from H and Ci-C6alkyl;
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, CrC6alkyl, F, CI, and -OH;
each R111 is independently selected from H, CrC6alkyl, F, CI, -NH2, -OCH3, -OCH2CH3, - N(CH3)2, -CN, -N02 and -OH; each R1 12 is independently selected from H, C1-6alkyl, fluoro, benzyloxy substituted with - C(=0)OH, benzyl substituted with -C(=0)OH, CMalkoxy substituted with -C(=0)OH and C alkyl substituted with -C(=0)OH;
and provided at least one of R20 or R21 is -NHUR15 or is substituted with -NHUR15, or at least one of R3, R4, R5, R7, R3a, R4a, R5a or R7a is -OL^ R15.
Embodiment 95. A compound of Formula (A-4), or a pharmaceutically acceptable salt thereof, wherein: R1, R1 a, R3, R3a, R6, R6a, Y3 and Y4 are as defined in Embodiment 94.
Embodiment 96. A compound of Formula (A-4a), Formula A-4b), Formula A-4c) or Formula A-4d), or a pharmaceutically acceptable salt thereof, wherein:
R1 , R1a, R3, R3a, R6 and R6a are as defined in Embodiment 94;
Y3 is OR9, N(R10)2, SH or S", and
Y4 is OR9, N(R10)2, SH or S".
Embodiment 97. 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 , R1a, R3, R3a, R6 and R6a are as defined in Embodiment 94;
Y3 is OR9, N(R10)2, SH or S", and
Y4 is OR9, N(R10)2, SH or S".
Embodiment 98. A compound of Formula (B-4), or a pharmaceutically acceptable salt thereof, wherein: R1, R1 a, R3a, R5, R6a, Y3 and Y4 are as defined in Embodiment 94.
Embodiment 99. 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 R6a are as defined in Embodiment 94;
Y3 is OR9, N(R10)2, SH or S", and
Y4 is OR9, N(R10)2, SH or S".
Embodiment 100. 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 94;
Y3 is OR10, N(R10)2, SH or S", and
Y4 is OR10, N(R10)2, SH or S\
Embodiment 101. A compound of Formula (C-4), or a pharmaceutically acceptable salt thereof, wherein: R1, R1 a, R3, R5a, R6, Y3 and Y4 are as defined in Embodiment 94.
Embodiment 102. A compound of Formula (C-4a), Formula (C-4b), Formula (C-4c) or
Formula (C-4d), or a pharmaceutically acceptable salt thereof, wherein: R1 , R1a, R3, R5a and R6 are as defined in Embodiment 94;
Y3 is OR10, N(R10)2, SH or S", and
Y4 is OR10, N(R10)2, SH or S".
Embodiment 103. A compound of Formula (C-4e), Formula (C-4f), Formula (C-4g) or Formula (C-4h), or a pharmaceutically acceptable salt thereof, wherein:
R1 , R1a and R5a are as defined in Embodiment 94;
Y3 is OR10, N(R10)2, SH or S", and
Y4 is OR10, N(R10)2, SH or S".
Embodiment 104. A compound of Formula (D-4), or a pharmaceutically acceptable salt thereof, wherein: R1, R1 a, R5, R5a, Y3 and Y4 are as defined in Embodiment 94.
Embodiment 105. 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 94;
Y3 is OR10, N(R10)2, SH or S", and
Y4 is OR10, N(R10)2, SH or S".
Embodiment 106. A compound of Formula (E-4), or a pharmaceutically acceptable salt thereof, wherein: R1, R1 a, R3, R3a, R4, R a, R5 and R7 are as defined in Embodiment 94. Embodiment 107. 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 94;
and
Y3 is OR10, N(R10)2, SH or S\
Embodiment 108. A compound of Formula (F-4), or a pharmaceutically acceptable salt thereof, wherein: R1, R1 a, R1 b, R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 94. Embodiment 109. 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 94;
and
each Y3 is independent selected from OR10, N(R10)2, SH and S".
Embodiment 110. The compound of any one of Embodiments 76 to 109, wherein R1 is
Embodiment 111. The compound of any one of Embodiments 76 to 109, wherein R1a is
Embodiment 112. The compound of any one of Embodiments 76 to 109, wherein R1 is
Embodiment 113. The compound of any one of Embodiments 76 to 109, wherein R1 is
Embodiment 114. The compound of any one of Embodiments 76 to 109, wherein R1a is
Em pound of any one of Embodiments 76 to 109, wherein R1 b is
Embodiment 116. The compound of any one of Embodiments 76 to 109, wherein R1 is
Embodiment 117. The compound of any one of Embodiments 76 to 109, wherein R1a is
Embodiment 118. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R21 is -UR15.
Embodiment 119. The compound of any one of Embodiments 76 to 109, wherein R1 is
. wherein R20 is -UR15.
Em mbodiments 76 to 109, wherein R1a is
Embodiment 121. The compound of any one of Embodiments 76 to 109, wherein R1 b is
, wherein R21 is -UR15.
Embodiment 122. The compound of any one of Embodiments 76 to 109, wherein R1
and R21 is H. Embodiment 123. The compound of any one of Embodiments 76 to 109, wherein R1 is
Embodiment 124. The compound of any one of Embodiments 76 to 109, wherein R1 is
Embodiment 125. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R20 is U R15 and R21 is H.
Embodiment 126. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R20 is H and R21 is LiR15.
Embodiment 127. The compound of any one of Embodiments 76 to 109, wherein R1 is
Embodiment 128. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R20 is H and R21 is U R15.
Embodiment 129. The compound of any one of Embodiments 76 to 109, wherein R1 is
and R1a is , wherein R20 is R15 and R21 is H. Embodiment 130. The compound of any one of Embodiments 76 to 109, wherein R1 is
R
Embodiment 131. The compound of any one of Embodiments 76 to 109, wherein R is
, wherein R20 is H and R21 is U R15.
Embodiment 132. The compound of any one of Embodiments 76 to 109, wherein R1 is
Embodiment 133. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R20 is LiR15 and R21 is Li R15.
Embodiment 134. The compound of any one of Embodiments 76 to 109, wherein R1 is
R20
HN' O
I and R1a is i , wherein R20 is L^R15 and R21 is H.
Embodiment 135. The compound of any one of Embodiments 76 to 109, wherein R1
and R1a is , wherein R20 is H and R21 is LiR15. Embodiment 136. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R20 is UR15 and R21 is UR15.
Embodiment 137. The compound of any one of Embodiments 76 to 109, wherein R is
is UR15 and each R21 is H.
Embodiment 138. The compound of any one of Embodiments 76 to 109, wherein R1 is
R21 of R1a is H.
Embodiment 139. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R20 is H, R21 of R1b is H and R:
R1a is RK.
Embodiment 140. The compound of any one of Embodiments 76 to 109, wherein R1a is
OUR15.
Embodiment 141. The compound of any one of Embodiments 76 to 109, wherein R1b is
, or wherein R21 is H and one of R3, R3a, R5 or R5a is -
OUR15. Embodiment 142. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R20 is H and one of R3, R3a, R5 or R5a is -OUR15.
Embodiment 143. The compound of any one of Embodiments 76 to 109, wherein R1a is
, wherein R21 is H and one of R3, R3a, R5 or R5a is -OUR
Embodiment 144. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R21 is H and one of R3, R3a, R5 or R5a is -OUR
Embodiment 145. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R20 is H, R21 is H and one of R3, R3a, R5 or R5a is - OU R15.
Embodiment 146. The compound of any one of Embodiments 76 to 109, wherein R1 is , wherein R20 is H, R21 is H and one of R3, R3a, R5 or R5a is -OUR15.
Embodiment 147. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R20 is H, R21 is H and one of R3, R3a, R5 or R5a
15
is -OU R Embodiment 148. The compound of any one of Embodiments 76 to 109, wherein R1 is
is -OU R15.
Embodiment 149. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R20 is H, R21 is H and one of R3, R3a, R5 or R5a is -
OU R15.
Embodiment 150. The compound of any one of Embodiments 76 to 109, wherein R1 is
, wherein R is H, each R is H and one of R3, R3a, R5 or R5a is -OL^R15.
Embodiment 151. The compound of any one of Embodiments 76 to 150, wherein:
Y3 is OH, 0", SH or S", and
Y4 is OH, O", SH or S".
Embodiment 152. The compound of any one of Embodiments 76 to 150, wherein:
Y3 is OH or O", and
Y4 is OH or 0".
Embodiment 153. The compound of any one of Embodiments 76 to 150, wherein:
Y3 is SH or S", and
Y4 is OH or 0".
Embodiment 154. The compound of any one of Embodiments 76 to 150, wherein:
Y3 is OH or 0", and
Y4 is SH or S'.
Embodiment 155. The compound of any one of Embodiments 76 to 150, wherein:
Y3 is SH or S" , and
Y4 is SH or S". Embodiment 156. The compound of any one of Embodiments 76 to 139 or Embodiments
151 to 155, wherein: R2, R2a, R4, R a, R6, RSa, R7 and R7a are each H.
Embodiment 157. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein: R3 is -OH, F or -NH2.
Embodiment 158. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein: R3 is -OH or F.
Embodiment 159. The compound of any one of Embodiments 76 to 139 or Embodiments
151 to 155, wherein: R3a is -OH, F or -NH2.
Embodiment 160. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein: R3a is -OH or F.
Embodiment 161. The compound of any one of Embodiments 76 to 139 or Embodiments
151 to 155, wherein: R5 is -OH, F or -NH2.
Embodiment 162. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein: R5 is -OH or F.
Embodiment 163. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein: R5a is -OH, F or -NH2.
Embodiment 164. The compound of any one of Embodiments 76 to 139 or Embodiments
151 to 155, wherein: R5a is -OH or F.
Embodiment 165. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is -OH, and
R3a is F.
Embodiment 166. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is F, and
R3a is -OH.
Embodiment 167. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R a, R6, R6a, R7 and R7a are each H;
R3 is F, and
R3a is F.
Embodiment 168. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is -OH, and R3a is -OH.
Embodiment 169. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R a, R6, R6a, R7 and R7a are each H;
R3a is -OH, and
R5 is F.
Embodiment 170. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3a is F, and
R5 is -OH.
Embodiment 171. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3a is F, and
R5 is F.
Embodiment 172. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R a, R6, R6a, R7 and R7a are each H;
R3a is -OH, and
R5 is -OH.
Embodiment 173. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is -OH, and
R5a is F.
Embodiment 174. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is F, and
R5a is -OH.
Embodiment 175. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R a, R6, R6a, R7 and R7a are each H;
R3 is F, and
R5a is F. Embodiment 176. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is -OH, and
R5a is -OH.
Embodiment 177. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R5 is -OH, and
R5a is F.
Embodiment 178. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R a, R6, R6a, R7 and R7a are each H;
R5 is F, and
R5a is -OH.
Embodiment 179. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R5 is F, and
R5a is F.
Embodiment 180. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R5 is -OH, and
R5a is -OH.
Embodiment 181. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein:
R3 is -OH or F;
R3a is -OH or F;
R5 is -OH or F;
R5a is -OH or F;
R6 is H, and
R6a is H.
Embodiment 182. The compound of any one of Embodiments 76 to 139 or Embodiments 151 to 155, wherein:
R3 is H, -OH or F;
R3a is H, -OCH3, -OH or F; R5 is -OH or F;
R4, R4a, R6, R6a, R7, R7a are H, and
R6a is H.
Embodiment 183. The compound of any one of Embodiments 76 to 182, wherein:
Li is -C(=0)0(CH2)mNR11C(=0)(CH2)m-**; -C(=0)0(CH2)mNR11C(=0)(CH2)mO(CH2)m- **; -C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; - C(=0)OC(R12)2(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; - C(=0)0(CH2)mNR8C(=0)X1X2C(=0)(CH2)mO(CH2)m-**; - C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-**; - C(=0)0(CH2)mNR11C(=0)X4C(=0)NR11 (CH2)mNR11C(=0)(CH2)mO(CH2)m-**; - C(=0)0(CH2)mNR11C(=0)XsC(=0)(CH2)mNR11C(=0)(CH2)m-**; - C(=0)0(CH2)mX6C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**; - (CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; - (CH2)m(CHOH)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m**; - C(=0)X6C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;- C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**; - C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; - C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-*i, or - C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**,
where the ** indicates the point of attachment to R15 and
where R11 , R12, X2, m and n are s defined in Embodiment 94.
Embodiment 184. The compound of any one of Embodiments 76 to 183, wherein:
280
Embodiment 185. A compound of Formula (A) selected from:
282
283
285
286
287
288
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 1 below:
Scheme 1 Ab-RG2 + y(R15-L-(D)m)n)
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 5). D, R15, L, Ab, y, m, n and R115 are as defined herein.
Scheme 2 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 2 shows the antibody having four RG2 groups.
(Ab > (Ab2)
In one aspect, Linker-Drug moieties are conjugated to antibodies via modified cysteine residues in the antibodies (see for example WO2014/124316). Scheme 3 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 maleimide) 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 3 shows the antibody chaving four free thiol groups.
Scheme 3
In another aspect, Linker-Drug moieties are conjugated to antibodies via lysine residues in the antibodies. Scheme 4 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 4 shows the antibody chaving four amine groups.
Scheme 4
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 initially 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-dichloroacetone). 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 WO2014/083505). Scheme 5 illustrates this approach.
Scheme 5
(AM) (Ab2)
(4 interchain disulfide modified (Ab1))
In yet another aspect, Linker-Drug moieties are conjugated to antibodies by inserting a peptide tag containing a serine residue, such as an S6, 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'-phosphopantetheinyl transferases (PPTase) enzymes wherein the PPTase posttranslationally 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 oxime linkage which attaches the Linker-Drug moiety to the antibody. Scheme 6 illustrates this approach. Scheme 6
PPTase Step 1
Immunoconjuqates 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 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 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 cleavable linker.
In one aspect, the immunoconjugates of the invention comprise 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 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 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 one or more linker(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 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 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 linker 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 linker 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 linker 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 (I) or Formula (II), D is an agonist of Stimulator of Interferon Genes (STING) receptor.
In an embodiment of Formula (I) or Formula (II), D is a cyclic dinucleotides which bind to Stimulator of Interferon Genes (STING) receptor.
In an embodiment of Formula (I) or Formula (II), D is a cyclic dinucleotide which is an agonist of Stimulator of Interferon Genes (STING) receptor. In one aspect, the immunoconjugates of the invention comprise 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 a linker.
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 a linker, wherein linker (L) is a cleavable linker.
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 linker.
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 linker, wherein linker (L) is a cleavable linker.
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 linker.
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 linker, wherein linker (L) is a cleavable linker.
The term "cleavage 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 toSTING 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 immunoconjugate has STING agonist activity.
In one embodiment, the immunoconjugates of the invention comprise Formula (I):
Ab-(L-(D)m)p (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 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 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;
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 cell 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-Dual assay, a TANK binding kinase 1 (TBK1) assay, or an interferon-Y-inducible protein (IP-10) secretion assay.
In one aspect the immunoconjugate of the invention, the immunoconjugate is selected from
-a) Formula (AA-b)
-e) Formula (AA-f)
Formula (BB-a) Formula (BB-b)
Formula (BB-e)
Formula (CC-c) Formula (CC-d)
300
301
Formula (FF-a) Formula (FF-b)
Formula (FF-e) Formula (FF-f)
Formula (FF-i) Formula (FF-j)
Formula (FF-k);
wherein:
each Gi is independently selected
where * indicates the point of attachment to -CR R -;
XA is C(=0)-, -C(=S)- or -C(=NR11)- and each Ζ is NR12;
XB is C, and each Z2 is N; , where * indicates the point of attachment to -CR8aR9a-;
Xc is C(=0)-, -C(=S)- or -C(=NR11)- and each Z3 is NR12;
YI is -0-, -S-, -S(=0)-, -SO2-, -CH2-, or -CF2-;
Y2 is -0-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2-;
Y3 is OH, 0", OR10, N(R10)2, SR10, SeH, Se", BH3, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SR10, SeH, Se , BH3, SH or S";
Ys is -CH2-, -NH-, -0- or -S;
Ye is -CH2-, -NH-, -0- or -S;
Y7 is 0 or S;
Ys is 0 or S; Y9 is -CH , -NH-, -0- or -S;
Y10 is -CH2-, -NH-, -0- or -S;
Yi i is -0-, -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 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 0, 1 , 2, 3 or 4 substituents independently selected from -NHUR115, F, CI, Br, OH, SH, NH2, D, CD3, CrCsalkyl, CrCsalkoxyalkyl, CrCehydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(Cr C6alkyl), -0(C3-C8cycloalkyl), -StCrCealkyl), -S(CrCsaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -NKd-Cealkyl^, -N(Cr C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), -
NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
R1a 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 -NHUR115, F, CI, Br, OH, SH, NH2, D, CD3, CrCsalkyl, CrCsalkoxyalkyl, CrCehydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(Cr C6alkyl), -0(C3-C8cycloalkyl), -StCrCealkyl), -S(CrCsaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -NKd-Cealkyl^, -N(Cr
C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
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 0, 1 , 2, 3 or 4 substituents independently selected from -NHUR115, F, CI, Br, OH, SH, NH2, D, CD3, CrCsalkyl, CrCsalkoxyalkyl, CrCehydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(Cr
C6alkyl), -0(C3-C8cycloalkyl), -StCrCealkyl), -S(CrCsaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -NKd-Cealkyl^, -N(Cr C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -(CH2)1.10C(=O)OH,- CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
each R2 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C3haloalkenyl, C2-
C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - 0C(0)0C2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl,
CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R3 is independently selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and the
CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R4 is independently selected from the group consisting of -OL^R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, CrCshaloalkynyl, -0(Ci-C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)d-C6alkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4 are substituted by
0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; each R5 is independently selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5 are substituted by
0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R6 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -
OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6 and the CrC6alkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R7 is independently selected from the group consisting of -OL^R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -
O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), -
0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R8 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C3haloalkenyl, C2-
Cshaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R8 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R9 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C3haloalkenyl, C2- Cehaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, -
O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R9 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, d-Cshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Ci-C6alkyl), -0(C2-C3alkenyl), -
0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R2a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr
C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-
C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3
R3a is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and the
CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4a and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C3alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R6aare substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; R7a is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - 0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)phenyl, -OC^C Cealkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7a and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC^OC Cealkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7a are substituted by
0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R8a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, -
OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R8a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R9a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -
O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R9a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C3haloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; each R10 is independently selected from the group consisting of H, d-C^alkyl, Cr
C6heteroalkyl, , wherein the CrC12alkyl and d-dheteroalkyl of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, C C12alkoxy, -S-C(=0)CrC6alkyl, halo, -CN, C C12alkyl, -O-aryl, _0-heteroaryl, -O-cycloalkyl, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, -OC(0)OC1-C6alkyland C(0)OCi-Cealkyl, wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is substituted by 0, 1 , 2 or 3 substituents independently selected from C Cu alkyl, 0-CrC12alkyl, CrC12heteroalkyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroaryl, -C(=0)C1-C12alkyl, -OC(=0)d- C12alkyl, -C(=0)OCrC12alkyl, -OC(=0)OCrC12alkyl, -C(=0)N(R11)-CrC12alkyl, - N(R11)C(=0)-C1-C12alkyl; -OC(=0)N(R11)-C1-C12alkyl, -C(=0)-aryl, -C(=0)-heteroaryl, -OC(=0)-aryl, -C(=0)0-aryl, -OC(=0)-heteroaryl, -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(0)-aryl, -N(R11)C(0)-heteroaryl, and S(0)2N(R11)-aryl;
each R1 1 is independently selected from H and Chalky!;
each R12 is independently selected from H and CrCsalkyl;
optionally R3 and R6 are connected to form d-C6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3 and R6 are connected, the 0 is bound at the R3 position
optionally R3a and R6a, are connected to form d-C8alkylene, d-dalkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3a and R6a are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form Crdalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2 and R3 are connected, the 0 is bound at the R3 position;
optionally R2a and R3a, are connected to form d-C8alkylene, d-dalkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form Crdalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-d-C6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4 and R3 are connected, the 0 is bound at the R3 position;
optionally R a and R3a, are connected to form d-C3alkylene, d-dalkenylene, C2- C6alkynylene, -0-d-C6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4a and R3a are connected, the O is bound at the R3a position; optionally R and R are connected to form C^Cealkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R6 are connected, the 0 is bound at the R5 position;
optionally R5a and R6a, are connected to form CrCsalkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R6a are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form C^Cealkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R7 are connected, the 0 is bound at the R5 position;
optionally R5a and R7a, are connected to form CrCsalkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R7a are connected, the O is bound at the R5a position;
optionally R8 and R9 are connected to form a d-Calkylene, C2-C6alkenylene, C2- C6alkynylene, and
optionally R8a and R9a are connected to form a CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene,
is a linker;
, -C(=0)-, -ON=***, -S-, -NHC(=0)CH2-
S(=0)2CH2CH2-***, -(CH2)2S(=0)2CH2CH2-***, -NHS(=0)2CH2CH2..., -NHC(=0)CH2CH2-
R13 is H or methyl;
R1 isH,-CH3 or phenyl;
each R110 is independently selected from H, d-C6alkyl, F, CI, and -OH;
each R111 is independently selected from H, CrC6alkyl, F, CI, -NH2, -OCH3, -OCH2CH3, -
N(CH3)2, -CN,-N02 and -OH;
each R112 is independently selected from H, Ci.6alkyl, fluoro, benzyloxy substituted with -
C(=0)OH, benzyl substituted with -C(=0)OH, CMalkoxy substituted with -C(=0)OH and
CMalkyl substituted with -C(=0)OH;
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 is substituted with -NHL^R115, or at least one of
R3, R4, R5, R7, R3a, Ra, R5a or R7a is -OL^115. Certain 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 188. The immunoconjugate of Formulas (AA-a to AA-f), Formulas (BB-a to BB- f), Formulas (CC-a to CC-f), Formulas (DD-a to DD-f), Formulas (EE-a to EE-h) or Formulas
(FF-a to FF-k), or stereoisomers or pharmaceutically acceptable salts thereof, wherein is a linker comprising one or more cleavage elements;
Embodiment 189. An immunoconjugate of Formulas (AA-a to AA-f), Formulas (BB-a to BB- f), Formulas (CC-a to CC-f), Formulas (DD-a to DD-f), Formulas (EE-a to EE-h) or Formulas -a to FF-k), or stereoisomers or pharmaceutically acceptable salts thereof selected from:
Formula (AA-1 e) Formula (AA-1f)
Formula (CC- 1a) Formula (CC- 1b)
317
318 -1a) -1b)
Formula (FF-1c) Formula (FF-1d)
Formula (FF-1e) Formula (FF-1f)
Formula (FF-1 i) Formula (FF-1j)
Formula (FF-1 k);
wherein y, Ab, R1 , R1a, R1 b, R2, R2a, R3, R3a, R4, R4a, R5, R5a, R8, R6a, R7, R7a, R8, R8a, R9, R9a, Yi > Y2, Y3, Y4, Y5, Ye> Y7, Ye, Y9, Y10 and Yn are as defined above for imunoconjugates of Formulas (AA-a to AA-f), Formulas (BB-a to BB-f), Formulas (CC-a to CC-f), Formulas (DD- a to DD-f), Formulas (EE-a to EE-h) and Formulas (FF-a to FF-k), and provided at least one of R1, R1a or R1 is substituted with -NHLiR115, or at least one of R3, R4, R5, R7, R3a, R4a, R5a or R7a is -OUR115.
Embodiment 190. The immunoconjugate of Embodiment 146, 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 is a pyrimidine or purine nucleic acid base or analogue thereof, each of which is substituted as described in R1 , R1a or R1 for immunoconjugates of
Formulas (AA-a to AA-f), Formulas (BB-a to BB-f), Formulas (CC-a to CC-f), Formulas (DD- a to DD-f), Formulas (EE-a to EE-h) and Formulas (FF-a to FF-k).
Formula (AA-2a) Formula (AA-2b)
323
Formula (CC-2e) Formula (CC-2f)
-2a) -2b)
Formula (EE-2g) Formula (EE-2h)
Formula (FF-2c) Formula (FF-2d)
Formula (FF-2g) Formula (FF-2h)
Formula (FF-2k)
wherein y, Ab, R1, R1a, R1 , R2, R2a, R3, R3a, R4, R4a, R5, R5a, R6, R8a, R7, R7a, R8, R8a, R9, R9a, Υ,, Y2> Y3, Y4, Y5, Ye, Y7, Ya, Y9, Y10 and Y are as defined above for imunoconjugates of Formulas (AA-a to AA-f), Formulas (BB-a to BB-f), Formulas (CC-a to CC-f), Formulas (DD-a to DD-f), Formulas (EE-a to EE-h) and Formulas (FF-a to FF-k), and provided at least one of R1, R1a or R1b is substituted with -NHUR115, or at least one of R3, R4, R5, R7, R3a, R a, R5a or R7a is - OUR115.
Embodiment 192. The immunoconjugate of Formula (AA-a to AA-f), Formula (AA-1 a to AA- 1f) or Formula (AA-2a to AA-2f), wherein R2 and R2a are H;
one of R3 and R4 is H and the other is selected from the group consisting of -OL^115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -
OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 or R4 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrCealkyI, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3 or R4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R5 and R5a are H;
R6 and R6a are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R3a and R4a is H and the other is selected from the group consisting of -OL^R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, d-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -
OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and R4a and the CrCsalkyl, C2-C6alkenyl and C2- C6alkynyl of the CrCealkyI, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3a or R4a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and provided at least one of R1 or R1a is substituted with -NHL^R115, or at least one of R3, R4, R3a or R4a is -OUR115.
Embodiment 193. The immunoconjugate of Formula (AA-a to AA-f), Formula (AA-1a to AA- 1f) or Formula (AA-2a to AA-2f), wherein:
Y1 and Y2 are O, CH2 or S;
Y3 is OH, O", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S; Y9 and Y10 are 0 or S;
R2, R2a, R6, R6a, R5 and R5a are H
one of R3a and R4a is H and the other is -OUR115, H, OH or F;
one of R3 and R4 is H and the other is -OU R115, H, OH or F; and
R8a, R9a, R8 and R9 are independently selected from H or CrCealk l,
and provided at least one of R1 or R1a is substituted with -NHUR115, or at least one of R3, R4, R3a or R a is -OUR115.
Embodiment 194. The immunoconjugate of Formula (BB-a to BB-f), Formula (BB-1a to BB- 1f) or Formula (BB-2a to BB-2f), wherein:
R2 and R2a are H;
one of R3a and R4a is H and the other is selected from the group consisting of -O R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrOsalkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -
OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and R4a and the CrC3alkyl, C2-C6alkenyl and C2- C6alkynyl of the Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl,
-0C(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3a or R4a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; R5a and R6a are H;
R6 and R4 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R5 and R7 is H and the other is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -
OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and R7 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl,
-0C(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R5 or R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and provided at least one of R1 or R1a is substituted with -NHUR115, or at least one of R5, R7, R3a or R4a is -OL,R115.
Embodiment 195. The immunoconjugate of Formula (BB-a to BB-f), Formula (BB-1a to BB- 1f) or Formula (BB-2a to BB-2f), wherein:
Yi and Y2 are 0, CH2 or S;
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S;
Y9 and Y10 are 0 or S;
R2, R2a, R5a, RSa, R6 and R4 are H;
one of R3a and R4a is H and the other is -OUR115, H, OH or F;
one of R5 and R7 is H and the other is -OU R115, H, OH or F, and
R8a, R9a, R8 and R9 are independently selected from H or Chalky!,
and provided at least one of R1 or R1a is substituted with -NHUR115, or at least one of R5,
R7, R3a or R a is -OUR115.
Embodiment 196. An immunoconjugate of Formula (CC-a to CC-f), Formula (CC-1 a to CC- 1f) or Formula (CC-2a to CC-2f), wherein:
R2 and R2a are H;
one of R3 and R4 is H and the other is selected from the group consisting of -OUR115, H,
-OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr Cehaloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and R4 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(Ci-C3alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3 or R4 are substituted by 0, 1 , 2 or
3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; R4a and R6a are H;
R6 and R5 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl;
one of R5a and R7a is H and the other is selected from the group consisting of -OUR115,
H, -OH, F, CI, Br, I, D, CD3, CN, N3, C Cealkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and R7a and the CrCsalkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-
C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R5a or R7a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and provided at least one of R1 or R1a is substituted with -NHUR115, or at least one of
R5a, R7a, R3a or R4a is -OUR115.
Embodiment 197. An immunoconjugate of Formula (CC-a to CC-f), Formula (CC-1 a to CC- 1f) or Formula (CC-2a to CC-2f), wherein:
Y! and Y2 are O, CH2 or S;
Y3 is OH, 0-, OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S;
Y9 and Y10 are 0 or S;
R2, R2a, R4a, RSa, R6 and R5 are H;
one of R3 and R4 is H and the other is -OU R115, H, OH or F;
one of R5a and R7a is H and the other is -OUR115, H, OH or F, and
R8a, R9a, R8 and R9 are independently selected from H or CrCsalkyl,
and provided at least one of R1 or R1a is substituted with -NHUR115, or at least one of R5a, R7a, R3a or R a is -OUR115.
Embodiment 198. An immunoconjugate of Formula (DD-a to DD-f), Formula (DD-1 a to DD- 1f) or Formula (DD-2a to DD-2f), wherein:
R2 and R2a are H;
one of R5a and R7a is H and the other is selected from the group consisting of -OU R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, C Csalkyl, C2-C6alkenyl, C2-C6alkynyl, Cr
C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and R7a and the CrC3alkyl, C2-C6alkenyl and C2-
C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R5a or R7a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R a and R6a are H;
R6 and R4 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R5 and R7 is H and the other is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10P(=O)(OH)2, -
OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and R7 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(Ci-C3alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -0C(0)C2-C3alkynyl of R5 or R7 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and provided at least one of R1 or R1a is substituted with -NHUR115, or at least one of R5a, R7a, R5 or R7 is -OUR115.
Embodiment 199. An immunoconjugate of Formula (DD-a to DD-f), Formula (DD-1 a to DD- 1f) or Formula (DD-2a to DD-2f), wherein:
ΥΪ and Y2 are 0, CH2 or S;
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SH or S";
Y5 and Y6 are 0 or S;
Y7 and Y8 are 0 or S;
R2, R2a, R4a, RSa, R6 and R4 are H;
one of R5a and R7a is H and the other is -OUR115, OH or F;
one of R5 and R7 is H and the other is -OUR115, H, OH or F, and
R8, R9, R8a and R9a are independently H or CrC6alkyl,
and provided at least one of R1 or R1a is substituted with -NHUR115, or at least one of R5a, R7a, R5 or R7 is -OUR115.
Embodiment 200. An immunoconjugate of Formula (EE-a to EE-h), Formula (EE-1 a to EE- 1 h) or Formula (EE-2a to EE-2h), wherein:
R2 and R2a are H; Re and Rea are H;
R7 is H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R3a and R a is H and the other is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, C Cealkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -0P(=0)(0H)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and R a and the CrCsalkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -OtCrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -0C(0)C2-C3alkynyl of R3a or R4a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
one of R3 and R4 is H and the other is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and R4 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -OtCrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3 or R4 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and one of R5 and R7 is H and the other is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -0P(=0)(0H)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and R7 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -OtCrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R5 or R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and provided at least one of R1 or R1a is substituted with -NHUR115, or at least one of R3a, R a, R3, R4, R5 or R7 is -OUR115.
Embodiment 201. An immunoconjugate of Formula (EE-a to EE-h), Formula (EE-1 a to EE- 1 h) or Formula (EE-2a to EE-2h), wherein:
Y3 is OH, 0", OR10, N(R10)2, SH or S";
Y5 is O or S;
Y9 is O or S;
R2, R2a, R5, R6a, R6 and R7 are H;
one of R3a, R a is H and the other is -OUR115, H, OH, OCH3 or F;
one of R3, R4 is H and the other is -OUR115, H, OH, OCH3 or F;
one of R5 and R7 is H and the other is -OU R115, H, OH, OCH3 or F, and
R8, R9, R8a and R9a are independently H or CrC6alkyl,
and provided at least one of R1 or R1a is substituted with -NHUR115, or at least one of R3a, R4a, R3, R4, R5 or R7 is -OUR115.
Embodiment 202. An immunoconjugate of Formula (FF-a to FF-k), Formula (FF-1 a to FF- 1 k) or Formula (FF-2a to FF-2k), wherein:
R2 and R2a are H;
each Rs and RSa are H;
R5a and R7 are H;
R8, R9, R8a and R9a are independently H or CrC6alkyl, and
one of R3a and R a is H and the other is selected from the group consisting of -OUR115,
H, -OH, F, CI, Br, I, D, CD3, CN, N3, C Cealkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and R a and the CrCsalkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3a or R4a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; one of R3 and R4 is H and the other is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, Cr C6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, - OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and R4 and the CrC6alkyl, C2-C6alkenyl and C2- C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrCsalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C3alkynyl of R3 or R4 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3, and
R5 is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3 C^Cealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrCealkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2- C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2- C6alkenyl), -0(C2-C6alkynyl), -OC^OCrCealkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2- C6alkynyl of R5 is substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3,
and provided at least one of R1, R1a or R1b is substituted with -NHUR115, or at least one of R3a, R4a, R3, R4, R5 or R7 is -OUR115.
Embodiment 203. An immunoconjugate of Formula (FF-a to FF-k), Formula (FF-1a to FF- 1 k) or Formula (FF-2a to FF-2k), wherein:
ΥΪ and Y2 are 0, CH2 or S;
each Y3 is independently OH, 0", OR10, N(R10)2, SH or S";
each Y5 is independently 0 or S;
each Y7 is independently 0 or S;
each Yg is independently 0 or S;
R2, R2a, R6, R6a, R5a, and R7a are H;
one of R3a and R4a is H and the other is -OUR115, H, OH, OCH3 or F;
one of R3 and R4 is H and the other is -OUR115, H, OH, OCH3 or F; one of R5 and R7 is H and the other is -OU R115, H, OH, OCH3 or F, and R8, R9, R8a and R9a are independently H or CrC6alkyl,
and provided at least one of R1 , R1a or R1 is substituted with -NHUR115, or at least one of R3a, R a, R3, R4, R5 or R7 is -OUR115.
Embodiment 204. An immunoconjugate of Formula (AA-a to AA-f), Formula (BB-a to BB-f), Formula (CC-a to CC-f), Formula (DD-a to DD-f), Formula (EE-a to EE-h), Formula (FF-a to FF-k or an immunoconjugate of any one of Embodiments 146 to 161 , wherein:
wherein: R1 is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, CrC6alkoxyalkyl, Cr
C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), - 0(C3-C8cycloalkyl), -S(CrC6alkyl), -S(CrC6aminoalkyl), -S(Cr
C6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3- C8cycloalkyl), -N(Ci-C6alkyl)2, -N(CrC6alkyl) (C3-C8cycloalkyl), -CN, - P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,-CH=CH(CH2)1. 10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2,
and
each R200 is independently selected from H and U R115;
340
wherein: R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, Ci-Cealkoxyalkyl, d- C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), - 0(C3-C8cycloalkyl), -S(Ci-C6alkyl), -S(Ci-C6aminoalkyl), -S(Cr
C6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3- C8cycloalkyl), -N(C1-C6alkyl)2, -N(CrC6alkyl) (C3-C8cycloalkyl), -CN, - P(=0)(OH)2, -(CH2)1.10C(=O)OH,-CH=CH(CH2)1. 10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2,
and
210 is independently selected from H and UR115,
342
wherein: R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, d-Cealkoxyalkyl, Cr
C6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), - 0(C3-C8cycloalkyl), -SiCrCealkyl), -SiCrCeaminoalkyl), -S(Cr
C6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3- C8cycloalkyl), -N(CrC6alkyl)2, -N(CrC6alkyl) (C3-C8cycloalkyl), -CN, - P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,-CH=CH(CH2)1.
10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), -
NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2,
and
each R210 is independently selected from H and UR115.
Embodiment 205. An immunoconjugate selected from:
Formula (AA-3a) Formula (AA-3b)
Formula (AA-3c) Formula (AA-3d)
Formula (AA-3e) Formula (AA-3f)
Formula (BB-3a) Formula (BB-3b)
Formula (BB-3c) Formula (BB-3d)
Formula (BB-3e) Formula (BB-3f)
-3c) Formula (CC-3d)
-3e) Formula (CC-3f)
Formula (EE-3c) Formula (EE-3d)
Formula (FF-3a) Formula (FF-3b)
Formula (FF-3e) Formula (FF-3f)
Formula (FF-3i) Formula (FF-3j)
Formula (FF-3k)
wherein:
Yi is -0-, -S- -S(=0)-, -SOr, -CH2-, or - CF2-
Y2 js -0-, -S- -S(=0)-, -SOr, -CHr, or - CF2-
Y3 is OH, 0-, OR10, N(R10)2, SH or S";
Y4 is OH, 0-, OR10, N(R10)2, SH or S";
Y7 is 0 or S;
Y8 is 0 or S;
Yii is -0-, -S -, -S(=0)-, -S02-, -CH2-, or -CF2
wherein: R1 is substituted with 0, 1 , 2 or 3 substituents independently selected from
F, CI, Br, OH, SH, NH2, D, CD3, Ci-C6alkyl, d-C6alkoxyalkyl, Ci-C8 ydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -O^-Cealkyl), -0(C3-C8cycloalkyl), - S(CrC6alkyl), -S(CrCsaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl),■ NH(Ci-Cealkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(CrCsalkyl) (C3- Cgcycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)i-i0C(=O)OH, -(CH2)1.10C(=O)OH,- CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2,
352
wherein: R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, C Cealkyl, CrCealkoxyalkyl, CrCshydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from 0, N and S, -0(CrC6alkyl), -0(C3-C8cycloalkyl), - S(CrC6alkyl), -S(CrCsaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), - NH(Ci-C3alkyl), -NH(C3-C8cycloalkyl), -N(d-C6alkyl)2, -N(Ci-C3alkyl) (C3- C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2, each R is inde endently selected from H and UR
wherein: R is substituted with 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, OH, SH, NH2, D, CD3, CrCealkyl, CrCealkoxyalkyl, CrCshydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from O, N and S, -C CrCealkyl), -0(C3-C8cycloalkyl), -
S(CrC6alkyl), -S(CrCsaminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), - NH(CrCsalkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(CrCsalkyl) (C3- Cacycloalkyl), -CN, -P(=0)(OH)2, -(CH2)1.10C(=O)OH,- CH=CH(CH2)i-ioC(=0)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2,
and
each R210 is independently selected from H and UR115; each R2 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C3haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - 0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, -0C(0)phenyl, -OC(0)CrC6alkyl, - 0C(0)C2-C6alkenyl and -0C(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -0C(0)C2-C6alkenyl and -0C(0)C2-C6alkynyl of R2 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R3 is independently selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and the Ci-C3alkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R4 is independently selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R5 is independently selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, CrCshaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -
OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R6 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C3haloalkenyl, C2- Cehaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, -
O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, d-Cshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Ci-C6alkyl), -0(C2-C3alkenyl), -
0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of Rs are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R7 is independently selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl,
C2-C3haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7 and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl,
CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R2a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C3alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3
is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C3alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4a and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(d-C6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and the CrCsalkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; FT is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C3alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC^Od-Cealkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R6aare substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R7a is selected from the group consisting of -OUR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCsalkyl, C2-C3alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C3alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7a and the Ci-C3alkyl, C2-C3alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCshaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C3alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -0C(0)0C2-C6alkenyl, -0C(0)0C2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
eac 10 is independently selected from the group consisting of H, CrC12alkyl, -
, wherein the CrC12alkyl of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, d- C12alkoxy, -S-C(=0)CrC6alkyl and C(0)OCrCsalkyl;
optionally R3 and R6 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3 and R6 are connected, the O is bound at the R3 position
optionally R3a and R6a, are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3a and R6a are connected, the O is bound at the R3a position; optionally R2 and R3 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2 and R3 are connected, the 0 is bound at the R3 position;
optionally R2a and R3a, are connected to form CrCsalkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4 and R3 are connected, the 0 is bound at the R3 position;
optionally R a and R3a, are connected to form CrCsalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form d-Calkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R6 are connected, the 0 is bound at the R5 position;
optionally R5a and R6a, are connected to form CrCsalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R6a are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, 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 CrCsalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R7a are connected, the O is bound at the R5a position;
U is -C(=0)0(CH2)mNR11C(=0)(CH2)m-**; -C(=0)0(CH2)mNR11C(=0)(CH2)mO(CH2)m-**; -C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;
-C(=0)OC(R12)2(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-**;
-C(=0)0(CH2)mNR11C(=0)X4C(=0)NR1 1(CH2)mNR11C(=0)(CH2)mO(CH2)m-**; - C(=0)0(CH2)mNR11C(=0)XsC(=0)(CH2)mNR1 1C(=0)(CH2)m-**;
-C(=0)X4C(=0)NR1 1(CH2)mNR11C(=0)(CH2)mO(CH2)m-";
-C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -C(=0)(CH2)mNR11C(=0)X2C(=0)(CH2)m-** -C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**,
-C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**, -C(=0)0(CH2)mX6C(=0)(CH2)m-**,
-C(=0)0(CH2)mXsC(=0)(CH2)mO(CH2)m-**, -C(=0)0(CH2)mX6C(=0)X,X2C(=0)(CH2)m-**, -C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)mO(CH2)m-**, -C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)- **,
-C(=0)0(CH2)mXeC(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**,
-C(=0)X4C(=0)X6(CH2)mNR11C(=0)(CH2)mO(CH2)m-**,
-C(=0)(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**, -
C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)
**.
m" i
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0))X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR1 ,C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)Il(CH2)m-**;
C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)
**.
m~ i
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mNR11((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR1 ,C(=0)X5C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR1 ,C(=0)X5(CH2)mX3(CH2)m-**;
-C(=0)0(CH2)m-**; -C(=0)0((CH2)mO)n(CH2)m-**; -C(=0)0(CH2)mNR11(CH2)m-**;
-C(=0)0(CH2)mNR11(CH2)mC(=0)X2X1C(=0)-**; -C(=0)0(CH2)mX3(CH2)m-**; -
C(=0)0(CH2)mX6C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mX3(CH2)m-**; -C(=0)0((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0(CH2)mX3(CH2)m-ii;
-C(=0)0((CH2)mO)n(CH2)mNR1 ,C(=0)(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)nX3(CH2)m-**; -C(=0)0((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mC(=0)NR11(CH2)m-**; -C(=0)0(CH2)mC(R12)2-*i;
-C(=0)OCH2)mC(R12)2SS(CH2)mNR11C(=0)(CH2)m-*i;
-C(=0)0(CH2)mC(=0)NR11(CH2)m-**; -C(=0)(CH2)m-**; -C(=0)((CH2)mO)n(CH2)m-**; -C(=0)(CH2)mNR11(CH2)m-**;
-C(=0)(CH2)mNR11(CH2)mC(=0)X2X1C(=0)-**; -C(=0)(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**; -C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**; -C(=0)(CH2)mNR11C(=0(CH2)mX3(CH2)m-**; -(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -
(CH2)m(CHOH)(CH2)mNR11C(=0)X,X2C(=0)(CH2)m **;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)nX3(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mC(=0)NR11(CH2)m-**; -C(=0)(CH2)mC(R12)2-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-
**.
-C(=0)((CH2)mO)n(CH2)mNR11C(=0))X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)„(CH2)mX3(CH2)m- **;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mNR11((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)m-**;
-C(=0)((CH2)mO)p(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**;
■C(=0)(CH2)mC(R12)2SS(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)(CH2)mC(=0)NR11(CH2)m-**; -C(=0)X1X2C(=0)(CH2)m-ii;
-C(=0)X1X2C(=0)(CH2)mNR11C(=0)(CH2)m-**; -C(=0)X1X2C(=0)(CH2)mX3(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**; =0)X1X2C(=0)(CH2)mNR11C(=0)((CH2)mO)„(CH2)m-**;
=0)X1X2C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)n
=0)X1X2(CH2)mX3(CH2)m-**; -C(=0)X1X2((CH2)mO)n(CH2)m-*
=0)X1X2((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
=0)X1X2((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
=0)X1X2((CH2)mO)n(CH2)mX3(CH2)m-**;
=0)X1X2(CH2)mNR11((CH2)mO)n(CH2)m-**;
=0)X1X2C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-**;
=0)NR1 (CH2)m-**; -C(=0)NR11(CH2)mX3(CH2)n
=0)NR1 (CH2)mNR C(=0)X1X2C(=0)(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)0(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)X1X2-**; -C(=0)NR11(CH2)mNR11C(=0)Xs-;
=0)NR1 (CH2)mNR C(=0)(CH2)mX5(CH2)m-**;
=0)X1C(=0)NR11(CH2)mX5(CH2)
=0)NR1 (CH2)mNR C(=0)(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)(CH2)mO(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)X1X2C(=0)(CH2)mO(CH2)m-ii;
=0)NR1 (CH2)mNR C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-**;
=0)NR1 (CH2)mNR C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)n =0)NR1 (CH2)mNR C(=0)X1X2C(=0)(CH2)n
=0)NR1 (CH2)mNR C(=0)X5C(=0)(CH2)„
=0)NR1 (CH2)mNR C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-*i =0)NR1 (CH2)mNR C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)n =0)NR1 (CH2)mNR C(=0)X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**; =0)NR1 (CH2)mNR C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)n =0)NR1 (CH2)mNR C(=0)X5(CH2)mX3(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)X5((CH2)mO)n(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-";
=0)NR1 (CH2)mNR C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**; =0)NR1 (CH2)mNR C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)X5(CH2)mNR11((CH2)mO)n(CH2)m-**;
=0)NR1 (CH2)mNR C(=0)X5C(=0)(CH2)mNR11((CH2)mO)n(CH2)mX3(CH2)m-**; =0)NR1 (CH2)mNR C(=0)X5(CH2)m-**; -C(=0)NR11(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)NR11(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**;
1 (CH2)mNR11 C(=0)(CH2)m-**;
-C(=0)X1C(=0)NR11(CH2)mX3(CH2)m-**; -C(=0)NR11(CH2)mNR11C(=0)(CH2)m-**; -C(=0)NR11(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**; -C(=0)NR11(CH2)mNR11C(=0)- -C(=0)X1X2(CH2)m-**; -C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)X1X2(CH2)mX3(CH2)m-**; -C(=0)NR11(CH2)mX3(CH2)m-**;
-C(=0)NR11((CH2)mO)n(CH2)mX3(CH2)m-**; -C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-*i -C(=0)X1X2C(=0)(CH2)m-**; -C(=0)X1C(=0)(CH2)mNR11C(=0)(CH2)m-**; and -C(=0)X1C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
CH2CH2-, -(CH2)2S(=0)2CH2CH2-, -NHS(=0)2CH2CH2, -NHC(=0)CH2CH
, where the * indicates the point of attachment to
X4 is -O(CH2)nSSC(R1 2(CH2)n- or -(CH2)nC(Fr)2SS(CH2)nO-
Xs is where the
Xs i or, where the ** indicates orientation toward the Drug moiety;
each R1 1 is independently selected from H and Ci-Cealkyl;
each R12 is independently selected from H and d-Cealkyl;
R13 is H or methyl;
R14 is H, -CH3 or phenyl;
each R1 10 is independently selected from H, CrC6alkyl, F, CI, and -OH;
each R1 11 is independently selected from H, CrC6alkyl, F, CI, -NH2, -OCH3, -OCH2CH3, -
N(CH3)2, -CN, -N02 and -OH;
each R1 12 is independently selected from H, C1-6alkyl, fluoro, benzyloxy substituted with - C(=0)OH, benzyl substituted with -C(=0)0H, d^alkoxy substituted with -C(=0)OH and CMalkyl substituted with -C(=0)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, 10, 1 1 , 12, 13, 14, 15, 16, 17 and 18. Ab is an antibody or fragment thereof, and each y is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10, and provided at least one of R200 or R210 is -UR115 or is substituted with -NHL,R115, or at least one of R3, R4, R5, R7, R3a, R4a, R5a or R7a is -OUR115. Embodiment 206. An immunoconjugate selected from:
Formula (AA-4a) Formula (AA-4a)
Formula (AA-4c) Formula (AA-4d)
wherein: Ab, y, R1, R1a, R3, R3a, Rs, RSa, Y3 and Y4 are as defined in Embodiment 205. Embodiment 207. An immunoconjugate selected from:
Formula (AA-4e) Formula (AA-4f)
-4 )
Formula (AA-4i) Formula (AA-4j)
Formula (AA-4k) Formula (AA-41)
Formula (AA-4m) Formula (AA-4n)
Formula (AA-4o) Formula (AA-4p)
Formula (AA-4s) Formula (AA-4t) wherein: Ab, y, R1, R1a, R3, R3a, R6 and R6a are as defined in Embodiment 205;
Y3 is OR9, N(R10)2, SH or S", and
Y4 is OR9, N(R10)2, SH or S".
Embodiment 208. An immunoconjugate selected from:
Formula (AA-5c) Formula (AA-5d)
Formula (AA-5e) Formula (AA-5f)
Formula (AA-5k) Formula (AA-51)
-5u) -5v)
Formula (AA-5y)
Formula (AA-5bb)
Formula (AA-5cc) Formula (AA-5dd)
Formula (AA-5ee) Formula (AA-5ff) wherein: Ab, y, R1, R1a, R3, R3a, R6 and R6a are as defined in Embodiment 205;
Y3 is OR9, N(R10)2, SH or S", and
Y4 is OR9, N(R10)2, SH or S".
Embodiment 209. An immunoconjugate selected from:
Formula (BB-4c) Formula (BB-4d) wherein: Ab, y, R1, R1a, R3, R3a, R5, R6a, Y3 and Y4 are as defined in Embodiment 205. Embodiment 210. An immunoconjugate selected from:
372
SUBSTITUTE SHEET (RLILE 26) -4e) -4f)
-4i) -4j)
Formula (BB-4m) Formula (BB-4n)
Formula (BB-4s) Formula (BB-4t) wherein: Ab, y, R1, R1a, R3a, R5 and R6a are as defined in Embodiment 205;
Y3 is OR9, N(R10)2, SH or S", and
Y4 is OR9, N(R10)2, SH or S".
Embodiment 211. An immunoconjugate selected from:
Formula (BB-5a) Formula (BB-5b)
Formula (BB-5i) Formula (BB-5j)
Formula (BB-5k) Formula (BB-51) wherein: Ab, y, R , R and R are as defined in Embodiment 205;
Y3 is OR9, N(R10)2, SH or S", and
Y4 is OR9, N(R10)2, SH or S".
Embodiment 212. An immunoconjugate selected from:
Formula (CC-4c) Formula (CC-4d) wherein: Ab, y, R1, R1a, R3, R5a, R6, R6 , Y3 and Y4 are as defined in Embodiment 205. Embodiment 213. An immunoconjugate selected from:
Formula (CC-4e) Formula (CC-4f)
-k Formula (CC-41)
Formula (CC-4o) Formula (CC-4p)
Formula (CC-4s) Formula (CC-4t) wherein: Ab, y R1 , R1a, R3, R5a, R6 and R6a are as defined in Embodiment 205;
Y3 is OR9, N(R10)2, SH or S", and
Y4 is OR9, N(R10)2, SH or S".
Embodiment 214. An immunoconjugate selected from:
Formula (CC-5c) Formula (CC-5d)
Formula (CC-5e) Formula (CC-5f)
-5g) -5 )
Formula (CC-5j)
Formula (CC-5k) Formula (CC-51) wherein: Ab, y, R , Rla, Rsa and RM are as defined in Embodiment 205;
Y3 is OR9, N(R10)2, SH or S", and
Y4 is OR9, N(R10)2, SH or SV
Embodiment 215. An immunoconjugate selected from:
Formula (DD-4a) Formula (DD-4b)
Formula (DD-4c) Formula (DD-4d) wherein: Ab, y R1 , R1a, R5, R5a, Y3 and Y4 are as defined in Embodiment 205. Embodiment 216. An immunoconjugate selected from:
Formula (DD-4e) Formula (DD-4f)
Formula (DD-4g) Formula (DD-4h)
Formula (DD-4m) Formula (DD-4n)
Formula (DD-4o) Formula (DD-4p) wherein: Ab, y, R1, R1a, R5 and R5a are as defined in Embodiment 205;
Y3 is OR9, N(R10)2, SH or S", and
Y4 is OR9, N(R10)2, SH or S".
Embodiment 217. An immunoconjugate selected from:
Formula (E-4a) Formula (E-4b)
Formula (EE-4c) Formula (EE-4d)
Formula (EE-4e) Formula (EE-4f)
Formula (EE-4g) Formula (EE-4h)
wherein: Ab, y, R1, R1a, R3, R3a, R4, R4a, R5, R7 and Y3 are as defined in Embodiment 205. Embodiment 218. An immunoconjugate selected from:
Formula (EE-4i) Formula (EE-4j)
Formula (EE-4k) Formula (EE-41)
Formula (EE-4m) Formula (EE-4n)
Formula (EE-4o) Formula (EE-4p)
Formula (EE-4q) Formula (EE-4q)
Formula (EE-4s) Formula (EE-4s)
Formula (EE-4u) Formula (EE-4v)
Formula (EE-4w) Formula (EE-4x)
wherein: Ab, y, R1, R1a, R3, R3a, R4, R4a, R5, R7 and Y3 are as defined in Embodiment 205; and Y3 is OR9, N(R10)2, SH or S".
Formula (FF-4i) Formula (FF-4j)
Formula (FF-4k)
wherein: Ab, y, R1, R1a, R1b, R3, R3a, R4, R4a, R5, R7 and Y3 are as defined in Embodiment 205,
Formula (FF-5a) Formula (FF-5b)
Formula (FF-5g) Formula (FF-5h)
Formula (FF-5k)
wherein: Ab, y, R1, R1a, R1 b, R3, R3a, R4, R a, R5 and R7 are as defined in Embodiment 205, and each Y3 is independently selected from OR10, N(R10)2, SH and S".
Embodiment 221. An immunoconjugate selected from:
Formula (FF-6e) Formula (FF-6f)
Formula (FF-6k) wherein: Ab, y, R1, R1a, R1b, R3, R3a, R4, R a, R5 and R7 are as defined in Embodiment 205, and each Y3 is independently selected from OR10, N(R10)2, SH and S".
Formula (FF-7c) Formula (FF-7d)
Formula (FF-7k)
wherein: Ab, y, R1, R1a, R1 , R3, R3a, R4, R4a, R5 and R7 are as defined in Embodiment 205, and each Y3 is independently selected from OR10, N(R10)2, SH and S'.
Embodiment 223. An immunoconjugate selected from:
Formula (FF-8a) Formula (FF-8b)
Formula (FF-8g) Formula (FF-8h)
Formula (FF-8k)
wherein: Ab, y, R1, R1a, R1 b, R3, R3a, R4, R a, R5 and R7 are as defined in Embodiment 205, and each Y3 is independently selected from OR10, N(R10)2, SH and S".
Embodiment 224. The immunoconjugate of any one of Embodiments 188 to 223, wherein
Embodiment 225. The immunoconjugate of any one of Embodiments 188 to 223, wherein
Embodiment 226. The immunoconjugate of any one of Embodiments 188 to 223, wherein
Embodiment 227. The immunoconjugate of any one of Embodiments 188 to 223, wherein
Embodiment 228. The immunoconjugate of any one of Embodiments 188 to 223, wherein
Embodiment 229. The immunoconjugate of any one of Embodiments 188 to 223, wherein
Embodiment 230. The compound of any one of Embodiments 188 to 223, wherein R is
wherein R200 is -UR115. Embodiment 231. The compound of any one of Embodiments 188 to 223, wherein R1a is
. wherein R210 is -UR115.
Em ne of Embodiments 188 to 223, wherein R is is
, wherein R210 is -L1 R115.
Embodiment 233. The compound of any one of Embodiments 188 to 223, wherein R1
, wherein R"uu is -L,R '
Embodiment 234. The compound of any one of Embodiments 188 to 223, wherein R1a is
. wherein R210 is -UR115.
Em und of any one of Embodiments 188 to 223, wherein R1 b is
wherein R210 is -UR115.
Embodiment 236. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is , wherein R200 is L R115 and R210 is H. Embodiment 237. The immunoconjugate of any one of Embodiments 188 to 223, wherein
and R1a is , wherein R200 is H and R210 is URm.
Embodiment 238. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R200 R210
HN HN'
R1 is and R1a is T" , wherein R200 is LiR115 and R210 is LiR115.
Embodiment 239. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is and R1a is wherein R200 is l_,R115 and R210 is H.
Embodiment 240. The immunoconjugate of any one of Embodiments 188 to 223, wherein
wherein R200 is H and R210 is Rm.
Embodiment 241. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is and R1a is wherein R200 is l_,R115 and R210 is UR115.
Embodiment 242. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R' is , wherein R200 is H and R210 is L,R115.
Embodiment 243. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is and R210 is H. Embodiment 244. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is , wherein R200 is UR115 and R210 is LiR115.
Embodiment 245. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is and R1a is , wherein R'uu is H and R'1U is LiR
Embodiment 246. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is and R1a is , wherein R200 is UR115 and R210 is H.
Embodiment 247. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is and R1a is , wherein R200 is LiR115 and R210 is LiR115.
Embodiment 248. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is and R1a is , wherein R200 is Li R115 and R210 is H.
Embodiment 249. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is is ^ R115 Embodiment 250. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is and R210 is UR115.
Embodiment 251. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is and each
R210 is H.
Embodiment 252. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is and R1a is , wherein R200 is H, R210 of R1 is l_iR115 and R21 of R1a is H.
Embodiment 253. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is , wherein R200 is H, R210 of R1 b is H and R210 of R1a is Li R115.
Embodiment 254. The compound of any one of Embodiments 188 to 223, wherein R1 is
, wherein R200 is H and one of R3, R3a, R5 or R5a is Embodiment 255. The compound of any one of Embodiments 188 to 223, wherein R1a is
, wherein R210 is H and one of R3, R3a, R5 or R5
-OUR115.
Embodiment 256. The compound of any one of Embodiments 188 to 223, wherein R1 b is is
, wherein R210 is H and one of R3, R3a, R5 or R5a is
-OUR115.
Embodiment 257. The compound of any one of Embodiments 188 to 223, wherein R1 is
wherein R200 is H and one of R3, R3a, R5 or R5a is -OUR115.
Embodiment 258. The compound of any one of Embodiments 188 to 223, wherein R1a is
. wherein R210 is is H and one of R3, R3a, R5 or R5a is -OU R115.
Embodiment 259. The compound of any one of Embodiments 188 to 223, wherein R1 is
, wherein R210 is is H and one of R3, R3a, R5 or R5a is -OU R115.
Embodiment 260. The immunoconjugate of any one of Embodiments 188 to 223, wherein R1 is , wherein R200 is H, R210 is H and one of R3, R3a, R5 or R5a is -OU R115.
Embodiment 261. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is , wherein R200 is H, R210 is H and one of R3, R3a, R5 or R5a is -OUR115.
Embodiment 262. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is , wherein R200 is H, R210 is H and one of R3, R3a,
R5 or R5a is -OLiR115.
Embodiment 263. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is and R1a is , wherein R200 is H, R210 is H and one of R3, R3a,
R5 or R5a is -OUR115.
Embodiment 264. The immunoconjugate of any one of Embodiments 188 to 223, wherein
R1 is R200 is H, R210 is H and one of R3, R3a, R5 or
R5a is -OU R115.
Embodiment 265. The immunoconjugate of an one of Embodiments 188 to 223, wherein
Embodiment 266. R1 is wherein R200 is H, R210 is H and one of R3, R3a, R5 or R5a is -OUR115. Embodiment 267. The immunoconjugate of any one of Embodiments 188 to 266, wherein: Y3 is OH, 0", SH or S", and
Y4 is OH, 0", SH or S".
Embodiment 268. The immunoconjugate of any one of Embodiments 188 to 266, wherein:
Y3 is OH or 0", and
Y4 is OH or 0".
Embodiment 269. The immunoconjugate of any one of Embodiments 188 to 266, wherein:
Y3 is SH or S", and
Y4 is OH or 0".
Embodiment 270. The immunoconjugate of any one of Embodiments 188 to 266, wherein:
Y3 is OH or 0", and
Y4 is SH or S".
Embodiment 271. The immunoconjugate of any one of Embodiments 188 to 266, wherein:
Y3 is SH or S" , and
Y4 is SH or S\
Embodiment 272. The compound of any one of Embodiments 188 to 253 or Embodiments
267 to 271 , wherein: R2, R2a, R4, R a, R6, RSa, R7 and R7a are each H.
Embodiment 273. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein: R3 is -OH, F or -NH2.
Embodiment 274. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 wherein: R3 is -OH or F.
Embodiment 275. The compound of any one of Embodiments 188 to 253 or Embodiments
267 to 271 , wherein: R3a is -OH, F or -NH2.
Embodiment 276. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein: R3a is -OH or F.
Embodiment 277. The compound of any one of Embodiments 188 to 253 or Embodiments
267 to 271 , wherein: R5 is -OH, F or -NH2.
Embodiment 278. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein: R5 is -OH or F.
Embodiment 279. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein: R5a is -OH, F or -NH2.
Embodiment 280. The compound of any one of Embodiments 188 to 253 or Embodiments
267 to 271 , wherein: R5a is -OH or F.
Embodiment 281. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is -OH, and R3a is F.
Embodiment 282. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is F, and
R3a is -OH.
Embodiment 283. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is F, and
R3a is F.
Embodiment 284. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is -OH, and
R3a is -OH.
Embodiment 285. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R a, R6, R6a, R7 and R7a are each H;
R3a is -OH, and
R5 is F.
Embodiment 286. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R8a, R7 and R7a are each H;
R3a is F, and
R5 is -OH.
Embodiment 287. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3a is F, and
R5 is F.
Embodiment 288. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3a is -OH, and
R5 is -OH. Embodiment 289. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is -OH, and
R5a is F.
Embodiment 290. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is F, and
R5a is -OH.
Embodiment 291. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R a, R6, R6a, R7 and R7a are each H;
R3 is F, and
R5a is F.
Embodiment 292. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R3 is -OH, and
R5a is -OH.
Embodiment 293. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R5 is -OH, and
R5a is F.
Embodiment 294. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R5 is F, and
R5a is -OH.
Embodiment 295. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present:
R2, R2a, R4, R4a, R6, R6a, R7 and R7a are each H;
R5 is F, and
R5a is F.
Embodiment 296. The compound of any one of Embodiments 188 to 253 or Embodiments 267 to 271 , wherein when present: R2, R2a, R4, R a, R6, R6a, R7 and R7a are each H;
R5 is -OH, and
R5a is -OH.
Embodiment 297. The immunoconjugate of any one of Embodiments 188 to 253 or
Embodiments 267 to 271 , wherein:
R3 is -OH or F;
R3a is -OH or F;
R5 is -OH or F;
R5a is -OH or F;
R6 is H, and
R6a is H.
Embodiment 298. The immunoconjugate of any one of Embodiments 188 to 253 or
Embodiments 267 to 271 , wherein:
R3 is H, -OH or F;
R5 is -OH or F;
R4, R4a, R6, R6a, R7, R7a are H, and
R6a is H.
Embodiment 299. The immunoconjugate of any one of Embodiments 188 to 298, wherein:
is -C(=0)0(CH2)mNR11C(=0)(CH2)m-**; -C(=0)0(CH2)mNR11C(=0)(CH2)mO(CH2)m-
**; -C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; - C(=0)OC(R12)2(CH2)mNR11C(=0)XlX2C(=0)(CH2)m-**; - C(=0)0(CH2)mNR8C(=0)X1X2C(=0)(CH2)mO(CH2)m-**; - C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-**; - C(=0)0(CH2)mNR11C(=0)X4C(=0)NR11 (CH2)mNR11C(=0)(CH2)mO(CH2)m-**; - C(=0)0(CH2)mNR11C(=0)XsC(=0)(CH2)mNR11C(=0)(CH2)m-**; - C(=0)0(CH2)mX6C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**; - (CH2)mNR11C(=0)X,X2C(=0)(CH2)m-**; - (CH2)m(CHOH)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m*i; - C(=0)X6C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-*i; -
C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**; - C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**, - C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**, or - C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
where the ** indicates the point of attachment to R115 and
where R11 , R12, X^ X2, m and n are s defined in Embodiment 205.
Embodiment 300. An immunoconjugate selected from:
410
411
412
413
414
415
416
417
418
5
420
421
422
423
424
425
426
427
428
429
430
431
432
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, immunogenicity, 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; 26(8):884-885; Singh, S.K., Pharm Res. 2015; 32(1 1):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 anti-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. 2007; 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 593-603 of human HER2 (Cho et al., Nature 421 : 756-760, 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 Engl 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 ADCC, the anti-HER2 antibody binds to tumor cells 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 metastic breast cancer in patients whose tumors overexpress HER2 and who received one or more chemotherapy regimens for their metastatic disease.
Pertuzumab (also called 2C4, Omnitarg, 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 1 1 . 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 al., 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 cell lines (Agus et al., 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 al., J Clin Oncol. 2005; 23(1 1):2534-43. Epub Feb 7, 2005). Pertuzumab received U.S. FDA approval for use in combination with trastuzumab and docetaxel for the treatment of patients with HER2-positive metastatic breast cancer who have not received anti-HER2 therapy or chemotherapy for metastic disease in June 2012.
Margetuximab (also called MGAH22) is another anti-HER2 monoclonal antibody (See http://www.macrogenics.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 al., Cancer Res. 2015; 75 B-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 1 1 ; 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 8. 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 8. 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 8. 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 8.
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 8. 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 8. 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 8. 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 8.
Table 8. Sequences of exemplary anti-HER2 monoclonal antibodies
AGATGGGGAGGGGACGGCTTCTATGCTATGGA
CTACTGGGGTCAAGGAACCCTGGTCACCGTCT CCTCG
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI
HWVRQAPGKGLEWVARIYPTNGYTRYADSVKGR
FTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPCPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA
SEQ ID NO: 9 Heavy Chain
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSV HEALHN
HYTQKSLSLSPGK
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCT
GGTGCAGCCAGGGGGCTCACTCCGTTTGTCCT
GTGCAGCTTCTGGCTTCAACATTAAAGACACCT
ATATACACTGGGTGCGTCAGGCCCCGGGTAAG
GGCCTGGAATGGGTTGCAAGGATTTATCCTAC
GAATGGTTATACTAGATATGCCGATAGCGTCAA
GGG CCGTTTCACTATAAG CG CAG ACACATCCA
AAAACACAGCCTACCTGCAGATGAACAGCCTG
CGTG CTG AG G ACACTG CCGTCTATTATTGTTCT
AGATGGGGAGGGGACGGCTTCTATGCTATGGA
CTACTGGGGTCAAGGAACCCTGGTCACCGTCT
CCTCGGCTAGCACCAAGGGCCCAAGTGTGTTT
CCCCTGGCCCCCAGCAGCAAGTCTACTTCCGG
CGGAACTGCTGCCCTGGGTTGCCTGGTGAAGG
ACTACTTCCCCTGTCCCGTGACAGTGTCCTGG
AACTCTGGGGCTCTGACTTCCGGCGTGCACAC
SEQ ID NO: 10 Heavy Chain DNA
CTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGT
ACAGCCTGAGCAGCGTGGTGACAGTGCCCTCC
AGCTCTCTGGGAACCCAGACCTATATCTGCAAC
GTGAACCACAAGCCCAGCAACACCAAGGTGGA
CAAGAGAGTGGAGCCCAAGAGCTGCGACAAGA
CCCACACCTGCCCCCCCTGCCCAGCTCCAGAA
CTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCC
CCCCAAGCCCAAGGACACCCTGATGATCAGCA
GGACCCCCGAGGTGACCTGCGTGGTGGTGGA
CGTGTCCCACGAGGACCCAGAGGTGAAGTTCA
ACTGGTACGTGGACGGCGTGGAGGTGCACAAC
GCCAAGACCAAGCCCAGAGAGGAGCAGTACAA
CAGCACCTACAGGGTGGTGTCCGTGCTGACCG
TGCTGCACCAGGACTGGCTGAACGGCAAAGAA
TACAAGTGCAAAGTCTCCAACAAGGCCCTGCC
AGCCCCAATCGAAAAGACAATCAGCAAGGCCA AGGGCCAGCCACGGGAGCCCCAGGTGTACAC
CCTGCCCCCCAGCCGGGAGGAGATGACCAAG
AACCAGGTGTCCCTGACCTGTCTGGTGAAGGG
CTTCTACCCCTGTGATATCGCCGTGGAGTGGG
AGAGCAACGGCCAGCCCGAGAACAACTACAAG
ACCACCCCCCCAGTGCTGGACAGCGACGGCA
GCTTCTTCCTGTACAGCAAGCTGACCGTGGAC
AAGTCCAGGTGGCAGCAGGGCAACGTGTTCAG
CTGCAGCGTGATGCACGAGGCCCTGCACAACC
ACTACACCCAG AAGTCCCTG AG CCTG AG CCCC
GGCAAG
SEQ ID NO 1 1 LCDR1 (Kabat) RASQDVNTAVA
SEQ ID NO 12 LCDR2 (Kabat) SASFLYS
SEQ ID NO 13 LCDR3 (Kabat) QQHYTTPPT
SEQ ID NO 14 LCDR1 (Chothia) SQDVNTA
SEQ ID NO 15 LCDR2 (Chothia) SAS
SEQ ID NO 16 LCDR3 (Chothia) HYTTPP
SEQ ID NO 1 1 LCDR1 (Combined) RASQDVNTAVA
SEQ ID NO 12 LCDR2 (Combined) SASFLYS
SEQ ID NO 13 LCDR3 (Combined) QQHYTTPPT
DIQ TQSPSSLSASVGDRVTITCRASQDVNTAVA WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG
SEQ ID NO: 17 VL
TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT KVEIK
GATATCCAGATGACCCAGTCCCCGAGCTCCCT
GTCCGCCTCTGTGGGCGATAGGGTCACCATCA
CCTGCCGTGCCAGTCAGGATGTGAATACTGCT
GTAGCCTGGTATCAACAGAAACCAGGAAAAGC
TCCGAAACTACTGATTTACTCGGCATCCTTCCT
SEQ ID NO: 18 VL DNA CTACTCTGGAGTCCCTTCTCGCTTCTCTGGATC
CAGATCTGGGACGGATTTCACTCTGACCATCA
GCAGTCTGCAGCCGGAAGACTTCGCAACTTAT
TACTGTCAGCAACATTATACTACTCCTCCCACG
TTCGGACAGGGTACCAAGGTGGAGATCAAA
DIQ TQSPSSLSASVGDRVTITCRASQDVNTAVA
WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG
TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
SEQ ID NO: 19 Light Chain KVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC
GATATCCAGATGACCCAGTCCCCGAGCTCCCT
GTCCGCCTCTGTGGGCGATAGGGTCACCATCA
CCTGCCGTGCCAGTCAGGATGTGAATACTGCT
GTAGCCTGGTATCAACAGAAACCAGGAAAAGC
SEQ ID NO: 20 Light Chain DNA
TCCGAAACTACTGATTTACTCGGCATCCTTCCT
CTACTCTGGAGTCCCTTCTCGCTTCTCTGGATC
CAGATCTGGGACGGATTTCACTCTGACCATCA
GCAGTCTGCAGCCGGAAGACTTCGCAACTTAT TACTGTCAGCAACATTATACTACTCCTCCCACG
TTCGGACAGGGTACCAAGGTGGAGATCAAACG
TACGGTGGCCGCTCCCAGCGTGTTCATCTTCC
CCCCCAGCGACGAGCAGCTGAAGAGTGGCAC
CGCCAGCGTGGTGTGCCTGCTGAACAACTTCT
ACCCCCGGGAGGCCAAGGTGCAGTGGAAGGT
G G AC AACG CC CTG C AG AG C G G CAAC AG CC AG
GAGAGCGTCACCGAGCAGGACAGCAAGGACT
CCACCTACAGCCTGAGCAGCACCCTGACCCTG
AGCAAGGCCGACTACGAGAAGCATAAGGTGTA
CGCCTGCGAGGTGACCCACCAGGGCCTGTCC
AGCCCCGTGACCAAGAGCTTCAACAGGGGCGA
GTGC
anti-HER2 mAbl-
D265A-P329A
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI
HWVRQAPGKGLEVWARIYPTNGYTRYADSVKGR
FTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPCPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA
SEQ ID NO: 33 Heavy Chain
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWAV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSV HEALHN HYTQKSLSLSPGK
DIQ TQSPSSLSASVGDRVTITCRASQDVNTAVA
WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG
TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
SEQ ID NO: 19 Light Chain KVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC
anti-HER2 mAb2
SEQ ID NO: 1 HCDR1 (Kabat) DTYIH
SEQ ID NO: 2 HCDR2 (Kabat) RIYPTNGYTRYADSVKG
SEQ ID NO: 3 HCDR3 (Kabat) WGGDGFYAMDY
SEQ ID NO: 4 HCDR1 (Chothia) GFNIKDT
SEQ ID NO: 5 HCDR2 (Chothia) YPTNGY
SEQ ID NO: 3 HCDR3 (Chothia) WGGDGFYAMDY
SEQ ID NO: 6 HCDR1 (Combined) GFNIKDTYIH
SEQ ID NO: 2 HCDR2 (Combined) RIYPTNGYTRYADSVKG
SEQ ID NO: 3 HCDR3 (Combined) WGGDGFYAMDY
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI
HWVRQAPGKGLEWVARIYPTNGYTRYADSVKGR
FTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
SEQ ID NO: 7 VH GDGFYAMDYWGQGTLVTVSS
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCT
GGTGCAGCCAGGGGGCTCACTCCGTTTGTCCT
GTGCAGCTTCTGGCTTCAACATTAAAGACACCT
SEQ ID NO: 8 VH DNA ATATACACTGGGTGCGTCAGGCCCCGGGTAAG GGCCTGGAATGGGTTGCAAGGATTTATCCTAC
GAATGGTTATACTAGATATGCCGATAGCGTCAA GGG CCGTTTCACTATAAG CG CAG ACACATCCA AAAACACAGCCTACCTGCAGATGAACAGCCTG CGTG CTG AG G ACACTG CCGTCTATTATTGTTCT AGATGGGGAGGGGACGGCTTCTATGCTATGGA CTACTGGGGTCAAGGAACCCTGGTCACCGTCT CCTCG
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI
HWVRQAPGKGLEVWARIYPTNGYTRYADSVKGR
FTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPCPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
SEQ ID NO: 21 Heavy Chain
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSV HEALHN
HYTQKSLSLSPGK
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCT
GGTGCAGCCAGGGGGCTCACTCCGTTTGTCCT
GTGCAGCTTCTGGCTTCAACATTAAAGACACCT
ATATACACTGGGTGCGTCAGGCCCCGGGTAAG
GGCCTGGAATGGGTTGCAAGGATTTATCCTAC
GAATGGTTATACTAGATATGCCGATAGCGTCAA
GGG CCGTTTCACTATAAG CG CAG ACACATCCA
AAAACACAGCCTACCTGCAGATGAACAGCCTG
CGTG CTG AG G ACACTG CCGTCTATTATTGTTCT
AGATGGGGAGGGGACGGCTTCTATGCTATGGA
CTACTGGGGTCAAGGAACCCTGGTCACCGTCT
CCTCGGCTAGCACCAAGGGCCCCAGCGTGTTC
CCCCTGGCCCCCAGCAGCAAGAGCACCAGCG
GCGGCACAGCCGCCCTGGGCTGCCTGGTGAA
SEQ ID NO: 22 Heavy Chain DNA GGACTACTTCCCTTGTCCCGTGACCGTGTCCT
GGAACAGCGGAGCCCTGACCTCCGGCGTGCA
CACCTTCCCCGCCGTGCTGCAGAGCAGCGGC
CTGTACAGCCTGTCCAGCGTGGTGACAGTGCC
CAGCAGCAGCCTGGGCACCCAGACCTACATCT
GCAACGTGAACCACAAGCCCAGCAACACCAAG
GTGGACAAGAAAGTGGAGCCCAAGAGCTGCGA
CAAGACCCACACCTGCCCCCCCTGCCCAGCCC
CAGAGCTGCTGGGCGGACCCTCCGTGTTCCTG
TTCCCCCCCAAGCCCAAGGACACCCTGATGAT
CAGCAGGACCCCCGAGGTGACCTGCGTGGTG
GTGGACGTGAGCCACGAGGACCCAGAGGTGA
AGTTCAACTGGTACGTGGACGGCGTGGAGGTG
CACAACGCCAAGACCAAGCCCAGAGAGGAGCA
GTACAACAGCACCTACAGGGTGGTGTCCGTGC TGACCGTGCTGCACCAGGACTGGCTGAACGGC
AAGGAATACAAGTGCAAGGTCTCCAACAAGGC
CCTGCCAGCCCCCATCGAAAAGACCATCAGCA
AGGCCAAGGGCCAGCCACGGGAGCCCCAGGT
GTACACCCTGCCCCCCTCCCGGGAGGAGATGA
CCAAGAACCAGGTGTCCCTGACCTGTCTGGTG
AAGGGCTTCTACCCCTGCGACATCGCCGTGGA
GTGGGAGAGCAACGGCCAGCCCGAGAACAAC
TACAAGACCACACCTCCAGTGCTGGACAGCGA
CG GCAG CTTCTTCCTGTAC AG CAAGCTG ACCG
TGGACAAGTCCAGGTGGCAGCAGGGCAACGT
GTTCAGCTGCAGCGTGATGCACGAGGCCCTGC
ACAACCACTACACCCAGAAGAGCCTGAGCCTG
TCCCCCGGCAAG
SEQ ID NO 1 1 LCDR1 (Kabat) RASQDVNTAVA
SEQ ID NO 12 LCDR2 (Kabat) SASFLYS
SEQ ID NO 13 LCDR3 (Kabat) QQHYTTPPT
SEQ ID NO 14 LCDR1 (Chothia) SQDVNTA
SEQ ID NO 15 LCDR2 (Chothia) SAS
SEQ ID NO 16 LCDR3 (Chothia) HYTTPP
SEQ ID NO 1 1 LCDR1 (Combined) RASQDVNTAVA
SEQ ID NO 12 LCDR2 (Combined) SASFLYS
SEQ ID NO 13 LCDR3 (Combined) QQHYTTPPT
DIQ TQSPSSLSASVGDRVTITCRASQDVNTAVA WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
SEQ ID NO: 17 VL KVEIK
GATATCCAGATGACCCAGTCCCCGAGCTCCCT
GTCCGCCTCTGTGGGCGATAGGGTCACCATCA
CCTGCCGTGCCAGTCAGGATGTGAATACTGCT
GTAGCCTGGTATCAACAGAAACCAGGAAAAGC
TCCGAAACTACTGATTTACTCGGCATCCTTCCT
CTACTCTGGAGTCCCTTCTCGCTTCTCTGGATC
CAGATCTGGGACGGATTTCACTCTGACCATCA
GCAGTCTGCAGCCGGAAGACTTCGCAACTTAT
TACTGTCAGCAACATTATACTACTCCTCCCACG
SEQ ID NO: 18 VL DNA TTCGGACAGGGTACCAAGGTGGAGATCAAA
DIQ TQSPSSLSASVGDRVTITCRASQDVNTAVA
WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG
TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
SEQ ID NO 19 Light Chain KVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC
GATATCCAGATGACCCAGTCCCCGAGCTCCCT
GTCCGCCTCTGTGGGCGATAGGGTCACCATCA
CCTGCCGTGCCAGTCAGGATGTGAATACTGCT
SEQ ID NO 928 Light Chain DNA GTAGCCTGGTATCAACAGAAACCAGGAAAAGC
TCCGAAACTACTGATTTACTCGGCATCCTTCCT
CTACTCTGGAGTCCCTTCTCGCTTCTCTGGATC
CAGATCTGGGACGGATTTCACTCTGACCATCA GCAGTCTGCAGCCGGAAGACTTCGCAACTTAT
TACTGTCAGCAACATTATACTACTCCTCCCACG
TTCGGACAGGGTACCAAGGTGGAGATCAAACG
AACGGTGGCCGCTCCCAGCGTGTTCATCTTCC
CCCCCAGCGACGAGCAGCTGAAGAGCGGCAC
CGCCAGCGTGGTGTGCCTGCTGAACAACTTCT
ACCCCCGGGAGGCCAAGGTGCAGTGGAAGGT
G G AC AACG CC CTG C AG AG C G G CAAC AG CC AG
GAGAGCGTCACCGAGCAGGACAGCAAGGACT
CCACCTACAGCCTGAGCAGCACCCTGACCCTG
AGCAAGGCCGACTACGAGAAGCATAAGGTGTA
CGCCTGCGAGGTGACCCACCAGGGCCTGTCC
AGCCCCGTGACCAAGAGCTTCAACAGGGGCGA
GTGC
anti-HER2 mAb3
SEQ ID NO: 1 HCDR1 (Kabat) DTYIH
SEQ ID NO: 2 HCDR2 (Kabat) RIYPTNGYTRYADSVKG
SEQ ID NO: 3 HCDR3 (Kabat) WGGDGFYAMDY
SEQ ID NO: 4 HCDR1 (Chothia) GFNIKDT
SEQ ID NO: 5 HCDR2 (Chothia) YPTNGY
SEQ ID NO: 3 HCDR3 (Chothia) WGGDGFYAMDY
SEQ ID NO: 6 HCDR1 (Combined) GFNIKDTYIH
SEQ ID NO: 2 HCDR2 (Combined) RIYPTNGYTRYADSVKG
SEQ ID NO: 3 HCDR3 (Combined) WGGDGFYAMDY
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI HWVRQAPGKGLEWVARIYPTNGYTRYADSVKGR FTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
SEQ ID NO: 7 VH GDGFYAMDYWGQGTLVTVSS
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCT
GGTGCAGCCAGGGGGCTCACTCCGTTTGTCCT
GTGCAGCTTCTGGCTTCAACATTAAAGACACCT
ATATACACTGGGTGCGTCAGGCCCCGGGTAAG
GGCCTGGAATGGGTTGCAAGGATTTATCCTAC
GAATGGTTATACTAGATATGCCGATAGCGTCAA
GGG CCGTTTCACTATAAG CG CAG ACACATCCA
AAAACACAGCCTACCTGCAGATGAACAGCCTG
CGTG CTG AG G ACACTG CCGTCTATTATTGTTCT
AGATGGGGAGGGGACGGCTTCTATGCTATGGA
CTACTGGGGTCAAGGAACCCTGGTCACCGTCT
SEQ ID NO: 8 VH DNA CCTCG
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI
HWVRQAPGKGLEWVARIYPTNGYTRYADSVKGR
FTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
SEQ ID NO: 23 Heavy Chain
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCT
GGTGCAGCCAGGGGGCTCACTCCGTTTGTCCT
GTGCAGCTTCTGGCTTCAACATTAAAGACACCT
ATATACACTGGGTGCGTCAGGCCCCGGGTAAG
GGCCTGGAATGGGTTGCAAGGATTTATCCTAC
GAATGGTTATACTAGATATGCCGATAGCGTCAA
GGG CCGTTTCACTATAAG CG CAG ACACATCCA
AAAACACAGCCTACCTGCAGATGAACAGCCTG
CGTG CTG AG G ACACTG CCGTCTATTATTGTTCT
AGATGGGGAGGGGACGGCTTCTATGCTATGGA
CTACTGGGGTCAAGGAACCCTGGTCACCGTCT
CCTCGGCTAGCACCAAGGGCCCCAGCGTGTTC
CCCCTGGCCCCCAGCAGCAAGAGCACCAGCG
GCGGCACAGCCGCCCTGGGCTGCCTGGTGAA
GGACTACTTCCCCGAGCCCGTGACCGTGTCCT
GGAACAGCGGAGCCCTGACCTCCGGCGTGCA
CACCTTCCCCGCCGTGCTGCAGAGCAGCGGC
CTGTACAGCCTGTCCAGCGTGGTGACAGTGCC
CAGCAGCAGCCTGGGCACCCAGACCTACATCT
GCAACGTGAACCACAAGCCCAGCAACACCAAG
GTGGACAAGAAAGTGGAGCCCAAGAGCTGCGA
CAAGACCCACACCTGCCCCCCCTGCCCAGCCC
SEQ ID NO: 24 Heavy Chain DNA
CAGAGCTGCTGGGCGGACCCTCCGTGTTCCTG
TTCCCCCCCAAGCCCAAGGACACCCTGATGAT
CAGCAGGACCCCCGAGGTGACCTGCGTGGTG
GTGGACGTGAGCCACGAGGACCCAGAGGTGA
AGTTCAACTGGTACGTGGACGGCGTGGAGGTG
CACAACGCCAAGACCAAGCCCAGAGAGGAGCA
GTACAACAGCACCTACAGGGTGGTGTCCGTGC
TGACCGTGCTGCACCAGGACTGGCTGAACGGC
AAGGAATACAAGTGCAAGGTCTCCAACAAGGC
CCTGCCAGCCCCCATCGAAAAGACCATCAGCA
AGGCCAAGGGCCAGCCACGGGAGCCCCAGGT
GTACACCCTGCCCCCCTCCCGGGAGGAGATGA
CCAAGAACCAGGTGTCCCTGACCTGTCTGGTG
AAGGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAACGGCCAGCCCGAGAACAAC
TACAAGACCACACCTCCAGTGCTGGACAGCGA
CG GCAG CTTCTTCCTGTAC AG C AAGCTG ACCG
TGGACAAGTCCAGGTGGCAGCAGGGCAACGT
GTTCAGCTGCAGCGTGATGCACGAGGCCCTGC
ACAACCACTACACCCAGAAGAGCCTGAGCCTG
TCCCCCGGCAAG
SEQ ID NO 1 1 LCDR1 (Kabat) RASQDVNTAVA
SEQ ID NO 12 LCDR2 (Kabat) SASFLYS
SEQ ID NO 13 LCDR3 (Kabat) QQHYTTPPT
SEQ ID NO 14 LCDR1 (Chothia) SQDVNTA
SEQ ID NO 15 LCDR2 (Chothia) SAS SEQ ID NO 16 LCDR3 (Chothia) HYTTPP
SEQ ID NO 1 1 LCDR1 (Combined) RASQDVNTAVA
SEQ ID NO 12 LCDR2 (Combined) SASFLYS
SEQ ID NO 13 LCDR3 (Combined) QQHYTTPPT
DIQ TQSPSSLSASVGDRVTITCRASQDVNTAVA WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
SEQ ID NO: 17 VL KVEIK
GATATCCAGATGACCCAGTCCCCGAGCTCCCT
GTCCGCCTCTGTGGGCGATAGGGTCACCATCA
CCTGCCGTGCCAGTCAGGATGTGAATACTGCT
GTAGCCTGGTATCAACAGAAACCAGGAAAAGC
TCCGAAACTACTGATTTACTCGGCATCCTTCCT
CTACTCTGGAGTCCCTTCTCGCTTCTCTGGATC
CAGATCTGGGACGGATTTCACTCTGACCATCA
GCAGTCTGCAGCCGGAAGACTTCGCAACTTAT
TACTGTCAGCAACATTATACTACTCCTCCCACG
SEQ ID NO: 18 VL DNA TTCGGACAGGGTACCAAGGTGGAGATCAAA
DIQ TQSPSSLSASVGDRVTITCRASQDVNTAVA
WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG
TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
SEQ ID NO: 19 Light Chain KVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC
GATATCCAGATGACCCAGTCCCCGAGCTCCCT
GTCCGCCTCTGTGGGCGATAGGGTCACCATCA
CCTGCCGTGCCAGTCAGGATGTGAATACTGCT
GTAGCCTGGTATCAACAGAAACCAGGAAAAGC
TCCGAAACTACTGATTTACTCGGCATCCTTCCT
CTACTCTGGAGTCCCTTCTCGCTTCTCTGGATC
CAGATCTGGGACGGATTTCACTCTGACCATCA
GCAGTCTGCAGCCGGAAGACTTCGCAACTTAT
TACTGTCAGCAACATTATACTACTCCTCCCACG
TTCGGACAGGGTACCAAGGTGGAGATCAAACG
SEQ ID NO: 928 Light Chain DNA AACGGTGGCCGCTCCCAGCGTGTTCATCTTCC
CCCCCAGCGACGAGCAGCTGAAGAGCGGCAC
CGCCAGCGTGGTGTGCCTGCTGAACAACTTCT
ACCCCCGGGAGGCCAAGGTGCAGTGGAAGGT
G G AC AACG CC CTG C AG AG C G G CAAC AG CC AG
GAGAGCGTCACCGAGCAGGACAGCAAGGACT
CCACCTACAGCCTGAGCAGCACCCTGACCCTG
AGCAAGGCCGACTACGAGAAGCATAAGGTGTA
CGCCTGCGAGGTGACCCACCAGGGCCTGTCC
AGCCCCGTGACCAAGAGCTTCAACAGGGGCGA
GTGC
anti-HER2 mAb4
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI
HWVRQAPGKGLEVWARIYPTNGYTRYADSVKGR
FTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAP
SEQ ID NO: 30 Heavy Chain
SSKSTSGGTAALGCLVKDYFPCPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSV HEALHN HYTQKSLSLSPGK
DIQ TQSPSSLSASVGDRVTITCRASQDVNTAVA
WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG
TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
SEQ ID NO: 19 Light Chain KVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC
anti-HER2 mAb5
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI
HWVRQAPGKGLEWVARIYPTNGYTRYADSVKGR
FTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
SEQ ID NO: 32 Heavy Chain
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPEGDSLDMLEWSLM
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK
DIQ TQSPSSLSASVGDRVTITCRASQDVNTAVA
WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG
TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
SEQ ID NO: 19 Light Chain KVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC
anti-HER2 mAb6
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI
HWVRQAPGKGLEVWARIYPTNGYTRYADSVKGR
FTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
SEQ ID NO: 23 Heavy Chain
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSV HEALHN
HYTQKSLSLSPGK
DIQ TQSPSSLSASVGDRVTITCRASQDVNTAVA
WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG
SEQ ID NO: 34 Light Chain TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
KVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN
NFYPREAKVQWKVDNALQSGNCQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC
anti-HER2 mAb7
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI
HVWRQAPGKGLEVWARIYPTNGYTRYADSVKGR
FTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
SEQ ID NO: 35 Heavy Chain
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPEDSLEFIASKLANN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK
DIQ TQSPSSLSASVGDRVTITCRASQDVNTAVA
WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG
TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
SEQ ID NO: 19 Light Chain KVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC
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 8. 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 Table 8.
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 Table 8. 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 Table 8. 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 8, 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 NOs: 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 8, or combinations thereof. The amino acid sequences of the VH CDR1 s of the antibodies are shown in SEQ ID NOs: 1 , 4, and 6. 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 NOs 12 and 15. The amino acid sequences of the VL CDR3s of the antibodies are shown in SEQ ID NOs: 13 and 16.
Given that each of the antibodies binds HER2 and that antigen-binding specificity is provided primarily by the CDR1 , CDR2 and CDR3 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, although each antibody must contain a VH CDR1 , CDR2 and CDR3 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. , ELISAs). When VH CDR sequences are mixed and matched, the CDR1 , CDR2 and/or CDR3 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 CDR3 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 CDR3 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 8.
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 (HCDR2) 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: 11 ; 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 557-561 , 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.
P-Cadherin Antibody
In some embodiments disclosed herein, antibody conjugates include an antibody or a fragment there of (e.g., an antigen binding fragment thereof) that specifically binds to human P- cadherin (anti-Pcad antibody). Antibodies or antibody fragments (e.g., antigen binding fragments) of the invention include, but are not limited to, the human monoclonal antibodies or fragments thereof, isolated as described in the Examples.
The present invention also provides anti-P-cadherin antibodies or antigen binding fragments thereof that comprise modifications in the constant regions of the heavy chain, light chain, or both the heavy and light chain wherein particular amino acid residues have mutated to cysteines, also referred to herein at "CysMab" or "Cys" antibodies. As discussed herein, drug moieties may be conjugated site specifically and with control over the number of drug moieties ("DAR Controlled") to cysteine residues on antibodies. Cysteine modifications to antibodies for the purposes of site specifically controlling immunoconjugation are disclosed, for example, in WO2014/124316, which is incorporated herein by reference in its entirety.
In some embodiments, the anti-P-cadherin antibodies have been modified at positions
152 and 375 of the heavy chain, wherein the positions are defined according to the EU numbering system. Namely, the modifications are E152C and S375C. In other embodiments, the anti-P-cadherin antibodies have been modified at position 360 of the heavy chain and position 107 of the kappa light chain, wherein the positions are defined according to the EU numbering system. Namely, the modifications are K360C and K107C. The positions of these mutations are illustrated, for example, in the context of human lgG1 heavy chain and kappa light chain constant regions in Table 8A. Throughout Table 8A, cysteine modifications from wild type sequences are shown with underlining.
The present invention also provides nucleic acid sequences that encode the VH, VL, the full length heavy chain, and the full length light chain of the antibodies that specifically bind to P- cadherin. Such nucleic acid sequences can be optimized for expression in mammalian cells. Table 8A. Examples of anti-P-cadherin Antibodies
P-Cad Mab2
SEQ ID NO. Description Sequence
SEQ ID NO: 37 HCDR1 SQSAAWN
(Kabat)
SEQ ID NO: 38 HCDR2 RIYYRSKWYNDYALSVKS
(Kabat)
SEQ ID NO: 39 HCDR3 GEGYGREGFAI
(Kabat) SEQ ID NO: 40 HCDR1 GDSVSSQSA
(Chothia)
SEQ ID NO: 41 HCDR2 YYRSKWY
(Chothia)
SEQ ID NO: 42 HCDR3 GEGYGREGFAI
(Chothia)
SEQ ID NO: 43 VH Q VQ LQQSGPG LVKPSQTLSLTCAI SG DSVSSQSAAWN
WIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPD TSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWG QGTLVTVSS
SEQ ID NO: 44 DNA VH CAGGTGCAGCTGCAGCAGTCAGGCCCTGGCCTGGTC
AAGCCTAGTCAGACCCTGAGCCTGACCTGCGCTATTA
GCGGCGATAGTGTGTCTAGTCAGTCAGCCGCCTGGA
ACTG G ATTAG ACAGTCACCCTCTAG GG G CCTG G AGT
GGCTGGGTAGAATCTACTATAGGTCTAAGTGGTATAA
CGACTACGCCCTGAGCGTGAAGTCTAGGATCACTATT
AACCCCGACACCTCTAAGAATCAGTTTAGCCTGCAGC
TGAATAGCGTGACCCCCGAGGACACCGCCGTCTACT
ACTGCGCTAGAGGCGAGGGCTACGGTAGAGAGGGCT
TCGCTATCTGGGGTCAGGGCACCCTGGTCACCGTGT
CTAGC
SEQ ID NO: 45 DNA VH CAGGTGCAATTGCAGCAGAGCGGTCCGGGCCTGGTG
AAACCGAGCCAGACCCTGAGCCTGACCTGCGCGATT
TCCGGAGATAGCGTGAGCTCTCAGTCTGCTGCTTGG
AACTGGATTCGTCAGAGCCCGAGCCGTGGCCTCGAG
TGGCTGGGCCGTATCTACTACCGTAGCAAATGGTACA
ACGACTATGCCTTGAGCGTGAAAAGCCGCATTACCAT
TAACCCGGATACTTCGAAAAACCAGTTTAGCCTGCAA
CTGAACAGCGTGACCCCGGAAGATACGGCCGTGTAT
TATTGCGCGCGTGGTGAAGGTTACGGTCGTGAAGGT
TTCGCTATCTGGGGCCAAGGCACCCTGGTGACTGTTA
GCTCA
SEQ ID NO: 46 Heavy Chain Q VQ LQQSG PG LVKPSQTLSLTCAI SG DSVSSQSAAWN
WIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPD
TSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
WTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREE TKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 47 DNA Heavy CAGGTGCAGCTGCAGCAGTCAGGCCCTGGCCTGGTC Chain AAGCCTAGTCAGACCCTGAGCCTGACCTGCGCTATTA
GCGGCGATAGTGTGTCTAGTCAGTCAGCCGCCTGGA
ACTG G ATTAG ACAGTCACCCTCTAG GG G CCTG G AGT
GGCTGGGTAGAATCTACTATAGGTCTAAGTGGTATAA
CGACTACGCCCTGAGCGTGAAGTCTAGGATCACTATT
AACCCCGACACCTCTAAGAATCAGTTTAGCCTGCAGC
TGAATAGCGTGACCCCCGAGGACACCGCCGTCTACT
ACTGCGCTAGAGGCGAGGGCTACGGTAGAGAGGGCT
TCGCTATCTGGGGTCAGGGCACCCTGGTCACCGTGT
CTAGCGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCT
GGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGC
TGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGA
GCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGAC
TTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAG
CAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGT
GCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGC
AACGTGAACCACAAGCCCAGCAACACCAAGGTGGAC
AAG AG AGTG G AG CCCAAG AGCTG CG ACAAG ACCC AC
ACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGA
GGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG
GACACCCTGATGATCAGCAGGACCCCCGAGGTGACC
TGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAG
GTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG
CACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTAC
AACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTG
CTGCACCAGGACTGGCTGAACGGCAAAGAAT ACAAG
TGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCAATC
G AAAAG AC AATC AG C AAG GCCAAGGGCCAGCCACGG
GAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGA
GGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCT
GGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGA
GTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAA
GACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTT
CTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAG
GTG G C AG C AG G G C AACG TG TTC AG CTG C AG CGTG AT
GCACGAGGCCCTGCACAACCACTACACCCAGAAGTC
CCTGAGCCTGAGCCCCGGCAAG
SEQ ID NO: 48 E152C/S375 QVQ LQQSGPG LVKPSQTLSLTC Al SG DSVSSQSAAWN
C CysMab WIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPD Mutated TSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWG Heavy Chain QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
WTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPC
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 49 DNA CAG GTG CAATTGCAG CAG AGCG GTCCG GG CCTGGTG
E152C/S375 AAACCGAGCCAGACCCTGAGCCTGACCTGCGCGATT C CysMab TCCGGAGATAGCGTGAGCTCTCAGTCTGCTGCTTGG Mutated AACTGGATTCGTCAGAGCCCGAGCCGTGGCCTCGAG Heavy Chain TGGCTGGGCCGTATCTACTACCGTAGCAAATGGTACA
ACGACTATGCCTTGAGCGTGAAAAGCCGCATTACCAT
TAACCCGGATACTTCGAAAAACCAGTTTAGCCTGCAA
CTGAACAGCGTGACCCCGGAAGATACGGCCGTGTAT
TATTGCGCGCGTGGTGAAGGTTACGGTCGTGAAGGT
TTCGCTATCTGGGGCCAAGGCACCCTGGTGACTGTTA
GCTCAGCCTCTACGAAAGGCCCAAGCGTA I I I CCCCT
GGCTCCTTCTAGTAAATCAACCTCAGGTGGTACAGCA
GCCCTTGGCTGCCTGGTCAAAGACTATTTCCCCTGTC
CGGTGACCGTCTCATGGAACTCAGGTGCTTTGACATC
TGGTGTGCATACATTCCCAGCTGTGCTGCAAAGTAGT
GGACTGTACAGCCTTTCCAGCGTGGTCACGGTGCCA
AGTAGCTCCTTGGGTACTCAGACTTATATCTGCAATG
TGAACCACAAGCCCTCTAACACGAAGGTGGACAAGC
GCGTGGAGCCCAAATCTTGCGATAAGACGCATACTTG
TCCCCCATGCCCTGCTCCTGAGCTGTTGGGAGGCCC
GTCAGTGTTCTTGTTCCCTCCGAAGCCTAAGGACACT
TTG ATG ATAAGTAG G ACACCAG AG GTG ACTTGCGTG G
TGGTTGATGTGTCCCATGAAGATCCCGAGGTCAAATT
TAATTGGTACGTAGATGGTGTCGAAGTTCACAATGCT
AAG ACTAAG CC AAG G G AAG AG CAG TAC AAC AG TAC AT
ATAGGGTAGTCTCCGTGCTGACAGTCCTCCACCAGG
ACTG GTTG AACGG CAAG G AATACAAATGTAAG GTGTC
AAACAAAGCTCTGCCTGCTCCCATTGAGAAAACAATC
TCTAAAGCCAAAGGCCAGCCGAGAGAGCCCCAAGTC
TACACTTTGCCCCCGAGCAGGGAGGAAATGACCAAG
AATCAGGTGAGTCTGACGTGCCTCGTCAAAGGATTTT
ATCCATGCGATATTGCAGTTGAATGGGAGAGCAATGG
CCAGCCAGAGAACAACTATAAAACCACACCACCCGTG
CTCGACTCTGATGGCAGCTTCTTCCTCTATAGCAAGC
TGACAGTCGATAAATCTCGCTGGCAGCAAGGCAATGT
GTTCTCCTGCTCCGTCATGCACGAGGCTTTGCATAAC
CATTATACTCAAAAATCTCTGTCCCTGTCACCTGGTAA
A
SEQ ID NO: 50 K360C QVQ LQQSGPG LVKPSQTLSLTC Al SG DSVSSQSAAWN
CysMab WIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPD Mutated TSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWG Heavy Chain QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
WTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTCNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 51 LCDR1 RASQTISNTLA
(Kabat)
SEQ ID NO: 52 LCDR2 AASNLQS
(Kabat)
SEQ ID NO: 53 LCDR3 QQYLSWFT (Kabat)
SEQ ID NO: 54 LCDR1 SQTISNT
(Chothia)
SEQ ID NO: 55 LCDR2 AAS
(Chothia)
SEQ ID NO: 56 LCDR3 YLSWF
(Chothia)
SEQ ID NO: 57 VL DIQ TQSPSSLSASVGDRVTITCRASQTISNTLAWYQQK
PGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQYLSWFTFGQGTKVEIK
SEQ ID NO: 58 DNA VL GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCG
CTAGTGTGGGCGATAGAGTGACTATCACCTGTAGAGC
CTCTCAG ACTATCTCTAACACCCTGG CCTG GTATCAG
CAG AAG CCCG GTAAAGCCCCTAAG CTG CTG ATCTAC
GCCGCCTCTAACCTGCAGTCAGGCGTGCCCTCTAGG
TTTAGCGGTAGCGGTAGTGGCACCGACTTCACCCTG
ACTATTAGTAGCCTGCAGCCCGAGGACTTCGCTACCT
ACTACTGTCAGCAGTACCTGAGCTGGTTCACCTTCGG
TCAGGGCACTAAGGTCGAGATTAAG
SEQ ID NO: 59 DNA VL GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAGC
GCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGA
GCCAGCCAGACTATTTCTAACACTCTGGCTTGGTACC
AGCAG AAACCG G GCAAAG CG CCG AAACTATTAATCTA
CGCTGCTTCTAACCTGCAAAGCGGCGTGCCGAGCCG
CTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCT
GACCATTAGCTCTCTGCAACCGGAAGACTTTGCGACC
TATTATTGCCAGCAGTACCTGTCTTGGTTCACCTTTGG
CCAG GG CACG AAAGTTG AAATTAAA
SEQ ID NO: 60 Light Chain DIQ TQSPSSLSASVGDRVTITCRASQTISNTLAWYQQK
PGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQYLSWFTFGQGTKVEIKRTVAAPSVFIF
PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC
SEQ ID NO:61 DNA Light GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCG
Chain CTAGTGTGGGCGATAGAGTGACTATCACCTGTAGAGC
CTCTCAG ACTATCTCTAACACCCTGG CCTG GTATCAG
CAG AAG CCCG GTAAAGCCCCTAAG CTG CTG ATCTAC
GCCGCCTCTAACCTGCAGTCAGGCGTGCCCTCTAGG
TTTAGCGGTAGCGGTAGTGGCACCGACTTCACCCTG
ACTATTAGTAGCCTGCAGCCCGAGGACTTCGCTACCT
ACTACTGTCAGCAGTACCTGAGCTGGTTCACCTTCGG
TCAGGGCACTAAGGTCGAGATTAAGCGTACGGTGGC
CGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGA
GCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCT
GCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCA
GTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAG
CCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTC
CACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAA
GGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGA
GGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAA
GAGCTTCAACAGGGGCGAGTGC
SEQ ID NO: 62 DNA Light GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAGC
Chain GCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGA
GCCAGCCAGACTATTTCTAACACTCTGGCTTGGTACC AGCAG AAACCG G GCAAAG CG CCG AAACTATTAATCTA
CGCTGCTTCTAACCTGCAAAGCGGCGTGCCGAGCCG
CTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCT
GACCATTAGCTCTCTGCAACCGGAAGACTTTGCGACC
TATTATTGCCAGCAGTACCTGTCTTGGTTCACCTTTGG
CCAGGGCACGAAAGTTGAAATTAAACGTACGGTGGC
CGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGA
GCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCT
GCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCA
GTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAG
CCAGGAAAGCGTCACCGAGCAGGACAGCAAGGACTC
CACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAA
GGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGA
GGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAA
GAGCTTCAACCGGGGCGAGTGT
SEQ ID NO: 63 K107C DIQMTQSPSSLSASVGDRVTITCRASQTISNTLAWYQQK
CysMab PGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSL Mutated Light QPEDFATYYCQQYLSWFTFGQGTKVEICRTVAAPSVFIF Chain PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGK
SEQ ID NO: 73 DNA Heavy GAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAA
Chain GAAGCCCGGCGAGTCACTGAAGATTAGCTGTAAAG
TCTCAGGCTACACCTTCACCGATCACACTATTCACT
GGATGAGACAGATGCCCGGTAAAGGCCTGGAGTGG
ATG GG CTATATCTACCCTAG ATCAGG CTCTATTAAC
T AT AACG AG AAGTTTAAG G G TC AG G TC AC AATTAG C
GCCGATAAGTCTAGCTCTACCGCCTACCTGCAGTG
GTCTAGCCTGAAGGCTAGTGACACCGCTATGTACTA
CTGCGCTAGACGTAACCTGTTCCTGCCTATGGAATA
CTGGGGTCAGGGCACCCTGGTCACCGTGTCTAGCG
CTAGCACTAAGGGCCCAAGTGTG I I I CCCCTGGCC
CCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGC
CCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGC
CCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACT
TCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAG
CAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAG
TGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCT
GCAACGTGAACCACAAGCCCAGCAACACCAAGGTG
GACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGAC
CCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGC
TGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAG
CCCAAGGACACCCTGATGATCAGCAGGACCCCCGA
GGTGACCTGCGTGGTGGTGGACGTGTCCCACGAG
GACCCAGAGGTGAAGTTCAACTGGTACGTGGACGG
CGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAG
AGGAGCAGTACAACAGCACCTACAGGGTGGTGTCC
GTG CTG ACCGTG CTG CACCAG G ACTGG CTG AACGG
CAAAG AATACAAGTG CAAAGTCTCCAACAAG G CCCT
GCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCA
AGGGCCAGCCACGGGAGCCCCAGGTGTACACCCT
GCCCCCCAGCCGGGAGGAGATGACCAAGAACCAG
GTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCC
CAGCGATATCGCCGTGGAGTGGGAGAGCAACGGC
CAGCCCGAGAACAACTACAAGACCACCCCCCCAGT
GCTG G ACAGCG ACG GCAG CTTCTTCCTGTACAG CA
AGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGG
CAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCC
TGCACAACCACTACACCCAGAAGTCCCTGAGCCTG
AGCCCCGGCAAG
SEQ ID NO:74 E152C/S375 EVQLVQSGAEVKKPGESLKISCKVSGYTFTDHTIHWM
C CysMab RQMPGKGLEWMGYIYPRSGSINYNEKFKGQVTISADK Mutated SSSTAYLQWSSLKASDTAMYYCARRNLFLPMEYWGQ Heavy Chain GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK
SEQ ID NO:75 K360C EVQLVQSGAEVKKPGESLKISCKVSGYTFTDHTIHWM
CysMab RQMPGKGLEWMGYIYPRSGSINYNEKFKGQVTISADK Mutated SSSTAYLQWSSLKASDTAMYYCARRNLFLPMEYWGQ Heavy Chain GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTCNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGK
SEQ ID NO: 76 LCDR1 RSSQSLLSSGDQKNYLT
(Kabat)
SEQ ID NO: 77 LCDR2 WASTRES
(Kabat)
SEQ ID NO: 78 LCDR3 QNDYRYPLT
(Kabat)
SEQ ID NO: 79 LCDR1 SQSLLSSGDQKNY
(Chothia)
SEQ ID NO: 80 LCDR2 WAS
(Chothia)
SEQ ID NO: 81 LCDR3 DYRYPL
(Chothia)
SEQ ID NO: 82 VL DIVMTQTPLSLPVTPGEPASISCRSSQSLLSSGDQKNY
LTWYLQKPGQSPQLLIYWASTRESGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCQNDYRYPLTFGQGTKLEI K
SEQ ID NO: 83 DNA VL GATATCGTGATGACTCAGACCCCCCTGAGCCTGCC
CGTGACCCCTGGCGAGCCTGCCTCTATTAGCTGTA
GATCTAGTCAGTCACTGCTGTCTAGCGGCGATCAGA
AGAACTACCTGACCTGGTATCTGCAGAAGCCCGGT
CAGTCACCTCAGCTG CTG ATCTACTG GG CCTCTACT
AGAGAATCAGGCGTGCCCGATAGGTTTAGCGGTAG
CGGTAGTGGCACCGACTTCACCCTGAAGATCTCTA
GGGTGGAAGCCGAGGACGTGGGCGTCTACTACTGT
CAGAACGACTATAGATACCCCCTGACCTTCGGTCAG
G G C ACT AAG CTG G AG ATTAAG
SEQ ID NO: 84 Light Chain DIVMTQTPLSLPVTPGEPASISCRSSQSLLSSGDQKNY
LTWYLQKPGQSPQLLIYWASTRESGVPDRFSGSGSG
TDFTLKISRVEAEDVGVYYCQNDYRYPLTFGQGTKLEI
KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 85 DNA Light GATATCGTGATGACTCAGACCCCCCTGAGCCTGCC
Chain CGTGACCCCTGGCGAGCCTGCCTCTATTAGCTGTA
GATCTAGTCAGTCACTGCTGTCTAGCGGCGATCAGA
AGAACTACCTGACCTGGTATCTGCAGAAGCCCGGT
CAGTCACCTCAGCTG CTG ATCTACTG GG CCTCTACT
AGAGAATCAGGCGTGCCCGATAGGTTTAGCGGTAG
CGGTAGTGGCACCGACTTCACCCTGAAGATCTCTA GGGTGGAAGCCGAGGACGTGGGCGTCTACTACTGT
CAGAACGACTATAGATACCCCCTGACCTTCGGTCAG
GGCACTAAGCTGGAGATTAAGCGTACGGTGGCCGC
TCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGC
AGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCT
GCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGC
AGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAAC
AGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGG
ACTCCACCTACAGCCTGAGCAGCACCCTGACCCTG
AGCAAGGCCGACTACGAGAAGCATAAGGTGTACGC
CTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCC
GTGACCAAGAGCTTCAACAGGGGCGAGTGC
SEQ ID NO: 86 K107C DIVMTQTPLSLPVTPGEPASISCRSSQSLLSSGDQKNY
CysMab LTWYLQKPGQSPQLLIYWASTRESGVPDRFSGSGSG Mutated TDFTLKISRVEAEDVGVYYCQNDYRYPLTFGQGTKLEI Light Chain CRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK
SEQ ID NO: 96 DNA Heavy GAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAA
Chain GAAGCCCGGCGAGTCACTGAAGATTAGCTGTAAAG
TCTCAGGCTACACCTTCACCGATCACACTATTCACT
GGATGAGACAGATGCCCGGTAAAGGCCTGGAGTGG
ATG GG CTATATCTACCCTAG ATCAGG CTCTATTAAC
T AT AACG AG AAGTTTAAG G G TC AG G TC AC AATTAG C
GCCGATAAGTCTAGCTCTACCGCCTACCTGCAGTG
GTCTAGCCTGAAGGCTAGTGACACCGCTATGTACTA
CTGCGCTAGACGTAACCTGTTCCTGCCTATGGAATA
CTGGGGTCAGGGCACCCTGGTCACCGTGTCTAGCG
CTAGCACTAAGGGCCCAAGTGTG I I I CCCCTGGCC
CCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGC
CCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGC
CCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACT
TCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAG
CAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAG
TGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCT
GCAACGTGAACCACAAGCCCAGCAACACCAAGGTG
GACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGAC
CCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGC
TGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAG
CCCAAGGACACCCTGATGATCAGCAGGACCCCCGA
GGTGACCTGCGTGGTGGTGGACGTGTCCCACGAG
GACCCAGAGGTGAAGTTCAACTGGTACGTGGACGG
CGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAG
AGGAGCAGTACAACAGCACCTACAGGGTGGTGTCC
GTG CTG ACCGTG CTG CACCAG G ACTGG CTG AACGG
CAAAG AATACAAGTG CAAAGTCTCCAACAAG GCCCT
GCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCA
AGGGCCAGCCACGGGAGCCCCAGGTGTACACCCT
GCCCCCCAGCCGGGAGGAGATGACCAAGAACCAG
GTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCC
CAGCGATATCGCCGTGGAGTGGGAGAGCAACGGC
CAGCCCGAGAACAACTACAAGACCACCCCCCCAGT
GCTG G ACAGCG ACG GCAG CTTCTTCCTGTACAG CA
AGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGG
CAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCC
TGCACAACCACTACACCCAGAAGTCCCTGAGCCTG
AGCCCCGGCAAG
SEQ ID NO: 97 E152C/S375 EVQLVQSGAEVKKPGESLKISCKVSGYTFTDHTIHWM
C CysMab RQMPGKGLEWMGYIYPRSGSINYNEKFKGQVTISADK Mutated SSSTAYLQWSSLKASDTAMYYCARRNLFLPMEYWGQ Heavy Chain GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGK SEQ ID NO: 98 K360C EVQLVQSGAEVKKPGESLKISCKVSGYTFTDHTIHWM CysMab RQMPGKGLEWMGYIYPRSGSINYNEKFKGQVTISADK Mutated SSSTAYLQWSSLKASDTAMYYCARRNLFLPMEYWGQ Heavy Chain GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTC VWD VS H ED P E VKF N WYVDG VE VH N AKT KP RE E
QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTCNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGK
SEQ ID NO: 99 LCDR1 RSSQSLLSSGNQKNYLT
(Kabat)
SEQ ID NO: 100 LCDR2 WASTRES
(Kabat)
SEQ ID NO: 101 LCDR3 QNDYSYPLT
(Kabat)
SEQ ID NO: 102 LCDR1 SQSLLSSGNQKNY
(Chothia)
SEQ ID NO: 103 LCDR2 WAS
(Chothia)
SEQ ID NO: 104 LCDR3 DYSYPL
(Chothia)
SEQ ID NO: 105 VL DIVMTQTPLSLPVTPGEPASISCRSSQSLLSSGNQKNY
LTWYLQKPGQSPQLLIYWASTRESGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCQNDYSYPLTFGQGTKLEI K
SEQ ID NO: 106 DNA VL GATATCGTGATGACTCAGACCCCCCTGAGCCTGCC
CGTGACCCCTGGCGAGCCTGCCTCTATTAGCTGTA
GATCTAGTCAGTCACTGCTGTCTAGCGGTAATCAGA
AGAACTACCTGACCTGGTATCTGCAGAAGCCCGGT
CAGTCACCTCAGCTG CTG ATCTACTG GG CCTCTACT
AGAGAATCAGGCGTGCCCGATAGGTTTAGCGGTAG
CGGTAGTGGCACCGACTTCACCCTGAAGATCTCTA
GGGTGGAAGCCGAGGACGTGGGCGTCTACTACTGT
CAGAACGACTATAGCTACCCCCTGACCTTCGGTCAG
G G C ACT AAG CTG G AG ATTAAG
SEQ ID NO: 107 Light Chain DIVMTQTPLSLPVTPGEPASISCRSSQSLLSSGNQKNY
LTWYLQKPGQSPQLLIYWASTRESGVPDRFSGSGSG
TDFTLKISRVEAEDVGVYYCQNDYSYPLTFGQGTKLEI
KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 108 DNA Light GATATCGTGATGACTCAGACCCCCCTGAGCCTGCC
Chain CGTGACCCCTGGCGAGCCTGCCTCTATTAGCTGTA
GATCTAGTCAGTCACTGCTGTCTAGCGGTAATCAGA
AGAACTACCTGACCTGGTATCTGCAGAAGCCCGGT
CAGTCACCTCAGCTG CTG ATCTACTG GG CCTCTACT
AGAGAATCAGGCGTGCCCGATAGGTTTAGCGGTAG
CGGTAGTGGCACCGACTTCACCCTGAAGATCTCTA
GGGTGGAAGCCGAGGACGTGGGCGTCTACTACTGT
CAGAACGACTATAGCTACCCCCTGACCTTCGGTCAG
GGCACTAAGCTGGAGATTAAGCGTACGGTGGCCGC TCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGC
AGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCT
GCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGC
AGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAAC
AGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGG
ACTCCACCTACAGCCTGAGCAGCACCCTGACCCTG
AGCAAGGCCGACTACGAGAAGCATAAGGTGTACGC
CTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCC
GTGACCAAGAGCTTCAACAGGGGCGAGTGC
SEQ ID NO: 109 K107C DIVMTQTPLSLPVTPGEPASISCRSSQSLLSSGNQKNY
CysMab LTWYLQKPGQSPQLLIYWASTRESGVPDRFSGSGSG Mutated TDFTLKISRVEAEDVGVYYCQNDYSYPLTFGQGTKLEI Light Chain CRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: DNA Heavy CAGATTCAGCTGGTGCAGTCAGGCGCCGAAGTGAA 1 19 Chain GAAACCCGGCTCTAGCGTGAAAGTCAGCTGTAAAG
TCTCAGGCTACACCTTCACCGATCACACCCTGCACT
G G ATG AG AC AG G CCC C AG G TC AG GGCCTGGAGTG
GATGGGCTATATCTACCCTAGATCAGGCTCTACTAA
GTATAACGAGAACTTTAGGGGTAGAGTGACTATCAC
CGCCGACACTAGCTCTAGCACCGCCTATATGGAACT
GTCTAGCCTGAGATCAGAGGACACCGCCGTCTACT
ACTG CG CTAG ACGGCTG CTGTTCCTG CCCCTG G AC
TACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAG
CGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGG
CCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCT
GCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGA
GCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGA
CTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAG
AGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGAC
AGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATAT
CTGCAACGTGAACCACAAGCCCAGCAACACCAAGG
TGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG
ACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACT
GCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCA
AGCCCAAGGACACCCTGATGATCAGCAGGACCCCC
GAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGA
GGACCCAGAGGTGAAGTTCAACTGGTACGTGGACG
GCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGA
GAG GAG CAGTAC AACAGC ACCTACAG GGTG GTGTC
CGTGCTGACCGTGCTGCACCAGGACTGGCTGAACG
GCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCC
TGCCAG CCCCAATCG AAAAG ACAATCAG CAAG G CC
AAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCT
GCCCCCCAGCCGGGAGGAGATGACCAAGAACCAG
GTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCC
CAGCGATATCGCCGTGGAGTGGGAGAGCAACGGC
CAGCCCGAGAACAACTACAAGACCACCCCCCCAGT
GCTG G ACAGCG ACG GCAG CTTCTTCCTGTACAG CA
AGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGG
CAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCC
TGCACAACCACTACACCCAGAAGTCCCTGAGCCTG
AGCCCCGGCAAG
SEQ ID NO: E152C/S375 QIQLVQSGAEVKKPGSSVKVSCKVSGYTFTDHTLHW 120 C CysMab MRQAPGQGLEWMGYIYPRSGSTKYNENFRGRVTITA
Mutated DTSSSTAYMELSSLRSEDTAVYYCARRLLFLPLDYWG Heavy Chain QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: K360C QIQLVQSGAEVKKPGSSVKVSCKVSGYTFTDHTLHW 121 CysMab MRQAPGQGLEWMGYIYPRSGSTKYNENFRGRVTITA Mutated DTSSSTAYMELSSLRSEDTAVYYCARRLLFLPLDYWG Heavy Chain QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTCNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: LCDR1 RSSQSLLSSGNQKSYLT
122 (Kabat)
SEQ ID NO: LCDR2 WASTRES
123 (Kabat)
SEQ ID NO: LCDR3 QNDYSYPFT
124 (Kabat)
SEQ ID NO: LCDR1 SQSLLSSGNQKSY
125 (Chothia)
SEQ ID NO: LCDR2 WAS
126 (Chothia)
SEQ ID NO: LCDR3 DYSYPF
127 (Chothia)
SEQ ID NO: VL EIVMTQSPATLSLSPGERATLSCRSSQSLLSSGNQKS 128 YLTWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSG
TDFTLTISSLQPEDFAVYYCQNDYSYPFTFGQGTKLEI K
SEQ ID NO: DNA VL GAGATCGTGATGACTCAGTCACCCGCTACCCTGAG 129 CCTGAGCCCTGGCGAGAGAGCTACACTGAGCTGTA
GATCTAGTCAGTCACTGCTGTCTAGCGGTAATCAGA
AGTCCTACCTGACCTGGTATCAGCAGAAGCCCGGT
CAGGCCCCTAGACTGCTGATCTACTGGGCCTCTACT
AGAGAGTCAGGGATCCCCGCTAGGTTTAGCGGTAG
CG GTAGTG G CACCGACTTCACCCTG ACTATCTCTAG
CCTGCAGCCCGAGGACTTCGCCGTCTACTACTGTC
AGAACGACTATAGCTACCCCTTCACCTTCGGTCAGG
GCACTAAGCTGGAGATTAAG
SEQ ID NO: Light Chain EIVMTQSPATLSLSPGERATLSCRSSQSLLSSGNQKS 130 YLTWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSG
TDFTLTISSLQPEDFAVYYCQNDYSYPFTFGQGTKLEI
KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: DNA Light GAGATCGTGATGACTCAGTCACCCGCTACCCTGAG 131 Chain CCTGAGCCCTGGCGAGAGAGCTACACTGAGCTGTA
GATCTAGTCAGTCACTGCTGTCTAGCGGTAATCAGA
AGTCCTACCTGACCTGGTATCAGCAGAAGCCCGGT
CAGGCCCCTAGACTGCTGATCTACTGGGCCTCTACT
AGAGAGTCAGGGATCCCCGCTAGGTTTAGCGGTAG
CGGTAGTGGCACCGACTTCACCCTGACTATCTCTAG
CCTGCAGCCCGAGGACTTCGCCGTCTACTACTGTC
AGAACGACTATAGCTACCCCTTCACCTTCGGTCAGG
GCACTAAGCTGGAGATTAAGCGTACGGTGGCCGCT
CCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCA
GCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTG
CTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCA
GTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA
GCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGA
CTCCACCTACAGCCTGAGCAGCACCCTGACCCTGA
G C AAG G C CG ACT AC G AG AAGC ATAAG G TG TACG C C
TGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGT
G ACC AAG AG CTTCAACAGG GG CG AGTGC
SEQ ID NO: K107C EIVMTQSPATLSLSPGERATLSCRSSQSLLSSGNQKS 132 CysMab YLTWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSG
Mutated Light TDFTLTISSLQPEDFAVYYCQNDYSYPFTFGQGTKLEI Chain CRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
P-Cad Mab5
SEQ ID NO: HCDR1 DHTLH
133 (Kabat)
SEQ ID NO: HCDR2 YIYPRSGSTKYNENFRG
134 (Kabat)
SEQ ID NO: HCDR3 RLLFLPLDY
135 (Kabat)
SEQ ID NO: HCDR1 GYTFTDH
136 (Chothia)
SEQ ID NO: HCDR2 YPRSGS
137 (Chothia)
SEQ ID NO: HCDR3 RLLFLPLDY
138 (Chothia)
SEQ ID NO: VH QIQLVQSGAEVKKPGSSVKVSCKVSGYTFTDHTLHW 139 MRQAPGQGLEWMGYIYPRSGSTKYNENFRGRVTITA
DTSSSTAYMELSSLRSEDTAVYYCVRRLLFLPLDYWG QGTLVTVSS
SEQ ID NO: DNA VH CAGATTCAGCTGGTGCAGTCAGGCGCCGAAGTGAA 140 GAAACCCGGCTCTAGCGTGAAAGTCAGCTGTAAAG
TCTCAGGCTACACCTTCACCGATCACACCCTGCACT
G G ATG AG AC AG G CCC C AG G TC AG GGCCTGGAGTG
GATGGGCTATATCTACCCTAGATCAGGCTCTACTAA
GTATAACGAGAACTTTAGGGGTAGAGTGACTATCAC
CGCCGACACTAGCTCTAGCACCGCCTATATGGAACT
GTCTAGCCTGAGATCAGAGGACACCGCCGTCTACT
ACTGCGTCAGACGGCTGCTGTTCCTGCCCCTGGAC
TACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAG
C
SEQ ID NO: Heavy Chain QIQLVQSGAEVKKPGSSVKVSCKVSGYTFTDHTLHW 141 MRQAPGQGLEWMGYIYPRSGSTKYNENFRGRVTITA
DTSSSTAYMELSSLRSEDTAVYYCVRRLLFLPLDYWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: DNA Heavy CAGATTCAGCTGGTGCAGTCAGGCGCCGAAGTGAA 142 Chain GAAACCCGGCTCTAGCGTGAAAGTCAGCTGTAAAG
TCTCAGGCTACACCTTCACCGATCACACCCTGCACT
G G ATG AG AC AG G CCC C AG G TC AG GGCCTGGAGTG
GATGGGCTATATCTACCCTAGATCAGGCTCTACTAA
GTATAACGAGAACTTTAGGGGTAGAGTGACTATCAC
CGCCGACACTAGCTCTAGCACCGCCTATATGGAACT
GTCTAGCCTGAGATCAGAGGACACCGCCGTCTACT
ACTGCGTCAGACGGCTGCTGTTCCTGCCCCTGGAC
TACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAG
CGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGG
CCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCT
GCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGA
GCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGA
CTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAG
AGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGAC
AGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATAT
CTGCAACGTGAACCACAAGCCCAGCAACACCAAGG
TGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG
ACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACT
GCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCA
AGCCCAAGGACACCCTGATGATCAGCAGGACCCCC
GAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGA
GGACCCAGAGGTGAAGTTCAACTGGTACGTGGACG
GCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGA
GAG GAG CAGTAC AACAGC ACCTACAG GGTG GTGTC
CGTGCTGACCGTGCTGCACCAGGACTGGCTGAACG
GCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCC
TGCCAG CCCCAATCG AAAAG ACAATCAG CAAG G CC
AAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCT
GCCCCCCAGCCGGGAGGAGATGACCAAGAACCAG
GTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCC
CAGCGATATCGCCGTGGAGTGGGAGAGCAACGGC
CAGCCCGAGAACAACTACAAGACCACCCCCCCAGT
GCTG G ACAGCG ACG GCAG CTTCTTCCTGTACAG CA
AGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGG
CAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCC
TGCACAACCACTACACCCAGAAGTCCCTGAGCCTG
AGCCCCGGCAAG
SEQ ID NO: E152C/S375 QIQLVQSGAEVKKPGSSVKVSCKVSGYTFTDHTLHW 143 C CysMab MRQAPGQGLEWMGYIYPRSGSTKYNENFRGRVTITA
Mutated DTSSSTAYMELSSLRSEDTAVYYCVRRLLFLPLDYWG Heavy Chain QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: K360C QIQLVQSGAEVKKPGSSVKVSCKVSGYTFTDHTLHW
144 CysMab MRQAPGQGLEWMGYIYPRSGSTKYNENFRGRVTITA
Mutated DTSSSTAYMELSSLRSEDTAVYYCVRRLLFLPLDYWG
Heavy Chain QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTCNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: LCDR1 RSSQSLLSSGNQKSYLT
145 (Kabat)
SEQ ID NO: LCDR2 WASTRES
146 (Kabat)
SEQ ID NO: LCDR3 QNDYSYPFT
147 (Kabat)
SEQ ID NO: LCDR1 SQSLLSSGNQKSY
148 (Chothia)
SEQ ID NO: LCDR2 WAS
149 (Chothia)
SEQ ID NO: LCDR3 DYSYPF
150 (Chothia)
SEQ ID NO: VL EIVMTQSPATLSLSPGERATLSCRSSQSLLSSGNQKS
151 YLTWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSG
TDFTLTISSLQPEDFAVYYCQNDYSYPFTFGQGTKLEI
K
SEQ ID NO: DNA VL GAGATCGTGATGACTCAGTCACCCGCTACCCTGAG
152 CCTGAGCCCTGGCGAGAGAGCTACACTGAGCTGTA
GATCTAGTCAGTCACTGCTGTCTAGCGGTAATCAGA
AGTCCTACCTGACCTGGTATCAGCAGAAGCCCGGT
CAGGCCCCTAGACTGCTGATCTACTGGGCCTCTACT
AGAGAGTCAGGGATCCCCGCTAGGTTTAGCGGTAG
CGGTAGTGGCACCGACTTCACCCTGACTATCTCTAG
CCTGCAGCCCGAGGACTTCGCCGTCTACTACTGTC
AGAACGACTATAGCTACCCCTTCACCTTCGGTCAGG
GCACTAAGCTGGAGATTAAG
SEQ ID NO:153 Light Chain EIVMTQSPATLSLSPGERATLSCRSSQSLLSSGNQKS
YLTWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSG
TDFTLTISSLQPEDFAVYYCQNDYSYPFTFGQGTKLEI
KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:154 DNA Light GAGATCGTGATGACTCAGTCACCCGCTACCCTGAG
Chain CCTGAGCCCTGGCGAGAGAGCTACACTGAGCTGTA GATCTAGTCAGTCACTGCTGTCTAGCGGTAATCAGA
AGTCCTACCTGACCTGGTATCAGCAGAAGCCCGGT
CAGGCCCCTAGACTGCTGATCTACTGGGCCTCTACT
AGAGAGTCAGGGATCCCCGCTAGGTTTAGCGGTAG
CG GTAGTG G CACCGACTTCACCCTG ACTATCTCTAG
CCTGCAGCCCGAGGACTTCGCCGTCTACTACTGTC
AGAACGACTATAGCTACCCCTTCACCTTCGGTCAGG
GCACTAAGCTGGAGATTAAGCGTACGGTGGCCGCT
CCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCA
GCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTG
CTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCA
GTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA
GCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGA
CTCCACCTACAGCCTGAGCAGCACCCTGACCCTGA
G C AAG G C CG ACT AC G AG AAGC ATAAG G TG TACG C C
TGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGT
G ACC AAG AG CTTCAACAGG GG CG AGTGC
SEQ ID NO:155 K107C EIVMTQSPATLSLSPGERATLSCRSSQSLLSSGNQKS
CysMab YLTWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSG Mutated Light TDFTLTISSLQPEDFAVYYCQNDYSYPFTFGQGTKLEI Chain CRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: DNA Heavy CAGATTCAGCTGGTGCAGTCAGGCGCCGAAGTGAA 165 Chain GAAACCCGGCTCTAGCGTGAAAGTCAGCTGTAAAG
TCTCAGGCTACACCTTCACCGATCACACCCTGCACT
G G ATG AG AC AG G CCC C AG G TC AG GGCCTGGAGTG
GATGGGCTATATCTACCCTAGATCAGGCTCTACTAA
GTATAACGAGAACTTTAAGGGTAGAGTGACTATCAC
CGCCGACACTAGCTCTAGCACCGCCTATATGGAACT
GTCTAGCCTGAGATCAGAGGACACCGCCGTCTACT
ACTGCGTCAGACGGCTGCTGTTCCTGCCCCTGGAC
TACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAG
CGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGG
CCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCT
GCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGA
GCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGA
CTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAG
AGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGAC
AGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATAT
CTGCAACGTGAACCACAAGCCCAGCAACACCAAGG
TGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG
ACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACT
GCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCA
AGCCCAAGGACACCCTGATGATCAGCAGGACCCCC
GAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGA
GGACCCAGAGGTGAAGTTCAACTGGTACGTGGACG
GCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGA
GAG GAG CAGTAC AACAGC ACCTACAG GGTG GTGTC
CGTGCTGACCGTGCTGCACCAGGACTGGCTGAACG
GCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCC
TGCCAG CCCCAATCG AAAAG ACAATCAG CAAG G CC
AAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCT
GCCCCCCAGCCGGGAGGAGATGACCAAGAACCAG
GTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCC
CAGCGATATCGCCGTGGAGTGGGAGAGCAACGGC
CAGCCCGAGAACAACTACAAGACCACCCCCCCAGT
GCTG G ACAGCG ACG GCAG CTTCTTCCTGTACAG CA
AGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGG
CAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCC
TGCACAACCACTACACCCAGAAGTCCCTGAGCCTG
AGCCCCGGCAAG
SEQ ID NO: E152C/S375 QIQLVQSGAEVKKPGSSVKVSCKVSGYTFTDHTLHW 166 C CysMab MRQAPGQGLEWMGYIYPRSGSTKYNENFKGRVTITA
Mutated DTSSSTAYMELSSLRSEDTAVYYCVRRLLFLPLDYWG Heavy Chain QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: K360C QIQLVQSGAEVKKPGSSVKVSCKVSGYTFTDHTLHW
167 CysMab MRQAPGQGLEWMGYIYPRSGSTKYNENFKGRVTITA
Mutated DTSSSTAYMELSSLRSEDTAVYYCVRRLLFLPLDYWG
Heavy Chain QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTCNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: LCDR1 RSSQSLLSSGNQKSYLT
168 (Kabat)
SEQ ID NO: LCDR2 WASTRES
169 (Kabat)
SEQ ID NO:170 LCDR3 QNDYSYPFT
(Kabat)
SEQ ID NO: LCDR1 SQSLLSSGNQKSY
125 (Chothia)
SEQ ID NO: LCDR2 WAS
171 (Chothia)
SEQ ID NO: LCDR3 DYSYPF
172 (Chothia)
SEQ ID NO: VL EIVMTQSPATLSLSPGERATLSCRSSQSLLSSGNQKS
173 YLTWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSG
TDFTLTISSLQPEDFAVYYCQNDYSYPFTFGQGTKLEI
K
SEQ ID NO: DNA VL GAGATCGTGATGACTCAGTCACCCGCTACCCTGAG
174 CCTGAGCCCTGGCGAGAGAGCTACACTGAGCTGTA
GATCTAGTCAGTCACTGCTGTCTAGCGGTAATCAGA
AGTCCTACCTGACCTGGTATCAGCAGAAGCCCGGT
CAGGCCCCTAGACTGCTGATCTACTGGGCCTCTACT
AGAGAGTCAGGGATCCCCGCTAGGTTTAGCGGTAG
CGGTAGTGGCACCGACTTCACCCTGACTATCTCTAG
CCTGCAGCCCGAGGACTTCGCCGTCTACTACTGTC
AGAACGACTATAGCTACCCCTTCACCTTCGGTCAGG
GCACTAAGCTGGAGATTAAG
SEQ ID NO: Light Chain EIVMTQSPATLSLSPGERATLSCRSSQSLLSSGNQKS
175 YLTWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSG
TDFTLTISSLQPEDFAVYYCQNDYSYPFTFGQGTKLEI
KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: DNA Light GAGATCGTGATGACTCAGTCACCCGCTACCCTGAG
176 Chain CCTGAGCCCTGGCGAGAGAGCTACACTGAGCTGTA
GATCTAGTCAGTCACTGCTGTCTAGCGGTAATCAGA AGTCCTACCTGACCTGGTATCAGCAGAAGCCCGGT CAGGCCCCTAGACTGCTGATCTACTGGGCCTCTACT
AGAGAGTCAGGGATCCCCGCTAGGTTTAGCGGTAG
CG GTAGTG G CACCGACTTCACCCTG ACTATCTCTAG
CCTGCAGCCCGAGGACTTCGCCGTCTACTACTGTC
AGAACGACTATAGCTACCCCTTCACCTTCGGTCAGG
GCACTAAGCTGGAGATTAAGCGTACGGTGGCCGCT
CCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCA
GCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTG
CTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCA
GTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA
GCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGA
CTCCACCTACAGCCTGAGCAGCACCCTGACCCTGA
G C AAG G C CG ACT AC G AG AAGC ATAAG G TG TACG C C
TGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGT
G ACC AAG AG CTTCAACAGG GG CG AGTGC
SEQ ID NO: K107C EIVMTQSPATLSLSPGERATLSCRSSQSLLSSGNQKS 177 CysMab YLTWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSG
Mutated Light TDFTLTISSLQPEDFAVYYCQNDYSYPFTFGQGTKLEI Chain CRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Modification of Framework or Fc Region
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 al.
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 alter one or more functional properties of the antibody, such as serum half-life, 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 glycosylation, 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 ef a/. 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 (Junutula JR, et al.: 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 al, (1990) Protein Eng., 3:703-708, WO 201 1/005481 , WO2014/124316, 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, 1 19, 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 EU 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 light 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 light 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 Pel (pyrroline-carboxy-lysine), pyrrolysine, 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 subformulae thereof. For example, the antibodies or antibody fragments can be modified to incorporate Pel or pyrrolysine (W. Ou et al. (201 1) PNAS 108 (26), 10437-10442; WO2014124258) or unnatural amino acids (J.Y. Axup, et al. Proc Natl 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 Chem 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 al. Chem Biol. 2013, 20(2): 161 -7; Rabuka D., Curr Opin Chem Biol. 2010 Dec; 14(6)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 (WO2013184514; 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 CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl 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 al. Allotypic amino acid residues include, but are not limited to, constant region of a heavy chain of the lgG1 , lgG2, and lgG3 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 lgG1 antibodies comprise the so-called LALA mutant comprising L234A and L235A mutation in the lgG1 Fc amino acid sequence. Another example of a silent lgG 1 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 cellular 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 FcyRIa, FcyRlla, FcyRllla, and FcyRlllb, and human inhibitory Fey receptor includes FcyRllb. This approach is described in, e.g., the PCT Publication WO 00/42072 by Presta. Moreover, binding sites on human lgG1 for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al. , J. Biol. Chem. 276:6591 -6604, 2001). Optimization of Fc-mediated effector functions of monoclonal antibodies such as increased ADCC/ADCP function has been described (see Strohl, 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, K290S, R292P, S298A, S298D, S298V, Y300L, V305I, 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 al. , 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 to 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 al.
Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated 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 cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells 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 cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked 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 al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g. , beta(1 ,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., 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 half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6, 121 ,022 by Presta et al.
Production of Antibodies
Antibodies and antibody fragments (e.g. , antigen binding fragments) thereof can be produced by any means known in the art, including 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 cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, 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.
Some polynucleotides disclosed herein encode a variable region of an anti-P-Cad antibody. Some polynucleotides disclosed herein encode both a variable region and a constant region of an anti-P-Cad antibody. Some polynucleotide sequences encode a polypeptide that comprises variable regions of both a heavy chain and a light chain of an anti-P-Cad 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-P-cad 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. Enzymol. 68:90, 1979; the phosphodiester method of Brown et ai , Meth. Enzymol. 68: 109, 1979; the diethylphosphoramidite method of Beaucage ef a/. , 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 ef a/. , PCR Methods and Applications 1 :17, 1991 .
Also provided are expression vectors and host cells 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 cell.
Nonviral vectors and systems include plasmids, episomal 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) cells 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. Useful 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 Semliki Forest virus (SFV). See, Brent ef a/. , 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 inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions. Inducible promoters include, e.g., arabinose, lacZ, metallothionein 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. Cell Differ. 20:125, 1994; and Bittner ef a/., eth. Enzymol., 153:516, 1987). For example, an SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.
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. coli 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 enterobacteriaceae, 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 typically 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 cells 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 cell line harboring an exogenous expression vector (e.g., the SP2/0 myeloma cells). These include any normal mortal or normal or abnormal immortal animal or human cell. For example, a number of suitable host cell 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 cell 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 al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation 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 modulatable or regulatable. Useful promoters include, but are not limited to, a metallothionein promoter, a constitutive adenovirus major late promoter, a dexamethasoneinducible MMTV promoter, a SV40 promoter, a MRP pollll 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 cells, 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, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 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, cell 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 tumorigenicity 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, blastomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, biliarintestinal (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 cell 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 CNS 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 lymphoproliferative 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 myelodysplasia syndromes (MDS, formerly known as "preleukemia") which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells 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-cell acute lymphoid leukemia ("BALL"), T-cell 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 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 lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom
macroglobulinemia, and "preleukemia" 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 cells 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, detecting 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 (EG FR); E-cadherin; P-cadherin; Cadherin 6; cathepsin D; estrogen receptor;
progesterone receptor; CA125; CA15-3; CA19-9; P- glycoprotein (CD243); CD2; CD19; CD20; CD22; CD24; CD27; CD30; CD37; CD38; CD40; CD44v6; CD45; CD47; CD52; CD56; CD70; CD71 ; CD79a; CD79b; CD72; CD97; CD179a; CD123; CD137; CD171 ; CS-1 (also referred to as CD2 subset 1 , CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); epidermal growth factor receptor variant III (EGFRvl ll); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1 -4)bDGIcp(1-1)Cer); TNF receptor family member B cell maturation (BC A); 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 (EPCAM); B7H3 (CD276); KIT (CD1 17); lnterleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 1 1 receptor alpha (IL-1 1 Ra); prostate stem cell 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 (NCA ); 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 (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2);
glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type- A receptor 2 (EphA2); Fucosyl GM 1 ; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1 -4)bDGIcp(1 -1)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM 1/CD248); tumor 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 61 (CXORF61); anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (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 cell 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 MART1); 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-Acetyl glucosaminyl- 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 Cells 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); legumain; human papilloma virus E6 (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 (CD300LF); 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; DLL3; CD133; FZD7; Dysadherin; PD-L1 ; SLITRK6; Nectin-4; FGFR2; FGFR3; FGFR4;
CEACAM 1 ; CEACAM5; CD74; STEAP-1 ; PMEL17; Mud 6; FcRH5; TENB2; Ly6E; ETBR; 158P1 D7; 161 P2F10B; 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 cell, an antigen presenting cell, or a myeloid-derived suppressor cell (MDSC). Stromal cells can secrete growth factors to promote cell division in the microenvironment. MDSC cells can inhibit T cell proliferation and activation. In some embodiments, the stromal cell antigen is chosen from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) and tenascin. In embodiments, the MDSC antigen is chosen from one or more of: CD33, CD1 1 b, C14, CD15, and CD66b. Accordingly, in some embodiments, the tumor-supporting antigen is chosen from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) 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-malignant diseases or disorders, such as inflammatory bowel disease (IBD), 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 burns, 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 Bordetella pertussis, thrombus caused by platelet aggregation, reproductive conditions such as endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding, or menometrorrhagia, acute and chronic nephropathies (including proliferative glomerulonephritis), hypertrophic scar formation, endotoxic shock and fungal infection, familial adenomatosis polyposis, myelodysplastic 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, intracerobrospinal administration, subcutaneous administration, intra-articular administration, intrasynovial 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, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 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-pentoxycarbonyl-5- deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil
(Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), 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, docetaxel (Taxotere®), doxorubicin hydrochloride
(Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5- fluorouracil (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 (Velban®), vincristine (Oncovin®), vinorelbine (Navelbine®), epirubicin (Ellence®), oxaliplatin (Eloxatin®), exemestane (Aromasin®), letrozole (Femara®), 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 sequentially, 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 Kadcyla®, or T-DM 1).
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, Erlotinib hydrochloride (Tarceva®); Linifanib (N-[4-(3-amino-1 H-indazol-4-yl)phenyl]-N'-(2-fluoro-5- methylphenyl)urea, also known as ABT 869, 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-606, 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-Chloro-4-fluorophenyl)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- methoxyphenyl)amino)pyrrolo[2, 1-f][1 ,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514);
Canertinib dihydrochloride (CI-1033); 6-[4-[(4-Ethyl-1-piperazinyl)methyl]phenyl]-N-[(1 R)-1 - phenylethyl]- 7H-Pyrrolo[2,3-d]pyrimidin-4-amine (AEE788, CAS 497839-62-0); Mubritinib (TAK165); Pelitinib (EKB569); Afatinib (Gilotrif®); Neratinib (HKI-272); N-[4-[[1 -[(3- Fluorophenyl)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- yl]-phenol (PKI 166, CAS 187724-61-4).
EGFR antibodies include but are not limited to, Cetuximab (Erbitux®); Panitumumab (Vectibix®); Matuzumab (EMD-72000); Nimotuzumab (hR3); Zalutumumab; TheraCIM h-R3; MDX0447 (CAS 339151 -96-1); and ch806 (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 ditosylate (Tykerb®); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2, 1 - f][1 ,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); (2E)-N-[4-[(3-Chloro-4- fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2- butenamide (BIBW-2992, CAS 850140-72-6); N-[4-[[1 -[(3-Fluorophenyl)methyl]-1 H-indazol-5- yl]amino]-5-methylpyrrolo[2, 1 -f][1 ,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinylmethyl ester (BMS 599626, CAS 714971-09-2); Canertinib dihydrochloride (PD183805 or CI-1033); and N- (3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aD ,5D,6aD)-octahydro-2- methylcyclopenta[c]pyrrol-5-yl]methoxy]- 4-quinazolinamine (XL647, CAS 781613-23-8).
HER3 inhibitors include but are not limited to, LJM716, MM-121 , AMG-888, RG71 16, REGN-1400, AV-203, MP-RM-1 , MM-1 1 1 , and MEHD-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
1000873-98-2); 1 -(2-Hydroxy-2-methylpropyl)-A/-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5- methyl-3-oxo-2-phenyl-2,3-dihydro-1 /-/-pyrazole-4-carboxamide (AMG 458); Cryzotinib (Xalkori®, PF-02341066); (3Z)-5-(2,3-Dihydro-1 H-indol-1 -ylsulfonyl)-3-({3,5-dimethyl-4-[(4- methylpiperazin-1 -yl)carbonyl]-1 H^yrrol-2-yl}methylene)-1 ,3-dihydro-2H
(SU1 1271); (3Z)-N-(3-Chlorophenyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1 -yl)carbonyl]-1 H- pyrrol-2-yl}methylene)-N-methyl-2-oxoindoline-5-sulfonamide (SU1 1274); (3Z)-N-(3- Chlorophenyl)-3-{[3,5-dimethyl-4-(3-morpholin-4-ylpro^
oxoindoline-5-sulfonamide (SU1 1606); 6-[Difluoro[6-(1-methyl-1 Hpyrazol-4-yl)-1 ,2,4-triazolo[4,3- b]pyridazin-3-yl]methyl]-quinoline (JNJ38877605, CAS 943540-75-8); 2-[4-[1 -(Quinolin-6- ylmethyl)-1 H-[1 ,2,3]triazolo[4,5-b]pyrazin-6-yl]-1 H-pyrazol-1-yl]ethanol (PF04217903, CAS 956905-27-4); N-((2R)-1 ,4-Dioxan-2-ylmethyl)-N-methyl-N'-[3-(1-methyl-1 H-pyrazol-4-yl)-5-oxo- 5H-benzo[4,5]cyclohepta[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[4,3-6]pyridazin 3-yl]thio]-quinoline (SGX523, CAS 1022150-57-7); and (3Z)-5-[[(2,6-Dichlorophenyl)methyl]sulfonyl]-3-[[3,5-dimethyl-4-[[(2R)-2-(1 - pyrrolidinylmet yl)-1-pyrrolidinyl]carbonyl]-1 -/-pyrrol-2-yl]methylene]-1 ,3-dihydro-2 - -indol-2-one (PHA665752, CAS 477575-56-7).
IGFR inhibitors include but are not limited to, BMS-754807, XL-228, OSI-906,
GSK0904529A, A-928605, AXL1717, KW-2450, MK0646, AMG479, IMCA12, MEDI-573, and BI836845. 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, EK inhibitors, BRAF inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and also mTOR inhibitors, and CDK inhibitors.
For example, mitogen-activated protein kinase (MEK) inhibitors include but are not limited to, XL-518 (also known as GDC-0973, Cas No. 1029872-29-4, 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 PCT Publication No. WO2000035436); N- [(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]- benzamide (also known as PD0325901 and described in PCT Publication No. WO2002006213); 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- methoxyphenyl]-1 -[(2R)-2,3-dihydroxypropyl]- cyclopropanesulfonamide (also known as RDEA1 19 or BAY869766 and described in PCT Publication No. WO200701401 1);
(3S,4R,5Z,8S,9S, 1 1 E)-14-(Ethylamino)-8,9, 16-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. WO2003076424); 2'-Amino-3'-methoxyflavone (also known as PD98059 available from Biaffin GmbH & Co., KG, Germany); Vemurafenib (PLX-4032, CAS 918504-65- 1); (R)-3-(2,3-Dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[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 Zelboraf®), GDC-0879, PLX-4720 (available from Symansis), Dabrafenib (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 MDX-010, DX-101 , or Yervoy).
Phosphoinositide 3-kinase (PI3K) inhibitors include, but are not limited to, 4-[2-(1 H- lndazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1 -yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC0941 , RG7321 , GNE0941 , Pictrelisib, or Pictilisib; and described in PCT Publication Nos. WO 09/036082 and WO 09/055730); 2-Methyl-2-[4-[3-methyl-2-oxo-8- (quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1 -yl]phenyl]propionitrile (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-BKM120, and described in PCT Publication No. WO2007/084786); Tozasertib (VX680 or MK-0457, CAS 639089-54-6); (5Z)-5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4- thiazolidinedione (GSK1059615, CAS 958852-01 -2); (1 E,4S,4aR,5R,6aS,9aR)-5-(Acetyloxy)-1- [(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-1 1 -hydroxy-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 (LY294002, CAS 154447-36-6); (S)-N1 -(4- methyl-5-(2-(1 , 1 , 1 -trifluoro-2-methylpropan-2-yl)pyridin-4-yl)thiazol-2-yl)pyrrolidine-1 ,2- dicarboxamide (also known as BYL719 or Alpelisib); 2-(4-(2-(1-isopropyl-3-methyl-1 H-1 ,2,4- triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1 ,2-d][1 ,4]oxazepin-9-yl)-1 H-pyrazol-1 -yl)-2- methylpropanamide (also known as GDC0032, RG7604, or Taselisib).
mTOR inhibitors include but are not limited to, Temsirolimus (Torisel®); Ridaforolimus (formally known as deferolimus, (1 R,2R,4S)-4-[(2R)-2
[(1 R,9S, 12S, 15R, 16E, 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 RAD001); Rapamycin (AY22989, Sirolimus®); Simapimod (CAS 164301 -51 -3); (5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin- 7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-Amino-8-[frans-4-(2-hydroxyethoxy)cyclohexyl]- 6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-c/]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and /V -[1 ,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1 -benzopyran-2-yl)morpholinium-4- yl]methoxy]butyl]-L-arginylglycyl-L-D-aspartylL-serine- (SEQ ID NO: 932), inner salt (SF1 126, CAS 936487-67-1). CDK inhibitors include but are not limited to, Palbociclib (also known as PD-0332991 , Ibrance®, 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- 35156, AT406, and TL3271 1 . Other examples of IAP inhibitors include but are not limited to those disclosed in WO04/005284, WO 04/007529, WO05/097791 , WO 05/069894, WO
05/069888, WO 05/094818, US2006/0014700, US2006/0025347, WO 06/069063, WO
06/0101 18, WO 06/017295, and WO08/134679, all 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-
[(phenylthio)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]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-cyclopentyl-4-(1-cyano-2-ethoxy- 2-oxoethyl)-4Hchromone-3-carboxylate (HA14 -1); Oblimersen (G3139, Genasense®); Bak BH3 peptide; (-)-Gossypol 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- nitrophenyl]sulfonyl]-benzamide (ABT-737, CAS 852808-04-9); and Navitoclax (ABT-263, CAS 923564-51-6).
Proapoptotic receptor agonists (PARAs) including DR4 (TRAILR1) and DR5 (TRAILR2), including but are not limited to, Dulanermin (AMG-951 , RhApo2L/TRAIL); Mapatumumab (HRS- ETR1 , CAS 658052-09-6); Lexatumumab (HGS-ETR2, CAS 845816-02-6); Apomab
(Apomab®); Conatumumab (AMG655, 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- piperidinylpyrazolo[1 ,5-a]pyrimidin-7-amine (SCH900776, CAS 891494-63-6); 5-(3- Fluorophenyl)-3-ureidothiophene-2-carboxylic acid N-[(S)-piperidin-3-yl]amide (AZD7762, CAS 860352-01-8); 4-[((3S)-1 -Azabicyclo[2.2.2]oct-3-yl)amino]-3-(1 H-benzimidazol-2-yl)-6- chloroquinolin-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-methyl-2-pyrazinyl)urea (LY2603618, CAS 91 1222-45-2); Sulforaphane (CAS 4478-93-7, 4-Methylsulfinylbutyl isothiocyanate); 9, 10,1 1 , 12-Tetrahydro- 9, 12-epoxy-1 H- diindolo[1 ,2,3-¾:3',2', 1 '-/i/]pyrrolo[3,4-/][1 ,6]benzodiazocine-1 ,3(2H)-dione (SB-218078, CAS 135897-06-2); and TAT-S216A (YGRKKRRQRRRLYRSPAMPENL (SEQ ID NO: 929)), 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 immunomodulators (e.g., one or more of an activator of a costimulatory molecule or an inhibitor of an immune checkpoint molecule).
In certain embodiments, the immunomodulator is an activator of a costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule is selected from an agonist (e.g., an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion) of OX40, CD2, CD27, CDS, ICAM-1 , LFA-1 (CD1 1 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 costimulatory molecule is a GITR agonist. In some embodiments, the GITR agonist is GWN323 (NVS), BMS-986156, MK-4166 or MK-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 molecule. 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 14 (e.g., from the heavy and light chain variable region sequences of MAB7 disclosed in Table 14), or encoded by a nucleotide sequence shown in Table 14. In some embodiments, the CDRs are according to the Kabat definition (e.g., as set out in Table 14). In some
embodiments, the CDRs are according to the Chothia definition (e.g. , as set out in Table 14). 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 14, or encoded by a nucleotide sequence shown in Table 14.
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: 909, a VHCDR2 amino acid sequence of SEQ ID NO: 91 1 , and a VHCDR3 amino acid sequence of SEQ ID NO: 913; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 914, a VLCDR2 amino acid sequence of SEQ ID NO: 916, and a VLCDR3 amino acid sequence of SEQ ID NO: 918, each disclosed in Table 14.
In one embodiment, the anti-GITR antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 901 , or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 901 . In one embodiment, the anti-GITR antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 902, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 902. In one embodiment, the anti-GITR antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 901 and a VL comprising the amino acid sequence of SEQ ID NO: 902.
In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 905, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 905. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 906, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 906. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 905 and a VL encoded by the nucleotide sequence of SEQ ID NO: 906.
In one embodiment, the anti-GITR antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 903, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 903. In one embodiment, the anti- GITR antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 904, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 904. In one embodiment, the anti-GITR antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 903 and a light chain comprising the amino acid sequence of SEQ ID NO: 904.
In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 907, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 907. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 908, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 908. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 907 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 908.
The antibody molecules described herein can be made by vectors, host cells, and methods described in WO 2016/057846, incorporated by reference in its entirety.
Table 14: Amino acid and nucleotide sequences of exemplary anti-GITR antibody molecule
SEQ ID NO: 903 Heavy EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDW Chain VRQAPGKGLEWVGVIWGGGGTYYASSLMGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCARHAYGHDGGFAM
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSV HEALHN
HYTQKSLSLSPGK
SEQ ID NO: 904 Light EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQ
Chain QRPGQAPRLLIYGASNRATGIPARFSGSGSGTDFTLTI
SRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIKRTVAA
PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWK
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY
EKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 905 DNA GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGG
VH TGCAGTCCGGCGGCTCTCTGAGACTGTCTTGCGCT
GCCTCCGGCTTCTCCCTGTCCTCTTACGGCGTGGA
CTGGGTGCGACAGGCCCCTGGCAAGGGCCTGGAA
TGGGTGGGAGTGATCTGGGGCGGAGGCGGCACCT
ACTACGCCTCTTCCCTGATGGGCCGGTTCACCATCT
CCCGGGACAACTCCAAGAACACCCTGTACCTGCAG
ATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTA
CTACTGCGCCAGACACGCCTACGGCCACGACGGC
GGCTTCGCCATGGATTATTGGGGCCAGGGCACCCT
GGTGACAGTGTCCTCC
SEQ ID NO: 906 DNA GAGATCGTGATGACCCAGTCCCCCGCCACCCTGTC
VL TGTGTCTCCCGGCGAGAGAGCCACCCTGAGCTGCA GAGCCTCCGAGTCCGTGTCCTCCAACGTGGCCTGG TATCAGCAGAGACCTGGTCAGGCCCCTCGGCTGCT GATCTACGGCGCCTCTAACCGGGCCACCGGCATCC CTGCCAGATTCTCCGGCTCCGGCAGCGGCACCGAC TTCACCCTGACCATCTCCCGGCTGGAACCCGAGGA CTTCG CCGTGTACTACTG CGGCCAGTCCTACTCATA CCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAAA TCAAG
SEQ ID NO: 907 DNA GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGG
Heavy TGCAGTCCGGCGGCTCTCTGAGACTGTCTTGCGCT
Chain GCCTCCGGCTTCTCCCTGTCCTCTTACGGCGTGGA
CTGGGTGCGACAGGCCCCTGGCAAGGGCCTGGAA
TGGGTGGGAGTGATCTGGGGCGGAGGCGGCACCT
ACTACGCCTCTTCCCTGATGGGCCGGTTCACCATCT
CCCGGGACAACTCCAAGAACACCCTGTACCTGCAG
ATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTA
CTACTGCGCCAGACACGCCTACGGCCACGACGGC
GGCTTCGCCATGGATTATTGGGGCCAGGGCACCCT
GGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCAA
GTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTT
CCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAG
GACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAA
CTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCC
CCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCT
GAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGG
GAACCCAGACCTATATCTGCAACGTGAACCACAAGC
CCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCC
AAGAGCTGCGACAAGACCCACACCTGCCCCCCCTG
CCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGT
TCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATG
ATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGT
GGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCA
ACTGGTACGTGGACGGCGTGGAGGTGCACAACGC
CAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCA
CCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCAC
C AG G ACTG G CTG AACGG C AAAG AATAC AAG TG C AA
AGTCTCCAACAAGGCCCTGCCAGCCCCAATCGAAA
AGACAATCAGCAAGGCCAAGGGCCAGCCACGGGA
GCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAG
GAGATGACCAAGAACCAGGTGTCCCTGACCTGTCT
GGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGG
AGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC
AAG ACCACCCCCCC AGTG CTG G AC AG CG ACG GCA
GCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGT
CC AG G TG G C AG C AG G G C AACG TG TTC AG CTG C AG C
GTGATGCACGAGGCCCTGCACAACCACTACACCCA
GAAGTCCCTGAGCCTGAGCCCCGGCAAG
SEQ ID NO: 908 DNA GAGATCGTGATGACCCAGTCCCCCGCCACCCTGTC Light TGTGTCTCCCGGCGAGAGAGCCACCCTGAGCTGCA Chain GAGCCTCCGAGTCCGTGTCCTCCAACGTGGCCTGG
TATCAG CAG AG ACCTG GTC AGG CCCCTCGG CTG CT
GATCTACGGCGCCTCTAACCGGGCCACCGGCATCC
CTGCCAGATTCTCCGGCTCCGGCAGCGGCACCGAC
TTCACCCTGACCATCTCCCGGCTGGAACCCGAGGA
CTTCG CCGTGTACTACTG CGGCCAGTCCTACTCATA
CCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAAA
TCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATC
TTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCAC
CGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACC
CCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAA
CGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTC
ACCGAGCAGGACAGCAAGGACTCCACCTACAGCCT
GAGCAGCACCCTGACCCTGAGCAAGGCCGACTACG
AGAAGCATAAGGTGTACGCCTGCGAGGTGACCCAC
CAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAA
CAGGGGCGAGTGC
SEQ ID NO: 909 HCDR1 SYGVD
(KABAT)
SEQ ID NO: 910 HCDR1 GFSLSSY
(CHOTHIA)
SEQ ID NO: 91 1 HCDR2 VIWGGGGTYYASSLMG
(KABAT)
SEQ ID NO: 912 HCDR2 WGGGG
(CHOTHIA)
SEQ ID NO: 913 HCDR3 HAYGHDGGFAMDY
(KABAT)
SEQ ID NO: 913 HCDR3 HAYGHDGGFAMDY
(CHOTHIA)
SEQ ID NO: 914 LCDR1 RASESVSSNVA
(KABAT)
SEQ ID NO: 915 LCDR1 SESVSSN
(CHOTHIA)
SEQ ID NO: 916 LCDR2 GASNRAT
(KABAT)
SEQ ID NO: 917 LCDR2 GAS
(CHOTHIA)
SEQ ID NO: 918 LCDR3 GQSYSYPFT
(KABAT)
SEQ ID NO: 919 LCDR3 SYSYPF
(CHOTHIA)
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 15. In one embodiment, the anti-GITR antibody molecule is MK-4166 or MK-1248 (Merck). MK-4166, MK-1248, and other anti-GITR antibodies are disclosed, e.g. , in US 8,709,424, WO 201 1/028683, WO 2015/026684, and Mahne et al. Cancer Res. 2017; 77(5): 1 108-1 1 18, 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 al. (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 INCAGN 1876 (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 INCAGN1876.
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-1 10 (Inhibrx). 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 GITR 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
(Medlmmune), also known as MEDI 1873. MEDI 1873 and other GITR agonists are disclosed, e.g. , in US 2017/0073386, WO 2017/025610, and Ross et al. Cancer Res 2016; 76(14 Suppl): 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 ligand (GITRL) of MEDI 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 15: Amino acid sequence of other exemplary anti-GITR antibody molecules
In certain embodiments, the immunomodulator 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, LAG 3, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4 and/or TGFRbeta. In one embodiment, the inhibitor of an immune checkpoint molecule 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 ligand (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, LAG 3, 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 , lgG2, lgG3, lgG4, IgM, lgA1 , lgA2, IgD, and IgE; particularly, selected from, e.g., the heavy chain constant regions of lgG1 , lgG2, lgG3, and lgG4, more particularly, the heavy chain constant region of lgG1 or lgG4 {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 molecule. 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 bispecific 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 bispecific 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 trispecific 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 immunomodulator is an inhibitor of PD-1 , e.g., human PD-1 .
In another embodiment, the immunomodulator 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 anti-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, LAG3, 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), Pembrolizumab (Merck & Co),
Pidilizumab (CureTech), MEDI0680 (Medimmune), REGN2810 (Regeneron), TSR-042
(Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), or AMP-224 (Amplimmune).
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 molecule as described in US
2015/0210769, 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 16 (e.g., from the heavy and light chain variable region sequences of BAP049-Clone-E or BAP049-Clone-B disclosed in Table 16), or encoded by a nucleotide sequence shown in Table 16. In some embodiments, the CDRs are according to the Kabat definition (e.g., as set out in Table 16). In some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 16). In some embodiments, the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g., as set out in Table 16). In one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GYTFTTYWMH (SEQ ID NO: 541). 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 16, or encoded by a nucleotide sequence shown in Table 16.
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: 501 , a VHCDR2 amino acid sequence of SEQ ID NO: 502, and a VHCDR3 amino acid sequence of SEQ ID NO: 503; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 510, a VLCDR2 amino acid sequence of SEQ ID NO: 51 1 , and a VLCDR3 amino acid sequence of SEQ ID NO: 512, each disclosed in Table 16.
In one embodiment, the antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 524, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 525, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 526; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 529, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 530, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 531 , each disclosed in Table 16.
In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 506, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 506. In one embodiment, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 520, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 520. In one embodiment, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 516, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 516. In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 506 and a VL comprising the amino acid sequence of SEQ ID NO: 520. In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 506 and a VL comprising the amino acid sequence of SEQ ID NO: 516.
In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 507, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 507. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 521 or 517, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 521 or 517. In one
embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 507 and a VL encoded by the nucleotide sequence of SEQ ID NO: 521 or 517.
In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 508, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 508. In one embodiment, the anti- PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 522, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 522. In one embodiment, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 518, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 518. In one embodiment, the anti- PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 508 and a light chain comprising the amino acid sequence of SEQ ID NO: 522. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 508 and a light chain comprising the amino acid sequence of SEQ ID NO: 518.
In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 509, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 509. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 523 or 519, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 523 or 519. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 509 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 523 or 519.
The antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0210769, incorporated by reference in its entirety.
Table 16. Amino acid and nucleotide sequences of exemplary anti-PD-1 antibody molecules
AGGCTACCGGTCAAGGCCTCGAGTGGATGGGTAATATC
TACCCCGGCACCGGCGGCTCTAACTTCGACGAGAAGTT
TAAGAATAGAGTGACTATCACCGCCGATAAGTCTACTAG
CACCGCCTATATGGAACTGTCTAGCCTGAGATCAGAGGA
CACCGCCGTCTACTACTGCACTAGGTGGACTACCGGCA
CAGGCGCCTACTGGGGTCAAGGCACTACCGTGACCGTG
TCTAGCGCTAGCACTAAGGGCCCGTCCGTGTTCCCCCT
GGCACCTTGTAGCCGGAGCACTAGCGAATCCACCGCTG
CCCTCGGCTGCCTGGTCAAGGATTACTTCCCGGAGCCC
GTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGG
AGTGCACACCTTCCCCGCTGTGCTGCAGAGCTCCGGGC
TGTACTCGCTGTCGTCGGTGGTCACGGTGCCTTCATCTA
GCCTGGGTACCAAGACCTACACTTGCAACGTGGACCAC
AAGCCTTCCAACACTAAGGTGGACAAGCGCGTCGAATC
GAAGTACGGCCCACCGTGCCCGCCTTGTCCCGCGCCG
GAGTTCCTCGGCGGTCCCTCGGTCTTTCTGTTCCCACC
G AAG CCCAAGG ACACTTTG ATG ATTTCCCG CACCCCTG A
AGTGACATGCGTGGTCGTGGACGTGTCACAGGAAGATC
CGGAGGTGCAGTTCAATTGGTACGTGGATGGCGTCGAG
GTGC AC AACG CCAAAACC AAG CCG AG GG AG G AGCAGTT
CAACTCCACTTACCGCGTCGTGTCCGTGCTGACGGTGC
TG CATC AG G ACTG G CTG AACG G G AAG GAG TAC AAG TG C
AAAGTGTCCAACAAGGGACTTCCTAGCTCAATCGAAAAG
ACCATCTCGAAAGCCAAGGGACAGCCCCGGGAACCCCA
AGTGTATACCCTGCCACCGAGCCAGGAAGAAATGACTAA
GAACCAAGTCTCATTGACTTGCCTTGTGAAGGGCTTCTA
CCCATCGGATATCGCCGTGGAATGGGAGTCCAACGGCC
AGCCGGAAAACAACTACAAGACCACCCCTCCGGTGCTG
GACTCAGACGGATCCTTCTTCCTCTACTCGCGGCTGACC
GTGG ATAAG AG CAG ATG GCAG G AGG G AAATGTGTTCAG
CTGTTCTGTGATGCATGAAGCCCTGCACAACCACTACAC
TCAGAAGTCCCTGTCCCTCTCCCTGGGA
BAP049-Clone-B LC
SEQ ID NO: 510
¾ (Kabat) LCDR1 KSSQSLLDSGNQKNFLT
SEQ ID NO: 51 1
(Kabat) LCDR2 WASTRES
SEQ ID NO: 512
(Kabat) LCDR3 QNDYSYPYT
SEQ ID NO: 513
(Chothia) LCDR1 SQSLLDSGNQKNF
SEQ ID NO: 514
¾ (Chothia) LCDR2 WAS
SEQ ID NO: 515
(Chothia) LCDR3 DYSYPY
EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTW YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTIS
SEQ ID NO: 516 VL SLQPEDIATYYCQNDYSYPYTFGQGTKVEIK
GAGATCGTCCTGACTCAGTCACCCGCTACCCTGAGCCT
GAGCCCTGGCGAGCGGGCTACACTGAGCTGTAAATCTA
GTCAGTCACTGCTGGATAGCGGTAATCAGAAGAACTTCC
TGACCTGGTATCAGCAGAAGCCCGGTAAAGCCCCTAAG
CTGCTGATCTACTGGGCCTCTACTAGAGAATCAGGCGTG
DNA CCCTCTAGG I I I AGCGGTAGCGGTAGTGGCACCGACTT
SEQ ID NO: 517 VL CACCTTCACTATCTCTAGCCTGCAGCCCGAGGATATCGC TACCTACTACTGTCAGAACGACTATAGCTACCCCTACAC
CTTCG GTCAAG GCACTAAG GTCG AG ATTAAG
EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTW
YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTIS
SLQPEDIATYYCQNDYSYPYTFGQGTKVEIKRTVAAPSVFIF
PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSG
Light NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
SEQ ID NO: 518 chain HQGLSSPVTKSFNRGEC
GAGATCGTCCTGACTCAGTCACCCGCTACCCTGAGCCT
GAGCCCTGGCGAGCGGGCTACACTGAGCTGTAAATCTA
GTCAGTCACTGCTGGATAGCGGTAATCAGAAGAACTTCC
TGACCTGGTATCAGCAGAAGCCCGGTAAAGCCCCTAAG
CTGCTGATCTACTGGGCCTCTACTAGAGAATCAGGCGTG
CCCTCTAGG I I I AGCGGTAGCGGTAGTGGCACCGACTT
CACCTTCACTATCTCTAGCCTGCAGCCCGAGGATATCGC
TACCTACTACTGTCAGAACGACTATAGCTACCCCTACAC
CTTCGGTCAAGGCACTAAGGTCGAGATTAAGCGTACGG
TGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGAC
GAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCC
TGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAG
TGG AAG GTG G ACAACGCCCTG CAG AGCG GC AACAG CCA
GGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCT
ACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGAC
DNA TACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCA
light CCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACA
SEQ ID NO: 519 chain GGGGCGAGTGC
BAP049-Clone-E HC
SEQ ID NO: 501
(Kabat) HCDR1 TYWMH
SEQ ID NO: 502
(Kabat) HCDR2 NIYPGTGGSNFDEKFKN
SEQ ID NO: 503
(Kabat) HCDR3 WTTGTGAY
SEQ ID NO: 504
(Chothia) HCDR1 GYTFTTY
SEQ lb NO: 505
(Chothia) HCDR2 YPGTGG
SEQ ID NO: 503
(Chothia) HCDR3 WTTGTGAY
EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQ ATGQGLEWMGNIYPGTGGSNFDEKFKNRVTITADKSTSTA
SEQ ID NO: 506 VH YMELSSLRSEDTAVYYCTRWTTGTGAYWGQGTTVTVSS
GAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAAGAA
GCCCGGCGAGTCACTGAGAATTAGCTGTAAAGGTTCAG
GCTACACCTTCACTACCTACTGGATGCACTGGGTCCGCC
AGGCTACCGGTCAAGGCCTCGAGTGGATGGGTAATATC
TACCCCGGCACCGGCGGCTCTAACTTCGACGAGAAGTT
TAAGAATAGAGTGACTATCACCGCCGATAAGTCTACTAG
CACCGCCTATATGGAACTGTCTAGCCTGAGATCAGAGGA
CACCGCCGTCTACTACTGCACTAGGTGGACTACCGGCA
DNA CAGGCGCCTACTGGGGTCAAGGCACTACCGTGACCGTG
SEQ ID NO: 507 VH TCTAGC
EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQ
Heavy ATGQGLEWMGNIYPGTGGSNFDEKFKNRVTITADKSTSTA
SEQ ID NO: 508 chain YMELSSLRSEDTAVYYCTRWTTGTGAYWGQGTTVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYT
CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE TKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
F F LYS RLTVD KS RWQ EG N VF SCS VM H EALH N H YTQ KSLS L
SLG
GAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAAGAA
GCCCGGCGAGTCACTGAGAATTAGCTGTAAAGGTTCAG
GCTACACCTTCACTACCTACTGGATGCACTGGGTCCGCC
AGGCTACCGGTCAAGGCCTCGAGTGGATGGGTAATATC
TACCCCGGCACCGGCGGCTCTAACTTCGACGAGAAGTT
TAAGAATAGAGTGACTATCACCGCCGATAAGTCTACTAG
CACCGCCTATATGGAACTGTCTAGCCTGAGATCAGAGGA
CACCGCCGTCTACTACTGCACTAGGTGGACTACCGGCA
CAGGCGCCTACTGGGGTCAAGGCACTACCGTGACCGTG
TCTAGCGCTAGCACTAAGGGCCCGTCCGTGTTCCCCCT
GGCACCTTGTAGCCGGAGCACTAGCGAATCCACCGCTG
CCCTCGGCTGCCTGGTCAAGGATTACTTCCCGGAGCCC
GTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGG
AGTGCACACCTTCCCCGCTGTGCTGCAGAGCTCCGGGC
TGTACTCGCTGTCGTCGGTGGTCACGGTGCCTTCATCTA
GCCTGGGTACCAAGACCTACACTTGCAACGTGGACCAC
AAGCCTTCCAACACTAAGGTGGACAAGCGCGTCGAATC
GAAGTACGGCCCACCGTGCCCGCCTTGTCCCGCGCCG
GAGTTCCTCGGCGGTCCCTCGGTCTTTCTGTTCCCACC
G AAG CCCAAGG ACACTTTG ATG ATTTCCCG CACCCCTG A
AGTGACATGCGTGGTCGTGGACGTGTCACAGGAAGATC
CGGAGGTGCAGTTCAATTGGTACGTGGATGGCGTCGAG
GTGC AC AACG CCAAAACC AAG CCG AG GG AG G AGCAGTT
CAACTCCACTTACCGCGTCGTGTCCGTGCTGACGGTGC
TG CATC AG G ACTG G CTG AACG G G AAG GAG TAC AAG TG C
AAAGTGTCCAACAAGGGACTTCCTAGCTCAATCGAAAAG
ACCATCTCGAAAGCCAAGGGACAGCCCCGGGAACCCCA
AGTGTATACCCTGCCACCGAGCCAGGAAGAAATGACTAA
GAACCAAGTCTCATTGACTTGCCTTGTGAAGGGCTTCTA
CCCATCGGATATCGCCGTGGAATGGGAGTCCAACGGCC
AGCCGGAAAACAACTACAAGACCACCCCTCCGGTGCTG
GACTCAGACGGATCCTTCTTCCTCTACTCGCGGCTGACC
DNA GTGG ATAAG AG CAG ATG GCAG G AGG G AAATGTGTTCAG
heavy CTGTTCTGTGATGCATGAAGCCCTGCACAACCACTACAC
SEQ ID NO: 509 chain TCAGAAGTCCCTGTCCCTCTCCCTGGGA
BAP049-Clone-E LC
SEQ ID NO: 510
(Kabat) LCDR1 KSSQSLLDSGNQKNFLT
SEQ ID NO: 51 1
(Kabat) LCDR2 WASTRES
SEQ ID NO: 512
(Kabat) LCDR3 QNDYSYPYT
SEQ ID NO: 513
(Chothia) LCDR1 SQSLLDSGNQKNF
SEQ ID NO: 514
(Chothia) LCDR2 WAS SEQ ID NO: 515
(Chothia) LCDR3 DYSYPY
EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTW
YQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTDFTFTI
SEQ ID NO: 520 VL SSLEAEDAATYYCQNDYSYPYTFGQGTKVEIK
GAGATCGTCCTGACTCAGTCACCCGCTACCCTGAGCCT
GAGCCCTGGCGAGCGGGCTACACTGAGCTGTAAATCTA
GTCAGTCACTGCTGGATAGCGGTAATCAGAAGAACTTCC
TGACCTGGTATCAGCAGAAGCCCGGTCAAGCCCCTAGA
CTGCTGATCTACTGGGCCTCTACTAGAGAATCAGGCGTG
CCCTCTAGG I I I AGCGGTAGCGGTAGTGGCACCGACTT
CACCTTCACTATCTCTAGCCTGGAAGCCGAGGACGCCG
DNA CTACCTACTACTGTCAGAACGACTATAGCTACCCCTACA
SEQ ID NO: 521 VL CCTTCGGTCAAGGCACTAAGGTCGAGATTAAG
EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTW
YQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTDFTFTI
SSLEAEDAATYYCQNDYSYPYTFGQGTKVEIKRTVAAPSVF
IFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQS
Light GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
SEQ ID NO: 522 chain THQGLSSPVTKSFNRGEC
GAGATCGTCCTGACTCAGTCACCCGCTACCCTGAGCCT
GAGCCCTGGCGAGCGGGCTACACTGAGCTGTAAATCTA
GTCAGTCACTGCTGGATAGCGGTAATCAGAAGAACTTCC
TGACCTGGTATCAGCAGAAGCCCGGTCAAGCCCCTAGA
CTGCTGATCTACTGGGCCTCTACTAGAGAATCAGGCGTG
CCCTCTAGGTTT AGCGGTAGCGGTAGTGGCACCGACTT
CACCTTCACTATCTCTAGCCTGGAAGCCGAGGACGCCG
CTACCTACTACTGTCAGAACGACTATAGCTACCCCTACA
CCTTCGGTCAAGGCACTAAGGTCGAGATTAAGCGTACG
GTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGA
CGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGC
CTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCA
GTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGC
CAG G AG AGCGTCACCG AG CAGG ACAG CAAGG ACTCCAC
CTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCG
DNA ACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACC
light CACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAA
SEQ ID NO: 523 chain CAGGGGCGAGTGC
BAP049-Clone-B HC
SEQ ID NO: 524
(Kabat) HCDR1 ACCTACTG G ATG CAC
SEQ ID NO: 525 AATATCTACCCCGGCACCGGCGGCTCTAACTTCGACGA (Kabat) HCDR2 GAAGTTTAAGAAT
SEQ ID NO: 526
(Kabat) HCDR3 TGGACTACCGGCACAGGCGCCTAC
SEQ ID NO: 527
^ (Chothia) HCDR1 GGCTACACCTTCACTACCTAC
SEQ lb NO: 528
(Chothia) HCDR2 TACCCCGGCACCGGCGGC
SEQ ID NO: 526
(Chothia) HCDR3 TGGACTACCGGCACAGGCGCCTAC
BAP049-Clone-B LC
SEQ ID NO: 529 AAATCTAGTCAGTCACTGCTGGATAGCGGTAATCAGAAG (Kabat) LCDR1 AACTTCCTGACC
(Chothia) LCDR3 GACTATAGCTACCCCTAC
Other Exemplary PD-1 Inhibitors
selected fromln 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 OPDIVO®. Nivolumab is a fully human lgG4 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 PCT Publication No.
WO2006/121 168, 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 17. 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 lgG4 monoclonal antibody that binds to PD1 . Pembrolizumab is disclosed, e.g., in Hamid, O. et al. (2013) New England Journal of Medicine 369 (2): 134-44, PCT Publication No. WO2009/1 14335, 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 all 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 17.
In some embodiments, the anti-PD-1 antibody is Pidilizumab. 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.
WO2009/10161 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 all 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 17.
Other anti-PD1 antibodies are disclosed in US Patent No. 8,609,089, US Publication No. 2010028330, and/or US Publication No. 201201 14649, 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. MEDI0680 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 all 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 anti-PD-1 antibody molecule is BGB-A317 or BGB-108
(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 INCSHR1210 (Incyte), also known as INCSHR01210 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-042 (Tesaro), also known as ANB01 1 . 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-042.
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 2015/2001 19, 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 201 1/066342, incorporated by reference in their entirety).
Table 17. Amino acid sequences of other exemplary anti-PD-1 antibody molecules
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Pembrolizumab
QVQLVQ^
QGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSL QFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSV FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
Heavy NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL SEQ ID NO: 537 chain HNHYTQKSLSLSLGK
E VLfoSPA LSLSPGER
GQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAV YYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA
Light SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SEQ ID NO: 538 chain SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Pidilizumab
QVQLVQSGSELK ^
GLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLT
AEDTGMYFCVRVGYDALDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCW
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
Heavy PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
SEQ ID NO: 539 chain QKSLSLSPGK
LWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQ QRSSFPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWC
Light LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
SEQ ID NO: 540 chain TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
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), Atezolizumab (Genentech/Roche), Avelumab (Merck Serono and Pfizer), Durvalumab (Medlmmune/AstraZeneca), or BMS-936559 (Bristol-Myers Squibb).
Exemplary PD-L1 Inhibitors
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 18 (e.g., from the heavy and light chain variable region sequences of BAP058-Clone 0 or BAP058-Clone N disclosed in Table 18), or encoded by a nucleotide sequence shown in Table 18. In some embodiments, the CDRs are according to the Kabat definition (e.g. , as set out in Table 18). In some embodiments, the CDRs are according to the Chothia definition (e.g. , as set out in Table 18). In some embodiments, the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g. , as set out in Table 18). In one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GYTFTSYW Y (SEQ ID NO: 647). 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 18, or encoded by a nucleotide sequence shown in Table 18.
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: 601 , a VHCDR2 amino acid sequence of SEQ ID NO: 602, and a VHCDR3 amino acid sequence of SEQ ID NO: 603; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 609, a VLCDR2 amino acid sequence of SEQ ID NO: 610, and a VLCDR3 amino acid sequence of SEQ ID NO: 61 1 , each disclosed in Table 18.
In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 628, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 629, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 630; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 633, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 634, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 635, each disclosed in Table 18.
In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 606, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 606. In one embodiment, the anti-PD-L1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 616, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 616. In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 620, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 620. In one embodiment, the anti-PD-L1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 624, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 624. In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 606 and a VL comprising the amino acid sequence of SEQ ID NO: 616. In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 620 and a VL comprising the amino acid sequence of SEQ ID NO: 624.
In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 607, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 607. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 617, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 617. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 621 , or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 621 . In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 625, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 625. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 607 and a VL encoded by the nucleotide sequence of SEQ ID NO: 617. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 621 and a VL encoded by the nucleotide sequence of SEQ ID NO: 625.
In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 608, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 608. In one embodiment, the anti- PD-L1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 618, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 618. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 622, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 622. In one embodiment, the anti-PD-L1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 626, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 626. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 608 and a light chain comprising the amino acid sequence of SEQ ID NO: 618. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 622 and a light chain comprising the amino acid sequence of SEQ ID NO: 626.
In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 615, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 615. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 619, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 619. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 623, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 623. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 627, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 627. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 615 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 619. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 623 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 627.
The antibody molecules described herein can be made by vectors, host cells, and methods described in US 2016/0108123, incorporated by reference in its entirety.
Table 18. Amino acid and nucleotide sequences of exemplary anti-PD-L1 antibody molecules
BAP058-Clone 0 HC
SEQ ID NO: 601 HCDR1 SYWMY
(Kabat)
SEQ ID NO: 602 HCDR2 RIDPNSGSTKYNEKFKN
(Kabat)
SEQ ID NO: 603 HCDR3 DYRKGLYAMDY
(Kabat)
SEQ ID NO: 604 HCDR1 GYTFTSY
(Chothia)
SEQ ID NO: 605 HCDR2 DPNSGS
(Chothia)
SEQ ID NO: 603 HCDR3 DYRKGLYAMDY
(Chothia)
SEQ ID NO: 606 VH EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWV
RQARGQRLEWIGRIDPNSGSTKYNEKFKNRFTISRDNS KNTLYLQMNSLRAEDTAVYYCARDYRKGLYAMDYWG QGTTVTVSS
SEQ ID NO: 607 DNA VH GAAGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAA
GAAACCCGGCGCTACCGTGAAGATTAGCTGTAAAGT
CTC AG G CT AC AC CTTC ACTAG CT ACTG G ATG TACTG
GGTCCGACAGGCTAGAGGGCAAAGACTGGAGTGGA
TCGGTAGAATCGACCCTAATAGCGGCTCTACTAAGTA
TAACGAGAAGTTTAAGAATAGGTTCACTATTAGTAGG
GATAACTCTAAGAACACCCTGTACCTGCAGATGAATA
GCCTGAGAGCCGAGGACACCGCCGTCTACTACTGC
GCTAGAGACTATAGAAAGGGCCTGTACGCTATGGAC
TACTGGGGTCAAGGCACTACCGTGACCGTGTCTTCA
SEQ ID NO: 608 Heavy EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWV chain RQARGQRLEWIGRIDPNSGSTKYNEKFKNRFTISRDNS
KNTLYLQMNSLRAEDTAVYYCARDYRKGLYAMDYWG
QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
WTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP
CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCW
VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
YRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS
KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSV HEALHNHYTQKSLSLSLG
SEQ ID NO: 615 DNA GAAGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAA heavy GAAACCCGGCGCTACCGTGAAGATTAGCTGTAAAGT chain CTC AG G CT AC AC CTTC ACTAG CT ACTG G ATG TACTG
GGTCCGACAGGCTAGAGGGCAAAGACTGGAGTGGA
TCGGTAGAATCGACCCTAATAGCGGCTCTACTAAGTA
TAACGAGAAGTTTAAGAATAGGTTCACTATTAGTAGG
GATAACTCTAAGAACACCCTGTACCTGCAGATGAATA
GCCTGAGAGCCGAGGACACCGCCGTCTACTACTGC
GCTAGAGACTATAGAAAGGGCCTGTACGCTATGGAC
TACTGGGGTCAAGGCACTACCGTGACCGTGTCTTCA
GCTAGCACTAAGGGCCCGTCCGTGTTCCCCCTGGCA
CCTTGTAGCCGGAGCACTAGCGAATCCACCGCTGCC
CTCGGCTGCCTGGTCAAGGATTACTTCCCGGAGCCC
GTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTC
CGGAGTGCACACCTTCCCCGCTGTGCTGCAGAGCTC
CGGGCTGTACTCGCTGTCGTCGGTGGTCACGGTGC
CTTCATCTAGCCTGGGTACCAAGACCTACACTTGCAA
CGTGGACCACAAGCCTTCCAACACTAAGGTGGACAA
GCGCGTCGAATCGAAGTACGGCCCACCGTGCCCGC
CTTGTCCCGCGCCGGAGTTCCTCGGCGGTCCCTCG
GTCTTTCTGTTCCCACCGAAGCCCAAGGACACTTTGA
TGATTTCCCGCACCCCTGAAGTGACATGCGTGGTCG
TGGACGTGTCACAGGAAGATCCGGAGGTGCAGTTCA
ATTGGTACGTGGATGGCGTCGAGGTGCACAACGCCA
AAACCAAG CCG AGG G AGG AG CAGTTCAACTCCACTT
ACCGCGTCGTGTCCGTGCTGACGGTGCTGCATCAG
G ACTG G CTG AACG G G AAGG AGTACAAGTGCAAAGTG
TCCAACAAGGGACTTCCTAGCTCAATCGAAAAGACC
ATCTCGAAAGCCAAGGGACAGCCCCGGGAACCCCA
AGTGTATACCCTGCCACCGAGCCAGGAAGAAATGAC
TAAGAACCAAGTCTCATTGACTTGCCTTGTGAAGGGC
TTCTACCCATCGGATATCGCCGTGGAATGGGAGTCC
AACG GCC AG CCG G AAAACAACTACAAG ACCACCCCT
CCGGTGCTGGACTCAGACGGATCCTTCTTCCTCTAC
TCGCGGCTGACCGTGGATAAGAGCAGATGGCAGGA
GGGAAATGTGTTCAGCTGTTCTGTGATGCATGAAGC
CCTGCACAACCACTACACTCAGAAGTCCCTGTCCCT
CTCCCTGGGA
BAP058-Clone 0 LC
SEQ ID NO: 609 LCDR1 KASQDVGTAVA
(Kabat)
SEQ ID NO: 610 LCDR2 WASTRHT
(Kabat)
SEQ ID NO: LCDR3 QQYNSYPLT
61 1 (Kabat)
SEQ ID NO: 612 LCDR1 SQDVGTA
(Chothia) SEQ ID NO: 613 LCDR2 WAS
(Chothia)
SEQ ID NO: 614 LCDR3 YNSYPL
(Chothia)
SEQ ID NO: 616 VL AIQLTQSPSSLSASVGDRVTITCKASQDVGTAVAWYLQ
KPGQSPQLLIYWASTRHTGVPSRFSGSGSGTDFTFTIS SLEAEDAATYYCQQYNSYPLTFGQGTKVEIK
SEQ ID NO: 617 DNA VL GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAGC
G CTAG TG TG GG CG ATAG AG TG ACTATC AC CTGT AAA
GCCTCTCAGGACGTGGGCACCGCCGTGGCCTGGTA
TCTGCAGAAGCCTGGTCAATCACCTCAGCTGCTGAT
CTACTGGGCCTCTACTAGACACACCGGCGTGCCCTC
TAGGTTTAGCGGTAGCGGTAGTGGCACCGACTTCAC
CTTCACTATCTCTTCACTGGAAGCCGAGGACGCCGC
TACCTACTACTGTCAGCAGTATAATAGCTACCCCCTG
ACCTTC G G TC AAG G CACTAAG G TCG AG ATT AAG
SEQ ID NO: 618 Light AIQLTQSPSSLSASVGDRVTITCKASQDVGTAVAWYLQ chain KPGQSPQLLIYWASTRHTGVPSRFSGSGSGTDFTFTIS
SLEAEDAATYYCQQYNSYPLTFGQGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 619 DNA light GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAGC chain GCTAGTGTGGGCGATAGAGTGACTATCACCTGTAAA
GCCTCTCAGGACGTGGGCACCGCCGTGGCCTGGTA
TCTGCAGAAGCCTGGTCAATCACCTCAGCTGCTGAT
CTACTGGGCCTCTACTAGACACACCGGCGTGCCCTC
TAGGTTTAGCGGTAGCGGTAGTGGCACCGACTTCAC
CTTCACTATCTCTTCACTGGAAGCCGAGGACGCCGC
TACCTACTACTGTCAGCAGTATAATAGCTACCCCCTG
ACCTTCGGTCAAGGCACTAAGGTCGAGATTAAGCGT
ACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCC
AGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGT
GGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGC
CAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGA
GCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGAC
AGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTG
ACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTG
TACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAG
CCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC
BAP058-Clone N HC
SEQ ID NO: 601 HCDR1 SYWMY
(Kabat)
SEQ ID NO: 602 HCDR2 RIDPNSGSTKYNEKFKN
(Kabat)
SEQ ID NO: 603 HCDR3 DYRKGLYAMDY
(Kabat)
SEQ ID NO: 604 HCDR1 GYTFTSY
(Chothia)
SEQ ID NO: 605 HCDR2 DPNSGS
(Chothia)
SEQ ID NO: 603 HCDR3 DYRKGLYAMDY
(Chothia)
SEQ ID NO: 620 VH EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWV
RQATGQGLEWMGRIDPNSGSTKYNEKFKNRVTITADK STSTAY ELSSLRSEDTAWYCARDYRKG LYAMDYWG
QGTTVTVSS
SEQ ID NO: 621 DNA VH GAAGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAA
GAAACCCGGCGCTACCGTGAAGATTAGCTGTAAAGT
CTC AG G CT AC AC CTTC ACTAG CT ACTG G ATG TACTG
GGTCCGACAGGCTACCGGTCAAGGCCTGGAGTGGA
TGGGTAGAATCGACCCTAATAGCGGCTCTACTAAGT
ATAACGAGAAGTTTAAGAATAGAGTGACTATCACCGC
CGATAAGTCTACTAGCACCGCCTATATGGAACTGTCT
AGCCTGAGATCAGAGGACACCGCCGTCTACTACTGC
GCTAGAGACTATAGAAAGGGCCTGTACGCTATGGAC
TACTGGGGTCAAGGCACTACCGTGACCGTGTCTTCA
SEQ ID NO: 622 Heavy EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWV chain RQATGQGLEW GRIDPNSGSTKYNEKFKNRVTITADK
STSTAYM ELSSLRSEDTAWYCARDYRKG LYAMDYWG
QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
WTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP
CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCW
VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
YRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS
KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
SEQ ID NO: 623 DNA GAAGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAA heavy GAAACCCGGCGCTACCGTGAAGATTAGCTGTAAAGT chain CTC AG G CT AC AC CTTC ACTAG CT ACTG G ATG TACTG
GGTCCGACAGGCTACCGGTCAAGGCCTGGAGTGGA
TGGGTAGAATCGACCCTAATAGCGGCTCTACTAAGT
ATAACGAGAAGTTTAAGAATAGAGTGACTATCACCGC
CGATAAGTCTACTAGCACCGCCTATATGGAACTGTCT
AGCCTGAGATCAGAGGACACCGCCGTCTACTACTGC
GCTAGAGACTATAGAAAGGGCCTGTACGCTATGGAC
TACTGGGGTCAAGGCACTACCGTGACCGTGTCTTCA
GCTAGCACTAAGGGCCCGTCCGTGTTCCCCCTGGCA
CCTTGTAGCCGGAGCACTAGCGAATCCACCGCTGCC
CTCGGCTGCCTGGTCAAGGATTACTTCCCGGAGCCC
GTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTC
CGGAGTGCACACCTTCCCCGCTGTGCTGCAGAGCTC
CGGGCTGTACTCGCTGTCGTCGGTGGTCACGGTGC
CTTCATCTAGCCTGGGTACCAAGACCTACACTTGCAA
CGTGGACCACAAGCCTTCCAACACTAAGGTGGACAA
GCGCGTCGAATCGAAGTACGGCCCACCGTGCCCGC
CTTGTCCCGCGCCGGAGTTCCTCGGCGGTCCCTCG
GTCTTTCTGTTCCCACCGAAGCCCAAGGACACTTTGA
TGATTTCCCGCACCCCTGAAGTGACATGCGTGGTCG
TGGACGTGTCACAGGAAGATCCGGAGGTGCAGTTCA
ATTGGTACGTGGATGGCGTCGAGGTGCACAACGCCA
AAACCAAGCCGAGGGAGGAGCAGTTCAACTCCACTT
ACCGCGTCGTGTCCGTGCTGACGGTGCTGCATCAG
G ACTG G CTG AACG G G AAGG AGTACAAGTGCAAAGTG
TCCAACAAGGGACTTCCTAGCTCAATCGAAAAGACC
ATCTCGAAAGCCAAGGGACAGCCCCGGGAACCCCA
AGTGTATACCCTGCCACCGAGCCAGGAAGAAATGAC
TAAGAACCAAGTCTCATTGACTTGCCTTGTGAAGGGC
TTCTACCCATCGGATATCGCCGTGGAATGGGAGTCC AACG GCC AG CCG G AAAACAACTACAAG ACCACCCCT
CCGGTGCTGGACTCAGACGGATCCTTCTTCCTCTAC
TCGCGGCTGACCGTGGATAAGAGCAGATGGCAGGA
GGGAAATGTGTTCAGCTGTTCTGTGATGCATGAAGC
CCTGCACAACCACTACACTCAGAAGTCCCTGTCCCT
CTCCCTGGGA
BAP058-Clone N LC
SEQ ID NO: 609 LCDR1 KASQDVGTAVA
(Kabat)
SEQ ID NO: 610 LCDR2 WASTRHT
(Kabat)
SEQ ID NO: LCDR3 QQYNSYPLT
61 1 (Kabat)
SEQ ID NO: 612 LCDR1 SQDVGTA
(Chothia)
SEQ ID NO: 613 LCDR2 WAS
(Chothia)
SEQ ID NO: 614 LCDR3 YNSYPL
(Chothia)
SEQ ID NO: 624 VL DWMTQSPLSLPVTLGQPASISCKASQDVGTAVAWYQ
QKPGQAPRLLIYWASTRHTGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCQQYNSYPLTFGQGTKVEIK
SEQ ID NO: 625 DNA VL GACGTCGTGATGACTCAGTCACCCCTGAGCCTGCCC
GTGACCCTGGGGCAGCCCGCCTCTATTAGCTGTAAA
GCCTCTCAGGACGTGGGCACCGCCGTGGCCTGGTA
TCAGCAGAAGCCAGGGCAAGCCCCTAGACTGCTGAT
CTACTGGGCCTCTACTAGACACACCGGCGTGCCCTC
TAGGTTTAGCGGTAGCGGTAGTGGCACCGAGTTCAC
CCTGACTATCTCTTCACTGCAGCCCGACGACTTCGC
TACCTACTACTGTCAGCAGTATAATAGCTACCCCCTG
ACCTTC G G TC AAG G CACTAAG G TCG AG ATT AAG
SEQ ID NO: 626 Light DWMTQSPLSLPVTLGQPASISCKASQDVGTAVAWYQ chain QKPGQAPRLLIYWASTRHTGVPSRFSGSGSGTEFTLTI
SSLQPDDFATYYCQQYNSYPLTFGQGTKVEIKRTVAAP
SVF I F PPSDEQ LKSGTASWCLLN N F YPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
KVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 627 DNA light GACGTCGTGATGACTCAGTCACCCCTGAGCCTGCCC chain GTGACCCTGGGGCAGCCCGCCTCTATTAGCTGTAAA
GCCTCTCAGGACGTGGGCACCGCCGTGGCCTGGTA
TCAG CAG AAG CCAG GG CAAGCCCCTAG ACTG CTG AT
CTACTGGGCCTCTACTAGACACACCGGCGTGCCCTC
TAGGTTTAGCGGTAGCGGTAGTGGCACCGAGTTCAC
CCTGACTATCTCTTCACTGCAGCCCGACGACTTCGC
TACCTACTACTGTCAGCAGTATAATAGCTACCCCCTG
ACCTTCGGTCAAGGCACTAAGGTCGAGATTAAGCGT
ACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCC
AGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGT
GGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGC
CAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGA
GCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGAC
AGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTG
ACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTG
TACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAG
CCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC BAP058-Clone 0 HC
SEQ ID NO: 628 HCDR1 agctactggatgtac
(Kabat)
SEQ ID NO: 629 HCDR2 agaatcgaccctaatagcggctctactaagtataacgagaagtttaagaat (Kabat)
SEQ ID NO: 630 HCDR3 gactatagaaagggcctgtacgctatggactac
(Kabat)
SEQ ID NO: 631 HCDR1 ggctacaccttcactagctac
(Chothia)
SEQ ID NO: 632 HCDR2 gaccctaatagcggctct
(Chothia)
SEQ ID NO: 630 HCDR3 gactatagaaagggcctgtacgctatggactac
(Chothia)
BAP058-Clone O LC
SEQ ID NO: 633 LCDR1 aaagcctctcaggacgtgggcaccgccgtggcc
(Kabat)
SEQ ID NO: 634 LCDR2 tgggcctctactagacacacc
(Kabat)
SEQ ID NO: 635 LCDR3 cagcagtataatagctaccccctgacc
(Kabat)
SEQ ID NO: 636 LCDR1 tctcaggacgtgggcaccgcc
(Chothia)
SEQ ID NO: 637 LCDR2 tgggcctct
(Chothia)
SEQ ID NO: 638 LCDR3 tataatagctaccccctg
(Chothia)
BAP058-Clone N HC
SEQ ID NO: 628 HCDR1 agctactggatgtac
(Kabat)
SEQ ID NO: 629 HCDR2 agaatcgaccctaatagcggctctactaagtataacgagaagtttaagaat (Kabat)
SEQ ID NO: 630 HCDR3 gactatagaaagggcctgtacgctatggactac
(Kabat)
SEQ ID NO: 631 HCDR1 ggctacaccttcactagctac
(Chothia)
SEQ ID NO: 632 HCDR2 gaccctaatagcggctct
(Chothia)
SEQ ID NO: 630 HCDR3 gactatagaaagggcctgtacgctatggactac
(Chothia)
BAP058-Clone N LC
SEQ ID NO: 633 LCDR1 aaagcctctcaggacgtgggcaccgccgtggcc
(Kabat)
SEQ ID NO: 634 LCDR2 tgggcctctactagacacacc
(Kabat)
SEQ ID NO: 635 LCDR3 cagcagtataatagctaccccctgacc
(Kabat)
SEQ ID NO: 636 LCDR1 tctcaggacgtgggcaccgcc
(Chothia)
SEQ ID NO: 637 LCDR2 tgggcctct
(Chothia)
SEQ ID NO: 638 LCDR3 tataatagctaccccctg
(Chothia) Other Exemplary PD-L1 Inhibitors
In some embodiments, the PD-L1 inhibitor is anti-PD-L1 antibody. In some
embodiments, the anti-PD-L1 inhibitor is selected from YW243.55.S70, MPDL3280A, MEDI- 4736, or MDX-1 105MSB-0010718C (also referred to as A09-246-2) 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-1 105. MDX-1 105, also known as BMS- 936559, is an anti-PD-L1 antibody described in PCT 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 2010/077634.
In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech / Roche) also known as Atezolizumabm, RG7446, R05541267, YW243.55.S70, or TECENTRIQ™. MDPL3280A is a human Fc optimized lgG 1 monoclonal antibody that binds to PD-L1 . MDPL3280A 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 collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Atezolizumab, e.g., as disclosed in Table 19.
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-DCIg; 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 Avelumab (Merck Serono and Pfizer), also known as MSB0010718C. Avelumab 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 all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Avelumab, e.g., as disclosed in Table 19.
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 19. In one embodiment, the anti-PD-L1 antibody molecule is BMS-936559 (Bristol-Myers Squibb), also known as MDX-1 105 or 12A4. BMS-936559 and other anti-PD-L1 antibodies are disclosed in US 7,943,743 and WO 2015/081 158, 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 19.
Further known anti-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 19. Amino acid sequences of other exemplary anti-PD-L1 antibody molecules
SYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLV
CLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLS
LTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
Durvalumab
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKG
LEVWANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAED
TAVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK
THTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD
WLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEM
SEQ ID NO: Heavy TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF 643 chain FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAP
RLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY
GSLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN
SEQ ID NO: Light NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS 644 chain KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
BMS-936559
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGL
SEQ ID NO: EWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTA 645 VH VYFCARKFHFVSGSPFGMDVWGQGTTVTVSS
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL
SEQ ID NO: LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSN 646 VL WPTFGQGTKVEIK
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-986016 (Bristol-Myers Squibb), or TSR-033 (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 20 (e.g., from the heavy and light chain variable region sequences of BAP050-Clone I or BAP050-Clone J disclosed in Table 20), or encoded by a nucleotide sequence shown in Table 20. In some embodiments, the CDRs are according to the Kabat definition (e.g. , as set out in Table 20). In some embodiments, the CDRs are according to the Chothia definition (e.g. , as set out in Table 20). In some embodiments, the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g. , as set out in Table 20). In one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GFTLTNYGMN (SEQ ID NO: 766). 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 20, or encoded by a nucleotide sequence shown in Table 20.
In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 701 , a VHCDR2 amino acid sequence of SEQ ID NO: 702, and a VHCDR3 amino acid sequence of SEQ ID NO: 703; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 710, a VLCDR2 amino acid sequence of SEQ ID NO: 71 1 , and a VLCDR3 amino acid sequence of SEQ ID NO: 712, each disclosed in Table 20.
In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 736 or 737, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 738 or 739, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 740 or 741 ; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 746 or 747, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 748 or 749, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 750 or 751 , each disclosed in Table 20. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 758 or 737, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 759 or 739, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 760 or 741 ; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 746 or 747, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 748 or 749, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 750 or 751 , each disclosed in Table 20.
In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 706, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 706. In one embodiment, the anti-LAG-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 718, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 718. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 724, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 724. In one embodiment, the anti-LAG-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 730, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 730. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 706 and a VL comprising the amino acid sequence of SEQ ID NO: 718. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 724 and a VL comprising the amino acid sequence of SEQ ID NO: 730.
In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 707 or 708, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 707 or 708. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 719 or 720, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 719 or 720. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 725 or 726, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 725 or 726. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 731 or 732, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 731 or 732. In one
embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 707 or 708 and a VL encoded by the nucleotide sequence of SEQ ID NO: 719 or 720. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 725 or 726 and a VL encoded by the nucleotide sequence of SEQ ID NO: 731 or 732.
In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 709, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 709. In one embodiment, the anti- LAG-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 721 , or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 721 . In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 727, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 727. In one embodiment, the anti-LAG-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 733, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 733. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 709 and a light chain comprising the amino acid sequence of SEQ ID NO: 721. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 727 and a light chain comprising the amino acid sequence of SEQ ID NO: 733.
In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 716 or 717, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 716 or 717. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 722 or 723, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 722 or 723. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 728 or 729, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 728 or 729. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 734 or 735, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 734 or 735. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 716 or 717 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 722 or 723. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 728 or 729 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 734 or 735.
The antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0259420, incorporated by reference in its entirety. Table 20. Amino acid and nucleotide sequences of exemplary anti-LAG-3 antibody molecules
BAP050-Clone 1 HC
SEQ ID NO: 701
(Kabat) HCDR1 NYGMN
SEQ ID NO: 702
¾ (Kabat) HCDR2 WINTDTGEPTYADDFKG
SEQ ID NO: 703
(Kabat) HCDR3 NPPYYYGTNNAEAMDY
SEQ ID NO: 704
(Chothia) HCDR1 GFTLTNY
SEQ ID NO: 705
(Chothia) HCDR2 NTDTGE
SEQ ID NO: 703
¾ (Chothia) HCDR3 NPPYYYGTNNAEAMDY
QVQLVQSGAEVKKPGASVKVSCKASGFTLTNYGMNWVRQ j ARGQRLEWIGWINTDTGEPTYADDFKGRFVFSLDTSVSTA i YLQISSLKAEDTAVYYCARNPPYYYGTNNAEAMDYWGQG !
SEQ ID NO:706 VH TTVTVSS
CAAGTGCAGCTGGTGCAGTCGGGAGCCGAAGTGAAGAA j GCCTGGAGCCTCGGTGAAGGTGTCGTGCAAGGCATCCG I GATTCACCCTCACCAATTACGGGATGAACTGGGTCAGAC ! AGGCCCGGGGTCAACGGCTGGAGTGGATCGGATGGATT j AACACCGACACCGGGGAGCCTACCTACGCGGACGATTT j CAAGGGACGGTTCGTGTTCTCCCTCGACACCTCCGTGT j CCACCGCCTACCTCCAAATCTCCTCACTGAAAGCGGAG i GACACCGCCGTGTACTATTGCGCGAGGAACCCGCCCTA ! CTACTACGGAACCAACAACGCCGAAGCCATGGACTACT j
SEQ ID NO: 707 DNA VH GGGGCCAGGGCACCACTGTGACTGTGTCCAGC
CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAA I ACCTGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCTG j GCTTCACCCTGACCAACTACGGCATGAACTGGGTGCGA j
SEQ ID NO: 708 DNA VH CAGGCCAGGGGCCAGCGGCTGGAATGGATCGGCTGGA j TCAACACCGACACCGGCGAGCCTACCTACGCCGACGAC
TTCAAGGGCAGATTCGTGTTCTCCCTGGACACCTCCGTG
TCCACCGCCTACCTGCAGATCTCCAGCCTGAAGGCCGA
GGATACCGCCGTGTACTACTGCGCCCGGAACCCCCCTT
ACTACTACGGCACCAACAACGCCGAGGCCATGGACTAT
TGGGGCCAGGGCACCACCGTGACCGTGTCCTCT
QVQLVQSGAEVKKPGASVKVSCKASGFTLTNYGMNWVRQ
ARGQRLEWIGWINTDTGEPTYADDFKGRFVFSLDTSVSTA
YLQISSLKAEDTAVYYCARNPPYYYGTNNAEA DYWGQG
TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS
SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF
LGGPSVFLFPPKPKDTL ISRTPEVTCWVDVSQEDPEVQF
NWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWL
NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
Heavy PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN
SEQ ID NO: 709 chain HYTQKSLSLSLG
CAAGTGCAGCTGGTGCAGTCGGGAGCCGAAGTGAAGAA
GCCTGGAGCCTCGGTGAAGGTGTCGTGCAAGGCATCCG
GATTCACCCTCACCAATTACGGGATGAACTGGGTCAGAC
AGGCCCGGGGTCAACGGCTGGAGTGGATCGGATGGATT
AACACCGACACCGGGGAGCCTACCTACGCGGACGATTT
CAAGGGACGGTTCGTGTTCTCCCTCGACACCTCCGTGT
CCACCGCCTACCTCCAAATCTCCTCACTGAAAGCGGAG
GACACCGCCGTGTACTATTGCGCGAGGAACCCGCCCTA
CTACTACGGAACCAACAACGCCGAAGCCATGGACTACT
GGGGCCAGGGCACCACTGTGACTGTGTCCAGCGCGTC
CACTAAGGGCCCGTCCGTGTTCCCCCTGGCACCTTGTA
GCCGGAGCACTAGCGAATCCACCGCTGCCCTCGGCTGC
CTGGTCAAGGATTACTTCCCGGAGCCCGTGACCGTGTC
CTGGAACAGCGGAGCCCTGACCTCCGGAGTGCACACCT
TCCCCGCTGTGCTGCAGAGCTCCGGGCTGTACTCGCTG
TCGTCGGTGGTCACGGTGCCTTCATCTAGCCTGGGTAC
CAAGACCTACACTTGCAACGTGGACCACAAGCCTTCCAA
CACTAAGGTGGACAAGCGCGTCGAATCGAAGTACGGCC
CACCGTGCCCGCCTTGTCCCGCGCCGGAGTTCCTCGGC
GGTCCCTCGGTCTTTCTGTTCCCACCGAAGCCCAAGGA
CACTTTGATGATTTCCCGCACCCCTGAAGTGACATGCGT
GGTCGTGGACGTGTCACAGGAAGATCCGGAGGTGCAGT
TCAATTGGTACGTGGATGGCGTCGAGGTGCACAACGCC
AAAACCAAGCCGAGGGAGGAGCAGTTCAACTCCACTTA
CCGCGTCGTGTCCGTGCTGACGGTGCTGCATCAGGACT
GGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAAC
AAGG G ACTTCCTAG CTC AATCG AAAAG ACCATCTCG AAA
GCCAAGGGACAGCCCCGGGAACCCCAAGTGTATACCCT
GCCACCGAGCCAGGAAGAAATGACTAAGAACCAAGTCT
CATTGACTTGCCTTGTGAAGGGCTTCTACCCATCGGATA
TCGCCGTGGAATGGGAGTCCAACGGCCAGCCGGAAAAC
AACTACAAG ACCACCCCTCCG GTGCTGG ACTCAG ACG G
ATCCTTCTTCCTCTACTCGCGGCTGACCGTGGATAAGAG
DNA CAGATGGCAGGAGGGAAATGTGTTCAGCTGTTCTGTGAT
heavy GCATGAAGCCCTGCACAACCACTACACTCAGAAGTCCCT
SEQ ID NO: 716 chain GTCCCTCTCCCTGGGA
DNA CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAG SEQ ID NO: 717 heavy ACCTGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCTG chain GCTTCACCCTGACCAACTACGGCATGAACTGGGTGCGA
CAGGCCAGGGGCCAGCGGCTGGAATGGATCGGCTGGA
TCAACACCGACACCGGCGAGCCTACCTACGCCGACGAC
TTCAAGGGCAGATTCGTGTTCTCCCTGGACACCTCCGTG
TCCACCGCCTACCTGCAGATCTCCAGCCTGAAGGCCGA
GGATACCGCCGTGTACTACTGCGCCCGGAACCCCCCTT
ACTACTACGGCACCAACAACGCCGAGGCCATGGACTAT
TGGGGCCAGGGCACCACCGTGACCGTGTCCTCTGCTTC
TACCAAGGGGCCCAGCGTGTTCCCCCTGGCCCCCTGCT
CCAGAAGCACCAGCGAGAGCACAGCCGCCCTGGGCTG
CCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGT
CCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACAC
CTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCC
TGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGG
CACCAAGACCTACACCTGTAACGTGGACCACAAGCCCA
GCAACACCAAGGTGGACAAGAGGGTGGAGAGCAAGTAC
GGCCCACCCTGCCCCCCCTGCCCAGCCCCCGAGTTCCT
GGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCA
AGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACC
TGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGT
CCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA
ACGCCAAGACCAAGCCCAGAGAGGAGCAGTTTAACAGC
ACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCA
G G ACTG G CTG AACG G C AAAG AG TAC AAG TG TAAG GTCT
CCAACAAGGGCCTGCCAAGCAGCATCGAAAAGACCATC
AGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGGTCTA
CACCCTGCCACCCAGCCAAGAGGAGATGACCAAGAACC
AGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCA
AGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGC
CCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGAC
AGCG ACG GCAG CTTCTTCCTGTACAG CAGG CTG ACCGT
G G AC AAGTCC AG ATG G C AG G AG G G C AACG TCTTTAG CT
GCTCCGTGATGCACGAGGCCCTGCACAACCACTACACC
CAGAAGAGCCTGAGCCTGTCCCTGGGC
BAP050-Clone 1 LC
SEQ ID NO: 710
(Kabat) LCDR1 SSSQDISNYLN
SEQ ID NO: 71 1
¾ (Kabat) LCDR2 YTSTLHL
SEQ ID NO: 712
(Kabat) LCDR3 QQYYNLPWT
SEQ ID NO: 713
(Chothia) LCDR1 SQDISNY
SEQ ID NO: 714
(Chothia) LCDR2 YTS
SEQ ID NO: 715
(Chothia) LCDR3 YYNLPW
DIQ TQSPSSLSASVGDRVTITCSSSQDISNYLNWYLQKPG QSPQLLIYYTSTLHLGVPSRFSGSGSGTEFTLTISSLQPDDF
SEQ ID NO: 718 VL ATYYCQQYYNLPWTFGQGTKVEIK
GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCT AGTGTGGGCGATAGAGTGACTATCACCTGTAGCTCTAGT CAGG ATATCTCTAACTACCTGAACTG GTATCTG CAG AAG CCCGGTCAATCACCTCAGCTGCTGATCTACTACACTAGC
SEQ ID NO: 719 DNA VL ACCCTGCACCTGGGCGTGCCCTCTAGGTTTAGCGGTAG CGGTAGTGGCACCGAGTTCACCCTGACTATCTCTAGCCT
GCAGCCCGACGACTTCGCTACCTACTACTGTCAGCAGTA CTATAACCTGCCCTGGACCTTCGGTCAAGGCACTAAGGT CGAGATTAAG
GACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGC
TTCCGTGGGCGACAGAGTGACCATCACCTGTTCCTCCA
GCCAGGACATCTCCAACTACCTGAACTGGTATCTGCAGA
AGCCCGGCCAGTCCCCTCAGCTGCTGATCTACTACACC
TCCACCCTGCACCTGGGCGTGCCCTCCAGATTTTCCGG
CTCTG GCTCTG GCACCG AGTTTACCCTG ACCATCAG CTC
CCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGC
AGTACTACAACCTGCCCTGGACCTTCGGCCAGGGCACC
SEQ ID NO: 720 DNA VL AAGGTGGAAATCAAG
DIQ TQSPSSLSASVGDRVTITCSSSQDISNYLNWYLQKPG
QSPQLLIYYTSTLHLGVPSRFSGSGSGTEFTLTISSLQPDDF
ATYYCQQYYNLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESV
Light TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
SEQ ID NO: 721 chain PVTKSFNRGEC
GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCT
AGTGTGGGCGATAGAGTGACTATCACCTGTAGCTCTAGT
CAGG ATATCTCTAACTACCTGAACTG GTATCTG CAG AAG
CCCGGTCAATCACCTCAGCTGCTGATCTACTACACTAGC
ACCCTGCACCTGGGCGTGCCCTCTAGGTTTAGCGGTAG
CGGTAGTGGCACCGAGTTCACCCTGACTATCTCTAGCCT
GCAGCCCGACGACTTCGCTACCTACTACTGTCAGCAGTA
CTATAACCTGCCCTGGACCTTCGGTCAAGGCACTAAGGT
CGAGATTAAGCGTACGGTGGCCGCTCCCAGCGTGTTCA
TCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACC
GCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCG
GGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTG
CAG AG CG GCAACAGCCAG G AG AG CGTCACCG AG CAGG
ACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTG
DNA ACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTA
light CGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCC
SEQ ID NO: 722 chain GTGACCAAGAGCTTCAACAGGGGCGAGTGC
GACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGC
TTCCGTGGGCGACAGAGTGACCATCACCTGTTCCTCCA
GCCAGGACATCTCCAACTACCTGAACTGGTATCTGCAGA
AGCCCGGCCAGTCCCCTCAGCTGCTGATCTACTACACC
TCCACCCTGCACCTGGGCGTGCCCTCCAGATTTTCCGG
CTCTG GCTCTG GCACCG AGTTTACCCTG ACCATCAG CTC
CCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGC
AGTACTACAACCTGCCCTGGACCTTCGGCCAGGGCACC
AAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGT
GTTCATCTTCCCCCCAAGCGACGAGCAGCTGAAGAGCG
GCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTAC
CCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACG
CCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGA
GCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCA
DNA CCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAG
light GTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAG
SEQ ID NO: 723 chain CCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC
B AP050-Clo ne J HC
SEQ ID NO: 701 HCDR1 NYGMN (Kabat)
SEQ ID NO: 702
(Kabat) HCDR2 WINTDTGEPTYADDFKG
SEQ ID NO: 703
v (Kabat) HCDR3 NPPYYYGTNNAEAMDY
SEQ ID NO: 704
(Chothia) HCDR1 GFTLTNY
SEQ ID NO: 705
(Chothia) HCDR2 NTDTGE
SEQ ID NO: 703
(Chothia) HCDR3 NPPYYYGTNNAEAMDY
QVQLVQSGAEVKKPGASVKVSCKASGFTLTNYGMNVWRQ APGQGLEWMGWINTDTGEPTYADDFKGRFVFSLDTSVST AYLQ I SSLKAEDTAVYYCARN PPYYYGTN N AEAM DYWG Q
SEQ ID NO: 724 VH GTTVTVSS
CAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAAGAA
ACCCGGCGCTAGTGTGAAAGTCAGCTGTAAAGCTAGTG
GCTTCACCCTGACTAACTACGGGATGAACTGGGTCCGC
CAGGCCCCAGGTCAAGGCCTCGAGTGGATGGGCTGGAT
TAACACCGACACCGGCGAGCCTACCTACGCCGACGACT
TTAAGGGCAGATTCGTGTTTAGCCTGGACACTAGTGTGT
CTACCGCCTACCTGCAGATCTCTAGCCTGAAGGCCGAG
GACACCGCCGTCTACTACTGCGCTAGAAACCCCCCCTA
CTACTACGGCACTAACAACGCCGAGGCTATGGACTACT
SEQ ID NO: 725 DNA VH GGGGTCAAGGCACTACCGTGACCGTGTCTAGC
CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAA
ACCTGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCTG
GCTTCACCCTGACCAACTACGGCATGAACTGGGTGCGA
CAGGCCCCTGGACAGGGCCTGGAATGGATGGGCTGGA
TCAACACCGACACCGGCGAGCCTACCTACGCCGACGAC
TTCAAGGGCAGATTCGTGTTCTCCCTGGACACCTCCGTG
TCCACCGCCTACCTGCAGATCTCCAGCCTGAAGGCCGA
GGATACCGCCGTGTACTACTGCGCCCGGAACCCCCCTT
ACTACTACGGCACCAACAACGCCGAGGCCATGGACTAT
SEQ ID NO: 726 DNA VH TGGGGCCAGGGCACCACCGTGACCGTGTCCTCT
QVQLVQSGAEVKKPGASVKVSCKASGFTLTNYGMNWVRQ
APGQGLEWMGWINTDTGEPTYADDFKGRFVFSLDTSVST
AYLQ I SSLKAEDTAVYYCARN PPYYYGTN N AEAM DYWG Q
GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS
SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE
FLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDW
LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS
QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
Heavy PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH
SEQ ID NO: 727 chain NHYTQKSLSLSLG
CAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAAGAA
ACCCGGCGCTAGTGTGAAAGTCAGCTGTAAAGCTAGTG
GCTTCACCCTGACTAACTACGGGATGAACTGGGTCCGC
CAGGCCCCAGGTCAAGGCCTCGAGTGGATGGGCTGGAT
TAACACCGACACCGGCGAGCCTACCTACGCCGACGACT
DNA TTAAGGGCAGATTCGTGTTTAGCCTGGACACTAGTGTGT
heavy CTACCGCCTACCTGCAGATCTCTAGCCTGAAGGCCGAG
SEQ ID NO: 728 chain GACACCGCCGTCTACTACTGCGCTAGAAACCCCCCCTA CTACTACGGCACTAACAACGCCGAGGCTATGGACTACT
GGGGTCAAGGCACTACCGTGACCGTGTCTAGCGCTAGC
ACTAAGGGCCCGTCCGTGTTCCCCCTGGCACCTTGTAG
CCGGAGCACTAGCGAATCCACCGCTGCCCTCGGCTGCC
TGGTCAAGGATTACTTCCCGGAGCCCGTGACCGTGTCC
TGGAACAGCGGAGCCCTGACCTCCGGAGTGCACACCTT
CCCCGCTGTGCTGCAGAGCTCCGGGCTGTACTCGCTGT
CGTCGGTGGTCACGGTGCCTTCATCTAGCCTGGGTACC
AAGACCTACACTTGCAACGTGGACCACAAGCCTTCCAAC
ACTAAGGTGGACAAGCGCGTCGAATCGAAGTACGGCCC
ACCGTGCCCGCCTTGTCCCGCGCCGGAGTTCCTCGGCG
GTCCCTCGGTCTTTCTGTTCCCACCGAAGCCCAAGGACA
CTTTGATGATTTCCCGCACCCCTGAAGTGACATGCGTGG
TCGTGGACGTGTCACAGGAAGATCCGGAGGTGCAGTTC
AATTGGTACGTGGATGGCGTCGAGGTGCACAACGCCAA
AACCAAGCCGAGGGAGGAGCAGTTCAACTCCACTTACC
GCGTCGTGTCCGTGCTGACGGTGCTGCATCAGGACTGG
CTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAA
GGGACTTCCTAGCTCAATCGAAAAGACCATCTCGAAAGC
CAAGGGACAGCCCCGGGAACCCCAAGTGTATACCCTGC
CACCGAGCCAGGAAGAAATGACTAAGAACCAAGTCTCAT
TGACTTGCCTTGTGAAGGGCTTCTACCCATCGGATATCG
CCGTGGAATGGGAGTCCAACGGCCAGCCGGAAAACAAC
TACAAGACCACCCCTCCGGTGCTGGACTCAGACGGATC
CTTCTTCCTCTACTCGCGGCTGACCGTGGATAAGAGCAG
ATGGCAGGAGGGAAATGTGTTCAGCTGTTCTGTGATGCA
TGAAGCCCTGCACAACCACTACACTCAGAAGTCCCTGTC
CCTCTCCCTGGGA
CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAA
ACCTGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCTG
GCTTCACCCTGACCAACTACGGCATGAACTGGGTGCGA
CAGGCCCCTGGACAGGGCCTGGAATGGATGGGCTGGA
TCAACACCGACACCGGCGAGCCTACCTACGCCGACGAC
TTCAAGGGCAGATTCGTGTTCTCCCTGGACACCTCCGTG
TCCACCGCCTACCTGCAGATCTCCAGCCTGAAGGCCGA
GGATACCGCCGTGTACTACTGCGCCCGGAACCCCCCTT
ACTACTACGGCACCAACAACGCCGAGGCCATGGACTAT
TGGGGCCAGGGCACCACCGTGACCGTGTCCTCTGCTTC
TACCAAGGGGCCCAGCGTGTTCCCCCTGGCCCCCTGCT
CCAGAAGCACCAGCGAGAGCACAGCCGCCCTGGGCTG
CCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGT
CCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACAC
CTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCC
TGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGG
CACCAAGACCTACACCTGTAACGTGGACCACAAGCCCA
GCAACACCAAGGTGGACAAGAGGGTGGAGAGCAAGTAC
GGCCCACCCTGCCCCCCCTGCCCAGCCCCCGAGTTCCT
GGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCA
AGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACC
TGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGT
CCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA
ACGCCAAGACCAAGCCCAGAGAGGAGCAGTTTAACAGC
ACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCA
DNA G G ACTG G CTG AACG G C AAAG AG TAC AAG TG TAAG GTCT
heavy CCAACAAGGGCCTGCCAAGCAGCATCGAAAAGACCATC
SEQ ID NO: 729 chain AGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGGTCTA CACCCTGCCACCCAGCCAAGAGGAGATGACCAAGAACC
AGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCA
AGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGC
CCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGAC
AGCGACGGCAGCTTCTTCCTGTACAGCAGGCTGACCGT
G G AC AAGTCC AG ATG G C AG G AG G G C AACG TCTTTAG CT
GCTCCGTGATGCACGAGGCCCTGCACAACCACTACACC
CAGAAGAGCCTGAGCCTGTCCCTGGGC
BAP050-Clone J LC
SEQ ID NO: 710
(Kabat) LCDR1 SSSQDISNYLN
SEQ ID NO: 71 1
v (Kabat) LCDR2 YTSTLHL
SEQ ID NO: 712
(Kabat) LCDR3 QQYYNLPWT
SEQ ID NO: 713
(Chothia) LCDR1 SQDISNY
SEQ ID NO: 714
(Chothia) LCDR2 YTS
SEQ ID NO: 715
¾ (Chothia) LCDR3 YYNLPW
DIQ TQSPSSLSASVGDRVTITCSSSQDISNYLNWYQQKP GKAPKLLIYYTSTLHLGIPPRFSGSGYGTDFTLTINNIESEDA
SEQ ID NO: 730 VL AYYFCQQ YYN LPWTFG QGTKVE I K
GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCT AGTGTGGGCGATAGAGTGACTATCACCTGTAGCTCTAGT CAGG ATATCTCTAACTACCTG AACTG GTATCAG CAG AAG CCCGGTAAAG CCCCTAAG CTG CTG ATCTACTACACTAG C ACCCTGCACCTGGGAATCCCCCCTAGGTTTAGCGGTAG CGGCTACGGCACCGACTTCACCCTGACTATTAACAATAT CGAGTCAGAGGACGCCGCCTACTACTTCTGTCAGCAGT ACTATAACCTGCCCTGGACCTTCGGTCAAGGCACTAAGG
SEQ ID NO: 731 DNA VL TC GAG ATT AAG
GACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGC
TTCCGTGGGCGACAGAGTGACCATCACCTGTTCCTCCA
GCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGA
AGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACC
TCCACCCTGCACCTGGGCATCCCCCCTAGATTCTCCGG
CTCTGGCTACGGCACCGACTTCACCCTGACCATCAACAA
CATCGAGTCCGAGGACGCCGCCTACTACTTCTGCCAGC
AGTACTACAACCTGCCCTGGACCTTCGGCCAGGGCACC
SEQ ID NO: 732 DNA VL AAGGTGGAAATCAAG
DIQ TQSPSSLSASVGDRVTITCSSSQDISNYLNWYQQKP
GKAPKLLIYYTSTLHLGIPPRFSGSGYGTDFTLTINNIESEDA
AYYFCQQYYNLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESV
Light TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
SEQ ID NO: 733 chain PVTKSFNRGEC
GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCT
AGTGTGGGCGATAGAGTGACTATCACCTGTAGCTCTAGT
CAGG ATATCTCTAACTACCTG AACTG GTATCAG CAG AAG
CCCGGTAAAG CCCCTAAG CTG CTG ATCTACTACACTAG C
DNA ACCCTGCACCTGGGAATCCCCCCTAGGTTTAGCGGTAG
light CGGCTACGGCACCGACTTCACCCTGACTATTAACAATAT
SEQ ID NO: 734 chain CGAGTCAGAGGACGCCGCCTACTACTTCTGTCAGCAGT ACTATAACCTGCCCTGGACCTTCGGTCAAGGCACTAAGG
TCGAGATTAAGCGTACGGTGGCCGCTCCCAGCGTGTTC
ATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCAC
CGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCC
GGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCT
GCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAG
GACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCT
GACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGT
ACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCC
CGTGACCAAGAGCTTCAACAGGGGCGAGTGC
GACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGC
TTCCGTGGGCGACAGAGTGACCATCACCTGTTCCTCCA
GCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGA
AGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACC
TCCACCCTGCACCTGGGCATCCCCCCTAGATTCTCCGG
CTCTGGCTACGGCACCGACTTCACCCTGACCATCAACAA
CATCGAGTCCGAGGACGCCGCCTACTACTTCTGCCAGC
AGTACTACAACCTGCCCTGGACCTTCGGCCAGGGCACC
AAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGT
GTTCATCTTCCCCCCAAGCGACGAGCAGCTGAAGAGCG
GCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTAC
CCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACG
CCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGA
GCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCA
DNA CCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAG
light GTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAG
SEQ ID NO: 735 chain CCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC
BAP050-Clone 1 HC
SEQ ID NO: 736
(Kabat) HCDR1 AATTACGGGATGAAC
SEQ ID NO: 737
¾ (Kabat) HCDR1 AACTACGGCATGAAC
SEQ ID NO: 738 TGGATTAACACCGACACCGGGGAGCCTACCTACGCGGA (Kabat) HCDR2 CGATTTCAAGGGA
SEQ ID NO: 739 TGGATCAACACCGACACCGGCGAGCCTACCTACGCCGA (Kabat) HCDR2 CGACTTCAAGGGC
SEQ ID NO: 740 AACCCGCCCTACTACTACGGAACCAACAACGCCGAAGC (Kabat) HCDR3 CATGGACTAC
SEQ ID NO: 741 AACCCCCCTTACTACTACG G CACCAACAACG CCG AGG C ¾ (Kabat) HCDR3 CATGGACTAT
SEQ ID NO: 742
(Chothia) HCDR1 GGATTCACCCTCACCAATTAC
SEQ ID NO: 743
(Chothia) HCDR1 GGCTTCACCCTGACCAACTAC
SEQ ID NO: 744
(Chothia) HCDR2 AACACCGACACCGGGGAG
SEQ ID NO: 745
(Chothia) HCDR2 AACACCGACACCGGCGAG
SEQ ID NO: 740 AACCCGCCCTACTACTACGGAACCAACAACGCCGAAGC v (Chothia) HCDR3 CATGGACTAC
SEQ ID NO: 741 AACCCCCCTTACTACTACG G CACCAACAACG CCG AGG C (Chothia) HCDR3 CATGGACTAT
BAP050-Clone 1 LC
SEQ ID NO: 746 LCDR1 AGCTCTAGTCAGGATATCTCTAACTACCTGAAC
1 SEQ ID NO: 748
j (Kabat) LCDR2 TACACTAGCACCCTGCACCTG
! SEQ ID NO: 749
! (Kabat) LCDR2 TACACCTCCACCCTGCACCTG
! SEQ ID NO: 750
i (Kabat) LCDR3 CAGCAGTACTATAACCTGCCCTGGACC
! SEQ ID NO: 751
I (Kabat) LCDR3 CAGCAGTACTACAACCTGCCCTGGACC
! SEQ ID NO: 752
i (Chothia) LCDR1 AGTCAGGATATCTCTAACTAC
! SEQ ID NO: 753
! (Chothia) LCDR1 AGCCAGGACATCTCCAACTAC
! SEQ ID NO: 754
[ (Chothia) LCDR2 TACACTAGC
! SEQ ID NO: 755
i (Chothia) LCDR2 TACACCTCC
i SEQ ID NO: 756
j (Chothia) LCDR3 TACTATAACCTG CCCTG G
i SEQ ID NO: 757
j (Chothia) LCDR3 TACTACAACCTGCCCTGG
Other 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 BMS-986016 (Bristol-Myers Squibb), also known as BMS986016. BMS-986016 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 all 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 1 1 .
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 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-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 all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of IMP731 , e.g., as disclosed in Table 1 1 . 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 2015/1 16539, 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 1 1. Amino acid sequences of other exemplary anti-LAG-3 antibody molecules
TIM-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 TIM-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 12 (e.g. , from the heavy and light chain variable region sequences of ABTIM3-hum1 1 or ABTIM3-hum03 disclosed in Table 12), or encoded by a nucleotide sequence shown in Table 12. In some embodiments, the CDRs are according to the Kabat definition (e.g. , as set out in Table 12). In some embodiments, the CDRs are according to the Chothia definition (e.g. , as set out in Table 12). 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 12, or encoded by a nucleotide sequence shown in Table 12.
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: 801 , a VHCDR2 amino acid sequence of SEQ ID NO: 802, and a VHCDR3 amino acid sequence of SEQ ID NO: 803; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 810, a VLCDR2 amino acid sequence of SEQ ID NO: 81 1 , and a VLCDR3 amino acid sequence of SEQ ID NO: 812, each disclosed in Table 12. 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: 801 , a VHCDR2 amino acid sequence of SEQ ID NO: 820, and a VHCDR3 amino acid sequence of SEQ ID NO: 803; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 810, a VLCDR2 amino acid sequence of SEQ ID NO: 81 1 , and a VLCDR3 amino acid sequence of SEQ ID NO: 812, each disclosed in Table 12.
In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 806, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 806. In one embodiment, the anti-TIM-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 816, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 816. In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 822, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 822. In one embodiment, the anti-TIM-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 826, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 826. In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 806 and a VL comprising the amino acid sequence of SEQ ID NO: 816. In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 822 and a VL comprising the amino acid sequence of SEQ ID NO: 826.
In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 807, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 807. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 817, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 817. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 823, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 823. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 827, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 827. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 807 and a VL encoded by the nucleotide sequence of SEQ ID NO: 817. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 823 and a VL encoded by the nucleotide sequence of SEQ ID NO: 827.
In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 808, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 808. In one embodiment, the anti- TIM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 818, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 818. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 824, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 824. In one embodiment, the anti-TIM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 828, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 828. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 808 and a light chain comprising the amino acid sequence of SEQ ID NO: 818. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 824 and a light chain comprising the amino acid sequence of SEQ ID NO: 828.
In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 809, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 809. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 819, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 819. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 825, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 825. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 829, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 829. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 809 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 819. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 825 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 829.
The antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0218274, incorporated by reference in its entirety.
Table 12. Amino acid and nucleotide sequences of exemplary anti-TIM-3 antibody molecules
TGG CTACACCTTCACTAG CTATAATATG CACTGG GTTC
GCCAGGCCCCAGGGCAAGGCCTCGAGTGGATGGGCG
ATATCTACCCCGGGAACGGCGACACTAGTTATAATCAG
AAGTTTAAGGGTAGAGTCACTATCACCGCCGATAAGTC
TACTAGCACCGTCTATATGGAACTGAGTTCCCTGAGGT
CTGAGGACACCGCCGTCTACTACTGCGCTAGAGTGGG
CG G AG CCTTCCCTATGG ACTACTG GG GTCAAG GCACT
ACCGTGACCGTGTCTAGC
SEQ ID NO: 808 Heavy QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVR chain QAPGQGLEWMGDIYPGNGDTSYNQKFKGRVTITADKST
STVY ELSSLRSEDTAVYYCARVGGAFPMDYWGQGTTV
TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS
SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE
FLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEV
QFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQ
DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
MHEALHNHYTQKSLSLSLG
SEQ ID NO: 809 DNA CAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAAG heavy AAACCCGGCTCTAGCGTGAAAGTTTCTTGTAAAGCTAG chain TGG CTACACCTTCACTAG CTATAATATG CACTGG GTTC
GCCAGGCCCCAGGGCAAGGCCTCGAGTGGATGGGCG
ATATCTACCCCGGGAACGGCGACACTAGTTATAATCAG
AAGTTTAAGGGTAGAGTCACTATCACCGCCGATAAGTC
TACTAGCACCGTCTATATGGAACTGAGTTCCCTGAGGT
CTGAGGACACCGCCGTCTACTACTGCGCTAGAGTGGG
CG GAG CCTTCCCTATGG ACTACTG GG GTCAAG GCACT
ACCGTGACCGTGTCTAGCGCTAGCACTAAGGGCCCGT
CCGTGTTCCCCCTGGCACCTTGTAGCCGGAGCACTAG
CGAATCCACCGCTGCCCTCGGCTGCCTGGTCAAGGAT
TACTTCCCGGAGCCCGTGACCGTGTCCTGGAACAGCG
GAGCCCTGACCTCCGGAGTGCACACCTTCCCCGCTGT
GCTGCAGAGCTCCGGGCTGTACTCGCTGTCGTCGGTG
GTCACGGTGCCTTCATCTAGCCTGGGTACCAAGACCT
ACACTTGCAACGTGGACCACAAGCCTTCCAACACTAAG
GTGGACAAGCGCGTCGAATCGAAGTACGGCCCACCGT
GCCCGCCTTGTCCCGCGCCGGAGTTCCTCGGCGGTC
CCTCGGTCTTTCTGTTCCCACCGAAGCCCAAGGACAC
TTTGATGATTTCCCGCACCCCTGAAGTGACATGCGTGG
TCGTGGACGTGTCACAGGAAGATCCGGAGGTGCAGTT
CAATTGGTACGTGGATGGCGTCGAGGTGCACAACGCC
AAAACC AAG CCGAG GG AG G AG CAGTTCAACTCCACTT
ACCGCGTCGTGTCCGTGCTGACGGTGCTGCATCAGGA
CTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTGTCC
AACAAGGGACTTCCTAGCTCAATCGAAAAGACCATCTC
GAAAGCCAAGGGACAGCCCCGGGAACCCCAAGTGTAT
ACCCTGCCACCGAGCCAGGAAGAAATGACTAAGAACC
AAGTCTCATTGACTTGCCTTGTGAAGGGCTTCTACCCA
TCGGATATCGCCGTGGAATGGGAGTCCAACGGCCAGC
CGGAAAACAACTACAAGACCACCCCTCCGGTGCTGGA
CTCAG ACG G ATCCTTCTTCCTCTACTCG CGG CTG ACC
GTGGATAAGAGCAGATGGCAGGAGGGAAATGTGTTCA
GCTGTTCTGTGATGCATGAAGCCCTGCACAACCACTAC
ACTCAGAAGTCCCTGTCCCTCTCCCTGGGA
(Kabat) SEQ ID NO: 804 HCDR1 GYTFTSY
(Chothia)
SEQ ID NO: 821 HCDR2 YPGQGD
(Chothia)
SEQ ID NO: 803 HCDR3 VGGAFPMDY
(Chothia)
SEQ ID NO: 822 VH QVQLVQSGAEVK PG
QAPGQGLEWIGDIYPGQGDTSYNQKFKGRAT TADKST STVY ELSSLRSEDTAVYYCARVGGAFPMDYWGQGTLV TVSS
SEQ ID NO: 823 DNA VH CAGGTGCAGC
AAACCCGGCGCTAGTGTGAAAGTTAGCTGTAAAGCTA
GTGGCTATAC I I I CACTTCTTATAATATGCACTGGGTC
CGCCAGGCCCCAGGTCAAGGCCTCGAGTGGATCGGC
GATATCTACCCCGGTCAAGGCGACACTTCCTATAATCA
GAAGTTTAAGGGTAGAGCTACTATGACCGCCGATAAGT
CTACTTCTACCGTCTATATGGAACTGAGTTCCCTGAGG
TCTGAGGACACCGCCGTCTACTACTGCGCTAGAGTGG
GCGGAGCCTTCCCAATGGACTACTGGGGTCAAGGCAC
CCTGGTCACCGTGTCTAGC
SEQ ID NO: 824 Heavy QVQLVQSGAEVkK
chain QAPGQGLEWIGDIYPGQGDTSYNQKFKGRAT TADKST
STVY ELSSLRSEDTAVYYCARVGGAFPMDYWGQGTLV
TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS
SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE
FLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEV
QFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQ
DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
MHEALHNHYTQKSLSLSLG
SEQ ID NO: 825 DNA C AG GTGCAG CT G GTGCAGT CAG GCG CCG AA
heavy AAACCCGGCGCTAGTGTGAAAGTTAGCTGTAAAGCTA chain GTGGCTATAC I I I CACTTCTTATAATATGCACTGGGTC
CGCCAGGCCCCAGGTCAAGGCCTCGAGTGGATCGGC
GATATCTACCCCGGTCAAGGCGACACTTCCTATAATCA
GAAGTTTAAGGGTAGAGCTACTATGACCGCCGATAAGT
CTACTTCTACCGTCTATATGGAACTGAGTTCCCTGAGG
TCTGAGGACACCGCCGTCTACTACTGCGCTAGAGTGG
GCGGAGCCTTCCCAATGGACTACTGGGGTCAAGGCAC
CCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCCG
TCCGTGTTCCCCCTGGCACCTTGTAGCCGGAGCACTA
GCGAATCCACCGCTGCCCTCGGCTGCCTGGTCAAGGA
TTACTTCCCGGAGCCCGTGACCGTGTCCTGGAACAGC
GGAGCCCTGACCTCCGGAGTGCACACCTTCCCCGCTG
TGCTGCAGAGCTCCGGGCTGTACTCGCTGTCGTCGGT
GGTCACGGTGCCTTCATCTAGCCTGGGTACCAAGACC
TACACTTGCAACGTGGACCACAAGCCTTCCAACACTAA
GGTGGACAAGCGCGTCGAATCGAAGTACGGCCCACC
GTGCCCGCCTTGTCCCGCGCCGGAGTTCCTCGGCGG
TCCCTCGGTCTTTCTGTTCCCACCGAAGCCCAAGGAC
ACTTTGATGATTTCCCGCACCCCTGAAGTGACATGCGT
GGTCGTGGACGTGTCACAGGAAGATCCGGAGGTGCA
GTTCAATTGGTACGTGGATGGCGTCGAGGTGCACAAC GCCAAAACCAAGCCGAGGGAGGAGCAGTTCAACTCCA
CTTACCGCGTCGTGTCCGTGCTGACGGTGCTGCATCA
GGACTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTG
TCCAACAAGGGACTTCCTAGCTCAATCGAAAAGACCAT
CTCGAAAGCCAAGGGACAGCCCCGGGAACCCCAAGT
GTATACCCTGCCACCGAGCCAGGAAGAAATGACTAAG
AACCAAGTCTCATTGACTTGCCTTGTGAAGGGCTTCTA
CCCATCGGATATCGCCGTGGAATGGGAGTCCAACGGC
CAG CCG G AAAACAACTACAAG ACCACCCCTCCG GTGC
TGGACTCAGACGGATCCTTCTTCCTCTACTCGCGGCT
GACCGTGGATAAGAGCAGATGGCAGGAGGGAAATGTG
TTCAGCTGTTCTGTGATGCATGAAGCCCTGCACAACCA
CTACACTCAGAAGTCCCTGTCCCTCTCCCTGGGA
SEQ ID NO: 810 LCDR1 R AS ES VEYYG TSLM Q
(Kabat)
SEQ ID NO: 81 1 LCDR2 AASNVES
(Kabat)
SEQ ID NO: 812 LCDR3 QQSRKDPSf
(Kabat)
SEQ ID NO: 813 LCDR1 SESVEYYGTSL
(Chothia)
SEQ ID NO: 814 LCDR2 AAS
(Chothia)
SEQ ID NO: 815 LCDR3 SRKDPS
(Chothia)
SEQ ID NO: 826 VL DIVLTQSPDSLAVSLGERATINCRASESVEYYCBTSLMQW
YQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTDFTLTI
SSLQAEDVAVYYCQQSRKDPSTFGGGTKVEIK
SEQ ID NO: 827 DNA VL GATATCGf CCTGACTCAGf
TCAGCCTGGGCGAGCGGGCTACTATTAACTGTAGAGC
TAGTGAATCAGTCGAGTACTACGGCACTAGCCTGATG
CAGTGGTATCAGCAGAAGCCCGGTCAACCCCCTAAGC
TGCTGATCTACGCCGCCTCTAACGTGGAATCAGGCGT
GCCCGATAGGTTTAGCGGTAGCGGTAGTGGCACCGAC
TTCACCCTGACTATTAGTAGCCTGCAGGCCGAGGACG
TGGCCGTCTACTACTGTCAGCAGTCTAGGAAGGACCC
TAGCACCTTCGGCGGAGGCACTAAGGTCGAGATTAAG
SEQ ID NO: 828 Light D IVLTQSP D STAVS LG E RAT I N C R AS ES VEYYG S L M Q W chain YQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTDFTLTI
SSLQAEDVAVYYCQQSRKDPSTFGGGTKVEIKRTVAAPS
VFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 829 DNA GAfAfCGTCCTGACTCA
light TCAG CCTG GG CG AGCG GG CTACTATTAACTGTAG AGC chain TAGTGAATCAGTCGAGTACTACGGCACTAGCCTGATG
CAGTGGTATCAGCAGAAGCCCGGTCAACCCCCTAAGC
TGCTGATCTACGCCGCCTCTAACGTGGAATCAGGCGT
GCCCGATAGGTTTAGCGGTAGCGGTAGTGGCACCGAC
TTCACCCTGACTATTAGTAGCCTGCAGGCCGAGGACG
TGGCCGTCTACTACTGTCAGCAGTCTAGGAAGGACCC
TAG C ACCTTCG GCG G AG G C ACT AAG GTC G AG ATT AAG
CGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCC
CCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCG
TGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGC CAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAG
CG G CAACAG CCAGG AG AG CGTCACCG AGCAGG ACAG CAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACC CTG AG CAAGG CCG ACTACG AG AAG CATAAG GTGTACG CCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCG TG ACC AAG AG CTTC AAC AG GGGCGAGTGC
Other Exemplary TIM-3 Inhibitors
In one embodiment, the anti-TIM-3 antibody molecule is TSR-022 (AnaptysBio/Tesaro). 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 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 13. APE5137, APE5121 , and other anti-TIM-3 antibodies are disclosed in WO 2016/161270, incorporated by reference in its entirety.
In one embodiment, the anti-TIM-3 antibody molecule is the antibody clone F38-2E2. 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 F38-2E2.
Further known anti-TIM-3 antibodies include those described, e.g. , in WO 2016/1 1 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 13. Amino acid sequences of other exemplary anti-TIM-3 antibody molecules
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, IL-2, IL-15, IL-7, or IL21 . In certain embodiments, antibody conjugate is administered in combination with an IL- 15/IL-15Ra complex. In some embodiments, the IL-15/IL-15Ra complex is selected from NIZ985 (Novartis), ATL-803 (Altor) or CYP0150 (Cytune). Exemplary IL- 15/1 L- 15Ra complexes
In one embodiment, the cytokine is IL-15 complexed with a soluble form of IL-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: 922 in Table 21 or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 922, and the soluble form of human IL-15Ra comprises an amino acid sequence of SEQ ID NO:923 in Table 21 , or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 923, as described in WO 2014/066527, incorporated by reference in its entirety. The molecules described herein can be made by vectors, host cells, and methods described in WO 2007084342, incorporated by reference in its entirety.
Table 21. Amino acid and nucleotide sequences of exemplary IL- 5/IL-15Ra complexes
NIZ985
SEQ I D NO: i Human IL-15 ! NWVNVISDLKKIEDLIQS HIDATLYTESDVHPSCKVTAMKCF I 922 I LLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKE \
\ CEELEEKNIKEFLQSFVHIVQMFINTS
SEQ I D NO: i Human I ITCPPP SVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLT j 923 I Soluble IL- I ECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGV \
15Ra \ TPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKS \
\ PSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQ j
I G
Other exemplary IL-15/IL-15Ra complexes
In one embodiment, the IL-15/IL-15Ra complex is ALT-803, an IL-15/IL-15Ra Fc fusion protein (IL-15N72D:IL-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 22.
In one embodiment, the IL-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-15Ra, and ending at the fourth cysteine residue after said signal peptide. The complex of IL-15 fused to the sushi domain of IL-15Ra is described in WO 2007/04606 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 22.
Table 22. Amino acid sequences of other exemplary IL- 5/IL-15Ra complexes
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 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 alaninate (BMS-582664, (S)-((R)-1 -(4-(4-Fluoro-2-methyl-1 H-indol-5-yloxy)- 5-methylpyrrolo[2,1 -/][1 ,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate); Sorafenib (Nexavar®); Pazopanib (Votrient®); Sunitinib malate (Sutent®); Cediranib (AZD2171 , CAS 288383-20-1); Vargatef (BIBF1 120, CAS 928326-83-4); 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 755037-03-7); Vatalanib dihydrochloride (PTK787, CAS 212141 -51-0); Brivanib (BMS-540215, CAS 649735-46-6); Vandetanib (Caprelsa® or AZD6474); Motesanib diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl- 1 H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-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 1 1 1358-88- 4); N-[5-[[[5-(1 , 1 -Dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7); (3R,4R)-4-Amino-1 -((4-((3-methoxyphenyl)amino)pyrrolo[2, 1- f][1 ,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); A/-(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); 4-Methyl-3-[[1-methyl-6-(3-pyridinyl)-1 H- pyrazolo[3,4-d]pyrimidin-4-yl]amino]-A/-[3-(trifluoromethyl)phenyl]-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-allylamino- 17-demethoxygeldanamycin, also known as KOS-953 and 17-AAG, available from SIGMA, and described in US Patent No. 4,261 ,989); Retaspimycin (IPI504), Ganetespib (STA-9090); [6- Chloro-9-(4-methoxy-3,5-dimethylpyridin-2-ylmethyl)-9H-purin-2-yl]amine (BIIB021 or CNF2024, CAS 848695-25-0); fA-ans-4-[[2-(Aminocarbonyl)-5-[4,5,6,7-tetrahydro-6,6-dimethyl-4-oxo-3- (trifluoromethyl)-1 /-/-indazol-1 -yl]phenyl]amino]cyclohexyl glycine ester (SNX5422 or
PF049291 13, CAS 9081 15-27-5); 5-[2,4-Dihydroxy-5-(1-methylethyl)phenyl]-/V-ethyl-4-[4-(4- morpholinylmethyl)phenyl]- 3-lsoxazolecarboxamide (AUY922, CAS 747412-49-3); or 17- Dimethylaminoethylamino-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 (Istodax®);
Treichostatin A (TSA); Oxamflatin; Vorinostat (Zolinza®, Suberoylanilide hydroxamic acid); Pyroxamide (syberoyl-3-aminopyridineamide hydroxamic acid); Trapoxin A (RF-1023A);
Trapoxin B (RF-10238); Cyclo[(aS,2S)-a-amino-n-oxo-2-oxiraneoctanoyl-0-methyl-D-tyrosyl-L- isoleucyl-L-prolyl] (Cyl-1); Cyclo[(aS,2S)-a-amino-n-oxo-2-oxiraneoctanoyl-0-methyl-D-tyrosyl- L-isoleucyl-(2S)-2-piperidinecarbonyl] (Cyl-2); Cyclic[L-alanyl-D-alanyl-(2S)-r|-oxo-L-C(- aminooxiraneoctanoyl-D-prolyl] (HC-toxin); Cyclo[(aS,2S)-a-amino-n,-oxo-2-oxiraneoctanoyl-D- phenylalanyl-L-leucyl-(2S)-2-piperidinecarbonyl] (WF-3161); Chlamydocin ((S)-Cyclic(2- methylalanyl-L-phenylalanyl-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, 1 1 -pentadeoxy- D-f/?reo-D-/cfo-Undeca-1 ,6- dienitol); 4-(Acetylamino)-N-(2-aminophenyl)-benzamide (also known as CI-994); N1-(2- Aminophenyl)-N8-phenyl-octanediamide (also known as BML-210); 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 PXD101 , Beleodaq®, or (2£)-/V-Hydroxy-3-[3- (phenylsulfamoyl)phenyl]prop-2-enamide), or chidamide (also known as CS055 or HBI-8000, (E)-N-(2-amino-5-fluorophenyl)-4-((3-(pyridin-3-yl)aci7lamido)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 histone demethylase 1A or KD 1A).
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 (also known as NLG-8189), a-Cyclohexyl-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-6 inhibitor or IL-6 receptor (IL-6R) inhibitors (e.g., tocilizumab or siltuximab), bazedoxifene, sgp130 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.
Tocilizumab is a humanized, immunoglobulin G 1 kappa anti-human IL-6R monoclonal antibody. Tocilizumab blocks binding of IL-6 to soluble and membrane bound IL-6 receptors (IL- 6Rs) and thus inhibitos classical and trans-IL-6 signaling. In embodiments, tocilizumab 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-6 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 siltuximab. In other embodiments, the inhibitor comprises a soluble gp130 (sgpl 30) or a fragment thereof that is capable of blocking IL-6 signalling. In some embodiments, the sgpl 30 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-6 signalling comprises an antibody, e.g., an antibody to the IL-6 receptor, such as sarilumab, olokizumab (CDP6038), elsilimomab, sirukumab (CNTO 136), ALD518/BMS-945429, ARGX-109, or F 101. 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-1 1 , endothelin-1 , alpha adrenergic agonists, rostanoids, phosphodiesterase inhibitors, endothelin antagonists, inotropes (e.g., adrenaline, dobutamine, isoprenaline, ephedrine), vasopressors (e.g., noradrenaline, vasopressin, metaraminol, vasopressin, methylene blue), inodilators (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 10 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 IL-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 H2 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,
immunomodulators, 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 a/., 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 al. (eds.), Pharmaceutical Dosage Forms:
Parenteral Medications, Marcel Dekker, NY, 1993; Lieberman, et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY, 1990; Lieberman, 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 lyophilisate preparation. In certain embodiments a pharmaceutical composition comprising the antibody conjugate is a lyophilisate in a vial containing an antibody conjugate, histidine, sucrose, and polysorbate 20. In certain
embodiments the pharmaceutical composition comprising the antibody conjugate is a lyophilisate in a vial containing an antibody conjugate, sodium succinate, and polysorbate 20. In certain embodiments the pharmaceutical composition comprising the antibody conjugate is a lyophilisate in a vial containing an antibody conjugate, trehalose, citrate, and polysorbate 8. The lyophilisate 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 several 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 cells 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 ai , New Engl. J. Med. 348:601-608, 2003; Milgrom et ai, New Engl. J. Med. 341 :1966-1973, 1999; Slamon et ai , New Engl. J. Med. 344:783-792, 2001 ; Beniaminovitz et ai , New Engl. J. Med. 342:613-619, 2000; Ghosh et ai, New Engl. J. Med. 348:24-32, 2003; Lipsky et ai , New Engl. 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, rectally, intramuscular, intracerebrally, 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 mg/kg and 10 mg/kg, 0.05 and 5 mg/kg, 0.1 mg/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. , aynard et a/. , A Handbook of SOPs for Good Clinical Practice, Interpharm 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, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional administration, or by sustained release systems or an implant (see, e.g. , Sidman et al. , Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15: 167-277, 1981 ; Langer, Chem. Tech. 12:98-105, 1982; Epstein et al. , Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et al. , Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980; U.S. Pat. Nos. 6,350,466 and 6,316,024). 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 al., (eds.) (2001) 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, Phila., 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 1 1 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 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 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 al.); mannosides (Umezawa et al. , (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (Bloeman et al. , (1995) FEBS Lett. 357:140; Owais et a/. , (1995) Antimicrob. Agents Chemother. 39: 180); surfactant Protein A receptor (Briscoe et al. , (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. 346: 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 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 1 1 hours apart, at about 1 1 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 prophylactic 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 included 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.
Example 1 : Synthesis of Linker Intermediates
Example 1 -1 : Synthesis of 5,5,9, 12, 15, 15-hexamethyl-8, 13-dioxo-14-oxa-3,4-dithia-9, 12- diazahexadecyl carbonochloridate (LI-1)
Step 1 : Acetic acid (0.025 ml, 1.3 mmol) was added to a solution of 4-mercapto-4- methylpentanoic acid (250 mg, 1 .69 mmol) and 2-(pyridin-2-yldisulfanyl)ethanol (380 mg, 2.02 mmol) in MeOH (15 mL) and the mixture was heated at 45°C for 5 days and then concentrated and purified by ISCO using 15 g C18 column, eluted with 5-40% acetonitrile (ACN) in water with 0.05% TFA. The fractions containing the desired product were concentrated to give 4-((2- hydroxyethyl)disulfanyl)-4-methylpentanoic acid (220 mg, 58.1 % yield). LCMS M+23= 247.1 , tr= 0.768 min. 1 H N R (500 MHz, Chloroform-d) δ 3.86 (t, J = 5.8 Hz, 1 H), 2.84 (t, J = 5.8 Hz, 2H), 2.49 - 2.37 (m, 2H), 2.00 - 1 .86 (m, 2H), 1.29 (s, 6H).
Step 2: DIEA (0.082 ml, 0.47 mmol) and tert-butyl methyl(2-(methylamino)ethyl)carbamate (44 mg, 0.23 mmol) were added to a solution of 4-((2-hydroxyethyl)disulfanyl)-4-methylpentanoic acid (35 mg, 0.16 mmol) in dichloromethane (DCM) (5 ml), followed by the addition of N1- ((ethylimino)methylene)-N3,N3-dimethylpropane-1 ,3-diamine hydrochloride (EDCI) (45 mg, 0.23 mmol). The mixture was stirred at room temperature for 16 hours, then quenched with water, extracted with DCM, dried, concentrated and purified by ISCO using 15g C18 column, eluted with ACN-water containing 0.05% TFA to obtain tert-butyl (2-(4-((2-hydroxyethyl)disulfanyl)-N,4- dimethylpentanamido)ethyl)(methyl)carbamate (34 mg, 50 % yield). LCMS M+ 1 = 395.2, tr= 1.044 min. 1H NMR (500 MHz, Chloroform-d) δ 3.84 (t, J = 6.0 Hz, 2H), 3.49 (s, 2H), 3.35 (t, J = 6.1 Hz, 2H), 3.03 (s, 2H), 2.94 (s, 1 H), 2.89 - 2.78 (m, 5H), 2.38 (d, J = 7.3 Hz, 2H), 2.01 - 1 .90 (m, 2H), 1 .83 (s, 3H), 1 .44 (s, 9H), 1 .30 (s, 6H).
Step 3: Pyridine (0.010 ml, 0.12 mmol) was added to a solution of tert-butyl (2-(4-((2- hydroxyethyl)disulfanyl)-N,4-dimethylpentanamido)ethyl)(methyl)carbamate (27 mg, 0.068 mmol) in DCM (4ml) at 0°C followed by addition of a 20% phosgene solution in toluene (0.3 ml). The reaction was stirred for 15 mins and then concentrated to give 5,5,9, 12, 15, 15-hexamethyl- 8, 13-dioxo-14-oxa-3,4-dithia-9, 12-diazahexadecyl carbonochloridate (LI-1) which was immediately used without purification.
Example 1 -2: Synthesis of 18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-5,5,9,12-tetramethyl-8,13- dioxo-16-oxa-3,4-dithia-9, 12-diazaoctadecyl (4-nitrophenyl) carbonate (LI-2)
Step 1 : Trifluoroacetic acid (TFA) (1 ml) was added to a flask containing tert-butyl (2-(4-((2- hydroxyethyl)disulfanyl)-N,4-dimethylpentanamido)ethyl)(methyl)carbamate (34 mg, 0.086 mmol) and the mixture was immediately concentrated to give 4-((2-hydroxyethyl)disulfanyl)-N,4- dimethyl-N-(2-(methylamino)ethyl)pentanamide as a TFA salt. LCMS M+ 1 =295.3, tr= 0.619 min. Step 2: Ν,Ν-diisopropyl ethylamine (DIEA) (0.075 ml, 0.431 mmol) was added to a solution of 3- (2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)propanoic acid (Mal-PEG 1-Acid) (18.4 mg, 0.086 mmol) in DMF (2 ml), followed by the addition of 3-[Bis(dimethylamino)methyliumyl]-3H- benzotriazol-1-oxide hexafluorophosphate (HBTU) (33 mg, 0.086 mmol). The mixture was stirred at room temperature for 5 mins and then added dropwise to a solution of 4-((2- hydroxyethyl)disulfanyl)-N,4-dimethyl-N-(2-(methylamino)ethyl)pentanamide TFA salt (35 mg, 0.086 mmol) in Ν,Ν-dimethyl formamide (DMF) (1 ml). The mixture was then stirred at room temperature for 2 hours and then purified by mass-triggered reverse phase HPLC using a C18 column, eluted with 10-40% acetonitrile-H20 containing 0.05% TFA. Fractions containing desired product were concentrated to obtain N-(2-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)ethoxy)-N-methylpropanamido)ethyl)-4-((2-hydroxyethyl)disulfanyl)-N,4-dimethylpentanamide (40.1 mg, 90 % yield). LCMS M+1 = 490.3 tr=0.841 min.
Step 3: To a solution of N-(2-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)-N- methylpropanamido)ethyl)-4-((2-hydroxyethyl)disulfanyl)-N,4-dimethylpentanamide (40.1 mg, 0.082 mmol) obtained in step 2 in DCM (3 ml) was added bis(4-nitrophenyl) carbonate (125 mg, 0.409 mmol) and then DIEA (0.043 mL, 0.246 mmol). It was stirred at room temperature for 4 days and the reaction was complete to form the desired product. It was concentrated and the residue was dissolved in ACN and purified by ISCO using 50g C18 column, eluted with 25-75% ACN in water with 0.035% TFA. Fractions containing the desired product were combined and lyophilized to give 18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol- 1 -yl)-5,5,9, 12-tetramethyl-8, 13-dioxo-16- oxa-3,4-dithia-9, 12-diazaoctadecyl (4-nitrophenyl) carbonate (LI-2) (44 mg, 73 % yield). LCMS M+1 =655.2, tr=1 .177 min. It is contaminated by a small amount of bis (4-nitrophenyl) carbonate and hydrolyzed alcohol by-product. Example 1 -3: 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 (4- nitrophenyl) carbonate (LI-3)
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 to 2,5-dioxopyrrolidin-1 -yl 3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)propanoate (77 mg, 0.29 mmol) 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 50g 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 M+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 MeOH: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-3) (20 mg, 0.029 mmol, 46 % yield). LCMS M+1 =696.3, tr= 1 .039 min.
Example 1 -4: Synthesis of (S)-4-(2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)-3- phenylpropanamido)benzyl (4-nitrophenyl) carbonate (LI-4)
Step 1 : N-Hydroxybenzotriazole (HOBT) (509 mg, 3.77 mmol) and DMF (6 ml) was added to a solution of BocPhe-OH (500 mg, 1 .89 mmol) and (4-aminophenyl)methanol (464 mg, 3.77 mmol) in DC (30 ml), followed by the addition of diisopropylcarbodiimide (476 mg, 3.77 mmol). The mixture was stirred at room temperature for 16 hours, concentrated to remove DCM and then purified by silica gel column eluted with 10% MeOH in DCM to give tert-butyl (S)-(1 -((4- (hydroxymethyl)phenyl)amino)-1 -oxo-3-phenylpropan-2-yl)carbamate (1.12g, 97% yield). LCMS M+1 = 275.2. tr= 0.561 min. 1 H NMR (500 MHz, Chloroform-d) δ 7.99 (s, 1 H), 7.88 (d, J = 7.1 Hz, 1 H), 7.39 - 7.18 (m, 9H), 5.17 (s, 1 H), 4.60 (s, 2H), 4.46 (s, 1 H), 3.12 (d, J = 6.9 Hz, 2H), 1.40 (s, 9H).
Step 2: TFA (5ml) and DCM (1 ml) were added to tert-butyl (S)-(1 -((4- (hydroxymethyl)phenyl)amino)-1 -oxo-3-phenylpropan-2-yl)carbamate (1.12g, 1 .82 mmol) and the mixture was concentrated immediately. The solid was then dissolved in MeOH-DCM (5%) and extracted from 2M Na2C03 aqueous solution, dried and concentrated to obtain (S)-2-amino- N-(4-(hydroxymethyl)phenyl)-3-phenylpropanamide (Phe-pab-OH), which was used in the next step without further purification. LCMS M+1 =271.3 tr= 0.618 min.
Step 3: HOBT (200 mg, 1.48 mmol) was added to a solution of Phe-pab-OH (400 mg, 1 .48 mmol) and 3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanoic acid (250 mg, 1 .480 mmol) in DCM-DMF (5:1 , 24 ml), followed by the addition of diisopropylcarbodiimide (187 mg, 1 .48 mmol). The mixture was stirred at room temperature for 16 hours, concentrated and purified by silica gel column, eluted with 5% MeOH in DCM. Fractions containing the desired product were combined and concentrated. The mixture was further purified by reverse phase ISCO using 50g C18 aq column, eluted with 10-50% acetonitrile-H20 containing 0.05% TFA. Fractions containing the desired product were concentrated to obtain (S)-2-(3-(2,5-dioxo-2,5-dihydro-1 H- pyrrol-1 -yl)propanamido)-N-(4-(hydroxymethyl)phenyl)-3-phenylpropanamide (MP-Phe-pab-OH) (0.214 g, 32.6 % yield) as free base. LCMS M+ 1 =422.2, tr= 0.851 min. 1 H NMR (500 MHz, Acetonitrile-d3) δ 8.40 (s, 1 H), 7.45 (d, J = 8.5 Hz, 2H), 7.25 (ddd, J = 20.2, 7.7, 3.3 Hz, 7H), 6.80 (d, J = 7.8 Hz, 1 H), 6.70 (s, 2H), 4.62 (td, J = 8.0, 6.2 Hz, 1 H), 4.51 (s, 2H), 3.64 (t, J = 7.0 Hz, 2H), 3.13 (dd, J = 13.9, 6.2 Hz, 1 H), 2.93 (dd, J = 13.9, 8.1 Hz, 1 H), 2.54 - 2.31 (m, 2H). Step 4: A solution of (S)-2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)-N-(4- (hydroxymethyl)phenyl)-3-phenylpropanamide (MP-Phe-pab-OH) (89.3 mg, 0.212 mmol), bis(4- nitrophenyl) carbonate (645 mg, 2.1 19 mmol) and DIEA (0.1 1 1 ml_, 0.636 mmol) was stirred at room temperature for 2 days, then concentrated and purified by silica gel column, eluted with 2- 6% MeOH:DCM. Fractions containing the desired product were collected and concentrated to give (S)-4-(2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)-3- phenylpropanamido)benzyl (4-nitrophenyl) carbonate (LI-4) (1 16 mg, 89 % yield). LCMS M+ 1 =587.2, tr= 1 .268min. 1 H N R (500 MHz, DMSO-d6) δ 10.21 (s, 1 H), 8.46 (d, J = 8.1 Hz, 1 H), 8.40 - 8.23 (m, 2H), 7.68 - 7.56 (m, 4H), 7.45 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 4.4 Hz, 4H), 7.01 (s, 2H), 5.28 (s, 2H), 4.68 (dt, J = 8.7, 4.4 Hz, 1 H), 3.63 - 3.48 (m, 2H), 3.36 (s, 4H), 3.05 (dd, J = 13.7, 5.5 Hz, 1 H), 2.92-2.83 (m, 2H), 2.44 - 2.34 (m, 2H).
Example 1 -5: Synthesis of 4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-
Step 1 : DIEA (204 mg, 1 .6 mmol) was added to a solution of Mal-PEG1-Acid (1 12 mg, 0.53 mmol) in DMF (10 ml), followed by the addition of 1 -[Bis(dimethylamino)methylene]-1 H-1 ,2,3- triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (200 mg, 0.53 mmol). The mixture was stirred at room temperature for 5 mins and then was added to a solution of (S)-2- ((S)-2-amino-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (valcit- pab-OH) (purchased from Levena Biopharma, San Diego) (200 mg, 0.527 mmol) in DMF (5 ml). The mixture was stirred at room temperature for 1 h and then concentrated and purified by reverse phase ISCO using 50 g C18 column, eluted with 10-40% acetonitrile-H20 containing 0.05% TFA. Fractions containing the desired product were concentrated to obtain (S)-2-((S)-2- (3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)propanamido)-3-methylbutanamido)-N-(4- (hydroxymethyl)phenyl)-5-ureidopentanamide (MPEG 1-vc-pab-OH) (190 mg, 57 % yield) as afree base. LCMS M+ 1 = 575.3, tr= 0.658 min.
Step 2: A solution of (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)ethoxy)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5- ureidopentanamide (MPEG1 -valcit-pabOH) (57.5 mg, 0.100 mmol), bis(4-nitrophenyl) carbonate (130 mg, 1.0 mmol) and DIEA (0.056 mL, 0.32 mmol) was stirred at room temperature for 2 days. The mixture was then concentrated and purified by silica gel column, eluted with 2- 6% MeOH:DCM and fractions containing the desired product were collected and concentrated to give 4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)propanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (LI-5) (59 mg, 80 % yield). LCMS M+1 =740.2, tr= 1 .02 min.
Example 1 -6: Synthesis of tert-butyl (2S,4S)-2-(((chlorocarbonyl)oxy)methyl)-4- fluoropyrrolidine-1 -carboxylate (LI-6)
To a dry flask was introduced potassium carbonate (257 mg, 1 .7 equiv), followed by toluene ( 5 ml_). Phosgene in toluene (2.4 ml_, 15% in toluene, 3.0 equiv) was added under nitrogen at - 35°C. To this vigorously stirred suspension was added dropwise a solution of (2S,4S)-tert-butyl 4-fluoro-2-(hydroxymethyl)pyrrolidine-1 -carboxylate (1 .093 mmol, 1 .0 equiv) in toluene (3.6 ml). Upon completion of the addition, the mixture was stirred at low temperature (~ -35°C to 0°C) for 30 mins. The cool bath was removed, and the mixture stirred for a further 1 h at room temperature and then filtered by syringe filters with 0.45 micron pore. The volatiles were removed under vacuum with rotary evaporator and the resultant clear pare yellow oil was used directly without further purification.
Example 1 -7: Synthesis of Ketone-Coenzyme A Analog (LI-7)
Coenzyme A trilithium salt (259 mg, Sigma, assay >93%) was dissolved in 2.0 mL of 100 mM phosphate buffer (pH 7.5) containing 5 mM EDTA, followed by addition of 3-buten-2-one (29.0 μΙ_, Aldrich, 99%). The reaction was carried out for 75 min at 20°C. Next, the reaction mixture was loaded onto a reverse phase RediSep Rf Gold® C18Aq column (Teledyne Isco), where the product eluted at 100% H20. Product-containing fractions were combined and lyophilized, affording linker intermediate (LI-7) as crystalline solid. MS (ESI+) m/z 838.2 (M+1). H-NMR (400 MHz, D20) δ 8.525 (s, 1 H), 8.235 (s, 1 H), 6.140 (d, 1 H, J=7.2Hz), 4.746 (m, 1 H), 4.546 (bs, 1 H), 4.195 (bs, 1 H), 3.979 (s, 1 H), 3.786 (dd, 1 H, J= 4.8, 9.6Hz), 3.510 (dd, 1 H, J=4.8, 9.6Hz), 3.429 (t, 2H, J= 6.6Hz), 3.294S (t, 2H, J=6.6Hz), 2.812 (t, 2H, J=6.8Hz), 2.676 (t, 2H, J=6.8Hz), 2.604 (t, 2H, J=6.8Hz), 2.420 (t, 2H, J=6.6Hz), 2.168 (s, 3H), 0.842 (s, 3H), 0.71 1 (s, 3H) (note: some peaks which overlap with D20 are not reported).
Example 1 -8: Synthesis of 4-((tert-butoxycarbonyl)amino)butanoic anhydride (LI-8)
A solution of DCC (0.53 g, 2.56 mmol) in anhydrous dichloromethane (5 ml) was added via syringe to a solution of 4-((tert-butoxycarbonyl)amino)butanoic acid (1.0 g, 4.9 mmol) in anhydrous dichloromethane (30 ml). After 1 hr of stirring, precipitation of urea was filtered through a syringe filter and the solvent was removed under vacuum. 4-((tert- butoxycarbonyl)amino)butanoic anhydride (LI-8) (1 g, 105 % yield) was obtained as a white solid and used without further purification.
Example 1 -9: l-1 -
DIEA (25.8 mg, 0.2 mmol) was added to glycine (16.7 mg, 0.06 mmol) dissolved in 1 mL DMF and Linker intermediate (LI-3) (34.8 mg, 0.05 mmol) was added, followed by HOAT (8.2 mg, 0.06 mmol). The mixture was then stirred at rt overnight. After completion DMF was removed under reduced pressure, and the crude product was purified by reverse phase ISCO, eluted with 5-50% acetonitrile-H20. Fractions containing the desired product were combined and lyophilized 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)oxy)carbonyl)glycine (LI-9) (16.4 mg, 49% yield). LCMS M+1 = 632.3, tr= 0.714 min. Example 2: Synthesis of cyclic dinucleotide (CDN) Intermediates
Example 2-1 : Synthesis of 2-(methylamino)ethyl (9-
((2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12R, 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-purin-6-yl)carbamate (CDNI-1 )
To a solution of phosgene 15% in toluene (14.4 ml, 21.7 mmol) in anhydrous DCM (30 ml) at - 78°C was added a solution of tert-butyl (2-hydroxyethyl)(methyl)carbamate (1 .76 g, 10.0 mmol) and pyridine (1 .85 ml, 23.4 mmol) in DCM (10 ml). The mixture was stirred at -78°C for 10 min, warmed to room temperature, stirred for an additional 20 mins and then concentrated and residual solvent was furher removed under vacuum. Compound (T1-1) Et3N salt (300 mg, 0.334 mmol) was dissolved in pyridine (5 ml) and then added to the residue and the mixture was stirred at room temperature for 1 hour resulting in approximately 60% conversion with -30% diadduct. Water was added to the mixture, and the mixture was stirred for 10 mins and then concentrated. The residue was suspended in DMSO and purified by ISCO using 15.5g C18 aq column, eluted with ACN-water 5-50%, aq phase containing 10mM HOAc-Et3N. Fractions containing the monoadduct Et3N salt and were collected and concentrated. (131 mg) LCMS M+1 =896.1 , tr=0.770 min. 1 H NMR (500 MHz, Methanol-c/4) δ 8.96 (d, J = 6.0 Hz, 1 H), 8.64 (s, 1 H), 8.57 (s, 1 H), 8.42 (s, 1 H), 8.18 (s, 1 H), 6.44 (d, J = 16.8 Hz, 1 H), 6.36 (d, J = 17.3 Hz, 1 H), 5.46 (ddd, J = 51 .9, 15.5, 3.8 Hz, 2H), 5.24 - 4.99 (m, 2H), 4.64 - 4.50 (m, 2H), 4.47 - 4.30 (m, 4H), 4.00 (dt, J = 10.3, 4.8 Hz, 2H), 3.64 (t, J = 5.9 Hz, 2H), 3.58 (s, 2H), 3.18 (q, J = 7.3 Hz, 22H), 3.01 - 2.83 (m, 7H), 1.46 (s, 8H), 1 .41 (d, J = 7.6 Hz, 10H), 1 .29 (t, J = 7.3 Hz, 35H).
Note: Fractions containing the diadduct were collected and concentrated (218 mg). LCMS M+1 =1097.1 , tr=0.958 min). Monoadduct and starting compound (T1 -1 ) were then obtained by treating the diadduct with NaOH. Specifically the diadduct was dissolved in ACN (10 ml) and then water (20 ml) was added, followed by 1 .2 g NaOH. The mixture was stirred at 50°C for 4h hours, neutralized with 10% HCI and then concentrated. The residue was purified by reverse phase ISCO C18 column, eluted with 10-40 acetonitrile-^O containing 10 mM Et3N HOAc to give monoadduct (106mg).
To a flask containing 4-methylbenzenethiol sodium salt (318 mg, 2.16 mmol) was added TFA (5 ml) and the mixture was stirred until near complete dissolution of the solid. This mixture was then added to a flask containing the monoadduct from Step 1 (237 mg, 0.216 mmol) and the mixture was stirred for 2 mins and then concentrated. LCMS showed full Boc deprotection, however approximately 1/3 of t-butylthio adduct remained. The residue was dissolved in DMSO and purified by ISCO using C18 aq column, eluted with 5-30% ACN-water containing 0.05% TFA. Fractions containing the desired product were collected and concentrated to give (CDNI-1) (107 mg, 39.2 % yield) (LCMS M+1=796.1 , tr=0.555 min). 1 H NMR (500 MHz, DMSO-d6) δ 10.34 (s, 1 H), 8.83 (b, 7H), 8.09 (s, 1 H), 6.41 (d, J = 15.2 Hz, 1 H), 6.30 (d, J = 15.2 Hz, 1 H), 5.70 - 5.51 (m, 1 H), 5.44 (d, J = 51.8 Hz, 1 H), 5.03 (d, J = 25.7 Hz, 2H), 4.49 - 4.33 (m, 4H), 4.27 (s, 2H), 3.90 - 3.55 (m, 2H), 3.10 (d, J = 51.8 Hz, 1 H), 2.91 - 2.57 (m, 2H)
Note: Fractions containing the t-butylthio adduct (LCMS M+1=852.1 , tr=0.792 min) were collected and after standing for 3 days the t-butylthio adduct converted to (CDNI-1) (37 mg, 0.029 mmol, 13% yield).
Example 2-1 : Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5- ureidopentanamido)benzyl (2-(((9-
((2R,3R,3aR,5R,7aR,9R,10R,10aR,12R,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-purin-6- yl)carbamoyl)oxy)ethyl)(methyl)carbamate (CDNI-2)
Step 1 : 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)benzyl 2-(4-nitrophenyl)acetate (Fmoc-Val-Cit-PABC-PNP) (23.18 mg, 0.030 mmol) (purchased from Levena Biopharma, San Diego), DIEA (0.024 mL, 0.137 mmol) and 3-Hydroxytriazolo[4,5-b]pyridine (HOAT) (3.74 mg, 0.027 mmol) were added to a round bottom flask containing (CDNI-1) (25 mg, 0.027 mmol) in DMF (2 mL). The reaction was stirred at room temperature for 4 hours and then heated to 45°C and stirred for an additional hour. The mixture was then concentrated and the residue purified by ISCO using 15.5 g C18 aq column, eluted with 5-60% ACN-water with 0.05% TFA. Fmoc-vc-pabc-(CDNI-2) (34.4 mg, 81 % yield) was obtained. LCMS M/2+1 =712.3, tr =1 .007 min.
Step 2: Piperidine (0.200 ml) was added to a solution of Fmoc-vc-pabc-(CDNI-2) (34.4mg, 0.022 mmol) TFA salt in DMF (5 mL) and the mixture was stirred at room temperature for 30 mins, and then concentrated. The residue was purified by reverse phase ISCO using C18 aq column, eluted with 5-35% acetonitrile-h^O containing 0.05% TFA. Fractions containing desired product were concentrated to (CDNI-2) (31 .1 mg, 92% yield) as TFA salt. LCMS M+ 1 = 1201 .2 tr = 0.671 min.
Example 2-3: Synthesis of (CDNI-3)
Step 1 : a) Et3N (1 ml) was added to Compound (T1 -2) ammonium salt (400 mg, 0.552 mmol) in pyridine (30 ml) and the mixture was concentrated. The procedure was repeated twice to obtain the triethylammonium salt of Compound (T1 -2).
b) A solution of tert-butyl (2-hydroxyethyl)(methyl)carbamate (290 mg, 1 .66 mmol) in DCM (10 ml) with pyridine (0.313 mL, 3.86 mmol) was added to a solution of 15% phosgene solution in toluene (4.4 ml) in DCM (20 ml) at -78°C and the mixture was stirred for 15 mins and then warmed to room temperature and concentrated to obtain 2-((tert- butoxycarbonyl)(methyl)amino)ethyl carbonochloridate.
Step 2: Compound (T1 -2) Et3N salt was resuspended in anhydrous pyridine (30 ml) and then added to 2-((tert-butoxycarbonyl)(methyl)amino)ethyl carbonochloridate from step 1 b) and the mixture was stirred at room temperature for 30 mins. Water was then added and the mixture was concentrated. The residue was suspended in DMSO-water and then purified by reverse phase ISCO using C18 column, 15.5 g aq column, eluted with 2-40% acetonitrile-h^O containing 10 mM Et3N HOAc. The fractions containing the desired Boc protected monoadduct (387 mg, 57.7 % yield) were collected and lyophilized. M+1 =892.2. tr= 0.770 min. 1 H NMR (500 MHz, Methanol-c/4) δ 8.83 (s, 1 H), 8.34 (s, 1 H), 8.24 (s, 1 H), 8.18 (s, 1 H), 6.33 (dd, J = 25.9, 6.9 Hz, 2H), 6.10 (s, 1 H), 5.51 (s, 1 H), 5.33 (s, 1 H), 4.68 (s, 1 H), 4.51 - 4.14 (m, 7H), 4.03 (d, J = 9.5 Hz, 1 H), 3.70 - 3.56 (m, 1 H), 3.45 (s, 2H), 3.17 (d, J = 7.3 Hz, 22H), 2.88 (s, 4H), 1 .40 (s, 4H), 1 .29 (t, J = 7.3 Hz, 33H).
Step 3: TFA (5 mL) was added to a flask containing 4-methylbenzenethiol sodium salt (200 mg, 1.36 mmol) and the mixture was stirred until complete dissolution. The mixture was then added to another flask containing the Boc protected mono-adduct from step 2 (250 mg, 0.228 mmol) and after 1 min at room temperature the TFA was removed. The mixture was then dissolved in DMSO and purified by reverse phase ISCO using 15 g C18 aq column, eluted with 2-20% acetonitrile-H20 containing 0.05% TFA. The fractions containing desired product were concentrated to obtain the de-protected monoadduct (CDNI-3) as aTFA salt. LCMS M+ 1 =792.0, tr= 0.61 1 min. 1H NMR (500 MHz, DMSO-d6) δ 9.37 (d, J = 41 .6 Hz, 2H), 8.89 (s, 1 H), 8.70 (s, 1 H), 8.43 (s, 1 H), 8.30 (s, 1 H), 6.33 (d, J = 7.8 Hz, 1 H), 6.21 (d, J = 8.2 Hz, 1 H), 5.51 - 5.24 (m, 2H), 4.72 - 4.62 (m, 1 H), 4.49 (s, 1 H), 4.41 (s, 1 H), 4.31 (s, 3H), 4.07 (s, 2H), 3.85 (s, 1 H), 3.43 (s, 1 H), 3.23 (s, 1 H), 2.67 (s, 2H).
Note: Fractions containing the t-butylthio adduct were collected and after standing for 3 days the t-butylthio adduct converted to (CDNI-3)
Example 2-4: Synthesis of (CDNI-4)
Step 2 ep
Step 1 : Di-t-butyl dicarbonate (4.26 g, 19.5 mmol) was added dropwise over 10 minutes to a mixture of 4-(methylamino)butyric acid hydrochloride (2.0 g, 13.0 mmol) in MeOH (25 mL) and Et3N (7.26 ml_,52.1 mmol). The reaction mixture was stirred at room temperature for 22 hrs and then concentrated. The residue was dissolved in EtOAc (100 mL), and washed with an ice-cold 0.1 N HCI solution (20.0 mL). The organic layer was then washed with water to neutral pH, and then washed with sat. NaCI. The EtOAc layer was dried over Na2S04 and concentrated to give 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (2.08 g, 70%). 1 H NMR (500 MHz, Chloroform-d) δ 3.28 (t, J = 6.9 Hz, 2H), 2.84 (s, 3H), 2.35 (t, J = 7.2 Hz, 2H), 1 .84 (p, J = 7.1 Hz, 2H), 1 .45 (s, 9H).
Step 2: A solution of dicyclohexylcarbodiimide (704 mg, 3.41 mmol) in 10 ml of anhydrous DCM was added drop wise under N2 to a flask containing 4-((tert- butoxycarbonyl)(methyl)amino)butanoic acid (1.43 g, 6.56 mmol) in anhydrous DCM (20 ml). The mixture was stirred for 2 hrs and then concentrated to about 15 mL, filtered and the solvent removed under vacuum. The crude was filtered through 0.45 micron filter twice to yield 4-((tert- butoxycarbonyl)(methyl)amino)butanoic anhydride as a clear pale yellow oil (1 .36 g, 99% yield). 1H NMR (500 MHz, Chloroform-d) δ 3.28 (t, J = 6.9 Hz, 2H), 2.84 (s, 3H), 2.46 (t, J = 7.3 Hz, 2H), 1 .87 (p, J = 7.2 Hz, 2H), 1 .45 (s, 9H).
Step 3: 4-((tert-butoxycarbonyl)(methyl)amino)butanoic anhydride (152.0 mg, 0.366 mmol) in DMF (1 .6 mL) was added to Compound (T1 -2) (63.1 mg, 0.091 mmol) in pyridine (0.8 mL). The reaction mixture was stirred at room temperature for 3 days and then the solvent was removed. The residue was purified by reverse phase ISCO using C18 column, 50 g aq column, eluted with 5-50 % MeCN/ water (containing 10 mM Et3N HOAc), . Fractions containing desired boc protected monoadduct were collected and lyophilized (45.3 mg, 56 % yield). LCMS
M+1 =890.20, tr= 0.787 min.
Step 4: TFA (2 mL) was added to a flask containing 4-methylbenzenethiol sodium salt and the mixture was stirred until complete dissolution and then added to another flask containing the boc protected monoadduct from step 3. TFA was immediately removed and the mixture was then dissolved in DMSO and purified by reverse phase ISCO C18 column, 15 g C18 aq column, eluted with 2-20% acetonitrile-^O containing 0.05% TFA. Fractions containing desired product were concentrated to obtain (CDNI-4) (35.0 mg, 89% yield) as TFA salt. LCMS M+1 =790.2, tr= 0.220 min.
Example 2-5: Synthesis of (CDNI-5)
Step 1 : a) Compound (T1 -2) (20 mg, 0.028 mmol) ammonium salt was dissolved in 5 ml pyridine and 0.06 ml Et3N was then added. The mixture was concentrated and the process repeated twice to obtain the Compound (T1 -2) triethylammonium salt.
b) A solution of tert-butyl (2-hydroxyethyl)(methyl)carbamate (84 mg, 0.44 mmol) in
DCM (3 ml) with pyridine (0.072 ml_, 0.88 mmol) was added to a solution of 15% phosgene solution in toluene (0.88 ml) in DCM (10 ml) at -78°C. The mixture was stirred for 15 mins, then warmed to room temperature and concentrated to give 1-((tert- butoxycarbonyl)(methyl)amino)propan-2-yl carbonochloridate.
Step 2: Compound (T1 -2) Et3N salt was resuspended in anhydrous pyridine (1 ml) and then added to 1-((tert-butoxycarbonyl)(methyl)amino)propan-2-yl carbonochloridate. The mixture was stirred for 30 mins and then water was added. The mixture was concentrated, dissolved in DMSO-water and purified by reverse phase ISCO using C18 column, 15.5 g aq column, eluted with 2-40% acetonitrile-H20 containing 10 mM Et3N HOAc. Fractions containing desired Boc protected monoadduct were collected and lyophilized (33 mg, 43 % yield). M+1 =906.1 , tr=0.785 min.
Step 3: TFA (2 mL) was added to a flask containing 4-methylbenzenethiol sodium salt and the mixture was stirred until complete dissolution and then added to another flask containing the boc protected monoadduct from step 3 (33 mg, 0.030 mmol. TFA was immediately removed and the mixture was then dissolved in DMSO and purified by reverse phase ISCO using 15.5 g C18 aq column, eluted with 2-20% acetonitrile-h^O containing 0.05% TFA. Fractions containing desired product were concentrated to obtain (CDNI-5) (21 mg, 55 % yield) as TFA salt. LCMS M+1 =806.0, tr= 0.586 min. Example 2-6: Synthesis of (CDNI-6)
Intermediate (CDNI-6) was prepared using the methods described for the synthesis of intermediate (CDNI-3), except Compound (T1 -5) was used in place of Compound (T1 -2). Intermediate (CDNI-6) (25.6 mg, 66.8 % yield) as TFA salt. LCMS M+1 =794.1 , tr= 0.518min.
Example 2-7: Synthesis of (CDNI-7)
Intermediate (CDNI-7) was prepared using the methods described for the synthesis of intermediate (CDNI-4), except Compound (T1 -5) was used in place of Compound (T1 -2). Intermediate (CDNI-7) (10.0 mg, 8% yield) as TFA salt. LCMS M+1 =792.2, tr= 0.381 min.
Intermediate (CDNI-8) was prepared using the methods described for the synthesis of intermediate (CDNI-3), except Compound (T1 -3) was used in place of Compound (T1 -2).
Example 2-9: Synthesis of (CDNI-9a) and of (CDNI-9b):
a) Synthesis of (CDNI-9a):
Intermediate (CDNI-9a) was prepared using the methods described for the synthesis of intermediate (CDNI-3), except Compound (T1 -6) was used in place of Compound (T1 -2). Intermediate (CDNI-9a) (32.1 mg, 39.0% yield) (LCMS M+1 =796.0, tr= 0.406 min).
However, Step 1 for the preparation of 2-((tert-butoxycarbonyl)(methyl)amino)ethyl carbonochloridate was modified as follows:
Tert-butyl (2-hydroxyethyl)(methyl)carbamate (175 mg, 0.736 mmol) and K2CO3 (43 mg, 0.626 mmol) were added to a flask under N2., and then toluene (10 mL) was added and the mixture cooled to -15 °C. The mixture was stirred and a solution of phosgene in toluene (1 .1 mmol, 15% in toluene) was added dropwise. The mixture was stirred at low temperature (-15°C to 0°C) for an additional 30 mins, warmer to room temperature and stirred for another hour. The mixture was filtered by syringe filter (0.45 micron pore), and the solvent was removed to give 2-((tert- butoxycarbonyl)(methyl)amino)ethyl carbonochloridate as a clear pare yellow oil which was used directly without further purification.
b) Synthesis of (CDNI-9b):
Intermediate (CDNI-9b) was also obtained during the synthesis of Intermediate (CDNI-9a). CDN intermediate (CDNI-9a) and CDN intermediate (CDNI-9b) could not separated. (CDNI-9a). CDN intermediate (CDNI-9a) and CDN intermediate (CDNI-9b) (32.1 mg, 39.0% yield) (LCMS M+ 1 =796.0, tr= 0.406 min).
Example 2-10: Synthesis of (2R,3R,3aR,5R,7aR,9R, 10R,10aR,12R, 14aR)-2-(6-amino-9H- purin-9-yl)-9-(6-((3-aminopropyl)amino)-9H-purin-9-yl)-3, 10-difluoro-5, 12- dimercaptooctahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine 5, 12-dioxide (CDNI-10)
Step 1 : HOAc (0.020 ml) and tert-butyl (3-oxopropyl)carbamate (10 mg, 0.058 mmol) were added to a suspension of Compound (T1-1) (5 mg, 0.0056 mmol) in MeOH (1 ml) and the mixture was heated to 50°C for 16 hours (LCMS showed slow imine formation M+1 850.2 tr=0.680min) NaBH3CN (0.35 mg, 0.0056 mmol) was then added and the reaction was stirred at room temperature for 2 hours. LCMS indicated -25% conversion. M+1 = 852.1 tr= 0.708 min. An additional 5 mg of tert-butyl (3-oxopropyl)carbamate was added and the mixture was heated at 50°C for 2 hours, followed by addition of 5 mg NaBH3CN. The mixture was stirred for 1 hour and conversion monitored by LCMS. The process of adding 5 mg additional tert-butyl (3- oxopropyl)carbamate and 5 mg additional NaBH3CN was repeated until ~50% conversion was achieved. The mixture was concentrated, the residue dissolved in 2 ml MeOH and purified by mass triggered reverse phase HPLC, using C18 column, eluted with 13-29% acetonitrile-H20 containing 0.05% TFA. Fractions containing desired product were concentrated to obtain tert- butyl (3-((9-((2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12R, 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-purin-6-yl)amino)propyl)carbamate as TFA salt. LCMS M+1 = 852.1 tr= 0.695 min.
Step 2: tert-butyl (3-((9-((2R,3R,3aR,5R,7aR,9R,10R, 10aR, 12R, 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-purin-6-yl)amino)propyl)carbamate (1 mg, 0.001 mmol) was treated with TFA (1 ml) and was immediately concentrated. H20 and ACN (1 :1) was added and the sample was lyophilized to give
(2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12R, 14aR)-2-(6-amino-9H-purin-9-yl)-9-(6-((3- aminopropyl)amino)-9H-purin-9-yl)-3,10-difluoro-5,12-dimercaptooctahydro-2H,7H-difuro[3,2- d:3',2'-j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine 5, 12-dioxide (0.9 mg, 30 % yield) as TFA salt. LCMS M+1 =748.0, tr= 0.227 min.
Example 2-11 :
a) Synthesis of ((2S,4S)-4-fluoropyrrolidin-2-yl)methyl (9-
((2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12S, 1 aR)-9-(6-amino-9H-purin-9-yl)-3, 10-difluoro-5, 12- dimercapto-5, 12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-
Step 1 : Compound (T1 -6) Et3N salt (224 mg, 0.25 mmol) with pyridine (88 uL, 7.0 equiv) in NMP (0.5 mL) and DCM (1 .5 mL) was added to (2S,4S)-tert-butyl 2- (((chlorocarbonyl)oxy)methyl)-4-fluoropyrrolidine-1 -carboxylate (LI-6) in DCM (1.5 mL) over 5 minutes. The mixture was stirred at room temperature for one hour. Water was added to the reaction and it was stirred for another 10 mins and then concentrated. The mixture was suspended in DMSO and purified by ISCO using 15.5g C18 aq column, eluted with ACN-water 5-50%, aq phase containing 10 mM HOAc-Et3N to give the diadduct, di-tert-butyl 5,5'- (((((((2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12S, 14aR)-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]diphosphacyclododecine-2,9- diyl)bis(9H-purine-9,6-diyl))bis(azanediyl))bis(carbonyl))bis(oxy))bis(methylene))(3S,3'S,5S,5'^ bis(3-fluoropyrrolidine-1-carboxylate), (149.5 mg). LCMS M+1 =1 185.1 , tr=0.944 min.
Step 2: The diadduct (149.5 mg) from step 1 was dissolved in ACN (5 ml) and then water (10 ml) was added, followed by 0.6 g NaOH. The mixture was stirred at 50 °C for 4 hours, then neutralized with 4M HCI and then concentrated. The residue was purified by reverse phase ISCO, C18 column, eluted with 10-50 acetonitrile-h^O containing 10 mM Et3N HOAc to give the protected monoadduct, tert-butyl (2S,4S)-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-purin-6- yl)carbamoyl)oxy)methyl)-4-fluoropyrrolidine-1 -carboxylate, (32.0 mg). LCMS +1 =940.1 , tr=0.750 min.
Step 3: TFA (2.0 ml) was added to a flask containing monoadduct from step 2 (32.0 mg, 0.028 mmol) and the mixture was stirred for 2 mins and then concentrated. The residue was dissolved in DMSO and purified by ISCO using C18 aq column, eluted with 5-30% ACN-water containing 0.05% TFA to give ((2S,4S)-4-fluoropyrrolidin-2-yl)methyl (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-purin-6-yl)carbamate (CDNI-11a) (13.1 mg, 44.0 % yield) (LCMS M+1 =840.0, tr=0.407 min).
b) Synthesis of ((2S,4S)-4-fluoropyrrolidin-2-yl)methyl (9-
((2R,3R,3aR,5S,7aR,9R, 10R, 10aR, 12R, 14aR)-9-(6-amino-9H-purin-9-yl)-3, 10-difluoro-5, 12- dimercapto- j][1 ,3,7,9]tet (CDNI-11 b)
Intermediate (CDNI-11 b) was also obtained during the synthesis of Intermediate (CDN a). CDN intermediate (CDNI-11a) and CDN intermediate (CDNI-11 b) could not separated. (CDNI- 1a). CDN intermediate (CDNI-11a) and CDN intermediate (CDNI-9b) (13.1 mg, 44.0 % yield) (LCMS M+1 =840.0, tr=0.407 min).
Example 2-12: Synthesis of N-(9-((2R,3R,5R,7aR,9R,10R, 12R,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-purin-6-yl)-4- (methylamino)butanamide (CDNI-12)
Step 1 : 4-((tert-butoxycarbonyl)(methyl)amino)butanoic anhydride (241 mg, 0.580 mmol) was added to a solution of Compound (T1-1) Et3N salt (40 mg, 0.045 mmol) in pyridine (5 ml) and heated to 50°C and stirred for 72 hours. DMAP (10 mg) and 50 mg more anhydride were added and the reaction was stirred at 50°C for 8 hours and then concentrated and purified using reverse phase ISCO with 15 g C18 aq column, eluted with 5-45% acetonitrile-h^O (aqueous phase containing 10 mM Et3N HOAc). Fractions containing desired product were collected and lyophilized to obtain boc-protected intermediate CDNI-12 as an Et3N salt (8 mg, 16 % yield). LCMS M+1 = 894.0, tr = 0.776 min.
Note: 4-((tert-butoxycarbonyl)(methyl)amino)butanoic anhydride was synthesized as described in the synthesis of CDNI-4.
Step 2: TFA (1 ml) was added to a flask containing boc-protected intermediate CDNI-12 Et3N salt (8 mg, 0.007 mmol) and then immediately concentrated. The residue was purified by reverse phase ISCO using 15 g C18 column, eluted with 5-45% acetonitrile-h^O containing 0.05% TFA. Fractions containing desired product were concentrated to obtain intermediate CDNI-12 as a TFA salt (3.7 mg, 49.6 % yield). LCMS M+1 = 794.0, tr= 0.636 min.
Example 2-13: Synthesis of 4-amino-N-(9-((2R,3R,5R,7aR,9R, 10R, 12R, 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-purin-6- yl)butanamide (CDNI-13)
Step 1 : 4-((tert-butoxycarbonyl)amino)butanoic anhydride (LI-8) was added to a solution of Compound (T1 -1 ) Et3N salt (30 mg, 0.033 mmol) in pyridine (5 ml) (390 mg, 1.00 mmol) and heated at 50°C for 3 days. The reaction mixture was then concentrated and the crude was purified by reverse phase ISCO using15 g C18 column, eluted with 5-60% acetonitrile-h^O
(aqueous phase containing 10 mM Et3N HOAc). Fractions containing the desired product were isolated and concentrated to obtain boc-protected intermediate CDNI-13 as Et3N salt (10 mg, 28% yield). LCMS M+1 =880.1 , tr= 0.731 min.
Step 2: TFA (2 ml) was added to a flask containing boc-protected intermediate CDNI-12 Et3N salt (10 mg, 0.009 mmol) and immediately concentrated. The crude was purified by reverse phase ISCO using 15g C18 aq column, eluted with 5-60% acetonitrile-h^O containing 0.05%
TFA. Fractions containing the desired product were combined and lyophilized to obtain intermediate CDNI-13 as TFA salt (1 1 .2 mg, 96% yield). LCMS M+1-H2O=762.0, tr=0.608 min.
Example 2-14: Synthesis of tert-butyl ((S)-1 -((4-((9- ((2R,3R,3aR,5R,7aR,9R, 10R,10aR,12R, 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-purin-6-yl)amino)-4- oxobutyl)amino)-1 -oxo-5-ureidopentan-2-yl)carbamate (CDNI-14)
Step 1 : To a solution of (S)-2-((tert-butoxycarbonyl)amino)-5-ureidopentanoic acid (Boc-Cit-OH purchased from Bachem) (2.7 mg, 0.01 mmol) in D F (1 ml) was added DIEA (0.017 ml_, 0.10 mmol) and then HATU (3.8 mg, 0.01 mmol). The reaction mixture was stirred at rt for 5 mins and then was added to a solution of CDN intermediate (CDNI-13) TFA salt (10 mg, 0.01 mmol) in DMF and this mixture was stirred at rt for 5 hrs and then concentrated. The residue was purified by reverse phase ISCO using 15g C18 aq column, eluted with 5-40% acetonitrile-h^O
(aqueous phase containing 10 mM Et3N HOAc). Fractions containing desired product were concentrated to obtain boc-protected intermediate CDNI-14 as an Et3N salt (2.9 mg, 24 % yield). LCMS M+1 =1037.1 , tr= 0.699 min.
Step 2: TFA (1 ml) was added to a flask containing boc-protected intermediate CDNI-14 Et3N salt (2.9 mg, 0.0028 mmol) and the solution was stirred for 1 min and then concentrated to give CDN intermediate (CDNI-14) as TFA salt (2.9 mg, 100% yield). LCMS M+1 =937.1 , tr=0.598min.
Example 2-15: Synthesis of (S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((9-
((2R,3R,3aR,5R,7aR,9R, 10R,10aR,12R, 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-purin-6-yl)amino)-4- oxobutyl)-5-ureidopentanamide (CDNI-15)
Step 1 : To a vial containing (tert-butoxycarbonyl)-L-valine (Boc-Val-OH purchased from
Novabiochem) (1 .2 mg, 0.0056 mmol) was added DMF (1 ml) and then HATU (2.1 mg, 0.0056 mmol) and DIEA (3.6 mg, 0.028 mmol) were added. The mixture was stirred for 2 mins and then added to a solution containing intermediate CDNI-14 TFA salt (2.9 mg, 0.0028 mmol) in DMF (1 ml). The reaction was stirred at rt for 1 day and then concentrated. The residue was purified by reverse phase ISCO using 15 g C18 aq column, eluted with 5-40% acetonitrile-h^O (aqueous phase containing 10 mM Et3N HOAc). Fractions containing desired product were concentrated to obtain boc-protected intermediate CDNI-15 as Et3N salt (1 .8 mg, 48 % yield). LCMS
M+1 =1 136.2, tr=0.791 min.
Step 2: TFA (1 ml) was added to a flask containing boc-protected intermediate CDNI-15 Et3N salt (1 .8 mg, 0.0013 mmol) and the soluton was stirred for 1 min and then concentrated to give intermediate CDNI-15 as TFA salt (1 .7 mg, 100%). The compound was used in the next step without further purification. LCMS M+1 =1036.1 , tr=0.621 min.
Example 2-16: Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((2-(((9-
((2R,3R,3aR,5R,7aR,9R, 10R,10aR,12R, 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-purin-6- yl)carbamoyl)oxy)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(3- aminopropanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (CDNI-16)
Step 1 : To a solution of intermediate CDNI-1 TFA salt (15 mg, 0.015 mmol) and
(2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((4- nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (see Bioconjugate Chem. 2006, 17, 831 -840) (16 mg, 0.018 mmol) in DMF (1 ml) was added DIEA (0.026 ml, 0.15 mmol) and HOAT (2.0 mg, 0.015 mmol). The reaction was stirred at rt for 16 hrs. Solvent was then removed by high vacuum and the crude was purified by reverse phase ISACO using 15g C18 column, eluted with 5-60% acetonitrile-H20 (aqueous phase containing 10 mM Et3N HOAc). Fractions containing desired product were concentrated to obtain Fmoc protected intermediate CDNI-16 as Et3N salt (20.2 mg, 78 % yield). LCMS
M/2+1 =785.8, tr=1 .094 min.
Step 2: A solution of LiOH (9.3 mg, 0.388 mmol) in water was added to a vial containing Fmoc protected intermediate CDNI-16 (20.2 mg, 0.01 1 mmol) Et3N salt and MeOH (4 ml.) and the mixture was stirred at rt for 16 hrs. It was then neutralized with HOAc and concentrated. The crude was purified by reverse phase ISCO using 43g C18 aq column, eluted with 5-35% acetonitrile-H20 (aqueous phase containing 10 mM Et3N HOAc). Fractions containing desired product were concentrated to obtain intermediate CDNI-16 as Et3N salt (23.2 mg, 135 % yield). LCMS M+1 =1207.9, tr= 0.81 1 min.
Example 2-17: Synthesis of ((2S,4S)-4-fluoropyrrolidin-2-yl)methyl (9- ((2R,3R,3aR,5R,7aR,9R, 10R,10aR,12R, 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-purin-6-yl)carbamate
Intermediate (CDNI-17) was synthesized using the method described for CDNI intermediate (CDNI-11 ), except Compound (T1 -6) Et3N salt was replaced with Compound (T1 -1 ) Et3N salt.
Example 2-18: Synthesis of 2-azidoethyl (9-((2R,3R,3aR,5R,7aR,9R,10R, 10aR, 12R, 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- purin-6-yl)carbamate (CDNI-18)
Step 1 : A solution of diphosgene (275 mg, 1 .41 mmol) was added to a solution of 2- azidoethanol (87 mg, 1 .00 mmol) in DCM (10 ml) at -78°C and the mixture was slowly warmed to rt. After 15 mins the solution became clear. The reaction was concentrated and solvent and other volatile reagents were removed under vacuum to obtain 2-azidoethyl carbonochloridate which was used in step 2 without further purification.
Step 2: 2-azidoethyl carbonochloridate (149mg, LOOmmol) in DCM (1 ml) was added in portions over 30 mins to Compound (T1 -1 ) Et3N salt (30 mg, 0.033 mmol) dissolved in pyridine (2 ml). Then Et3N (0.03 ml) was added and the mixture was stirred at rt for 2 hrs. The solution was concentrated and water and acetonitrile were then added. 1 N NaOH (5ml) was then added and the reaction was stirred at 60°C for 2 hrs, Both mono- and diadduct were formed. The reaction was neutralized with HOAc, concentrated and then suspended in DMSO and purified by reverse phase ISCO using 43g C18 aq column, eluted with 5-35%, acetonitrile-water (aqueous phase containing 10mM Et3N HOAc). Fractions containing mono-adduct were collected and concentrated to give CDNI intermediate (CDNI-18) as Et3N salt (20mg, 45% yield). LCMS M+1 =808.0, tr= 0.764 min.
Example 2-19: Synthesis of N-(9-((2R,3R,3aR,5R,7aR,9R, 10R,10aR, 12R, 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-purin-6- yl)-4-azidobutanamide (CDNI-19)
Step 1 : 4-azidobutanoic acid (259 mg, 2.01 mmol) was dissolved in DCM (5ml) and oxalyl chloride (190 mg, 1 .5 mmol) was added, followed by DMF (0.005 ml). The reaction was stirred at rt for 1 hr, and then concentrated to obtain 4-azidobutanoyl chloride, which was used in the next step without further purification.
Step 2: 4-azidobutanoyl chloride (94 mg, 0.64 mmol) was dissolved in DCM (0.32 ml) and added to a solution of di-2'-F-RR-CDA (R277) Et3N salt (30 mg, 0.033 mmol) in pyridine (3ml). The reaction was stirred at 70°C for 0.5 h and then quenched with 2 drops of water and concentrated. The crude was purified by reverse phase ISCO using 50g C18 aq column, eluted with 5-50% acetonitrile-H20 (aqueous phase containing 10 mM Et3N HOAc). Fractions containing the desired product were isolated and lyophilized to give CDN intermediate (CDNI- 19) as Et3N salt (19.7 mg, 58.4% yield). LCMS +1 =806.0, tr= 0.807min.
Example 2-20: Synthesis of 3-azidopropyl (9-((2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12R, 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- purin-6-yl)carbamate (CDNI-20)
CDN intermediate (CDNI-20) was synthesized using the method described for the synthesis of CDN intermediate (CDNI-18) except 3-azidopropan-1 -ol was used in place of 2-azidoethanol. CDN intermediate (CDNI-20) Et3N salt (16.3mg, 47% yield). LCMS M+1 =822.0, tr=0.830 min.
Example 2-21 : Synthesis of a mixture of 4-amino-N-(9-((2R,3R,5S,7aR,9R, 10R, 12R, 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 purin-6-yl)butanamide (CDNI-21a) and N-(9-
((2R,3R,5R,7aR,9R,10R, 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-purin-6-yl)-4- (methylamino)butanamide (CDNI-21 b)
Step 1 : NaH (60% dispersion in oil, 38.5mg, 0.962 mmol) was added to a solution of Compound (T1 -6) Et3N salt (86.3 mg, 0.096 mmol) in DMF (3ml) and the mixture was stirred for 1 min before the addition of 4-((tert-butoxycarbonyl)amino)butanoic anhydride (347 mg, 0.894 mmol). The reaction was stirred at rt for 1 hr and then quenched with HOAc (0.2 ml). The reaction was concentrated and purified using reverse phase ISCO with 15 g C18 aq column, eluted with 5- 45% acetonitrile-H20 (aqueous phase containingl O mM Et3N HOAc). Fractions containing desired product were collected and lyophilized to obtain boc protected CDN intermediate (CDNI- 21a) and boc protected CDN intermediate (CDNI-21 b) as Et3N salt (20 mg, 19% yield). LCMS M+1 = 880.0, tr= 0.782 min. The mixture was not separated. Note: 4-((tert- butoxycarbonyl)(methyl)amino)butanoic anhydride was synthesized as described in the synthesis of CDNI-4.
Step 2: To a flask containing boc protected CDN intermediate (CDNI-21a) and boc protected CDN intermediate (CDNI-21 b) Et3N salt (20 mg, 0.018 mmol) was added acetonitrile (5 ml) and TFA (1 ml) and the mixture was stirred for 30 mins and then concentrated. The residue was purified by reverse phase ISCO using 15 g C18 column, eluted with 5-50% acetonitrile-h^O containing 0.05% TFA. Fractions containing desired product were concentrated to obtain a mixture of CDN intermediate (CDNI-21a) and CDN intermediate (CDNI-21 b) as TFA salt (13.4 mg, 72 % yield). LCMS M+1 -H20= 762, tr= 0.268 min.
Example 2-22: Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5- ureidopentanamido)benzyl (2S,4S)-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-purin-6- yl)carbamoyl)oxy)methyl)-4-fluoropyrrolidine-1 -carboxylate (CDNI-22a) and 4-
((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (2S,4S)-2-((((9-((2R,3R,3aR,5S,7aR,9R, 10R, 10aR, 12R, 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-purin-6- yl)carbamoyl)oxy)methyl)-4-fluoropyrrolidine-1 -carboxylate (CDNI-22b)
Stepl : Fmoc-Val-Cit-PABC-PNP (25.2 mg, 0.033 mmol) was added to a solution of CDN intermediate (CDNI-11a) and (CDNI-11 b) (31 .1 mg, 0.030 mmol) in DMF (1 ml), followed by the adition of DIEA (26.0 uL, 19.3 mg, 0.149 mmol) and HOAT (4.1 mg, 0.030 mmol). The reaction was stirred at rt overnight, water (1 .0 mL) was then added and the solution concentrated. The residue was dissolved in DMSO and purified by ISCO by using 50.0 g C18 aq column, eluted with 5-60% ACN in water with 10 mM TEA-HOAc. Fractions containing desired product were concentrated to obtain compound Fmoc protected CDN intermediate (CDNI-22a and CDNI-22b) (42.2 mg, 80 % yield) as TEA salt. LCMS M/2+1 =734.30, tr=1.002 min.
Step2: Piperidine (180.0 uL, 0.19 mmol) was added to a solution of Fmoc protected CDN intermediate (CDNI-22) (32.0 mg, 0.019 mmol ) TEA salt in DMF (Volume: 3.0 mL) and the mixture was stirred at rt for 30 mins and then concentrated. The residue was purified by reverse phase ISCO 50 g C18 aq column, eluted with 5-35% acetonitrile-H20 containing 0.05% TFA.
Fractions containing desired product were concentrated to obtain CDN intermediate (CDNI-22a and CDN22b) (20.0 mg, 67.8%) as TFA salt. LCMS M/2+ 1 =623.3, tr=0.790min.
Example 2-23: Synthesis of 2-(methylamino)ethyl (9- ((1 S,3R,6R,8R,9S, 1 1 R, 14R, 16R, 17R, 18R)-16-(6-amino-9H-purin-9-yl)-17, 18- difluoro-3, 1 1 -dimercapto-3, 1 1 -dioxido-2,4,7, 10,12, 15-hexaoxa-3, 1 1 - diphosphatricyclo[12.2.1.16,9]octadecan-8-yl)-9H-purin-6-yl)carbamate (CDNI- 23)
CDN intermediate (CDNI-23) was synthesized using the method described for the synthesis of CDN intermediate (CDNI-1 ) except Compound (T1-1) Et3N salt was replaced with Compound (T2-46) Et3N salt.
Boc-protected CDN intermediate (CDNI-23): LCMS M+ 1 =796.0, tr=0.625 min. 1H NMR (500 MHz, DMSO-d6) δ 10.71 (s, 1 H), 9.36 (d, J = 6.1 Hz, 2H), 8.92 (s, 1 H), 8.73 (s, 2H), 8.39 (s, 1 H), 6.27 (dd, J = 44.7, 8.4 Hz, 2H), 5.79 - 5.33 (m, 4H), 4.75 - 4.55 (m, 3H), 4.38 (s, 1 H), 4.00 (dd, J = 12.5, 5.4 Hz, 4H), 3.35 (dd, J = 10.3, 6.4 Hz, 1 H), 3.25 (s, 1 H), 3.12 (tt, J = 7.4, 3.7 Hz, 1 H).
CDN intermediate (CDNI-23) TFA salt (8.2 mg, 55.0 % yield). LCMS M+1 =796.0, tr=0.625 min. 1H NMR (500 MHz, DMSO-cf6) δ 10.71 (s, 1 H), 9.36 (d, J = 6.1 Hz, 2H), 8.92 (s, 1 H), 8.73 (s, 2H), 8.39 (s, 1 H), 6.27 (dd, J = 44.7, 8.4 Hz, 2H), 5.79 - 5.33 (m, 4H), 4.75 - 4.55 (m, 3H), 4.38 (s, 1 H), 4.00 (dd, J = 12.5, 5.4 Hz, 4H), 3.35 (dd, J = 10.3, 6.4 Hz, 1 H), 3.25 (s, 1 H), 3.12 (tt, J = 7.4, 3.7 Hz, 1 H).
Example 2-24: Synthesis of (2R,3R,3aR,5R,7aR,9R, 10R,10aR,12S, 14aR)-2-(2-amino-6-oxo- 1 ,6-dihydro-9H-purin-9-yl)-9-(6-amino-9H-purin-9-yl)-10-fluoro-5,12- dimercapto-5, 12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecin-3-yl (2-(methylamino)ethyl) carbonate (CDNI-24)
CDN intermediate (CDNI-24) was synthesized using the method described for the synthesis of CDN intermediate (CDNI-3) except Compound (T1-2) Et3N salt was replaced with Compound (T1-13) Et3N salt.
Boc-protected CDN intermediate (CDNI-24): LCMS M+1=910.1 , tr=0.731 min.1H NMR (500 MHz, Methanol-c/4) δ 8.46 (s, 1H), 8.20 (d, J= 7.6 Hz, 2H), 6.36 (d, J= 17.1 Hz, 1H), 6.07 (d, J = 11.8 Hz, 1H), 5.77-5.56 (m, 2H), 5.34 (s, 1H), 5.24-5.04 (m, 1H), 4.60 (dt, J = 12.3, 2.7 Hz, 1H), 4.42 (d, J = 10.2 Hz, 3H), 4.32 (d, J= 8.0 Hz, 3H), 4.08 - 3.95 (m, 2H), 3.64 (t, J = 5.9 Hz, 5H), 3.58 (s, 2H), 3.03 (q, J= 7.3 Hz, 31 H), 2.96 (s, 4H), 2.92 (s, 9H), 1.22 (t, J= 7.3 Hz, 42H). CDN intermediate (CDNI-24) TFA salt (8.1 mg, 71.7 % yield). LCMS M+1=810.2, tr=0.346 min. 1H NMR (500 MHz, DMSO-c/6) δ 10.80 (s, 1H), 9.36 (d, J= 42.0 Hz, 2H), 8.48 (d, J= 45.8 Hz, 2H), 8.27 (s, 1H), 6.70 (s, 2H), 6.41 (d, J= 16.4 Hz, 1H),6.06 (d, J= 7.3 Hz, 1H), 5.70-5.38 (m, 2H), 5.16 (dtd, J = 26.2, 9.3, 4.6 Hz, 1 H), 4.90 (ddd, J = 11.5, 5.4, 2.9 Hz, 1H), 4.59 (ddd, J = 12.9,6.7,2.4 Hz, 1H), 4.40 (dd, J= 11.4, 5.3 Hz, 2H), 4.26 (ddd, J= 17.0, 8.5, 5.9 Hz, 1H), 4.23- 4.06 (m, 1H), 3.92 - 3.71 (m, 2H), 3.43 - 3.17 (m, 2H), 3.13 (td, J = 7.3, 4.8 Hz, 1H),2.67 (t, J= 5.2 Hz, 3H).
Example 2-25: Synthesis (2R,3R,3aR,5R,7aR,9R,10R,10aS,12R,14aR)-2,9-bis(2-amino-6-oxo- 1,6-dihydro-9H-purin-9-yl)-10-hydroxy-5,12-dimercapto-5,12-dioxidooctahydro- n-3-yl (2-
CDN intermediate (CDNI-24) was synthesized using the method described for the synthesis of CDN intermediate (CDNI-3) except Compound (T1-2) Et3N salt was replaced with Compound (T1-16) Et3N salt.
Boc-protected CDN intermediate (CDNI-25): LCMS M+1=924.2. tr=0.813 min. CDN intermediate (CDNI-25) TFA salt (5.9 mg, 46.2 % yield). LCMS M+1 = 824.0 tr= 0.410 min. 1H NMR (500 MHz, DMSO-cf6) δ 10.64 (d, J = 12.1 Hz, 1 H), 9.26 (d, J = 105.9 Hz, 1 H), 8.04 (d, = 5.7 Hz, 1 H), 6.59 (s, 2H), 5.96 (d, = 7.8 Hz, 1 H), 5.80 - 5.61 (m, 1 H), 4.81 (ddd, J = 72.1 , 9.8, 4.4 Hz, 1 H), 4.57 - 4.43 (m, 1 H), 4.29 - 3.88 (m, 3H), 3.28 - 2.97 (m, 1 H.
Example 2-26: Synthesis ((2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12S, 14aR)-2,9-bis(6-amino-9H- purin-9-yl)-10-fluoro-5,12-dimercapto-5, 12-dioxidooctahydro-2H,7H-difuro[3,2- d:3',2'-j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecin-3-yl D-prolinate (CDNI-26)
Step 1 : A solution of dicyclohexylcarbodiimide (0.51 eq) in 5 ml of anhydrous DCM is added under nitrogen drop wise, with stirring, to a solution of (R)-1 -(tert-butoxycarbonyl)pyrrolidine-2- carboxylic acid (purchased from Combi-Blocks) (2.152 g, 10 mmol) in anhydrous
dichloromethane (45 ml). The solution was stirred for 150 min and the resulting urea precipitate was removed by filtration and the filtrate was concentrated to about 5ml, and then filtered through syringe filter. The solvent was removed under vacuum to give (R)-1-(tert- butoxycarbonyl)pyrrolidine-2-carboxylic anhydride as a sticky oil (2.169 g, 100% yield).
Step 2 : (R)-1 -(tert-butoxycarbonyl)pyrrolidine-2-carboxylic anhydride (501 mg, 1 .1 17 mmol) in NMP (3 mL) was added to Compound (T1 -20) sodium salt (55 mg, 0.074 mmol) in pyridine (1 .5 mL) and the mixture was stirred at rt for two days. n-Butylamine (0.1 mL) in water (1 .0 mL) was then added and the mixture was stirred at rt for 10 mins. The pyridine and water were then removed under vacuum and the NMP was removed by lyophilization. The crude was purified by reverse phase ISCO using 50g C18 aq column, eluted with 5-55% acetonitrile-H20 (aqueous phase containing 10 mM Et3N HOAc) to the boc-protected diadducts of CDN intermediate (CDNI-26). All diadducts were collected, dried by lyophilization. Step 3: The boc-protected diadduct was dissolved in MeOH (5 mL) in a 30 mL pressure vessel equipped with a Teflon valve. The vessel was placed in an oil bath heated at 1 10 °C for 5 hours. Volatiles were evaporated, and the residues was purified by reverse phase ISCO using 50g C18 aq. column, eluted with 5-55% acetonitrile-h^O (aqueous phase containing 10 mM Et3N HOAc). Fractions containing the desired product were combined and lyophilized to obtain boc-protected CDN intermediate (CDNI-26) as Et3N salt (18.9 mg). LCMS M+1 = 890.0, tr= 0.722 min.
Step4: To a vial containing boc-protected CDN intermediate (CDNI-26) Et3N salt (30.0 mg, 0.034 mmol) was added TFA (2 ml). The mixture was concentrated immediately and then concentrated. The crude was purified by reverse phase ISCO using 50g C18 column, eluted with 5-40% acetonitrile-H20 (aqueous phase containing 10 mM Et3N HOAc). Fractions containing desired product were concentrated to obtain CDN intermediate (CDNI-26) as TEA salt (12.4 mg, 37.1 % yield). LCMS M+1 = 790.1 , tr= 0.350 min.
The mixture of CDN Intermediate (CDNI-27a) and CDN Intermediate (CDNI-27b) was prepared using the methods described for the synthesis of intermediate (CDNI-3), except Compound (T1 - 56) was used in place of Compound (T1 -2), the reaction mixture of step was stirred for 2 hours instead of 30 mins and in step 1 purification used 5-50% acetonitrile-l-^O (aqueous phase containing 10 mM Et3N HOAc).
CDN Intermediate (CDNI-27a) and CDN Intermediate (CDNI-27b) as TEA salt (3.7 mg, 55.9 % yield). LCMS M+1 = 822.0, tr= 0.319 min.
Note: The mixture was not separated and 2-((tert-butoxycarbonyl)(methyl)amino)ethyl carbonochloridate was synthesized as described in the synthesis of CDNI-9 except the initial temperature was -30°C instead of -15°C.
Example 2-28: Synthesis (2R,3R,3aR,5S,7aR,9R,10R, 10aR, 12R, 14aR)-9-(2-amino-6-oxo-1 ,6- dihydro-9H-purin-9-yl)-2-(6-amino-9H-purin-9-yl)-10-fluoro-5,12-dimercapto- 5, 12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecin-3-yl (2-(methylamino)ethyl)
CDN intermediate (CDNI-28) was synthesized using the method described for the synthesis of CDN intermediate (CDNI-3) except Compound (T1-2) Et3N salt was replaced with Compound (T1 -11) Et3N salt, the reaction time in Step 2 was 2 hrs rather than 30 mins and purification of CDN intermediate (CDNI-28) was by reverse phase ISCO using 15g C18 column, eluted with 5- 40% acetonitrile-H20 (aqueous phase containing 10 mM Et3N HOAc).
Boc-protected CDN intermediate (CDNI-28) as Et3N salt (8.9 mg, 52.1 % yield). LCMS
M+1 =910.1 . tr=0.731 min..
CDN intermediate (CDNI-28) as TEA salt (6.5 mg, 62.4 % yield). LCMS M+1 = 810.0 tr= 0.350 min.
Example 2-29: Synthesis (2R,3R,3aR,7aR,9R, 10R, 10aR,14aR)-2-(6-((3-amino-2- hydroxypropyl)amino)-9H-purin-9-yl)-9-(6-amino-9H-purin-9-yl)-3, 10-difluoro- 5, 12-dihydroxyoctahydro-2H,7H-difuro[3,2-d:3',2'- j][1 ,3,7,9]tetraoxa[2,8]diphosphacyclododecine 5,12-dioxide (CDNI-29)
Step 1 : To a solution of Compound (T1-1) Et3N salt (30 mg, 0.033 mmol) in DMF (3 ml) was added tert-butyl (oxiran-2-ylmethyl)carbamate (57.9 mg, 0.334 mmol) and DIEA (43.2 mg, 0.334 mmol). The mixrure was heated to 100°C for 4 hours and the solvent was removed. The crude product was purified by reverse phase ISCO using 50g C18 aq column, eluted with 5-45% acetonitrile-H20 (aqueous phase containing 10 mM Et3N HOAc). Fractions containing boc protected CDN intermediate (CDNI-29) were isolated and lyophilized to obtain Boc protected CDN intermediate (CDNI-29) as Et3N salt (20mg, 58% yield). LCMS M+1 =836.0, tr =0.538 min.
Step 2: To a 25 ml round-bottom flask containing boc protected CDN intermediate (CDNI- 29) Et3N salt (20 mg, 0.019 mmol) was added TFA (1 ml, 13 mmol). The mixture was stirred for 1 min and then concentrated. The residue was purified by reverse phase ISCO using 50g C18 aq column, eluted with 5-35% acetonitrile-H20 (aqueous phase containing
10 mM Et3N HOAc). Fractions containing desired product were concentrated to obtain CDN intermediate (CDNI-29) as Et3N salt (11.1 mg, 62% yield). LCMS M+1= 736.0, tr= 0.235 min. Example 3: Synthesis of Exemplary Linker-Drug Compounds
Step 1 : Compound (T1 -2) (5 mg, 0.007 mmol) disodium salt was dissolved in anhydrous pyridine (1 ml) followed by the addition of Et3N (0.005 ml). The mixture was sonicated and then linker intermediate (LI-1) (30 mg, 0.068 mmol) was added. The reaction mixture was stirred for 30 mins at room temperature and monitored by LCMS. The mixture was concentrated and then dissolved in MeOH-water, followed by purification by mass triggered reverse phase HPLC, using C18 column, eluted with 5-55% acetonitrile-h^O containing 0.05% TFA. Fractions containing the desired boc-protected carbonate (2 mg, 22%) were collected LCMS M+1 =1 1 1 1 .1 , tr=0.898 min.
Step 2: TFA (1 ml) was added to a vial containing the carbonate from step 1 (2 mg, 0.0015 mmol) and then immediately concentrated. The residue was then dissolved in MeOH and purified by ISCO using 1 g C18 column, eluted with 5-50% ACN-water containing 0.05% TFA. Fractions containing the desired product were combined and lyophilized to give the de-protected carbonate (1.0 mg, 1 1 % yield) as TFA salt. LCMS M/2+1 =506.2, tr=0.669 min.
Step 3: DIEA (15 mg, 0.1 16 mmol) and then HATU (3.4 mg, 0.0089 mmol) were added to a solution of 3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)propanoic acid (Mal-PEG1-Acid) (1 .9 mg, 0.0089 mmol) in DMF (1 ml) and the reaction mixture was stirred at room temperature for 5 mins. 10% of this reaction mixture was then added to a flask containing the de-protected carbonate obtained in step 2 (1 .0 mg, 0.00089 mmol) in 0.5 ml DMF. The reaction was stirred at room temperature for 2 hours and then purified by mass-triggered reverse phase HPLC, using C18 column, eluted with 5-37% acetonitrile-h^O containing 0.05% TFA. The fractions containing desired product were concentrated to obtain Compound (C12) (0.7 mg, 57 % yield) as TFA salt. LCMS M+1 =1206.3, M/2+1 =603.7, tr=0.784 min.
Example 3-2: Synthesis of Compound 13 (C13)
18-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1 -yl)-5,5,9, 12-tetramethyl-8, 13-dioxo-16-oxa-3,4-dithia- 9, 12-diazaoctadecyl (4-nitrophenyl) carbonate (LI-2) (2.5 mg, 0.0039 mmol) and DIEA (0.013 ml_, 0.077 mmol) were added to a solution of CDN intermediate (CDNI-3) (3.5 mg, 0.0039 mmol) in DMF (1 ml) and the mixture was stirred at room temperature for 5 hours. The crude was purified by mass-triggered reverse phase HPLC, using C18 column, eluted with 20-33% acetonitrile-H20 containing 0.05% TFA. Fractions containing desired product were concentrated to compound A2 (2.2 mg, 38.1 % yield) as TFA salt. LCMS M/2+ 1 =654.2, tr= 0.799 min.
CDN intermediate (CDNI-3) ( (7.4 mg, 0.0073 mol) TFA salt was dissolved in anhydrous DMF (2ml) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (MC-vc-pab-PNP purchased from Levena Biopharma, San Diego) (6.3 mg, 0.009 mmol) was added, followed by addition of DIEA (1 1 mg, 0.084 mmol) and HOAT (4 mg, 0.029 mmol). The mixture was stirred at room temperature for 3 days and monitored by LCMS until completion of the reaction. The mixture was then purified by mass triggered reverse phase HPLC, using C18 column, eluted with 5-35% acetonitrile-H20 containing 0.05% TFA. Fractions containing the desired product were combined and concentrated to obtain Compound (C14) (3.6 mg, 25.8 % yield) as a TFA salt. LCMS M/2+ 1 = 695.8, tr=0.783 min.
Example 3-4: Synthesis of Compound 15 (C15)
CDN intermediate (CDNI-4) (13.5 mg, 0.015 mmol) TFA salt in DMF was added to a solution of linker intermediate (LI-3) (10.5 mg, 0.015 mmol, 1 .0 equiv), followed by the addition of DIEA (7.75 mg, 0.060 mmol) and HOAT (2.45 mg, 0.018 mmol). The mixture was stirred at room temperature for 16 hrs and then concentrated. The residue was dissolved in DMSO and purified by ISCO by using 15.5 gram, C18 aq column, eluted with 5-40% ACN in water with 10 mM TFA- HOAc. Fractions containing desired product were concentrated to obtain Compound (C15) (12.2 mg, 50% yield) as TEA salt. M+1 =1346.20, tr=0.732 min.
Example 3-5: Synthesis of Compound 16 (C16)
Compound (C16) was synthesized using the methods describe for the synthesis of Compound (C15), except CDN intermediate (CDNI-5) TFA salt was used in place of CDN intermediate (CDNI-4).
Compound (C16) (7.6 mg, 31.3 % yield) as TFA salt. LCMS M/2+1 =681.8, tr= 1 .025 min. Example 3-6: Synthesis of Compound 17 (C17)
TEA (6.7 mg, 0.066 mmol) and HATU (5.0 mg, 0.013 mmol) was added to a solution of 3-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1-yl)propanoic acid (2.2 mg, 0.013 mmol)) in DMF (1 ml.) and the mixture was stirred for 5 mins. CDN intermediate (CDNI-3) (15 mg, 0.013 mmol) in DMF (1 ml) was then added and the mixture was stirred for 18 hrs at room temperature and then concentrated. The residue was dissolved in DMSO (2 ml) and then purified by mass triggered reverse phase HPLC, using C18 column, eluted with 5-25% acetonitrile-H20 containing 0.05% TFA. Fractions containing desired product were lyophilized to obtain Compound (C17) (14.3 mg, 88 % yield) as TFA salt. LCMS M+1 =943.1 tr= 0.561 min.
Example 3-7: Synthesis of Compound 18 (C18)
CDN intermediate (CDNI-3) (20 mg, 0.018 mmol), DIEA (23 mg, 0.18 mmol) and HOAT (2.4 mg, 0.018 mmol) were added to a solution of linker intermediate (LI-3) (13.5 mg, 0.019 mmol) in DMF (1 mL) and the mixture was stirred for 18 hours at room temperature and then
concentrated. The residue was dissolved in DMSO (2 ml) and then was pre-purified by ISCO using 15.5 g C18 column, eluted with 5-35% ACN-water containing 0.05% TFA. Fractions containing the desired product were combined and then purified by mass triggered reverse phase HPLC, C18 column, eluted with 10-30% acetonitrile^O containing 0.05% TFA.
Fractions containing the desired product were combined, and lyophilized to obtain Compound (C18) (12.3 mg, 39.8 % yield) as TFA salt. LCMS M+1 =1348.2, M/2+1 =674.8, tr= 0.842 min.
Example 3-8: Synthesis of Compound 1 (C1) Linker intermediate (LI-3) (36.7 mg, 0.053 mmol) was added to a solution of CDN intermediate (CDNI-1) (60 mg, 0.053 mmol) in DMF (5 ml), followed by the addition of DIEA (68.2 mg, 0.527 mmol) and HOAT (7.2 mg, 0.053 mmol). The mixture was stirred at room temperature for 16 hrs and then concentrated. The residue was dissolved in DMSO and pre-purified by ISCO by using 15.5g C18 aq column, eluted with 5-35% ACN in water with 0.05% TFA. After purification, fractions were concentrated and then purified by mass triggered reverse phase HPLC, C18 column, eluted with 5-33% acetonitrile-H20 containing 0.05% TFA. Fractions containing desired product were concentrated to obtain Compound (C1) (55.4 mg, 68.1 % yield) as TFA salt. LCMS /2+ 1=676.8, M+1=1352.3, tr=0.753 min.1H N R (500 MHz, DMSO-c/6) δ 10.01 (s, 1H), 9.42 (b, 1H), 8.56 (d, J= 15.2 Hz, 1H), 8.31 (s, 1H), 8.16 (dd, J = 13.1, 7.4 Hz, 1H), 8.04 (d, J=8.4 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.48 (d, J=8.0 Hz, 1H), 7.32 (d, J= 8.1 Hz, 1H), 7.18 (s, 1H), 7.02 (s, 2H), 6.43 (d, J= 16.6 Hz, 2H),6.18(s, 2H), 5.61 (s, 1H), 5.50 (s, 1H), 5.13 (m, 3H), 5.02 (s, 1H), 4.93 (s, 1H), 4.55-4.34 (m, 6H), 4.27 (t, J= 5.3 Hz, 2H), 4.19 (dd, J = 8.5, 6.7 Hz, 1H), 3.87 (d, J= 12.1 Hz, 2H), 3.63 (q, J = 7.0, 6.6 Hz, 2H), 3.54 (s, 2H), 3.19— 2.88 (m, 5H), 2.48 (q, J =7.4 Hz, 1H), 2.07 - 1.94 (m, 1H), 1.75 (m, 1H), 1.65 (m, 1H), 1.46 (m, 3H),0.87 (dd, J= 13.9, 6.8 Hz, 6H).
Example 3-9: Synthesis of Compound 2 (C2)
TEA (1.3 mg, 0.013 mmol) and HATU (5 mg, 0.013 mmol) were added to a solution of 3-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (2.2 mg, 0.013 mmol) in DMF (1 mL) and the mixture was stirred for 5 mins. A solution of CDN intermediate (CDNI-1) TFA salt (15 mg, 0.013 mmol) in DMF (1 ml) was then added and the mixture was stirred for 18 hrs at room
temperature and then concentrated. The residue was dissolved in DMSO (2 ml) and then purified by mass triggered reverse phase HPLC using C18 column, eluted with 5-25% acetonitrile-H20 containing 0.05% TFA. Fractions containing desired product were lyophilized to obtain Compound (C2) (8.7 mg, 59 % yield) as TFA salt. LCMS M+1 =947.1 , tr= 0.646 min.
Example 3-10: Synthesis of Compound 3 (C3)
Compound (C3) was synthesized using the methods describe for the synthesis of Compound (C2), except linker intermediate (LI-4) was used in place of 3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol- 1-yl)propanoic acid.
Compound (C3) (4.5 mg, 26 % yield) as TFA salt. LCMS M+1 =1243.3, tr= 0.924 min.
Example 3-11 : Synthesis of Compound 4 (C4)
Compound (C4) was synthesized using the methods describe for the synthesis of Compound (C2), except bis(perfluorophenyl) 3,3'-oxydipropionate (purchased from Broadpharm, San Diego) was used in place of 3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanoic acid.
Compound (C4) (10.5 mg, 46.5 % yield) as TFA salt. LCMS M+1 = 1 106.0, tr=0.930 min.
Example 3-12: Synthesis of Compound 5 (C5)
Step 1 : DIEA (0.033 mL, 0.186 mmol) was added to a solution of CDN intermediate (CDNI-2) (26.6 mg, 0.019 mmol) and 2,5-dioxopyrrolidin- 1 -yl 2-(((((9H-fluoren-9- yl)methoxy)carbonyl)amino)oxy)acetate (15.28 mg, 0.037 mmol) in DMF (1 ml). The mixture was stirred at room temperature for 1 h and then concentrated. The residue was purified by reverse phase ISCO C18 50g column, eluted with 10-50% acetonitrile-h^O aqueous containing 10 m
HOAc Et3N. Fractions containing desired product were concentrated to obtain 4-((9S,12S)-1 - (9H-fluoren-9-yl)-9-isopropyl-3,7, 10-trioxo-12-(3-ureidopropyl)-2,5-dioxa-4,8, 1 1 -triazatridecan- 13-amido)benzyl (2-(((9-((2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12R, 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-purin-6- yl)carbamoyl)oxy)ethyl)(methyl)carbamate (6mg, 25% yield) as Et3N salt. LCMS M/2+ 1 =748.8, tr= 0.966 min.
Step 2: 4-((9S, 12S)-1 -(9H-fluoren-9-yl)-9-isopropyl-3,7, 10-trioxo- 12-(3-ureidopropyl)-2,5-dioxa- 4,8, 1 1 -triazatridecan-13-amido)benzyl (2-(((9-((2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12R, 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-purin-6- yl)carbamoyl)oxy)ethyl)(methyl)carbamate (6.0 mg, 0.0035 mmol) triethylammonium salt was dissolved in ACN (2 ml) and water (2ml) and LiOH (20 mg) was added. The mixture was stirred at room temperature for 4 hrs, neutralized with HOAc (0.06 ml) and then concentrated. The residue was purified by reverse phase ISCO 15.5g C18 aq column, eluted with 5-40% acetonitrile-H20 containing 0.05% TFA. Fractions containing desired product were concentrated to obtain Compound (C5) (2.8 mg, 36.9 % yield) as TFA salt. LCMS M/2+1 = 637.8 tr= 0.676 min.
Example 3-13: Synthesis of Compound 6 (C6)
Compound (C6) was synthesized using the methods describe for the synthesis of Compound (C14), except CDN intermediate (CDNI-1) was used in place of CDN intermediate (CDNI-3). Compound (C6) (1 .2 mg, 24 % yield) as TFA salt. LCMS M/2+1 = 697.8, M+1 =1394.5, tr=0.782 min.
Example 3-14: Synthesis of Compound 7 (C7) Η,Ν 0
Compound (C7) was synthesized using the methods describe for the synthesis of Compound (C4), except CDN intermediate (CDNI-2) was used in place of CDN intermediate (CDNI-1). Compound (C7) (5.3 mg, 55.3 % yield) as TFA salt. LCMS M/2+ 1 =756.3, tr=0.975 min.
DIEA (0.01 ml, 0.056 mmol) was added to a solution of CDN intermediate (CDNI-2) (8 mg, 0.0056 mmol) and bis(2,5-dioxopyrrolidin-1-yl) 3,3'-oxydipropionate (5.98 mg, 0.017 mmol) ((Bis-PEG1 -NHS ester purchased from Broadpharm, San Diego) in DMF (1 ml). The mixture was stirred at room temperature for 2 hours and then concentrated. The residue was purified by mass triggered reverse phase HPLC, using C18 column, eluted with 10-33% acetonitrile-h^O containing 0.05% TFA. Fractions containing desired product were lyophilized to obtain
Compound (C8) (5.7 mg, 62.2 % yield) as TFA salt. LCMS M/2+1 =721 .8, tr= 0.755 min.
Compound (C9) was synthesized using the methods describe for the synthesis of Compound (C1 ), except linker intermediate (LI-5) was used in place of linker intermediate (LI-3).
Compound (C9) (6.8mg, 52.6 % yield) as TFA salt. LCMS M/2+1 =698.8, tr = 0.758 min.
Example 3-17: Synthesis of Compound 10 (C10)
Compound (C10) was synthesized using the methods describe for the synthesis of Compound (C1 ), except linker intermediate (LI-2) was used in place of linker intermediate (LI-3).
Compound (C10) (7.3 mg, 55.3 % yield) as TFA salt. LCMS M+1 = 131 1 .2, M/2+1 =656.2, tr= 0.845 min.
Example 3-18: Synthesis of Compound 1 1 (C11)
Compound (C11 ) was synthesized using the methods describe for the synthesis of Compound (C1 ), except 1-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-3,6,9, 12-tetraoxapentadecan-15-oic acid (MPEG4-acid purchased from Broadpharm, San Diego) was used in place of linker intermediate (LI-3).
Compound (C11 ) 10.9 mg (37.6% yield) LCMS M+1 =1 123.1 , tr=0.722 min.
Example 3-19: Synthesis of Compound 19 (C19)
Compound (C19) was synthesized using the methods describe for the synthesis of Compound (C2), except CDN intermediate (CDNI-10) was used in place of CDN intermediate (CDNI-1) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamido)-3-methylbutanamido)-5- ureidopentanamido)benzyl (4-nitrophenyl) carbonate (MC-vc-pab-PNP purchased from Levena Biopharma, San Diego) was used in place of 3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)propanoic acid.
Compound (C19) (1.1 mg, 20 % yield) as TFA salt. LCMS M/2+1 = 675.8 tr= 0.776 min.
Example 3-20: Synthesis of Compound 20 (C20)
Compound (C20) was synthesized using the methods describe for the synthesis of Compound (C1 ), except CDN intermediate (CDNI-6) was used in place of CDN intermediate (CDNI-1). Compound (C20) (4.2 mg, 30 % yield) as TFA salt. LCMS M/2+ 1 =675.8, M+1 =1350.3, tr=0.751 min. 1 H NMR (500 MHz, DMSO-d6) δ 9.99 (s, 1 H), 9.28 (s, 2H), 8.98 (s, 3H), 8.14 (d, J = 7.4 Hz, 2H), 8.04 (d, J = 8.3 Hz, 2H), 7.99 (s, 1 H), 7.64 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.03 (s, 2H), 6.49 (d, J = 46.4 Hz, 2H), 6.03 (s, 1 H), 5.70 (d, J = 49.8 Hz, 2H), 5.21 - 4.83 (m, 5H), 4.68 - 4.32 (m, 9H), 4.28 - 4.13 (m, 2H), 3.13 (qd, J = 7.3, 4.9 Hz, 2H), 3.02 (d, J = 1 1 .7 Hz, 6H), 1 .97 (dt, J = 12.7, 6.2 Hz, 1 H), 1 .86 - 1.55 (m, 2H), 1 .45 (d, J = 32.2 Hz, 2H), 1.31 - 1.1 1 (m, 4H), 0.86 (dd, J = 16.0, 6.7 Hz, 8H).
Compound (C21 ) was synthesized using the methods describe for the synthesis of Compound (C1 ), except CDN intermediate (CDNI-7) was used in place of CDN intermediate (CDNI-1). Compound (C21 ) (12.2 mg, 50% yield) as TEA salt. M+1 =1348.20, tr= 0.721 min.
Example 3-22: Synthesis of Compound 22 (C22)
Compound (C22) was synthesized using the methods describe for the synthesis of Compound (C19), except CDN intermediate (CDNI-8) was used in place of CDN intermediate (CDNI-10). Compound (C22) (0.9 mg, 34.1 % yield) as TFA salt. LCMS M/2+1 = 695.8, M+1 =1391 , tr=0.695 min. Example 3-23:
Synt
Compound (C23a) was synthesized using the methods describe for the synthesis of Compound (C1 ), except CDN intermediate (CDNI-9) was used in place of CDN intermediate (CDNI-1). Compound (C23a) (12.7 mg, 51 .7 % yield) as TFA salt. LCMS M/2+1 =676.7, tr= 0.700 min. b)
Compound (23b) was obtained during the synthesis of Compound (23a). Compound (C23a) and Compound (23b) were not separated. (12.7 mg, 51 .7 % yield) as TFA salt. LCMS
M/2+ 1 =676.7, tr= 0.700 min.
Example 3-24: Synthesis of Compound 24 (C24)
HATU (1.9 mg, 0.005 mmol) was added to a mixture of (Z)-6-(((1 - ethoxyethylidene)amino)oxy)hexanoic acid (1 .2 mg, 0.0056 mmol) and DIEA (2.2 mg, 0.017 mmol) in DMF (1 ml). The mixture was then stirred at room temperature for 5 min and then added to a solution of CDN intermediate (CDNI-2) (4 mg, 0.0028 mmol) in DMF (1 ml). The mixture was then stirred for 5 hours at room temperature for 16 hours and then concentrated to give the protected derivative ethyl (Z)-N-((6-(((S)-1 -(((S)-1 -((4-((((2-(((9- ((2R,3R,3aR,5R,7aR,9R, 10R, 10aR, 12R, 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-purin-6- yl)carbamoyl)oxy)ethyl)(methyl)carbamoyl)oxy)methyl)phenyl)amino)-1 -oxo-5-ureidope yl)amino)-3-methyl-1 -oxobutan-2-yl)amino)-6-oxohexyl)oxy)acetimidate. LCMS M/2+ 1 =700.8, tr =0.890 min.
Purification of the residue by reverse phase HPLC, ISCO C18 50g column, eluted with 10-50% acetonitrile-H20 containing 0.05% TFA resulted in loss of the protecting group. Fractions containing desired product Compound (C-24) were concentrated further purified by reverse phase ISCO C18 column, eluted with 5-40% acetonitrile-h^O containing 0.05% TFA to obtain
Compound (C-24) (2.2 mg, 47.9 % yield) as TFA salt. LCMS M/2+1 =665.8, tr=0.697 min.
Note: Z)-6-(((1-ethoxyethylidene)amino)oxy)hexanoic acid was prepared from ethyl-(N- hedroxyacetimidate and 6-bromohexanoic acid in the presence of LiOH using the method described in Biomacromolecules 6(5) 2648, 2005.
Example 3-25:
a) Synthesis of Compound 25a (C25a)
Compound (C25a) was synthesized using the methods describe for the synthesis of Compound (C1 ), except CDN intermediate (CDNI-11) was used in place of CDN intermediate (CDNI-1). Compound (C25a) (7.5 mg, 37.1 % yield) as TFA salt. LCMS M/2+1 =698.8, tr=0.715 min. b)
Compound (25b) was obtained during the synthesis of Compound (25a). Compound (C23a) and Compound (25b) were not separated. (7.5 mg, 37.1 % yield) as TFA salt. LCMS
M/2+1 =698.8, tr=0.715 min Example 3-26: Synthesis of Compound 26 (C26)
DIEA (0.019 mL, 0.1 10 mmol) and HATU (9.2 mg, 0.024 mmol) were added to a solution of 1- (2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oic acid (Mal-PEG4-acid) (8.4 mg, 0.024 mmol) in DMF (1 ml) and the mixture was stirred for 5 mins and then added a solution of CDN intermediate (CDNI-7) (25 mg, 0.022 mmol) in DMF (1 ml). The reaction was then stirred at room temperature for 16 hrs and then concentrated. The residue was purified by reverse phase ISCO C18 column, eluted with 5-40% acetonitrile-h^O with the aqueous phase containing 10 mM Et3N HOAc. Fractions containing desired product were lyophilized to obtain Compound (C-26) (23.2 mg, 76 % yield) as TEA salt. LCMS +1 =1121.1 tr=0.733 min. 1 H NMR (500 MHz, DMSO-d6) δ 8.66 (d, J = 3.7 Hz, 2H), 7.96 - 7.75 (m, 2H), 7.06 (s, 2H), 6.32 (d, J = 14.0 Hz, 1 H), 6.26 (d, J = 3.1 Hz, 1 H), 5.81 (t, J = 5.8 Hz, 1 H), 5.63 (d, J = 52.4 Hz, 1 H), 5.24 - 5.00 (m, 2H), 4.58 - 4.26 (m, 6H), 3.89 - 3.72 (m, 3H), 3.72 - 3.63 (m, 2H), 3.64 - 3.54 (m, 3H), 3.54 - 3.47 (m, 12H), 3.16 (s, 2H), 3.01 (q, J = 7.2 Hz, 15H), 2.95 (s, 1 H), 2.74 - 2.61 (m, 2H), 1.94 (s, 1 H), 1.13 (t, J = 7.2 Hz, 21 H).
Exa
Compound (C27) was synthesized using similar methods describe for the synthesis of
Compound (C15), except CDN intermediate (CDNI-12) was used in place of CDN intermediate (CDNI-4) and the C18 column was eluted with 5-50% acetonitrile-H20 (aqueous phase containing 10 mM Et3N HOAc). Fractions containing the desired product were concentrated to obtain Compound (C27) as Et3N salt (1 mg, 1 1% yield). LCMS M/2+1 = 675.8, tr= 0.758 min.
Example 3-28: Synthesis of Compound 28 (C28)
Compound (C28) was synthesized using similar methods describe for the synthesis of Compound (C15), except CDN intermediate (CDNI-13) was used in place of CDN intermediate (CDNI-4). Compound (C28) (5.8 mg, 30 % yield). LCMS /2+1 =668.8, tr=0.724 min.
Examp -29: Synthesis of Compound 29 (C29)
2,5-dioxopyrrolidin-1 -yl 3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)propanoate (purchased from Combi-Blocks)(0.5 mg, 0.002 mmol) and DIEA (1 .7 mg, 0.013 mmol) were added to a solution of intermediate CDNI-15 TFA salt (1 .7 mg, 0.0013 mmol ) in DMF (1 ml) and the reaction was stirred at rt for 72 hrs and then concentrated. The crude was purified by reverse phase ISCO using 15 g C18 aq column, eluted with 5-40% acetonitrile-h^O (aqueous phase containing 10 mM Et3N HOAc). Fractions containing desired product were concentrated to obtain Compound 29 (C29) as an Et3N salt (2.3 mg, 1 1 1 % yield). LCMS M+1 = 1187.1 , tr=0.675 min.
Example 3-30: Synthesis of Compound 30 (C30)
(C30)
Compound (C30) was synthesized using similar methods describe for the synthesis of Compound (C29), except CDN intermediate (CDNI-16) was used in place of CDN intermediate (CDNI-15), the reaction mixture was stirred for 16 hrs and the crude was purified by reverse phase ISCO with 50g C18 aq column and eluted with 5-35% acetonitrile-water (aqueous phase containing 10 mM Et3N HOAc). Fractions with the desired product were combined and lyophilized to give Compound 30 (C30) as Et3N salt (3.8 mg, 14% yield). LC S M/2+1 =680.2, tr=0.705 min.
Examp -31 : Synthesis of Compound 31 (C31 )
Compound (C31 ) was synthesized using the methods describe for the synthesis of Compound (C1 ), except CDN intermediate (CDNI-17) TFA salt was used in place of CDN intermediate (CDNI-1 ), the reaction was stirred at rt for 20 hours and purification was by ISCO using 15.5 C18 aq column, eluted with 5-40% acetonitrile-h^O containing 10 mM Et3N-HOAc. Fractions containing desired product were concentrated to obtain Compound 31 (C31) (4.3 mg, 76% yield) as TEA salt. LCMS M/2+1 =698.8, tr=0.800min.
Example 3-32: Synthesis of Compound 32 (C32)
A solution of CDN intermediate (CDNI-18) Et3N salt (20 mg, 0.022 mmol) and 1-(prop-2-yn-1 -yl)- 1 H-pyrrole-2,5-dione (1 1 .7 mg, 0.087 mmol) in 1 :2 mixture of water-t-BuOH (4.5 ml) was degassed with N2, and a degassed solution of sodium L-ascobate (21.5 mg, 0.109 mmol) in water was added, followed by a degassed solution CuS04 (10.4 mg, 0.065 mmol) in water. The reaction mixture was stirred at rt for 1 hr and then lyophilized. The crude was purified by reverse phase ISCO using 50g C18 column, eluted with 10-30% acetonitrile-H^O (aqueous phase containing 10 mM Et3N HOAc). Fractions containing the desired product were combined and lyophilized and repurify with reverse phase ISCO using 50g C18 column, eluted with 10-30% acetonitrile-H20 containing 0.05% TFA. Fractions containing desired product were lyophilized to obtain Compound 32 (C32) as TFA salt (1 .9 mg, 6% yield). LC S M+1 =943.0, tr= 0.725 min.
Example -33: Synthesis of Compound 33 (C33)
Compound (C33) was synthesized using the methods describe for the synthesis of Compound (C32), except CDN intermediate (CDNI-19) TFA salt was used in place of CDN intermediate (CDNI-18). Compound (C33) TFA salt (2.7 mg, 10% yield). LCMS M+1 =941 .0, tr=0.725 min.
Example 3-34: Synthesis of Compound 34 (C34)
Compound (C34) was synthesized using the methods describe for the synthesis of Compound (C32), except CDN intermediate (CDNI-20) TFA salt was used in place of CDN intermediate (CDNI-18). LCMS M+1 = 957.1 , tr= 0.693 min.
Exampl -35: Synthesis of Compound 35 (C35)
Compound (C35) was synthesized using the methods describe for the synthesis of Compound (C1 ), except CDN intermediate (CDNI-10) TFA salt was used in place of CDN intermediate (CDNI-1 ), the reaction was stirred at rt for 1 day and purification was reverse phase ISCO using C18 column, eluted with 5-35% acetonitrile-h^O (aqueous phase containing 10 mM Et3N HOAc). Fractions containing desired product were concentrated to obtain Compound 35 (C35) as Et3N salt (4.0 mg, 120% yield). LCMS +1 = 1308.1 , tr= 0.761 min.
Example 3-36: Synthesis of a mixture of Compound 36a (C36a) and Compound 36b (C36b)
The mixture of Compound 36a (C36a) and Compound 36b (C36b) was obtained using the methods describe for the synthesis of Compound (C1 ), except the mixture of CDN intermediates (CDNI-21a) and (CDNI-21 b) TFA salt was used in place of CDN intermediate (CDNI-1), and an initial purification was by reverse phase ISCO using 15g C18 column, eluted with 5-45% acetonitrile-H20 (aqueous phase containing 10 mM Et3N HOAc). Fractions containing desired product were concentrated and further purified by reverse phase ISCO by using 50 g C18 aq column, eluted with 5-35% acetonitrile-water with 0.05% TFA. Fractions containing desired product were concentrated and lyophilized to obtain to obtain the mixture of Compound 36a (C36a) and Compound 36b (C36b) as TFA salt (8.3 mg, 41 % yield). LCMS M+1 = 1336.1 , tr= 0.799 min.
Example 3-37: Synthesis of a mixture of Compound 37a (C37a) and Compound 37b (C367b)
(C37b)
DIEA (1 1 .0 mg, 0.086 mmol) was added to a solution of CDN intermediate (CDNI-22a and CDI- 22b) (12.6 mg, 0.0086 mmol) and bis(perfluorophenyl) 3,3'-oxydipropionate (Bis-PEG1 -PFP ester purchased from Broadpharm) (12.7 mg, 0.026 mmol) in DMF (1 ml). The reaction was stirred at rt for 2 hours and then concentrated. The residue was purified by reverse phase ISCO by using 30 g C18 aq column, eluted with 5-100% acetonitrile-water with 0.05% TFA. Fractions containing desired product were concentrated and lyophilized to obtain mixture of Compound 37a and 37b (C37a and C37b) as TFA salt (6.2 mg, 38.6% yield). LCMS M/2+1 = 778.3, tr= 0.974 min.
Example 3-38: Synthesis of Compound 38 (C38)
Compound (C38) was synthesized using similar methods describe for the synthesis of Compound (C15), except CDN intermediate (CDNI-12) was used in place of CDN intermediate (CDNI-23) and the C18 column was eluted with 5-50% acetonitrile-h^O (aqueous phase containing 10 mM Et3N HOAc). Fractions containing the desired product were concentrated to obtain Compound (C38) as Et3N salt (1 1.6 mg, 88 % yield) as Et3N salt. LCMS M/2+ 1 =676.8, tr= 0.742 min. 1 H NMR (500 MHz, DMSO-d6) δ 10.80 (s, 1 H), 9.99 (s, 1 H), 9.37 (s, 1 H), 8.97 (s, 1H),8.68 (s, 1H), 8.23 (s, 1H), 8.13 (d, J= 7.5 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.62 (t, J = 10.0 Hz, 2H), 7.44 (s, 2H), 7.34 (t, J = 9.9 Hz, 2H), 7.03 (s, 1H), 6.27 (d, J = 8.8 Hz, 1H), 6.17 (d, J =8.8 Hz, 1H), 6.02 (s, 1H), 5.72-5.55 (m, 1H), 5.55 - 5.39 (m, 3H), 5.05 (s, 1H), 4.54 (ddd, J = 27.3, 20.2, 2.4 Hz, 2H), 4.41 (td, J = 8.1, 5.2 Hz, 1 H), 4.31 (s, 2H), 4.19 (dd, J = 8.5, 6.7 Hz, 1H), 4.05 -3.91 (m, 3H), 3.72 - 3.60 (m, 1H), 3.59 (d, J= 5.9 Hz, 2H), 3.11 - 3.02 (m, 1H), 3.00 (d, J= 9.6 Hz, 3H), 2.80 (qd, J= 13.5, 6.4 Hz, 16H), 2.52 - 2.42 (m, 1H), 1.94 (s, 3H), 1.73 (s, 1 H), 1.69 - 1.57 (m, 1 H), 1.52 - 1.34 (m, 2H), 1.02 (t, J = 7.2 Hz, 20H), 0.86 (dd, J = 15.8, 6.8 Hz, 5H).
Example -39: Synthesis of Compound 39 (C39)
Compound (C39) was synthesized using similar methods describe for the synthesis of Compound (C18), except CDN intermediate (CDNI-24) was used in place of CDN intermediate (CDNI-3) and the C18 column was eluted with 5-40% acetonitrile^O (aqueous phase containing 10 mM Et3N HOAc). Fractions containing the desired product were concentrated to obtain Compound (C39) as Et3N salt: (4.9 mg, 41.6 % yield). LCMS M/2+1 =683.8, tr= 0.709 min.1H NMR (500 MHz, DMSO-c/6) δ 9.99 (s, 1H), 8.35 (s, 1H), 8.21 (s, 1H), 8.17 - 7.99 (m, 3H), 7.68 - 7.52 (m, 2H), 7.33 (s, 5H), 7.03 (s, 2H), 6.57 (s, 2H), 6.30 (d, J= 16.6 Hz, 1H), 6.02 (dd, J= 55.5, 30.4 Hz, 2H), 5.60 (dd, J= 52.2, 3.8 Hz, 1H), 5.42 (d, J= 30.9 Hz, 3H), 5.01 (d, J = 12.8 Hz, 2H), 4.39 (d, J= 12.6 Hz, 2H), 4.30 (d, J= 10.7 Hz, 4H), 4.27 - 4.06 (m, 4H), 3.92- 3.74 (m, 2H), 3.69 - 3.50 (m, 3H), 3.14 - 2.83 (m, 5H), 2.69 (q, J = 7.2 Hz, 33H), 1.86 - 1.56 (m, 1H), 1.56- 1.31 (m,2H), 1.05 (t, J = 7.2 Hz, 44H), 0.86 (dd, J= 15.5, 6.8 Hz, 6H).
Example 3-40: Synthesis of Compound 40 (C40)
Compound (C40) was synthesized using similar methods describe for the synthesis of Compound (C18), except CDN intermediate (CDNI-25) was used in place of CDN intermediate (CDNI-3). Compound (C40) as Et3N salt: (8.0mg, 74% yield). LCMS M/2+1 = 690.8, tr= 0.771 min. 1H NMR (500 MHz, DMSO-cf6) δ 10.02 (s, 1 H), 8.14 (d, J = 7.5 Hz, 1 H), 8.04 (t, J = 8.9 Hz, 3H), 7.63 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 9.4 Hz, 2H), 7.03 (s, 2H), 6.71 (d, J = 67.7 Hz, 5H), 6.03 (s, 2H), 5.78 (d, J = 7.4 Hz, 1 H), 5.59 (s, 1 H), 5.45 (s, 2H), 5.15 (dt, J = 9.2, 4.2 Hz, 1 H), 5.06 - 4.83 (m, 3H), 4.58 (t, J = 6.3 Hz, 1 H), 4.42 (d, J = 6.6 Hz, 1 H), 4.33 - 4.09 (m, 6H), 4.06 - 3.86 (m, 2H), 3.65 (td, J = 8.1 , 6.7 Hz, 1 H), 3.15 - 2.82 (m, 4H), 2.66 (q, J = 7.2 Hz, 33H), 1.80 - 1.54 (m, 1 H), 1 .54 - 1 .34 (m, 2H), 1.04 (t, J = 7.2 Hz, 45H), 0.86 (dd, J = 16.3, 6.8 Hz, 5H).
Example 3-41 : Synthesis of Compound 41 (C41 )
Compound (C41 ) was synthesized using similar methods describe for the synthesis of Compound (C18), except CDN intermediate (CDNI-26) TEA salt was used in place of CDN intermediate (CDNI-3) and Linker intermediate (LI-9) was used in place of Linker intermediate (LI-3). Fractions containing the desired product were combined and lyophilized to obtain Compound (C41 ) as Et3N salt (2.3mg, 1 1 % yield). LCMS M/2+1 = 702.3, tr= 0.691 min.
Example 3-42: Synthesis of the mixture of Compound 42a (C42a) and Compound 42b (C42b)
The mixture of Compound (C42a) and Compound (C42b) was synthesized using similar methods describe for the synthesis of Compound (C18), except the mixture of CDN
intermediate (CDNI-27a) and CDN intermediate (CDNI-27b) was used in place of CDN intermediate (CDNI-3) and the C18 column was eluted with 5-40% acetonitrile-h^O (aqueous phase containing 10 mM Et3N HOAc). The mixture of Compound (C42a) and Compound (C42b) was obtained as Et3N salt (2.0mg, 33% yield). LCMS M/2+1 = 689.8, tr= 0.694 min.
Example 3
Compound (C43) was synthesized using similar methods describe for the synthesis of
Compound (C18), except CDN intermediate (CDNI-28) TEA salt was used in place of CDN intermediate (CDNI-3). Fractions containing the desired product were combined and lyophilized to obtain Compound (C43) as Et3N salt (3.3 mg, 31 .1 % yield). LCMS M/2+1 = 683.8, tr= 0.813 min.
Example 3-44: Synthesis of a mixture of Compound 44a (C44a) and Compound 44b (C44b)
Compound (C1 ) (20 mg, 0.013 mmol) was dissolved in 3:7 MeOH and DMSO (1 ml) and maintained at rt for 1 month. The mixture was purified by reverse phase ISCO using 50g C18 aq column, eluted with 5-40% ACN-water with 0.05% TFA. Fractions containing Compound (C44a) and Compound (C44b) were isolated and lyophilized to obtain the mixture of Compound (C44a) and Compound (C44b) as TFA salt (4.5 mg, 21 % yield). LCMS M/2+1 =668.8, tr=0.694 min.
Exampl -45: Synthesis of Compound 45 (C45)
Compound (C45) was synthesized using similar methods describe for the synthesis of
Compound (C18), except CDN intermediate (CDNI-29) TEA salt was used in place of CDN intermediate (CDNI-3). Fractions containing the desired product were combined and lyophilized to obtain Compound (C45) as Et3N salt (7.2 mg, 38% yield). LCMS M+1 =1292.1 , tr = 0.631 min.
Example 4: RNAseq Analysis of Tumor Cells Treated with STING Agonists
THP1 dual cells were obtained from InvivoGen and prepared according to
manufacturer's protocol without PMA induced differentiation (www.invivogen.com/thp1-dual). THP1 dual cells were then treated with 20uM T1 -2 for 6 hours. HCC1954 cells were obtained from ATCC (Catalogue No. CRL-2338) and were cultured in 10% FBS/90% RPMI. Two million cells were seeded in 6 well plates (2 mL culture volume) and allowed to attach overnight. Cells were treated with 2'3'-cGAMP (100 uM) or T1 -1 (5 uM) for 6 hr and harvested in buffer RLT (RNeasy Plus Kit, Qiagen).
Total RNA was prepared using a Qiagen RNeasy kit. Poly-A mRNA was isolated using poly-T oligos attached to magnetic beads, followed by fragmentation using divalent cations at elevated temperature. A cDNA library was prepared using random primers. Sequencing was performed on an lllumina HiSeq 1000 with between 22 and 70 million mapped reads per sample which were aligned to a human transcriptome file. Results are reported as reads per million. Fold changes were calculated by dividing the output from treated samples by the control sample for each gene.
FIG. 1A-10 lists the transcripts in HCC1954 cells that were found to increase in expression by at least 5-fold 6 hours after treatment with either 2'3'-cGA P or T1-1. FIG. 2A-2L lists the transcripts in THP1 dual cells whose expression increased by at least 5-fold after 6 hours of treatment with T1 -2.
Example 5: Preparation of anti-HER2-STING agonist conjugates
A) Preparation of anti-HER2 antibody with specific Cysteine (Cys) mutations
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, DNA 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 IgG 1 heavy chain and human kappa light chain. The heavy chain vector encodes the constant region of the human lgG1 antibody, includes a signal peptide (MPLLLLLPLLWAGALA) (SEQ ID NO: 28), a CMV promoter to drive expression of the heavy chain, and appropriate signal and selection sequences for stable transfection into CHO cells. The light chain vector encodes the constant region of the human kappa light chain, includes a signal peptide (MSVLTQVLALLLLWLTGTRC)
(SEQ 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 mAb1 , 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 cell supernatants by standard Protein A affinity chromatography.
Alternatively, CHO 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 cells 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 cell 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, et al., Biotechnol Bioeng. 75: 197- 203 (2001)). The expressed antibodies were purified from the cell 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 Cys 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 al., Nature
Biotechnology 26:925-932 (2008).
Because engineered Cys residues in antibodies expressed in mammalian cells 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 maleimido 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 dithiothreitol (DTT) followed by reoxidation of all 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 tetramer 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 ml/min. The elution of proteins from the column was monitored at 280 nm. Incubation was allowed to continue until reoxidation was complete. After reoxidation, maleimide-containing compounds were 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 HCI 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 μΜ 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 5-10 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 0.5 M sodium phosphate pH 8.0 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.
Properties of the anti-HER2-STING 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-300 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 are washed with PBS again and then eluted in 1 % formic acid. Samples were reduced with 133 mM TCEP, 0.67 M ammonium acetate pH 5.0 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 9, below). The majority of the conjugates contain less than 10% dimeric and oligomeric material (Table 9). In mouse PK studies, the majority of conjugates also have detectable payload retention 336 h post-injection (FIG. 3 and Table 9). These results suggest that conjugates can be made efficiently and have favorable characteristics.
B) Generation of anti-HER2 -STING agonist conjugates through partial reduction of native disulfide bonds of non-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) were first partially reduced in PBS pH 8.0 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) were reacted overnight at 4°C with 5:1 or 10:1 molar ratio of C1 . Resulting conjugates were purified by Protein A chromatography by standard methods and buffer exchanged to PBS, and profiled by MS and AnSEC as described above. Measured compound-to-antibody ratio and aggregation data are summarized in Table 9 and suggest that conjugates can be made efficiently and have favorable characteristics.
C) Generation of anti-HER2 -STING agonist conjugates using 1 ,3-dichloropropan-2-one to reconnect native interchain disulfide bonds of non-engineered anti-HER2 antibodies In an alternative method (United States Patent Application 20150150998), interchain disulfides bonds of a non-engineered, recombinant anti-HER2 antibody can be modified and conjugated to an agonist compound of the invention using the following two steps.
Scheme 15a
Two step conjugation to native cysteine residues using 1 ,3 dichloropropan-2-one bridging followed by addition to the introduced ketones.
anti-HER2 mAb3 anti-HER2 mAb3-KB
(4 interchain disulfide modified anti-HER2 mAb3
Step 1 : Reduction of interchain disulfide bridges and re-bridging using 1 ,3-dichloropropan-2- one: 1 ,3-dichloroacetone (DCA) was dissolved in DMSO to a final concentration of 1 M. Next, 1 1 .9 μΙ_ of the resulting solution was added to 22.0 mL of 27 μΜ anti-HER2 antibody (90 mg) in 0.1 M HEPES buffer (pH 8.0) corresponding to a 20-fold molar excess of DCA over antibody. Ketone bridge formation was then initiated by supplementing the solution with 36.5 μΙ_ of 200 mM TCEP/HCI in water to a final concentration of 0.33 mM. After gently shaking the reaction mixture at 4°C for approximately 16 hours, the antibody construct was purified using 6 mL of settled rmp Protein A Sepharose Fast Flow resin (GE Healthcare). Ketone bridge incorporation was verified by capillary electrophoresis on a PA 800 plus Pharmaceutical Analysis System (SCIEX). Under reducing conditions, fully cross-linked antibody containing two heavy chains and two light chains was the dominating species with an abundance of 86% by peak area. The yield of modified anti-HER2 antibody was quantified by absorbance measurements at 280 nm on a NanoDrop 1000 spectrophotometer (Thermo Scientific). Following Protein A affinity chromatography, 77 mg (86%) of modified antibody was obtained.
Step 2: To initiate the second step of the conjugation process, the re-bridged anti-HER2 antibody (anti-Her2 mAb3-KB) from Step 1 was concentrated using Amicon Ultra Centrifugal Filter Units with 50 kDa cut-off (EMD Millipore). Oxime ligation was performed with 5 mg of modified antibody at a final concentration of 180 μΜ and 15-fold molar excess (2.7 mM) of aminooxy-functionalized STING agonist C5 in 0.1 M anilinium acetate buffer (pH 4.4) supplemented with 14% (v/v) DMSO. After preforming the coupling reaction for 14 hours at 37°C, the resulting conjugate was purified by size-exclusion chromatography on a HiLoad 26/600 Superdex 200 pg column (GE Healthcare) to remove aggregated material. According to absorbance measurements at 280 nm on a NanoDrop 1000 spectrophotometer, 1 .7 mg of anti- HER2(mAb3)-KB-C5 conjugate was obtained. The conjugate was profiled by mass spectrometry, analytical size-exclusion chromatography and analytical hydrophobic interaction chromatography as summarized in Table 9. The example conjugate achieved a high compound-to-antibody ratio and was mainly monomeric.
D) Generation of anti-HER2 -STING agonist conjugates by conjugation to native lysine residues of wild type anti-HER2 antibody
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/ml was mixed with 20-fold molar excess of C7. The reaction was incubated at room temperature overnight, and then buffer exchanged by standard methods to PBS pH 7.2. The average compound to antibody ratio of the resulting conjugate was determined to be 3.5 (Table 9).
E) Generation of anti-HER2 -STING agonist conjugates using two-step conjugation of an ybbR-tagged anti-HER2 mutant antibody with agonist compounds containing an amino- oxy reactive group
Post-translational 4'-phosphopantetheinylation is a versatile method for the site-specific labeling of recombinant proteins with structurally diverse small molecules (Yin J, et al., Proc. Natl. Acad. Sci. U.S.A. 102:15815-15820, 2005; Zhou Z, et al., ACS Chem. Biol. 2:337-346, 2007). This enzymatic approach, which is based on the catalytic action of promiscuous 4'- phosphopantetheinyl transferases (PPTases), was adopted for the preparation of highly homogeneous antibody conjugates (see WO2013184514; Gruenewald et al., Bioconj Chem 2015, 26:2554-62). Enzymatic labeling is accomplished by incorporating 1 1 or 12-mer S6, ybbR, and A1 peptide sequences at various sites of the constant region of an antibody. For example, a ybbR tag of sequence DSLEFIASKLA (SEQ ID NO: 36) can be incorporated after residue E388 (EU numbering) to produce anti-HER2 mAb7, which has a light chain sequence of SEQ ID NO: 19 and a heavy chain sequence of SEQ ID NO: 35.
One strategy is a two-step method to prepare site-specific antibody-compound conjugates by post-translational 4'-phosphopantetheinylation (see WO2013184514). The first step of this approach is based on the PPTase-catalyzed labeling of a peptide-tagged antibody with a CoA analog containing a bioorthogonal group, such as an azido, alkene, alkyne, ketone, or aldehyde moiety. Following affinity purification of the bioorthogonally labeled antibody, the second step of the two-step method involves the conjugation of a compound comprising a moiety reactive with the bioorthogonal group. As way of example, the following section describes the two-step method for an anti-HER2 mutant antibody containing a ybbR tag insertion at a specific site within the constant region of the heavy chain. In addition, although the two-step method is exemplified for oxime ligation chemistry, this strategy can be extended to other bioorthogonal chemistries, such as click chemistry, including copper-free click chemistry, Staudinger ligation, isonitrile-based click chemistry, and tetrazine ligation.
Scheme 15b
Oxime ligation chemistry has been used by several research groups as an efficient, bioorthogonal method for the preparation of site-specific protein conjugates (Axup JY, et al., Proc Natl Acad Sci U S A. 109: 16101-16106, 2012; Rabuka D, et al., Nat Protoc. 7: 1052-1067 2012). In order to combine post-translational 4'-phosphopantetheinylation with oxime ligation, ketone-modified CoA analog LI-7 was prepared as discussed above. Next, PPTase catalysis was used to enzymatically conjugate the bioorthogonal ketone group site-specifically onto the embedded ybbR tag of an anti-HER2 antibody. Specifically, 2.5 μΜ of anti-HER2 mAb7 was conjugated with 100 μΜ of ketone-CoA analog (LI-7) (40 molar equivalents relative to antibody) in the presence of 2 μΜ of Sfp PPTase from Bacillus subtilis for 23 hours at 23°C in 75 mM Tris- HCI buffer (pH 8.0) supplemented with 12.5 mM MgCI2 and 20 mM NaCI. Labeling of the anti- HER2 mAb7 antibody with the ketone-CoA analog (LI-7) was verified by obtaining a
deconvoluted ESI-MS spectrum of the reduced and deglycosylated sample. The observed mass was in agreement with the calculated molecular weight of the corresponding ketone- functionalized heavy chain. After removing PPTase enzyme and excess ketone-CoA analog by Protein A affinity chromatography (rmp Protein A Sepharose Fast Flow, GE Healthcare Life Sciences), the ketone-activated antibody, anti-HER2 mAb7-(LI-7) was eluted with Pierce™ IgG Elution Buffer (Thermo Fisher Scientific) followed by immediate neutralization with 1 M Tris-HCI buffer (pH 8.0). The neutralized antibody solution was buffer-exchanged into PBS and
concentrated using a 50K Amicon filter.
Site-specific attachment of a ketone group enabled subsequent oxime ligation of an agonist compound to ketone-activated anti-HER2 mAb7-(LI-7) as the second step of the two-step method. Specifically, 25 μΜ of ketone-functionalized antibody was reacted with 40-fold molar excess (1 mM) of the aminooxy-agonist C5 in 100 mM anilinium acetate buffer (pH 4.5) containing 5% (v/v) DMSO. After approximately 14 hours of incubation at room temperature, insoluble material was removed by centrifugation. The supernatant was purified on a HiLoad 26/600 Superdex 200 pg column (GE Healthcare) pre-equilibrated with PBS. The conjugate was profiled by MS and AnSEC as described above. The measured compound-to-antibody ratio and aggregation behavior are summarized in Table 9. The example conjugate achieved compound-to-antibody ratio of 1 .4 and was mainly monomeric following purification.
Conjugation through this method results in a conjugation efficiency of approximately 72%. This suggests that conjugates can be made efficiently and have favorable characteristics.
Table 9. Properties of anti-HER2-STING agonist conjugates
Compound
Drug
-to- Aggregation AUC(Conjugate) 1-336,1
Conjugate retention
antibody (%)b /AUC(hlgG)1-336h'e in vivo0
ratio3
anti-HER2 mAb1 -C14 3.6 BLQ Moderate ND anti-HER2 mAb6-C14 1 .4 BLQ Moderate ND anti-HER2 mAb1-C13 3.4 4 ND ND anti-HER2 mAb1 -C1 3.8 1 High ND anti-HER2 mAb1 -C2 3.8 1 Highd 1 .4 anti-HER2 mAb1 -C3 3.8 2 Highd 1 .0 anti-HER2 mAb1-C15 3.8 5 Moderate ND anti-HER2 mAb1 -C20 4.2 BLQ Moderate ND Compound
Drug
-to- Aggregation AUC(Conjugate) 1-336,1
Conjugate retention
antibody (%)b /AUC(hlgG)1-336h e in vivo
ratio3
anti-HER2 mAb1 -C16 3.8 BLQ Moderate ND anti-HER2 mAb1 -C17 3.8 BLQ Moderate ND
Isotype lgG-C1 3.8 ND 0.9
anti-HER2 mAb1 (DAPA)-C1 4.0 ND ND
anti-HER2 mAb1-C18 3.4 Moderate ND anti-HER2 mAb1 -C9 3.9 TBD TBD anti-HER2 mAb1 -C23 3.8 TBD TBD anti-HER2 mAb1 -C10 4.1 TBD TBD anti-HER2 mAb3-KB-C5 TBD TBD TBD anti-HER2 mAb7-LI-7-C5 1 .4 BLQ TBD
anti-HER2 mAb3-C7 3.5 ND
anti-HER2 mAb3-C1 5.3 BLQ ND ND anti-HER2 mAb3-C1 3.1 ND ND anti-HER2-mAb1 -C38 3.8 ND ND anti-HER2-mAb1 -C40 3.7 ND ND anti-HER2-mAb1 -C42 3.4 ND ND anti-HER2-mAb1 -C36 3.6 BLQ ND ND anti-HER2-mAb1 -C44 3.6 ND ND anti-HER2-mAb1 -C39 3.7 ND ND anti-HER2-mAb1 -C28 3.8 ND ND anti-HER2-mAb1 -C29 12 ND ND anti-HER2-mAb1 -C30 3.6 9.5 ND ND anti-HER2-mAb1 -C34 3.8 6.7 ND ND anti-HER2 mAb7-LI-7-C5 1.4 1 ND ND
Compound-to-antibody ratio determined by LC-MS.
" Aggregation measured by analytical size exclusion chromatography; includes dimeric and oligomeric species. BLQ = below limit of quantitation.
c Samples were assessed by LC-MS as described in the text to determine compound-to-antibody ratio for each timepoint over the course of a 3 week PK study in CD1 mice. Compounds were scored based on the %compound retention during the study. Measurements were performed on samples pooled from 3 animals.
d Based on measurement of terminal samples at 504 h post-dose, average of 3 animals.
e Based on Gyros assay measurements, average of 3 animals for each timepoint between 1 and
336 h. The AUC for the hlgG and the conjugate detection are used to calculate a unitless ratio representing relative drug retention over that timecourse.
ND: not determined.
TBD: to be determined.
Example 6: Anti-HER2-STING agonist conjugates induce IP-10 secretion from HER2+ HCC1954 breast cancer cells in a target dependent manner. HCC1954 cells were suspended into 384 well plates and allowed to attach overnight. Cells were treated with anti-HER2-STING agonist conjugates or unconjugated payloads the next day and incubated for approximately 3 days. Cell culture supernatants were harvested and human IP-10 levels in the media were measured using a Human IP-10 Tissue Culture Kit from Mesoscale Devices. The EC50 values (triplicates) were calculated by performing logistic regression on measured dose-response curves. Data were curve fitted with the following formula to obtain EC50 values:
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. EC50 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 Matlab.
The maximum activity was compared to 2',3'-cGAMP (100 μΜ) treated cells to determine
%Efficacy. Values reported are the average of independent experiments when the conjugates drugs were tested multiple times. Representative data are summarized in the Table 10A and FIG.44.
Table 10A. IP-10 secretion from HER2+ HCC1954 breast cancer cells
In addition the maximum activity of certain conjugates was compared to cells treated with the conjugate anti-HER2-mAb1 -C1 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 10B. Table 10B. IP-10 secretion from HER2+ HCC1954 breast cancer cells
Example 7: In vivo testing of anti-HER2 mAb1-C1 in N87 gastric tumor xenograft model
Materials and Methods
For in vivo testing of the anti-Her2 mAb1 -C1 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 lot#7686255) were grown in sterile conditions in a 37°C incubator with 5% C02 for two weeks. Cells were grown in RPMI 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 RP 11640 serum-free media at a concentration of 5 x 106 cells and 50% matrigel/100 μΙ. 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 106 cells /100 μΙ injection volume per mouse). After cell implantation, tumors were measured by caliper and mice were weighed three times per week once tumors were palpable. Tumors were measured in two dimensions. Caliper measurements were calculated using (L x W2)/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 mm3, mice were administered by intravenous route 0.1 mg/kg, 0.3mg/kg, 1 mg/kg, or 3 mg/kg of anti-HER2 mAb1-C1 or 3 mg/kg of an Isotype lgG-C1. 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 carry 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-C1 at 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg, or Isotype control lgG-C1 at 3 mg/kg (9 mice per group). Anti-HER2 mAb1-C1 showed dose-dependent efficacy on N87 model in SCID mice. Inhibition of tumor growth was observed in mice treated with anti-HER2 mAb1-C1 at 0.3 mg/kg, 1 mg/kg, or 3 mg/kg compared to the untreated mice (FIG. 5). Tumor growth in amimals dosed with 0.1 mg/kg of Anti-HER2 mAb1-C1 or 3 mg/kg Isotype control lgG-C1 was statistically not any different than tumor growth in the vehicle control group. 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-C1 (FIG. 6).
Example 8: In vivo testing of anti-HER2 mAb1-C1 in a HCC1954 breast tumor xenograft model
Materials and Methods
For /n vivo testing of the anti-Her2 mAb1-C1 conjugate in the HCC1954 breast cancer xenograft mouse model, female SCID-beige mice at 6-8 weeks of age (purchased from Harlan Laboratories) were used for implantation. HCC1954 cells (obtained from ATCC,
(CATALOG#CRL-2338; LOT#5107643)) were grown in sterile conditions in a 37°C incubator with 5% C02 for two weeks. Cells were grown in RPMI medium with 10% fetal bovine serum. Cells were passaged every 3-4 days with 0.05% Trypsin/EDTA. On the day of implantation,
HCC1954 cells were (harvested) lifted (passage x15) and re-suspended in RPM11640 serum- free media at a concentration of 5 x 106 cells and 50% matrigel/100 μΙ. Cells were Radii tested to assure that they are free of mycoplasma and murine viruses.
HCC1954 cells were implanted to the left 4th mammary fat pad using a 28 ½ G needle (5 x 10s cells /100 μΙ injection volume per mouse). After implant, tumors were measured by caliper and mice were weighed three times per week once tumors were palpable. Tumors were measured in two dimensions. Caliper measurements were calculated using (L x W2)/2. Mice were fed with normal diet and housed in 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 140 mm3, mice were administered by intravenous route 0.3 mg/kg, 1 mg/kg or 3 mg/kg of anti-HER2 mAb1-C1 or 3 mg/kg of Isotype lgG-C1 . 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 carry on normal activities (feeding, mobility), was impaired, mice were euthanized.
Results
HCC1954 breast tumor xenograft mice were treated intravenously with a single dose of anti-HER2 mAb1 -C1 at 0.3 mg/kg, 1 mg/kg or 3 mg/kg, or Isotype lgG-C1 at 3 mg/kg (10 mice per group). Treatment with a single dose of at 0.3 mg/kg, 1 mg/kg or 3 mg/kg anti-HER2 mAbl - C1 led to complete regression of human HCC1954 xenograft tumors (FIG. 7). Tumor regression was not observed in mice treated with 3 mg/kg of Isotype control lgG-C1 (FIG. 7).
These data show that tumor regression can be achieved in high HER2 expressing HCC1954 breast tumor xenograft by a single low dose treatment with anti-HER2 mAb1-C1 .
The treatments were well tolerated by all mice in all dosing groups except for slight and short time body weight loss post initial dosing observed for mice treated with 3 mg/kg anti-HER2 mAb1 -C1 and Isotype control lgG-C1 (FIG. 8).
Example 9: In vivo testing of anti-HER2 mAb1-C1 in a SKOV3 ovarian carcinoma xenograft model
Materials and Methods
For in vivo testing of the anti-Her2 mAb1 -C1 conjugate in the SKOV3 ovarian carcinoma xenograft mouse model, female SCID-beige mice at 6-8 weeks of age (purchased from Harlan Laboratories) were used for implantation. SKOV3 cells (obtained from ATCC, (CATALOG #HTB- 77; LOT #7349765)) were grown in sterile conditions in a 37°C incubator with 5% C02 for two weeks. Cells were grown in McCoy's5A medium with 10% fetal bovine serum. Cells were passaged every 3-4 days with 0.05% Trypsin/EDTA. On the day of implantation, SKOV3 cells were (harvested) lifted (passage x10) and re-suspended in McCoy's5A serum-free media at a concentration of 5 x 10s cells and 50% matrigel/100 μΙ. Cells were Radii tested to assure that they are free of mycoplasma and murine viruses.
SKOV3 cells were implanted with a subcutaneous injection into the lower flank using a
28 Vi G (5 x 10s cells /100 μΙ injection volume per mouse). After implant, tumors were measured by caliper and mice were weighed three times per week once tumors were palpable. Tumors then were measured three times a week in two dimensions. Caliper measurements were calculated using (L x W2)/2. Mice were fed with normal diet and housed in 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 200 mm3, mice were administered by intravenous route 0.3mg/kg, 1 mg/kg, or 3 mg/kg of anti-HER2 mAb1 -C1 or 3 mg/kg of Isotype lgG-C1. 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 carry on normal activities (feeding, mobility), was impaired, mice were euthanized.
Results
SKOV3 ovarian carcinoma xenograft mice were treated intravenously with a single dose of anti-HER2 mAb1 -C1 , at 0.3 mg/kg, 1 mg/kg and 3 mg/kg or 3 mg/kg of Isotype lgG-C1 . While treatment with a single dose of 3 mg/kg anti-HER2 mAb1 -C1 led to tumor regression of human SKOV3 carcinoma xenograft model, tumor regression was not observed in mice treated with 3 mg/kg of Isotype control lgG-C1 when compared to untreated animals (FIG. 9A). The treatments were well tolerated by all mice except slight and short time body weight loss post initial dosing was observed for mice dosed with 3 mg/kg anti-HER2 mAb1-C1 and Isotype lgG-C1 (FIG. 9B).
Example 10: Preparation of P-Cad mAb1 -C1 conjugate
Anti-P-Cad antibody with site-specific cysteine mutations was prepared as
described in Example 5. Briefly, DNA encoding variable regions of the heavy and light chains of anti-P-Cad mAb1 were cloned into a single bicistronic vector that contains constant regions of human lgG1 heavy chain with cysteine introduced at positions 152 and 375 (EU numbering) and human kappa light chain. A CHO stable line was produced by transfection of cells followed by selection, and stably transfected cells were then cultured under conditions optimized for antibody production. Antibody was purified from the cell supernatants by Protein A affinity chromatography (ToyoPearl AF-rProtein A-650F resin, Tosoh Bioscience). Antibody was further purified by SEC (Superdex-200 resin, GE), and the peak corresponding to the antibody monomer was collected for conjugation.
Antibody was reduced and reoxidized through similar methods as described in
Example 5. Antibody at 14 mg/ml was incubated in 50 mM sodium phosphate pH 8.0, 1 mM EDTA, 5 mM DTT for 30 minutes at room temperature. DTT was removed by SEC on a 50 ml G25 superfine column equilibrated in PBS pH 8.0. Antibody was adjusted to 5 mg/ml and incubated at room temperature for 24 hours. Reoxidation was monitored by
RP-HPLC as described above.
After reoxidation was complete, maleimide-containing compound C1 was added to re- oxidized antibody in PBS buffer (pH 7.2) at a molar ratio of 8: 1. Incubation was carried out for 30 minutes. Excess free compound was removed by purification over Protein A resin by standard methods followed by buffer exchange into PBS.
The resulting conjugate anti-P-Cad mAb1-C1 was analyzed as described above and had a compound to antibody ratio of 3.8 and aggregation below limit of quantitation. This sample was used in examples below.
Example 11 : In vivo efficacy of P-Cad mAB1 -C1 conjugate against the MC38 murine colon adenocarcinoma model in mice
Female C57BL/6 mice were implanted subcutaneously with 5x105 C38 cells overexpressing murine CDH3 in a suspension containing Dulbecco's modified MEM (Life Technologies). The total injection volume containing cells in suspension was 200 μΙ. At time of implant, mice were separated into treatment groups (n= 8/group) and dosed with a single IV administration of either P-Cad mAb1 -C1 (2.5 mg/kg), unconjugated P-Cad mAb1 (10 mg/kg), or vehicle control (PBS) as per Table 10C. Doses were adjusted to individual mouse body weights. The IV dose volume was 10 ml/kg.
Treatment with 2.5 mg/kg P-Cad mAb1 -C1 (26 %AT/AC) resulted in significantly higher anti-tumor activity compared to the vehicle control (PBS) treated group on Day 21 (One way ANOVA; Tukey's Test, p < 0.05). Treatment with 10 mg/kg P-Cad mAb1 (48 %AT/AC) was not significantly different from the PBS treated group on Day 21 (FIG. 13A). All treatments were well tolerated and no overt clinical symptoms of toxicities were observed (FIG. 13B). Table 10C. In vivo activity of P-Cad mAB1 -C1 against the MC38 murine colon adenocarcinoma model in mice on Day 21 . The effect of the treatment on tumor volumes and body weights are presented as means ± SEM. Day 21 , *p<0.05 P-Cad mAB1 -C1 versus PBS (One way
ANOVA/Tukey's Test).
13.1 ±
Single
P-Cad mAbl 10 48.4 - 474 ±97.5 1.34 8/8
dose/IV
Example 12: Preparation of Target B Abs and Target B mAb1 -C1 conjugate
Target B mAbl with site-specific cysteine mutations was prepared as described in Example 5. Briefly, DNA encoding variable regions of the heavy and light chains of Target B mAbl were cloned into two mammalian expression vectors that contain constant regions of human lgG 1 heavy chain with cysteine introduced at positions 152 and 375 (EU
numbering) and human kappa light chain. Vectors were co-transfected into a CHO cell line. Cells underwent selection, and stably transfected cells were then cultured under conditions optimized for antibody production. Antibody was purified from the cell
supernatants by standard Protein A affinity chromatography (MabSelect SuRe resin, GE Healthcare).
Target B mAbl was reduced, reoxidized, and conjugated using an on-resin method as described in Example 5. Antibody was loaded to protein A resin with 1 ml resin per 10 mg protein, reduced in the presence of 20 mM cysteine for 30 minutes, washed with PBS and then reoxidized in presence of 1 μΜ copper chloride. Once reoxidation progressed to desired completeness, conjugation was initiated by addition of 2.5 molar equivalent of compound C1 over engineered cysteines and allowing the mixture to react at room temperature before the column was washed with 20 column volumes of PBS. Antibody conjugate was eluted with IgG elution buffer, neutralized with 0.1 volumes 0.5 M sodium phosphate pH 8.0 and buffer exchanged to PBS.
The resulting Target B mAb1-C1 conjugate was analyzed as described above and had a compound to antibody ratio of 3.8 and 1 .2 % aggregate content. This sample was used in examples below. Example13: The Target B mAb1 -C1 conjugate regresses tumor growth in a xenograft tumor model expressing Target B.
Female SCID/Beige mice were implanted with 5 x 106 human breast cancer cells subcutaneously in the hind flank. Tumors were measured 3 times weekly throughout the course of the study using digital calipers and tumor volume calculated using the equation (LxW2)/2. When tumors reached an average size of 100 mm3, mice were randomized and dosed with a single intravenous injection of vehicle control (PBS), 10 mg/kg of Target B mAbl , 10 mg/kg of Target B mAb1 -C1 conjugate, or isotype control lgG-C1 conjugate. Mice were sacrificed when tumors in control groups reached an average size of 1000 mm3. As shown in FIG. 14, treatment with 10 mg/kg of Target B mAb1 -C1 induced regressions of the human breast cancer tumors, whereas vehicle control and Target B mAb1 did not have any effect on tumor growth. In contrast, treatment with lgG-C1 , an isotype antibody control-C1 conjugate, delayed the growth kinetics of the human breast cancer tumors, however, this delay was not significant compared with vehicle control. (FIG. 14, **** denotes p value of <0.0001 in an unpaired Student's t test).
Example 14:. Preparation of Target C mAb1 , and Target C mAb1 -C1 Conjugate
Target C mAb1 with site-specific cysteine mutations was prepared as described in Example 5. Briefly, DNA encoding variable regions of the heavy and light chains of Target C mAb1 were cloned into two mammalian expression vectors that contain constant
regions of human lgG1 heavy chain with cysteine introduced at positions 152 and 375 (EU numbering) and human kappa light chain. Alternatively, the variable region of the heavy chain of Target C mAbl was cloned into an additional mammalian expression vector that contains the constant region of human lgG1 heavy chain with cysteine introduced at positions 152 and 375 and mutations D265A and P329A (EU numbering). One heavy chain vector and one light chain vector at a time were co-transfected into a CHO cell line.
Cells underwent selection, and stably transfected cells were then cultured under
conditions optimized for antibody production. Antibody was purified from the cell
supernatants by standard Protein A affinity chromatography.
Target C mAbl and mAbl (DAPA) were reduced, reoxidized and conjugated using an on-resin method as described in Example 5. Antibodies were loaded to protein A resin with 1 ml resin per 10 mg protein, reduced in the presence of 15 mM cysteine for 40 minutes, washed with PBS and then reoxidized in presence of 1 μΜ copper chloride. Once reoxidation progressed to desired completeness, conjugations were initiated by addition of 2.5 molar equivalent of compound C1 over engineered cysteines and allowing the mixtures to react at room temperature before the columns were washed with 20 column volumes of PBS. Antibody conjugates were eluted with IgG elution buffer and neutralized with 0.1 volumes 0.5 M sodium phosphate pH 8.0 and buffer exchanged to PBS.
The resulting conjugates were analyzed as described above. The resulting conjugate Target C mAb1-C1 had a compound to antibody ratio of 3.8 and aggregation below limit of quantitation. The resulting conjugate Target C mAb1 (DAPA)-C1 had a compound to antibody ratio of 3.7 and aggregation below limit of quantitation. These samples were used in examples below.
Example 15. The Target C mAb1 -C1 conjugate delays tumor growth in a xenograft tumor model expressing Target C.
Female SCID Beige mice were implanted with 1 x 106 lung cancer cells expressing Target C subcutaneously in the hind flank. Tumors were measured 3 times weekly throughout the course of the study using digital calipers and tumor volume calculated using the equation (LxW2)/2. When tumors reached an average size of 1 15 mm3, mice were randomized and dosed with a single intravenous injection of vehicle control (PBS) or 8 mg/kg of Target C mAb1 -C1 , Target C mAb1 (DAPA)-C1 , or isotype lgG-C1 conjugate. As shown in FIG. 15, treatment with 8 mg/kg of Target C mAb1 -C1 and Target C mAb1 (DAPA)-C1 significantly delayed tumor growth compared to Vehicle control. lgG-C1 conjugate also showed significant anti-tumor efficacy compared to Vehicle control, though was somewhat less efficacious than Target C mAb1-C1 and Target C mAb1 (DAPA)-C1 .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 included within the spirit and purview of this application and the scope of the appended claims.

Claims

WE CLAIM:
1 . 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.
2. The immunoconjugate of claim 1 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 has agonist activity against STING receptor;
m is an integer from 1 to 8; and
n is an integer from 1 to 20.
3. The immunoconjugate of claim 1 comprising 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 D, or a cleavage product thereof, that is released from the immunoconjugate has STING agonist activity.
The immunoconjugate of claim 1 comprising 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.
5. The immunoconjugate of claim 1 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 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.
6. The immunoconjugate of claim 1 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 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.
7. The immunoconjugate of claim 1 for delivery of a STING receptor agonist to a cell, 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 cell 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 -Dual assay, a TANK binding kinase 1 (TBK1) assay, or an interferon-v-inducible protein 10 (IP-10) secretion assay.
8. The immunoconjugate of any one of the preceding claims, wherein 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.
9. The immunoconjugate of claim 8, wherein the STING-dependent cytokine is selected from interferon, type 1 interferon, IFN-a, IFN-β, type 3 interferon, IFN , IP10, TNF, IL-6, CXCL9, CCL4, CXCL1 1 , CCL5, CCL3, or CCL8.
10. The immunoconjugate of any one of claims 1 -7, wherein 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-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.
1 1 . The immunoconjugate of any one of claims 1 -7, wherein D, or the cleavage product thereof, has STING agonist activity if it binds to STING and is able to stimulate expression of a STING- dependent transcript selected from any one of the transcripts listed in Fig. 1A - Fig. 10 and Fig. 2A - Fig. 2L in a STING-expressing cell at least 5-fold or greater than the expression level in an untreated STING-expressing cell.
12. The immunoconjugate of claim 1 1 , wherein expression of the STING-dependent transcript is increased 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 700-fold or greater.
13. The immunoconjugate of any one of claims 1 -7, wherein D, or the cleavage product thereof, has STING agonist activity if it binds to STING and is able to stimulate expression of a luciferase reporter gene controlled by interferon (IFN)-stimulated response elements in a STING-expressing cell at an EC50 of 20 micromolar (μΜ), 15 μΜ , 10 μΜ, 9 μΜ , 8 μΜ , 7 μΜ, 6 μΜ , 5 μΜ , 4 μΜ, 3 μΜ , 2 μΜ , 1 μΜ , or less.
14. The immunoconjugate of any one of claims 1 -7, wherein D, or the cleavage product thereof, has STING agonist activity if it binds to STING and is able to stimulate expression of a luciferase reporter gene controlled by interferon (IFN)-stimulated response elements in a STING-expressing cell to a level equal to or greater than the level of stimulation of 50 μΜ of 2'3'-cGAMP.
15. The immunoconjugate of claim 13 or 14, wherein the STING-expressing cell is THP1 -Dual cell, and the luciferase reporter gene is the IRF-Lucia reporter gene in THP1 -Dual cell, and optionally the STING agonist activity is determined by the THP1 -Dual assay described for Table 7.
16. The immunoconjugate of claim 13 or 14, wherein the luciferase reporter gene is the
5xlSRE-mlFNb-GL4 reporter gene and the STING-expressing cell is a cell expressing wild-type human STING protein, and optionally the STING agonist activity is determined by the hSTING wt assay described in Table 7.
17. The immunoconjugate of any one of claims 1 -7, wherein the immunoconjugate stimulates IP-10 secretion from a STING-expressing cell targeted by the Ab at an EC50 of 5 nanomolar
(nM) or less in an IP-10 secretion assay.
18. The immunoconjugate of anyone of the preceding claims, wherein the immunoconjugate is parenterally administered.
19. The immunoconjugate of any one of the preceding claims, wherein the Ab specifically binds a target antigen.
20. The immunoconjugate of claim 19, wherein the target antigen is a tumor antigen.
21 . The immunoconjugate of any one of the preceding claims, wherein the Ab is human or humanized.
22. The immunoconjugate of any one of the preceding claims, wherein the Ab is a monoclonal antibody.
23. The immunoconjugate of any one of the preceding claims, wherein the Ab comprises a modified Fc region.
24. The immunconjugate of claim 23, 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.
25. The immunconjugate of any one of the preceding claims, wherein L is attached to the Ab via conjugation to one or more modified cysteine residues in the Ab.
26. The immunoconjugate of claim 25, wherein 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.
27. The immunconjugate of claims 24 to 26, wherein L is conjugated via a maleimide linkage to the cysteine.
28. The immunoconjugate of any one of the preceding claims, wherein D is a dinucleotide.
29. The immunoconjugate of any one of the preceding claims, wherein D is a cyclic dinucleotide (CDN).
30. The immunoconjugate of any of the preceding claims, wherein D is a compound selected from any one of the compounds of Table 1 , Table 2, Table 3, or Table 4.
31. The immunoconjugate of any of the preceding claims, wherein D is a compound selected from
and
32. The immunoconjugate of any of the preceding claims, wherein L is a cleavable linker comprising one or more cleavage elements.
33. The immunoconjugate of claim 32, wherein L comprises two or more cleavage elements, and each cleavage element is independently selected from a self-immolative spacer and a group that is susceptible to cleavage.
34. The immunoconjugate of claim 33, wherein 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.
35. The immunoconjugate of claim 32, where L has a structure selected from: I (Lc)x CE D _ -HLc)x-CE— (Lc)y-CE-D ^ and ~^((Lc)x "CE)p-"(Lc)y ~CE ~D ^ wherein:
Lc is a linker component and each Lc is independently selected from a linker component as shown in Embodiments 70 to 75;
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 of claims 28 to 31 ;
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.
36. The immunoconjugate of any one of the preceeding claims, wherein L comprises a structure selected from:
and o
37. The immunoconjugate of any one of the preceding claims, wherein the immunoconjugate is selected from the following:
Formula (BB-a) Formula (BB-b)
637
Formula (CC-e) Formula (CC-f)
639
Formula (FF-a) Formula (FF-b)
Formula (FF-e) Formula (FF-f)
Formula (FF-i) Formula (FF-j)
Formula (FF-k);
wherein:
each Gi is independently selected from and
where * indicates the point of attachment to -CR8R9-;
XA is C(=0)-, -C(=S)- or -C(=NR11)- and each Ζ is NR12;
XB is C, and each Z2 is N;
G2 is , where * indicates the point of attachment to -CR8aR9a-;
Xc is C(=0)-, -C(=S)- or -C(=NR11)- and each Z3 is NR12;
YI is -0-, -S-, -S(=0)-, -SO2-, -CH2-, or -CF2-;
Y2 is -0-, -S-, -S(=0)-, -SO2-, -CH2-, or -CF2-;
Y3 is OH, 0", OR10, N(R10)2, SR10, SeH, Se", BH3, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SR10, SeH, Se", BH3, SH or S";
Ys is -CH2-, -NH-, -0- or -S;
Y6 is -CH2-, -NH-, -0- or -S;
Y8 is 0 or S; Y9 is -CH2-, -NH-, -O- or -S;
Y10 is -CH2-, -NH-, -O- or -S;
Y11 is -0-, -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 0, 1 , 2, 3 or 4 substituents independently selected from -NHL,R115, F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, CrC6alkoxyalkyl, CrC6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from O, N and S, -0(Cr C6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), -S(CrC6aminoalkyr), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(C C6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(C C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -(CH2)1.10C(=O)OH,- -NHC(0)(Ci-C6alkyl), -NHC(0)(C3-C8cycloalkyl), -
NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
R1a 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 -NHLiR115, F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, CrCealkoxyalkyl, CrC6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from O, N and S, -0(Cr C6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), -S(CrC6aminoalkyr), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(C C6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(C
C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- CH=CH(CH2)1.10C(=O)OH, -NHC(0)(C C6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
R1b 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 -NHLiR115, F, CI, Br, OH, SH, NH2, D, CD3, CrC6alkyl, CrCealkoxyalkyl, CrC6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from O, N and S, -0(Cr
C6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(Cr C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2,
-NHC(0)(C C6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
each R2 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, C Cealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-
C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl,
CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R3 is independently selected from the group consisting of -OI_iR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and the
CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R4 is independently selected from the group consisting of -OLiR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Ci-C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -
OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4 are substituted by
0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; each R5 is independently selected from the group consisting of -OLiR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R6 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R6 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R7 is independently selected from the group consisting of -OLiR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R8 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R8 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R9 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, C Cealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R9 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Ci-C6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R2a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3
R3a is selected from the group consisting of -OL^R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R4a is selected from the group consisting of -OL^R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R5a is selected from the group consisting of -OI_iR115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the C Cealkyl, C2-C6alkenyl, C2-C6alkynyl, C Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R6a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, C C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr Csalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)C C6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6a and the C C6alkyl, C2-C6alkenyl and C2-C6alkynyl of the C Cealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R6aare substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; R7a is selected from the group consisting of -OL^R115, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R8a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, C C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)C C6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R8a and the C C6alkyl, C2-C6alkenyl and C2-C6alkynyl of the Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)C C6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R9a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, C C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)C C6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R9a and the Ci-C6alkyl, C2-C6alkenyl and C2-C6alkynyl of the C Cealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3; each R10 is independently selected from the group consisting of H, CrC12alkyl, Cr
C6heteroalkyl, , wherein the
C12alkyl and CrC6heteroalkyl of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, CrC12alkoxy, -S-C(=0)C1-C6alkyl, halo, -CN, Cr C12alkyl, -O-aryl, _0-heteroaryl, -O-cycloalkyl, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, -OC(0)OCrC6alkyland C(0)OCrC6alkyl, wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is substituted by 0, 1 , 2 or 3 substituents independently selected from CrC12 alkyl, 0-CrC12alkyl, CrCeheteroalkyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroaryl,
C12alkyl, -OC(=0)OC1-C12alkyl, -C(=0)N(R11)-C1-C12alkyl, -N(R11)C(=0)-C1-C12alkyl; - OC(=0)N(R1 1)-C1-C12alkyl, -C(=0)-aryl, -C(=0)-heteroaryl, -OC(=0)-aryl, -C(=0)0-aryl, OC(=0)-heteroaryl, -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(0)-aryl, - N(R11)C(0)-heteroaryl, and S(0)2N(R11)-aryl;
each R1 1 is independently selected from H and CrC6alkyl;
each R12 is independently selected from H and C Cealkyl;
optionally R3 and R6 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3 and R6 are connected, the O is bound at the R3 position
optionally R3a and R6a, are connected to form CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3a and R6a are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4a and R3a are connected, the O is bound at the R3a position; optionally R5 and R6 are connected to form CrCalkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R6 are connected, the O is bound at the R5 position;
optionally R5a and R6a, are connected to form CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R6a are connected, the O is bound at the R5a position;
optionally R5 and R7 are connected to form C Cealkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R7 are connected, the O is bound at the R5 position;
optionally R5a and R7a, are connected to form C Cealkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R7a are connected, the O is bound at the R5a position;
optionally R8 and R9 are connected to form a CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene, and
optionally R8a and R9a are connected to form a CrC6alkylene, C2-C6alkenylene, C2-
, , , -C(=0)-, -ON=***, -S-, -NHC(=0)CH2-***, -
S(=0)2CH2CH2-***, -(CH2)2S(=0)2CH2CH2-***, -NHS(=0)2CH2CH2..., -NHC(=0)CH2CH2- piio
where *** indicates the point of attachment to Ab;
R13 is H or methyl;
R14 is H, -CH3 or phenyl;
each R1 10 is independently selected from H, Ci-C6alkyl, F, CI, and -OH;
each R1 11 is independently selected from H, C C6alkyl, F, CI, -NH2, -OCH3, -OCH2CH3, -
N(CH3)2, -CN, -N02 and -OH;
each R1 12 is independently selected from H, Ci-6alkyl, fluoro, benzyloxy substituted with -
C(=0)OH, benzyl substituted with -C(=0)OH, substituted with -C(=0)OH and
C1-4alkyl substituted with -C(=0)OH;
Ab is an antibody or a functional fragment thereof; and
y is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. The immunoconjugate of any one of the preceding claims comprising a structure selected from:
654
656
658
664
665
666
667
668
670
673
676
39. The immunoconjugate of any one of the preceding claims, wherein the immunoconjugate has in vivo anti-tumor activity.
40. A pharmaceutical composition comprising the immunconjugate according to any one of the preceding claims and a pharmaceutically acceptable excipient.
41 . A composition comprising the immunoconjugate according to any one of the preceding claims in combination and one or more additional therapeutic agents.
42. The composition of claim 41 , 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 cell therapy.
43. The composition of claim 41 , 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-inhibitory molecule is selected from Programmed death-1 (PD-1), Programmed death-ligand 1 (PD-L1), Lymphocyte activation gene-3 (LAG-3), or T-cell immunoglobulin domain and mucin domain 3 (TIM-3),
(ii) the co-stimulatory molecule is Glucocorticoid-induced TNFR-related protein (GITR), and
(iii) the cytokine is IL-15 complexed with a soluble form of IL-15 receptor alpha (IL-
15Ra).
44. 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 -39, the pharmaceutical composition of claim 40, or the composition of claims 41 -43.
45. Use of the immunconjugate of any one of claims 1 -39, the pharmaceutical composition of claim 40, or the composition of claims 41 -43 for treatment of a cancer in a subject in need thereof.
46. The immunconjugate of any one of claims 1 -39, the pharmaceutical composition of claim 40, or the composition of claims 41 -43, for use in the treatment of cancer.
47. Use of the immunconjugate of any one of claims 1 -39, the pharmaceutical composition of claim 40, or the composition of claims 41 -43 in the manufacture of a medicament for treatment of a cancer in a subject in need thereof.
48. The method according to claim 43, the use according to claim 44 or 46, or the
immunoconjugate of claim 45, wherein the cancer is selected from sarcomas,
adenocarcinomas, blastomas, 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 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 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), myelodysplasia syndromes, B-cell acute lymphoid leukemia ("BALL"), T-cell 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 lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplasia syndrome, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.
49. The method according to claim 43, the use according to claim 44 or 45, or the
immunoconjugate of claim 45, wherein the immunoconjugate is administered to the subject intravenously, intratumorally, or subcutaneously.
50. The immunconjugate of any one of claims 1-39, the pharmaceutical composition of claim 40, or the composition of claims 41-43, for use as a medicament.
51. A method of manufacturing the immunoconjugate of any one of claims 1-38 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)m)n (Formula (I)).
52. 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,
Formula (C) Formula (D)
Formula (E) Formula (F) wherein:
each Gi is independently selected from
indicates the point of attachment to -CR8R9-;
XA is C(=0)-, -C(=S)- or -C(=NR11)- and each is NR12;
wwhheerree ** indicates the point of attachment to -CR8aR9a-;
Xc is C(=0)-, -C(=S)- or -C(=NR11)- and each Z3 is NR12;
YI is -0-, -S-, -S(=0)-, -SO2-, -CH2-, or -CF2-;
Y2 is -0-, -S-, -S(=0)-, -S02-, -CH2-, or -CF2-;
Y3 is OH, 0", OR10, N(R10)2, SR10, SeH, Se", BH3, SH or S";
Y4 is OH, 0", OR10, N(R10)2, SR10, SeH, Se", BH3, SH or S";
Ys is -CH2-, -NH-, -0- or -S;
Y6 is -CH2-, -NH-, -0- or -S;
Y8 is 0 or S;
Y9 is -CH2-, -NH-, -0- or -S; Y10 is -CH2-, -NH-, -O- or -S;
Y11 is -0-, -S-, -S(=0)-, -SO2-, -CHr, 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 0, 1 , 2, 3 or 4 substituents independently selected from -NHL,R15, F, CI, Br, OH, SH, NH2, D, CD3, C C6alkyl, CrC6alkoxyalkyl, CrC6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from O, N and S, -0(Cr
C6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), -S(CrC6aminoalkyr), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(C C6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(C C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2,
CH=CH(CH2)1.10C(=O)OH, -NHC(0)(C C6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
R1a 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 -NHLiR15, F, CI, Br, OH, SH, NH2, D, CD3, C
C6alkyl, CrCealkoxyalkyl, CrCehydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from O, N and S, -0(Cr C6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl), -S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(Cr C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,-
CH=CH(CH2)1.10C(=O)OH, -NHC(0)(CrC6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
R1b 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 -NHLiR15, F, CI, Br, OH, SH, NH2, D, CD3, Cr C6alkyl, CrC6alkoxyalkyl, CrC6hydroxyalkyl, C3-C8cycloalkyl, a 3 to 6 membered heterocyclyl having 1 to 2 heteroatoms independently selected from O, N and S, -0(Cr C6alkyl), -0(C3-C8cycloalkyl), -S(CrC6alkyl), -S(CrC6aminoalkyl), -S(CrC6hydroxyalkyl),
-S(C3-C8cycloalkyl), -NH(CrC6alkyl), -NH(C3-C8cycloalkyl), -N(CrC6alkyl)2, -N(Cr C6alkyl) (C3-C8cycloalkyl), -CN, -P(=0)(OH)2, -O(CH2)1.10C(=O)OH, -(CH2)1.10C(=O)OH,- CH=CH(CH2)i-i0C(=O)OH,-NHC(O)(C1-C6alkyl), -NHC(0)(C3-C8cycloalkyl), - NHC(0)(phenyl), and -N(C3-C8cycloalkyl)2;
each R2 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2- Cehaloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -
O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -
0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R3 is independently selected from the group consisting of -OLiR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl,
C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)Ci-C6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3 and the CrCealkyl, C2-C6alkenyl and C2-C6alkynyl of the C C6alkyl, C2-C6alkenyl, C2-C6alkynyl,
CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R4 is independently selected from the group consisting of -OLiR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - -OC(0)Ophenyl, -OC(0)OCi-C6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4 and the
CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R5 is independently selected from the group consisting of -OLiR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R6 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrCealkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCi-C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R6 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R7 is independently selected from the group consisting of -OLiR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Ci-C6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R8 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R8 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
each R9 is independently selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, C Cealkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6haloalkyl, C2-C6haloalkenyl, C2- C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, - O(CH2)1.10C(=O)OH, -O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, - OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R9 and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, C Cehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), - 0(C2-C6alkynyl), -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, - OC(0)Ci-C6alkyl, -OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R2a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, C C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)C C6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R2a and the C C6alkyl, C2-C6alkenyl and C2-C6alkynyl of the C Cealkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)C C6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R2a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3
R3a is selected from the group consisting of -OI_iR15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Ci C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, 0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R3a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R3a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R4a is selected from the group consisting of -OL^R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, C C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - OCd-Csalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, -
O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)C C6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R4a and the C C6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- Cehaloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -
OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)C C6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R4a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R5a is selected from the group consisting of -OL^R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, C C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -
OCd-Csalkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)Ci-C6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R5a and the C C6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-
C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)C C6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R5a are substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R6a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, C C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R6a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R6aare substituted by 0,1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R7a is selected from the group consisting of -OL^R15, H, -OH, F, CI, Br, I, D, CD3, CN, N3, Cr C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, - 0(Ci-Cealkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)C C6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R7a and the C C6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(C C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)C C6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R7a are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R8a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, C C6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OC C6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)C C6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R8a and the Ci-C6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrCehaloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(CrC6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R8 are substituted by 0, 1 , 2 or 3 substituents independently selected from F, CI, Br, I, OH, CN, and N3;
R9a is selected from the group consisting of H, -OH, F, CI, Br, I, D, CD3, CN, N3, CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2-C6haloalkenyl, C2-C6haloalkynyl, -0(Cr C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), -OP(=0)(OH)2, -O(CH2)1.10C(=O)OH, - O(CH2)1.10P(=O)(OH)2, -OC(0)Ophenyl, -OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, - OC(0)OC2-C6alkynyl, -OC(0)phenyl, -OC(0)CrC6alkyl, -OC(0)C2-C6alkenyl and - OC(0)C2-C6alkynyl, wherein the -OC(0)Ophenyl of R9a and the CrC6alkyl, C2-C6alkenyl and C2-C6alkynyl of the CrC6alkyl, C2-C6alkenyl, C2-C6alkynyl, CrC6haloalkyl, C2- C6haloalkenyl, C2-C6haloalkynyl, -0(Ci-C6alkyl), -0(C2-C6alkenyl), -0(C2-C6alkynyl), - OC(0)OCrC6alkyl, -OC(0)OC2-C6alkenyl, -OC(0)OC2-C6alkynyl, -OC(0)CrC6alkyl, - OC(0)C2-C6alkenyl and -OC(0)C2-C6alkynyl of R9a are substituted by 0, 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 of H, CrC12alkyl, Cr
Ceheteroalkyl, , wherein the Ci
C12alkyl and CrC6heteroalkyl of R10 is substituted by 0, 1 , 2 or 3 substituents independently selected from -OH, CrC12alkoxy, -S-C(=0)C1-C6alkyl, halo, -CN, Cr C12alkyl, -O-aryl, _0-heteroaryl, -O-cycloalkyl, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, -OC(0)OCrC6alkyland C(0)OCrC6alkyl, wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is substituted by 0, 1 , 2 or 3 substituents independently selected from CrC12 alkyl, 0-CrC12alkyl, CrC12heteroalkyl, halo, CN, OH, oxo, aryl, heteroaryl, O-aryl, O-heteroaryl, -C(=0)C1-C12alkyl, -OC(=0)C1-C12alkyl, -C(=0)OC C12alkyl, -OC(=0)OC1-C12alkyl, -C(=0)N(R11)-C C12alkyl, -N(R11)C(=0)-C1-C12alkyl; - OC(=0)N(R1 1)-C C12alkyl, -C(=0)-aryl, -C(=0)-heteroaryl, -OC(=0)-aryl, -C(=0)0-aryl, - OC(=0)-heteroaryl, -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(0)-aryl, -
N(R11)C(0)-heteroaryl, and S(0)2N(R11)-aryl;
each R1 1 is independently selected from H and CrC6alkyl;
each R12 is independently selected from H and C Cealkyl;
optionally R3 and R6 are connected to form CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene, -0-Ci-C6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3 and R6 are connected, the O is bound at the R3 position
optionally R3a and R6a, are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-Ci-C6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R3a and R6a are connected, the O is bound at the R3a position;
optionally R2 and R3 are connected to form C Cealkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2 and R3 are connected, the O is bound at the R3 position;
optionally R2a and R3a, are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R2a and R3a are connected, the O is bound at the R3a position;
optionally R4 and R3 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4 and R3 are connected, the O is bound at the R3 position;
optionally R4a and R3a, are connected to form CrC6alkylene, C2-C6alkenylene, C2- C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R4a and R3a are connected, the O is bound at the R3a position;
optionally R5 and R6 are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R6 are connected, the O is bound at the R5 position;
optionally R5a and R6a, are connected to form CrC6alkylene, C2-C6alkenylene, C2-
C6alkynylene, -0-CrC6alkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R6a are connected, the O is bound at the R5a position; optionally R5 and R7 are connected to form Ci-Cealkylene, C2-C6alkenylene, C2-
C6alkynylene, -O-C Cealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5 and R7 are connected, the O is bound at the R5 position;
optionally R5a and R7a, are connected to form C Cealkylene, C2-C6alkenylene, C2-
C6alkynylene, -O-CrCealkylene, -0-C2-C6alkenylene, -0-C2-C6alkynylene, such that when R5a and R7a are connected, the O is bound at the R5a position;
optionally R8 and R9 are connected to form a Ci-Cealkylene, C2-C6alkenylene, C2- C6alkynylene, and
optionally R8a and R9a are connected to form a C Cealkylene, C2-C6alkenylene, C2- C6alkynylene,
-C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;
-C(=0)OC(R12)2(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-**;
-C(=0)0(CH2)mNR11C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**; -
C(=0)0(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**;
-C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -C(=0)(CH2)mNR11C(=0)X2C(=0)(CH2)m-**; -C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**,
-C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**,
-C(=0)0(CH2)mXsC(=0)(CH2)m-**,
-C(=0)0(CH2)mX6C(=0)(CH2)mO(CH2)m-**,
-C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**,
-C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)mO(CH2)m-**,
-C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)- **,
-C(=0)0(CH2)mXeC(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**,
-C(=0)X4C(=0)X6(CH2)mNR11C(=0)(CH2)mO(CH2)m-**,
-C(=0)(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**,
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR1 C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-
C(=0)0((CH2)mO)n(CH2)mNR11C(=0))X5C(=0)((CH2)mO)n(CH2)mX3(CH2)n -C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)m-
**.
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR1 1 C(=0)X5((CH2)mO)n(CH2)mNR1 1 C(=0)(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mNR11 ((CH2)mO)n(CH2)m-**; - C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11 ((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**; -C(=0)0(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)m-**; -C(=0)0(CH2)mNR1 1(CH2)m-**;
-C(=0)0(CH2)mNR11(CH2)mC(=0)X2XiC(=0)-**;
-C(=0)0(CH2)mX3(CH2)m-**; -C(=0)0(CH2)mX6C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**, -
C(=0)0((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)0(CH2)mNR11C(=0(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)nX3(CH2)m-**; -C(=0)0((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)0((CH2)mO)n(CH2)mC(=0)NR11 (CH2)m-**; -C(=0)0(CH2)mC(R12)2-**;
-C(=0)OCH2)mC(R12)2SS(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)0(CH2)mC(=0)NR11 (CH2)m-**; -C(=0)(CH2)m-**; -C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)(CH2)mNR11 (CH2)m-**; -C(=0)(CH2)mNR11 (CH2)mC(=0)X2X1C(=0)-**;
-C(=0)(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)(CH2)mNR11C(=0)(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mNR1 1C(=0)(CH2)m-**; -
C(=0)(CH2)mNR11C(=0(CH2)mX3(CH2)m-**; -(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;
-(CH2)m(CHOH)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)nX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mC(=0)NR11 (CH2)m-**; -
C(=0)(CH2)mC(R12)2-**; -C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-** -C(=0)((CH2)mO)n(CH2)mNR11C(=0))X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11 C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11 C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11 C(=0)(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mNR11 C(=0)(CH2)mX3(CH2)m-**; -C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mNR1 1 ((CH2)mO)n(CH2)m-**; - C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11 ((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)m-**; - C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)((CH2)mO)n(CH2)mNR11C(=0)X5(CH2)mX3(CH2)m-**; -
C(=0)(CH2)mC(R12)2SS(CH2)mNR11 C(=0)(CH2)m-**; -C(=0)(CH2)mC(=0)NR11 (CH2)m-**; -C(=0)X1X2C(=0)(CH2)m-**; -C(=0)X1X2C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X1X2C(=0)(CH2)mX3(CH2)m-**; -C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C(=0)X1X2C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)X1X2C(=0)(CH2)mNR11 C(=0)((CH2)mO)n(CH2)m-**;
-C(=0)X1X2C(=0)(CH2)mNR11 C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)X1X2(CH2)mX3(CH2)m-**; -C(=0)X1X2((CH2)mO)n(CH2)m-**;
-C(=0)X1X2((CH2)mO)n(CH2)mNR11 C(=0)(CH2)m-**;
-C(=0)X1X2((CH2)mO)n(CH2)mNR11 C(=0)(CH2)mX3(CH2)m-**;
-C(=0)X1X2((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)X1X2(CH2)mNR11 ((CH2)mO)n(CH2)m-**; -
C(=0)X1X2C(=0)(CH2)mNR11 ((CH2)mO)n(CH2)mX3(CH2)m-**;
-C(=0)NR11 (CH2)m-**; -C(=0)NR11 (CH2)mX3(CH2)m-**;
-C(=0)NR11 (CH2)mNR11 C(=0)X1X2C(=0) (CH2)m-**;
-C(=0)NR11 (CH2)mNR11 C(=0)0(CH2)m-**; -C(=0)NR11 (CH2)mNR11C(=0)X1X2-**; -
C(=0)NR11 (CH2)mNR11C(=0)X5-; -C(=0)NR11 (CH2)mNR11 C(=0)(CH2)mX5(CH2)m-**; -
C(=0)X1 C(=0)NR11 (CH2)mXs(CH2)m-**; -C(=0)X6C(=0)(CH2)mNR11 C(=0)X1X2C(=0)(CH2)m-
**; -C(=0)NR11 (CH2)mNR11 C(=0)(CH2)m-**; -C(=0)NR11 (CH2)mNR11 C(=0)(CH2)mO(CH2)m-**;
-C(=0)NR11 (CH2)mNR11 C(=0)X1X2C(=0)(CH2)mO(CH2)m-**;
-C(=0)NR11 (CH2)mNR11 C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-**;
-C(=0)NR11 (CH2)mNR11 C(=0)X4C(=0)NR11 (CH2)mNR11 C(=0)(CH2)mO(CH2)m-**; -C(=0)NR11 (CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -
C(=0)NR11 (CH2)mNR11 C(=0)X5C(=0)(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5C(=0)(CH2)mX3(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5C(=0)((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5C(=0)((CH2)mO)n(CH2)mX3(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)mX3(CH2)m-
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5(CH2)mX3(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5((CH2)mO)n(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5((CH2)mO)n(CH2)mNR11C(=0)(CH2)mX3(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5((CH2)mO)n(CH2)mX3(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1 C(=0)X5(CH2)mNR11 ((CH2)mO)n(CH2)m-**; -
C(: =0)NR11 (CH2)mNR11 C(=0)X5C(=0)(CH2)mNR11 ((CH2)mO)n(CH2)mX3(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5(CH2)m-**; -
C(: =0)NR11 (CH2)mNR11 C(=0)X5C(=0)((CH2)mO)n(CH2)m-**;
-C( =0)NR1 1 (CH2)mNR1 1C(=0)X5(CH2)mX3(CH2)m-**; -
C(: =0)X1C(=0)NR11 (CH2)mNR1 1C(=0)(CH2)m-**;
=0)X1C(=0)NR11(CH2)mX3(CH2)m-**; -C(=0)NR11(CH2)mNR11C(=0)(CH2)n
=0)NR11(CH2)mNR11C(=0)(CH2)mX3(CH2)m-* -C(=0)NR11(CH2)mNR11C(=0)-*
(=0)X1X2(CH2)m-**; -C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**;
(=0)X1X2(CH2)mX3(CH2)m-**; -C(=0)NR11(CH2)mX3(CH2)m-**; - C(=0)NR11((CH2)mO)n(CH2)mX3(CH2)m-**; -C(=0)X1X2C(=0)((CH2)mO)n(CH2)m- -C(=0)X1X2C(=0)(CH2)m-**; -C(=0)X1C(=0)(CH2)mNR11C(=0)(CH2)m-**; and
=0)X1C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2),
S(=0)2(CH=CH2), -(CH2)2S(=0)2(CH=CH2), -NHS(=0)2(CH=CH2), -NHC(=0)CH2Br, - =0)CH2l, , -C(0)NHNH2
or where the * indicates the point of attachment to x2; X2 i
* indicates the point of attachment to Xi
-0(CH2)nSSC(R12)2(CH2)n- or -(CH2)nC(R12)2SS(CH2)nO-;
Xs is where the indicates orientation toward the Drug moiety; X6 is , ¾ , or or, where the ** indicates orientation toward the Drug moiety;
R17 is 2-pyridyl or 4-pyridyl;
each R1 1 is independently selected from H and CrCealkyl;
each R12 is independently selected from H and Ci-C6alkyl;
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 R1 10 is independently selected from H, C Cealkyl, F, CI, and -OH;
each R1 11 is independently selected from H, C C6alkyl, F, CI, -NH2, -OCH3, -OCH2CH3, - N(CH3)2, -CN, -N02 and -OH;
each R1 12 is independently selected from H, C1-6alkyl, fluoro, benzyloxy substituted with - C(=0)OH, benzyl substituted with -C(=0)OH, d.4alkoxy substituted with -C(=0)OH and C1-4alkyl substituted with -C(=0)OH;
and provided at least one of R1 , R1a or R1 b is substituted with -NHL^R15, or at least one of R3, R4, R5, R7, R3a, R4a, R5a or R7a is -OLiR15.
53. The com ound of claim 52 wherein:
**; -C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; - C(=0)OC(R12)2(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -
C(=0)0(CH2)mNR8C(=0)X1X2C(=0)(CH2)mO(CH2)m-**; - C(=0)0(CH2)mNR11C(=0)X1X2C(=0)(CH2)mO(CH2)mC(=0)-**; - C(=0)0(CH2)mNR11C(=0)X4C(=0)NR11 (CH2)mNR11C(=0)(CH2)mO(CH2)m-**; - C(=0)0(CH2)mNR11C(=0)X5C(=0)(CH2)mNR11C(=0)(CH2)m-**; - C(=0)0(CH2)mX6C(=0)X1X2C(=0)((CH2)mO)n(CH2)m-**; -
(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; - (CH2)m(CHOH)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m**; - C(=0)X6C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**;- C(=0)X4C(=0)NR11(CH2)mNR11C(=0)(CH2)mO(CH2)m-**; - C(=0)(CH2)mNR11C(=0)X1X2C(=0)(CH2)m-**; -
C(=0)0(CH2)mX6C(=0)X1X2C(=0)(CH2)m-**, or - C(=0)(CH2)mNR11C(=0)((CH2)mO)n(CH2)m-**,
where the ** indicates the point of attachment to R15.
54. The compound of claim 52 or claim 53 selected from:
(C23b)
698 (C38)
(C45).
55. The compound of claim 52 or claim 53 selected from:
(C42a)
56. The compound of claim 52 or claim 53 selected from:
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