WO2026044149A2 - Synthèse de combinaisons médicament-lieur pour conjugués anticorps-médicament - Google Patents

Synthèse de combinaisons médicament-lieur pour conjugués anticorps-médicament

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Publication number
WO2026044149A2
WO2026044149A2 PCT/US2025/043044 US2025043044W WO2026044149A2 WO 2026044149 A2 WO2026044149 A2 WO 2026044149A2 US 2025043044 W US2025043044 W US 2025043044W WO 2026044149 A2 WO2026044149 A2 WO 2026044149A2
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formula
compound
protecting group
configuration
attorney reference
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English (en)
Inventor
Dae-Shik Kim
Kenzo ARAI
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Eisai R&D Management Co Ltd
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Eisai R&D Management Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H99/00—Subject matter not provided for in other groups of this subclass
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length

Definitions

  • ADCs can deliver up to 1,000-fold more drug to tumor cells when conjugated to an antigen-specific antibody when compared to the amount delivered by the free, non-conjugated drug.
  • the use of ADCs in cancer treatment can also reduce the onset of pleiotropic drug resistance, increase the sensitivity of drug-insensitive tumor types to chemotherapeutics and reduce the onset of dose-dependent toxicities.
  • a specific cancer that has been challenging to treat is prostate cancer.
  • Prostate cancer is the second most common type of cancer and the second leading cause of cancer death in men.
  • Prostate-specific membrane antigen is a cell-surface antigen that is highly expressed in prostate cancer. Expression levels of PSMA increase along with prostate cancer progression, with high expression of PSMA maintained at metastatic sites.
  • Anti-PSMA antibodies have previously been generated, including modified antibodies with reduced 1 Attorney Reference: 0080171-000886 immunogenicity in humans. See, e.g., U.S. Patent No.7,045,605 and U.S. Patent No.11,059,903. Examples of antibodies that bind PSMA are J591 and deimmunized J591 (deJ591). However, clinical trials using this antibody have shown undesirable effects of immunogenicity, including myelosuppression and liver enzyme abnormalities.
  • STING stimulator of interferon genes
  • IFN- ⁇ interferon- ⁇
  • TNF ⁇ tumor necrosis factor alpha
  • CXCL10 C-X-C Motif Chemokine Ligand 10
  • IL-6 interleukin-6
  • STING cancer protection activity is also supported by evidence obtained from mouse model studies. STING knockout mice have shown defective tumor control. (Woo SR, et al. "STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors” Immunity 2014;41:830-42).
  • First-generation STING agonists e.g., cyclic dinucleotides, often require intra-tumoral injection and show only modest systemic efficacy. These STING agonists are also poorly membrane permeable, which may limit their ability to engage STING inside the cell. Thus, while uses of STING agonists for treating infection or disease have been reported in the art, there remains unsolved challenges in improving the delivery of these compounds specific tumor sites.
  • the present application provides a solution to these challenges by providing methods for preparing drug-linker combinations that can be conjugated with anti-antibodies and/or antigen-binding fragments thereof to form ADCs that can be used to treat prostate 2 Attorney Reference: 0080171-000886 cancers.
  • the ADCs prepared by the methods disclosed herein can bind to PSMA with improved efficiencies and selectivities, thereby allowing higher payloads of STING agonists to be delivered to cancerous cells or tissues expressing PSMA.
  • One aspect of the present disclosure is a method (or process) for preparing a compound of Formula (V): an isomer thereof, a deuterated derivative thereof; or a salt thereof, wherein: P a and P b are each independently a phosphorus atom that is either achiral, has a (R)- configuration, or has a (S)-configuration; each of X a and X b are independently selected from OH and SH; each of Y a and Y b are independently selected from O and S; L 2 is a first self-immolative linker unit, where e and d denote attachment points to moieties adjacent to L 2 ; L 1 is a linker moiety having a structure of: 3 Attorney Reference: 0080171-000886 , wherein: W is a second self-immolative linker unit; U is a peptide moiety; and K is a spacer unit; m is selected from 1 and 2;
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (E): wherein: G 1 is a first protecting group; G 2 is a second protecting group; G 3 is a third protecting group, wherein one of G1, G2, and G3 can be selectively removed under conditions that render the others stable; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); m is selected from 1 and 2; 11 Attorney Reference: 0080171-000886 o is selected from 1 and 2; and O N , the the method including: reacting a compound of Formula (D) with a diol with the following structure: in suitable reaction conditions , to orm t e compound o ormula (E), for example, in the presence of triphenylphosphine and an azodicarboxylate, wherein the compound of Formula (D) has the following structure: 12 Attorney Reference: 0080
  • Another aspect of the present disclosure is a method of producing a compound of Formula (G): e G 3 P L R 2 2a wherein: P R is a phosphorus group possessing a leaving group; G 1 is a first protecting group; G 2 is a second protecting group; G 3 is a third protecting group; G 4 is a fourth protecting group; G 2a is a fifth protecting group, wherein one of G 1 , G 2 , G 2a , G 3 , and G 4 can be selectively removed under conditions that render the others stable; 16 Attorney Reference: 0080171-000886 m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); m is selected from 1 and 2; o is selected from 1 and 2; and O N L 2 is selected from the group includin , O N O O O N N N N N O d stereoisomers thereof, wherein the terminal nitrogen the method including: react
  • the first phosphorus reagent is selected from the group consisting S S (S) P Br S S (R) P Br nd Formula (K): Formula (K), wherein: P a is a phosphorus atom that is either achiral, has a (R)-configuration or has a (S)-configuration; X a is selected from OH and SH; 26 Attorney Reference: 0080171-000886 Y a is selected from O and S; G 3 is a third protecting group; G 4 is a fourth protecting group, wherein one of G 3 and G 4 can be selectively removed under conditions that render the other stable; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); m is selected from 1 and 2; o is selected from 1 and 2; and O N L 2 is selected from the group includin , O N O O O N N N N N O d
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (Va): , wherein: P a and P b are each independently a phosphorus atom that has a (R)- configuration or a (S)-configuration; each of Q a and Q b are independently selected from NH and O; each of V a and V b are independently selected from F and OH; 43 Attorney Reference: 0080171-000886 Y is selected from H and NH 2 ; Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; and m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); W is a self-immolative linker; and L 3 is a linker moiety having a structure of: , U is a peptide moiety; and K is a
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (b): , wherein: P a is a phosphorus atom having either a (R)-configuration or (S)- configuration; Q a is selected from NH and O; Q c is selected from NH 2 and OH; 48 Attorney Reference: 0080171-000886 V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); and G 5 is a first protecting group; the method including: reacting a compound of Formula (a) with a first protecting group installation reagent, wherein the compound of Formula (a) has the following structure: O
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (c): wherein: P a is a phosphorus atom having either a (R)-configuration or (S)- configuration; Q a is selected from NH and O; Q c is selected from NH 2 and OH; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); G 5 is a first protecting group; and G 6 is a second protecting group, wherein one of G 5 and G 6 can be selectively removed under conditions that render the other stable; the method including: reacting a compound of Formula (c): wherein: reacting a compound of Formula (
  • Another aspect of the present disclosure is a method of preparing a compound of Formula (d): 51 Attorney Reference: 0080171-000886 wherein: P a is a phosphorus atom having either a (R)-configuration or (S)- configuration; P R is a phosphorus group possessing a leaving group; Q a and Q b are independently selected from NH and O; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); G 5 is a first protecting group; and G 6 is a second protecting group, wherein one of G 5 and G 6 can be selectively removed under conditions that render the other stable; the method including
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (e): 53 Attorney Reference: 0080171-000886 wherein: P a and P b are each independently a phosphorus atom having either a (R)- configuration or a (S)-configuration; Q a and Q b are independently selected from NH and O; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); and G 5 is a first protecting group; the method including: deprotecting a compound of Formula (d) under a specific set of conditions that selectively removes G 6 from the compound of Formula (d); and subsequently subjecting
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (f): 55 Attorney Reference: 0080171-000886 wherein: P a and P b are each independently a phosphorus atom having either a (R)- configuration or a (S)-configuration; Q a and Q b are independently selected from NH and O; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); G 5 is a first protecting group; W is a self-immolative unit; U is a peptide moiety; and G 7 is a third protecting group, wherein one of G 5 and G 7 can be selective
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (g): wherein: P a and P b are each independently a phosphorus atom having either a (R)- configuration or a (S)-configuration; Q a and Q b are independently selected from NH and O; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); and W is a self-immolative unit; U is a peptide moiety; and G 7 is a third protecting group; the method including: 58 Attorney Reference: 0080171-000886 deprotecting a compound of Formula (f) under a specific
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (gi): er a (R)- configuration or a (S)-configuration; Q a and Q b are independently selected from NH and O; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); W is a self-immolative linker; U is a peptide moiety; and Nu is a nucleophilic group; the method including: deprotecting a compound of Formula (g) under a specific set of conditions that selectively removes G 7 from the compound of Formula (g)
  • Another aspect of the present disclosure is the compounds that are useful in the preparation of the drug-linker of an N-linked drug linker.
  • Another aspect of the present disclosure is a compound selected from the following structures: 61 Attorney Reference: 0080171-000886 Attorney Reference: 0080171-000886 e G 3 L P 2 R d F O Formula (K); 63 Attorney Reference: 0080171-000886 , 64 Attorney Reference: 0080171-000886 or a salt thereof, wherein: P a and P b are independently a phosphorus atom that is either achiral, has a (R)- configuration or has a (S)-configuration; X a and X b are independently selected from OH and SH; Y a and Y b are independently selected from O and S; L 2 is a self-immolative linker unit, where e and d denote attachment points to moieties adjacent to L 2 ; m eans that the bond is selected from a single
  • L 2 if present, is selected from the group consisting of: 65 Attorney Reference: 0080171-000886 O N O N O N N , rs [0035]
  • G 1 is selected from DMTr and TES;
  • G 2 is selected from TBS, TIPS, and TBDPS;
  • G 3 is Boc;
  • G 4 is Bz; and
  • G 2a is selected from TBS, TIPS, and TBDPS.
  • the compounds useful in the preparation of the drug-linker of an N-linked drug linker are: 66 Attorney Reference: 0080171-000886 , Attorney Reference: 0080171-000886 , , Attorney Reference: 0080171-000886 . , , Attorney Reference: 0080171-000886 , , 70 Attorney Reference: 0080171-000886 , , Attorney Reference: 0080171-000886 , stereoisomers thereof, or a salt thereof.
  • Yet another aspect of the present disclosure are compounds that are useful in the preparation of the drug-linker of an S-linked drug linker.
  • Another aspect of the present disclosure is a compound selected from the following structures: 72 Attorney Reference: 0080171-000886 , 73 Attorney Reference: 0080171-000886 , 74 Attorney Reference: 0080171-000886 or a salt thereof, wherein: P a and P b are each independently a phosphorous atom that is achiral or has either a (R)- configuration or a (S)-configuration; Q a is selected from NH and O; Q b is selected from NH and O Q c is selected from NH 2 and OH; each of V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; G 5 is a first protecting group, G 6 is a second protecting group, and G 7 is a third protecting group, wherein one of G 5 , G 6 and G 7 can be selectively removed under conditions that render the other stable; m is selected from 1
  • the peptide moiety U as appearing in all applicable formulas of compounds is Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Asn, Ala-(NMe)Ala- Asn, Asn, Gly-Gly-Phe-Gly, Glu-Val-Ala, or Gly-Val-Ala.
  • U comprises Val-Cit.
  • the U comprises Val-Ala.
  • spacer unit K comprises at least one polyethylene glycol (PEG) moiety, in some embodiments, the at least one PEG moiety comprises -(PEG) m - and m is an integer from 1 to 10, or from 2 to 8, from 2 to 5, or m is 2.
  • PEG polyethylene glycol
  • K is PEG 2 -Lys( ⁇ -PEG 8 -OMe)-PEG 2 . O .
  • compositions, methods, compounds and ADCs that are, for brevity, 80 Attorney Reference: 0080171-000886 described in the context of a single embodiment, may also be provided separately or in any sub- combination.
  • Definitions [0046] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein. [0047] As used herein, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise.
  • agent is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
  • therapeutic agent refers to an agent that is capable of modulating a biological process and/or has biological activity.
  • aliphatic or “aliphatic group” means a straight (i.e., unbranched) or branched hydrocarbon chain that can be either substituted with one or more heteroatoms or unsubstituted, and/or be completely saturated or contains one or more units of unsaturation.
  • the term “ambient conditions” means room temperature, open air condition and uncontrolled humidity condition.
  • the terms “room temperature” and “ambient temperature” mean 15 °C to 30 °C.
  • the linker L can include a cleavable moiety between the antibody or antigen-binding fragment and the therapeutic compound. In some embodiments, the linker L can include a cleavable moiety that can be attached to either or both the antibody or antigen-binding fragment and to the therapeutic compound, e.g., by spacer unit(s). Exemplary cleavable linkers are described and exemplified herein.
  • antibody is used in the broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • the heavy chain (HC) of an antibody is composed of a heavy chain variable domain (VH) and a heavy chain constant region (CH).
  • the light chain (LC) is composed of a light chain variable domain (VL) and a light chain constant domain (CL).
  • the terms “domain” and “region” may be used interchangeably (e.g., the term “variable domain” may be used interchangeably with the term “variable region” and understood to refer to the same part of the antibody).
  • the mature heavy chain and light chain variable domains each comprise three complementarity determining regions (CDR1, CDR2, and CDR3; also referred to as “hypervariable regions”) within four framework regions (FR1, FR2, FR3, and FR4) arranged from N terminus to C terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • CDRs may be identified according to the Kabat and/or IMGT numbering systems (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991); International ImMunoGeneTics Information System (IMGT®)).
  • An “antibody” can be naturally occurring or man-made, such as monoclonal antibodies produced by conventional hybridoma technology.
  • the term “antibody” includes full-length monoclonal antibodies and full- length polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fv, and single chain antibodies.
  • An antibody can be any one of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g., isotypes IgG1, IgG2, IgG3, IgG4).
  • An antibody of any of the aforementioned classes or subclasses can also comprise one of two functionally similar classes of light chains: Ig ⁇ (also referred to herein as “Ig kappa” or “kappa”) and Ig ⁇ (also referred to herein as “Ig lambda” or “lambda”).
  • antibody encompasses human antibodies, chimeric antibodies, humanized antibodies, and any modified 82 Attorney Reference: 0080171-000886 immunoglobulin molecule containing an antigen recognition site, so long as it demonstrates the desired biological activity.
  • chimeric antibody refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species.
  • the variable regions of both heavy and light chains correspond to the variable regions of antibodies derived from one species with desired specificity, affinity, and activity characteristics, while the constant regions are homologous to antibodies derived from another species (e.g., human) to minimize an immune response in the latter species.
  • human antibody refers to an antibody produced by a human or an antibody having an amino acid sequence of an antibody produced by a human.
  • humanized antibody refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the humanized antibody can be further modified by the substitution of residues, either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or activity.
  • the humanized antibody can also be further modified by the substitution of residues in the Fc domain to reduce its binding to various cell receptors, such as an Fc ⁇ receptor (Fc ⁇ R), and other immune molecules.
  • Fc ⁇ R Fc ⁇ receptor
  • the term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially 83 Attorney Reference: 0080171-000886 homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods. See, e.g., U.S. Pat. No.4,816,567.
  • Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, e.g., Clackson et al. (1991) Nature 352:624-8, and Marks et al. (1991) J. Mol. Biol.222:581-97.
  • the monoclonal antibodies described herein specifically include “chimeric” antibodies, in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind the target antigen and/or exhibit the desired biological activity.
  • antigen-binding fragment or “antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PSMA). Antigen-binding fragments preferably also retain the ability to internalize into an antigen-expressing cell. In some embodiments, antigen-binding fragments also retain immune effector activity. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding fragment” or “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment, which comprises a single variable domain, e.g., a VH domain (see, e.g., Ward et al.
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” or “antigen-binding portion” of an antibody, and are known in the art as an exemplary type of binding fragment that can internalize into cells upon binding. See, e.g., Zhu et al. (2010) 9:2131-41; He et al. (2010) J. Nucl. Med. 51 :427-32; and Fitting et al. (2015) MAbs 7:390-402.
  • scFv molecules may be incorporated into a fusion protein.
  • Other forms of single chain antibodies e.g., diabodies, are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites. See, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-8; and Poljak et al. (1994) Structure 2: 1121-3).
  • Antigen-binding fragments are obtained using conventional techniques known to those of skill in the art, and the binding fragments are screened for utility (e.g., binding affinity, internalization) in the same manner as are intact antibodies.
  • Antigen-binding fragments may be prepared, e.g., by cleavage of the intact protein, e.g., by protease or chemical cleavage.
  • anti-PSMA antibody or “antibody that specifically binds PSMA” refers to any form of antibody or fragment thereof that specifically binds PSMA and encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they specifically bind PSMA.
  • the anti-PSMA antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.
  • the terms “specific,” “specifically binds,” and “binds specifically” refer to the selective binding of the antibody to the target antigen or epitope over alternative antigens or epitopes.
  • Antibodies can be tested for specificity of binding by comparing binding to an appropriate antigen to binding to an irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen with at least 2-fold, or preferably at least 50-fold, at least 100-fold, or at least 1000-fold, greater affinity than to an irrelevant antigen or antigen mixture, then it is considered to be specific, e.g., as measured by surface plasmon resonance, e.g., BIAcore® analysis.
  • a specific antibody is one that binds the PSMA antigen but does not bind (or exhibits minimal binding) to other antigens.
  • aryl refers to a group or substituent derived from an aromatic ring and encompasses monocyclic aromatic rings and bicyclic, tricyclic, and fused ring systems having a total of six to fourteen ring members, wherein at least one ring in the system is aromatic. An aryl group may be optionally substituted with one or more substituents.
  • heteroaryl refers to a cyclic group comprising at least one ring atom that is a heteroatom, such as O, N, or S.
  • Heteroaryl groups encompass monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms, and wherein each ring in the system contains three to seven ring members.
  • the term “at least one” refers to one or more.
  • the term “bridge” refers to a grouping of atoms in a macrocycle-bridged STING agonist compound of the disclosure that extends from a first nucleic acid base in the macrocycle-bridged STING agonist compound to a second nucleic acid base in the macrocycle-bridged STING agonist compound.
  • cancer refers to the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth.
  • examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, bile duct cancer (e.g., cholangiocarcinoma), esophageal cancer, nasopharyngeal cancer, cancer of the peritoneum, hepatocellular cancer (e.g., hepatocellular carcinoma), gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, osteosarcoma, skin cancer (e.g., melanoma), colon cancer, colorectal cancer, endometrial or uterine cancer, ovarian cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer (e.g., advanced prostate cancer, metastatic castration-resistant prostate cancer), vulval cancer, thyroid cancer, hepatic carcinoma, bone cancer and various types of head and
  • cancer cell and “tumor cell” refer to individual cells or the total population of cells derived from a tumor, including both non-tumorigenic cells and cancer stem cells.
  • tumor cell will be modified by the term “non-tumorigenic” when 86 Attorney Reference: 0080171-000886 referring solely to those tumor cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.
  • tumor and “neoplasm” refer to any mass of tissue that results from excessive cell growth or proliferation, either benign or malignant, including precancerous lesions.
  • chemotherapeutic agent or “anti-cancer agent” is used herein to refer to a chemical compound that is effective in treating cancer regardless of mechanism of action. Inhibition of metastasis or angiogenesis is frequently a property of a chemotherapeutic agent. Stimulation of an antitumor immune response may also be a property of a chemotherapeutic agent.
  • chemotherapeutic agents include stimulatory agents, e.g., STING agonists.
  • chemotherapeutic agents include antibodies, biological molecules, and small molecules.
  • a chemotherapeutic agent may be a cytotoxic or cytostatic agent.
  • cytotoxic agent refers to a substance that causes cell death either by interfering with a cell’s expression activity and/or functioning or by stimulating a response that causes cell death, e.g., an immune response.
  • examples of cytotoxic agents include, but are not limited to, any STING agonist disclosed herein.
  • An “effective amount” of an ADC as disclosed herein is an amount sufficient to perform a specifically stated purpose, for example to produce a therapeutic effect after administration, such as a reduction in tumor growth rate or tumor volume, a reduction in a symptom of cancer, or some other indicia of treatment efficacy. An effective amount can be determined in a routine manner in relation to the stated purpose.
  • a therapeutically effective amount refers to an amount of an ADC effective to treat a disease or disorder in a subject.
  • a therapeutically effective amount of ADC can reduce the number of cancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and/or relieve one or more symptoms.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • epitope refers to the portion of an antigen capable of being recognized and specifically bound by an antibody.
  • the antigen is a polypeptide
  • epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of the polypeptide.
  • the epitope bound by an antibody may be identified using any epitope mapping 87 Attorney Reference: 0080171-000886 technique known in the art, including X-ray crystallography for epitope identification by direct visualization of the antigen-antibody complex, as well as monitoring the binding of the antibody to fragments or mutated variations of the antigen, or monitoring solvent accessibility of different parts of the antibody and the antigen.
  • Exemplary strategies used to map antibody epitopes include, but are not limited to, array-based oligo-peptide scanning, limited proteolysis, site- directed mutagenesis, high-throughput mutagenesis mapping, hydrogen-deuterium exchange, and mass spectrometry. See, e.g., Gershoni et al. (2007) 21:145-56; and Hager-Braun and Tomer (2005) Expert Rev. Proteomics 2:745-56).
  • Fc ⁇ receptor “Fc-gamma receptor,” or “Fc ⁇ R” refers to a cell surface protein generally found on immune cells of various types, e.g., neutrophils.
  • halogen means F, Cl, Br, or I.
  • homolog refers to a molecule which exhibits homology to another molecule, by, for example, having sequences of chemical residues that are the same or similar at corresponding positions.
  • inhibitor means to reduce by a measurable amount, and can include but does not require complete prevention or inhibition.
  • Internalizing refers to an antibody or antigen-binding fragment that is capable of being taken through the cell’s lipid bilayer membrane to an internal compartment (i.e., “internalized”) upon binding to the cell, preferably into a degradative compartment in the cell.
  • an internalizing anti-PSMA antibody is one that is capable of being taken into the cell after binding to PSMA on the cell membrane.
  • KD refers to the equilibrium dissociation constant of a particular antibody- antigen interaction. KD is calculated by k a /k d .
  • the rate can be determined using standard assays, such as a BIAcore® or ELISA assay.
  • the term “k on " or “k a ” refers to the on-rate constant for association of an antibody to the antigen to form the antibody/antigen complex.
  • the rate can be determined using standard assays, such as a BIAcore® or ELISA assay.
  • the term “k off ” or “k d ” refers to the off-rate constant for dissociation of an antibody from the antibody/antigen complex.
  • the rate can be determined using standard assays, such as a BIAcore® or ELISA assay.
  • a “linker” or “linker moiety” is any chemical moiety that is capable of covalently joining a compound, usually a drug moiety such as a chemotherapeutic agent, to another moiety such as an antibody moiety.
  • Linkers can be susceptible to or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and/or disulfide bond cleavage, at conditions under which the compound or the antibody remains active.
  • a “cleavable linker” is any linker that comprises a cleavable moiety and can thus be susceptible to cleavage.
  • a cleavable moiety can be a cleavable peptide moiety.
  • cleavable peptide moiety refers to any chemical bond linking amino acids (natural or synthetic amino acid derivatives) that can be cleaved by an agent that is present in the intracellular environment.
  • the use of “or” will mean “and/or” unless the specific context of its use dictates otherwise.
  • the term “p” or “antibody:drug ratio” or “drug-to-antibody ratio” or “DAR” refers to the number of drug moieties per antibody moiety, i.e., drug loading, or the number of L-D moieties per antibody or antigen-binding fragment (Ab) in ADCs of Formula I.
  • compositions comprising multiple copies of ADCs of Formula I, “p” refers to the average number of L-D moieties per antibody or antigen-binding fragment, also referred to as average drug loading.
  • a “pharmaceutical composition” refers to a preparation which is in such form as to permit administration and subsequently provide the intended biological activity of the active ingredient(s) and/or to achieve a therapeutic effect, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • the pharmaceutical composition may be sterile.
  • a “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH- adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like.
  • “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, for use in animals, and more particularly in humans.
  • pi bond means a covalent bond formed by the p orbitals of adjacent atoms.
  • PSMA state-specific membrane antigen
  • the term encompasses full-length PSMA (e.g., NCBI Reference Sequence: NP_004467.1), as well as any form of human PSMA that results from cellular processing.
  • the term may also encompass naturally occurring variants of PSMA, including but not limited to splice variants, allelic variants, and isoforms.
  • PSMA can be isolated from a human or may be produced recombinantly or by synthetic methods.
  • PSMA and “prostate-specific membrane antigen” are interchangeable with “glutamate carboxypeptidase II (GCPII),” “folate hydrolase 1,” “N-acetylated-alpha-linked acidic dipeptidase I (NAALADase I)” and any other name for proteins encoded by FOLH1 known in the art.
  • protecting group refers to any chemical group introduced into a molecule by chemical modification of a functional group to obtain chemoselectivity in a subsequent chemical reaction.
  • Methods of adding (a process generally referred to as “protecting”) and removing (process generally referred to as “deprotecting”) protecting groups are well-known in the art and available, for example, in P. J. Kocienski, Protecting Groups, 3rd edition (Thieme, 2005), and in Greene and Wuts, Protective Groups in Organic Synthesis, 4th edition (John Wiley & Sons, New York, 2007), both of which are hereby incorporated by reference in their entirety.
  • Non-limiting examples of useful protecting groups for amines include monovalent protecting groups, for example, t-butyloxycarbonyl (Boc), benzyl (Bn), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl (Ac), trifluoroacetyl (TFA), and p-toluenesulfonyl (Ts); and divalent protecting groups, for example, benzylidene, N-phthalimide, N-dithiasuccinimide, N-2,3-diphenylmaleimide, N-2,3- dimethylmaleimide, and N-2,5-dimethylpyrrole.
  • monovalent protecting groups for example, t-butyloxycarbonyl (Boc), benzyl (Bn), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), for
  • Non-limiting examples of useful protecting groups for alcohols include, for example, acetyl (Ac), benzoyl (Bz), benzyl (Bn), ⁇ -methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p- 90 Attorney Reference: 0080171-000886 methoxybenzyl (PMB), pivaloyl (Piv), tetrahydropyranyl (THP), trityl (Tr), 4-nitrophenyl carbonate, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), t-butyldimethylsilyl (TBS), and t-butyldiphenylsilyl (TBDPS).
  • Non-limiting examples of useful protecting groups for carboxylic acids include, for example, methyl or ethyl esters, substituted alkyl esters such as 9- fluorenylmethyl, methoxymethyl (MOM), tetrahydropyranyl (THP), tetrahydrofuranyl, ⁇ - methoxyethoxymethyl (MEM), 2-(trimethylsilyl)ethoxymethyl (SEM), benzyloxymethyl (BOM), acetyl (Ac), phenacyl, substituted phenacyl esters, t-butyl, allyl, phenyl (Ph), silyl esters, benzyl and substituted benzyl esters, 2,6-dialkylphenyl, and pentafluorophenyl (PFP).
  • methyl or ethyl esters substituted alkyl esters such as 9- fluorenylmethyl, methoxymethyl (MOM), tetrahydropyranyl (THP
  • Non-limiting examples of amine bases include, for example, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N-methylmorpholine (NMM), triethylamine (Et 3 N; TEA), diisopropylethyl amine (i-Pr 2 EtN; DIPEA), pyridine, 2,2,6,6- tetramethylpiperidine, 1,5,7- triazabicyclo[4.4.0]dec-5-ene (TBD), 7-methyl-1,5,7- triazabicyclo[4.4.0]dec-5-ene (MTBD), t-Bu-tetramethylguanidine, 1,5-diazabicyclo[4.3.0]non- 5-ene (DBN), lithium bis(trimethylsilyl)amide (LiHMDS), and potassium bis(trimethylsilyl)amide (KHMDS).
  • DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
  • NMM N-methyl
  • Non-limiting examples of carbonate bases that may be used in this disclosure include, for example, sodium carbonate (Na 2 CO 3 ), potassium carbonate (K 2 CO 3 ), cesium carbonate (Cs 2 CO 3 ), lithium carbonate (Li 2 CO 3 ), sodium bicarbonate (NaHCO 3 ), and potassium bicarbonate (KHCO 3 ).
  • Non-limiting examples of phosphate bases that may be used in this disclosure include, for example, sodium phosphate tribasic (Na 3 PO 4 ), potassium phosphate tribasic (K 3 PO 4 ), potassium phosphate dibasic (K 2 HPO 4 ), and potassium phosphate monobasic (KH 2 PO 4 ).
  • Non-limiting examples of acids include, for example, acetic acid (AcOH), trifluoroacetic acid (TFA), hydrochloric acid (HCl), camphorsulfonic acid (CSA), methanesulfonic acid (MsOH), formic acid (FA), phosphoric acid (H 3 PO 4 ), and sulfuric acid (H 2 SO 4 ).
  • Non-limiting examples of peptide coupling reagents include, for example, N,N'- dicyclohexylcarbodiimide (DCC), 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDCI), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium chloride (DMT-MM), 1- ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-[bis(dimethylamino)methylene]-1H- 91
  • HATU 1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
  • HOBT 1-hydroxybenzotriazole
  • TSTU N,N,N,N'-tetramethyl-O- (N-succinimidyl)uronium tetrafluoroborate
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein.
  • subject and patient are used interchangeably herein to refer to any animal, such as any mammal, including but not limited to, humans, non-human primates, rodents, and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.
  • target-negative or “target antigen-negative” refers to the absence of target antigen expression by a cell or tissue.
  • target-positive or “target antigen- positive” refers to the presence of target antigen expression.
  • a cell or a cell line that does not express a target antigen may be described as target-negative, whereas a cell or cell line that expresses a target antigen may be described as target-positive.
  • solvent refers to any liquid in which the product is at least partially soluble (solubility of product >1 g/L).
  • the term “isomer” refers to compounds with identical molecular formula but distinct spatial arrangement of atoms or bonds. Isomers include stereoisomers, cis- trans isomers, atropisomers, and tautomers.
  • stereoisomer refers to both enantiomers and diastereomers.
  • certain compounds of this invention may exist as separate stereoisomers or enantiomers and/or mixtures of those stereoisomers or enantiomers.
  • a stereogenic atom that is notated with an (R) or (S) indicates the stereochemical designation of the stereogenic atom under the Cahn-Ingold-Prelog convention.
  • a (“straight”) bond to a stereogenic atom indicates where there is a mixture (e.g., a racemate o enrichment).
  • two (“straight”) bonds to a double-bonded carbon indicates that the double bond possesse s the E/Z stereochemistry as drawn. 92 Attorney Reference: 0080171-000886 [00105]
  • a salt of a compound of this disclosure is formed between an acid and basic group(s) of the compound, such as an amino functional group, or a base and acidic group(s) of the compound, such as a carboxyl functional group.
  • acid and basic group(s) of the compound such as an amino functional group
  • a base and acidic group(s) of the compound such as a carboxyl functional group.
  • one compound may form salt with one or more molecular units of the acid/base, or multiple units of the compound may form salt with one unit of the acid/base.
  • the salt is a sodium salt.
  • the salt is a diammonium salt.
  • the salt is a dialkylammonium salt. In some embodiments, the salt is a bis(triethylammonium) salt.
  • to treat or “therapeutic,” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality. As is readily appreciated in the art, full eradication of disease is preferred but albeit not a requirement for a treatment act.
  • Treatment or “treat,” as used herein, refers to the administration of a described ADC or antibody to a subject, e.g., a patient.
  • the treatment can be to cure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improve, or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder, e.g., a cancer.
  • unsaturated means that a moiety has one or more units of unsaturation.
  • leaving group means an atom or group of atoms capable of breaking away from a molecule during a reaction, taking with it an electron pair that made the bond between it and the molecule. Non-limiting examples of leaving groups are 93 Attorney Reference: 0080171-000886 discussed in Fujishima, S. et al J. Am. Chem.
  • Examples of leaving groups also include, but are not limited to, halides (such as chloride, bromide, or iodide), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), arylcarbonyloxy, aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl, haloformates, and the like.
  • halides such as chloride, bromide, or iodide
  • the leaving group is a sulfonic acid ester, such as toluenesulfonate (tosylate, TsO), methanesulfonate (mesylate, MsO), or trifluoromethanesulfonate (triflate, TfO).
  • the leaving group may be a brosylate, such as p-bromobenzenesulfonyl.
  • the leaving group may be a nosylate, such as 2-nitrobenzenesulfonyl.
  • the leaving group may also be a phosphineoxide (e.g., those formed during a Mitsunobu reaction) or an internal leaving group such as an epoxide or cyclic sulfate.
  • the leaving group is a sulfonate-containing group. Examples of sulfonates include, but are not limited to, nonaflate, triflate, fluorosulfonate, tosylate, mesylate or besylate.
  • the leaving group is a O-acylisourea, such as those formed from the reaction of a carboxylic acid with a carbodiimide (e.g., DCC, EDC, CMCT, or CMC).
  • the leaving group is a hydroxybenzotriazole.
  • the leaving group is an activated carboxylic acid formed from a reaction between a carboxylic acid and a coupling agent (e.g., BOP, PyBOP, PyAOP, PyBrOP, BOP-Cl, HATU, HBTU, HCTU, TATU, or TBTU).
  • a coupling agent e.g., BOP, PyBOP, PyAOP, PyBrOP, BOP-Cl, HATU, HBTU, HCTU, TATU, or TBTU.
  • One aspect of the present disclosure is a method for preparing a compound of Formula (V): 94 Attorney Reference: 0080171-000886 an isomer thereof, a deuterated derivative thereof; or a salt thereof, wherein: P a and P b are each independently a phosphorus atom that are either achiral, have a (R)- configuration, or have a (S)-configuration; each of X a and X b are independently selected from OH and SH; each of Y a and Y b are independently selected from O and S; L 2 is a first self-immolative linker unit, where e and d denote attachment points to moieties adjacent to L2; L 1 is a linker moiety having a structure of: , wherein: W is a second self-immolative linker unit; U is a peptide moiety; and K is a spacer unit; m is selected from 1 and 2; o
  • the compounds produced during the method containing a L 2 unit have a L 2 unit selected from the group including: O N O N O N , ula (V), Formula (D), Formula (E), Formula (F), Formula (G), Formula (H), Formula (J), Formula (K), Formula (L), Formula (M), and Formula (N).
  • the method includes step (iii) of performing the substitution reaction on the compound of Formula (C) to produce the compound of Formula (D), wherein this step further includes: (i) reacting the compound of Formula (C) with a base to 106 Attorney Reference: 0080171-000886 produce a first nucleophile; and (ii) reacting the first nucleophile with a compound having a structure according to Formula (iiia): LG 2 -L 2 -G 3 Formula (iiia), to produce the compound of Formula (D), wherein LG 2 is a leaving group, L 2 is a first self- immolative linker unit and G 3 is a third protecting group.
  • LG 2 in Formula (iiia) includes reacting the compound of Formula (C) with lithium bis(trimethylsilyl)amide to produce a first nucleophile.
  • the method includes step (iv) of performing the second substitution reaction on the compound of Formula (D) to produce the compound of Formula (E), wherein this step further includes reacting the compound of Formula (D) with a diol in the presence of triphenylphosphine and an azodicarboxylate, wherein the reacting of Formula (D) with the diol in the presence of triphenylphosphine and the azodicarboxylate forms a nucleophile of Formula (ivb): , wherein: G 1 is a first protecting group; G 2 is a second protecting group; and G 3 is a third protecting group, wherein one of G 1 , G 2 , and G 3 can be selectively removed under conditions that render the others stable.
  • the diol that reacts with the compound of Formula (D) has the following structure: , wherein: m is selected from 1 and 2; o is selected from 1 and 2; and m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ).
  • the azodicarboxylate that is present during the reacting of the compound of Formula (D) with the diol is selected from, but not limited to, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), potassium azodicarboxylate, di-tert-butyl-azodicarboxylate (Di-tert-butyl-AD), bis(2,2,2- trichloroethyl)azodicarboxylate (TCEAD), dibenzyl-azodicarboxylate (DBAD), di(4- chlorobenzyl)-azodicarboxylate (Di(4-chlorobenzyl)-AD) and/or (1,1′-azodicarbonyl)- azodicarboxylate (ADDP).
  • DEAD diethyl azodicarboxylate
  • DIAD diisopropyl azodicarboxylate
  • DIAD di-tert-butyl-AD
  • the method includes step (v) of performing the third substitution reaction on the compound of Formula (E) to produce the compound of Formula (F), wherein this step further includes reacting the compound of Formula (E) with a nucleophile of Formula (vb) in the presence of triphenylphosphine and an azodicarboxylate, wherein the nucleophile of compound of Formula (vb) has the following structure: F OH N N 2 a wherein: G 2a is a fifth protecting group; and N u is G 4 -N-, wherein G 4 is a fourth protecting group.
  • the fourth protecting group G 4 in Formula (vb) can be selectively removed under conditions that render G 1 , G 2 , G 2a , and G 3 stable.
  • the azodicarboxylate that is present during the reacting of the compound of Formula (E) with the nucleophile of Formula (vb) is selected from, but not limited to, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), potassium azodicarboxylate, di-tert-butyl-azodicarboxylate (Di-tert-butyl-AD), bis(2,2,2- trichloroethyl)azodicarboxylate (TCEAD), dibenzyl-azodicarboxylate (DBAD), di(4- chlorobenzyl)-azodicarboxylate (Di(4-chlorobenzyl)
  • the method includes step (vi) of reacting the compound of Formula (F) with the first phosphorus-containing reagent to produce the compound of Formula (G), wherein this step further includes reacting the compound of Formula (F) with the first phosphorus-containing reagent in an organic solvent with a base for at least 15 minutes.
  • the first phosphorus-containing reagent that reacts with the compound of Formula (F) is selected from, but not limited to, (2S,3aS,6R,7aS)-3a- methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide: ((-) PSI), (2R,3aR,6S,7aR)-3a-methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2- yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide: 109 Attorney Reference: 0080171-000886 ((+) PSI), a the following formula: , wherein X is a leaving group tuted or unsubstituted –S-Ar, -O-Ar
  • the first phosphorus-containing reagent that reacts with the compound of Formula (F) is selected from, but not limited to, (-) PSI, (+) PSI, S S Br S S Br S S NO2 , . Attorney Reference: 0080171-000886
  • the method includes step (vii) of performing the first deprotection reaction on the compound of Formula (G) to produce the compound of Formula (H), wherein this step further includes subjecting the compound of Formula (G) to a specific set of conditions that only removes the first protecting group (G 1 ) from the compound of Formula (G).
  • G 1 in the compound of Formula (G) is dimethoxytrityl and the compound of Formula (G) is subjected to an acid to remove the first protecting group (G 1 ) from the compound of Formula (G).
  • G 1 in the compound of Formula (G) is dimethoxytrityl and the compound of Formula (G) is subjected to dichloroacetic acid and triethylsilane to remove the first protecting group (G 1 ) from the compound of Formula (G).
  • the method includes step (viii) of cyclizing the compound of Formula (H) to produce the compound of Formula (J), wherein this step further includes subjecting the compound of Formula (H) to a non-nucleophilic base in an organic solvent for at least 30 minutes.
  • a non- nucleophilic base is a base that is capable of extracting or removing a proton (H) from a compound with little to no risk of participating in a nucleophilic substitution or addition with the compound.
  • Non-limiting examples of non-nucleophilic bases include N,N-diisopropylethylamine (DIPEA), 1,8-diazabicycloundec-7-ene (DBU), 1,5-diazabicyclo(4.3.0)non-5-ene (DBN), and 2,6-di-tert-butylpyridine.
  • DIPEA 1,8-diazabicycloundec-7-ene
  • DBN 1,5-diazabicyclo(4.3.0)non-5-ene
  • 2,6-di-tert-butylpyridine 2,6-di-tert-butylpyridine.
  • the subjecting the compound of Formula (H) to the non-nucleophilic base occurs in dichloromethane.
  • the subjecting the compound of Formula (H) to the non-nucleophilic base occurs for at least 1 hour.
  • the subjecting the compound of Formula (H) to the non-nucleophilic base occurs at a temperature within the range of 0°C to 25°C.
  • the method includes step (ix) of performing the second deprotection reaction on the compound of Formula (J) to produce the compound of Formula (K), wherein this step further includes subjecting the compound of Formula (J) to a specific set of conditions that only removes the second protecting group (G 2 ) from the compound of Formula (J).
  • the second protecting group (G 2 ) in the compound of Formula (J) is tert-butyl(dimethyl)silyl (TBS) and the compound of Formula (J) is subjected to triethylamine trishydrofluoride in a solvent system including triethylamine and pyridine.
  • the solvent system includes a 5:1 molar ratio of triethylamine and pyridine. It will be appreciated by those of ordinary skill in the art that reagents besides triethylamine trishydrofluoride can be used to selectively remove a TBS protecting group from the compounds disclosed herein.
  • the method includes step (x) of reacting the compound of Formula (K) with the second phosphorus reagent to produce the compound of Formula (L), wherein this step further includes reacting the compound of Formula (K) with the second phosphorus reagent in an organic solvent with a base for at least 15 minutes.
  • the second phosphorus reagent can be selected from, but is not limited to, (-) PSI, (+) PSI, and a phosphorus reagent having the following formula: Attorney Reference: 0080171-000886 wherein X is a leaving group selected from substituted or unsubstituted –S-Ar, -O-Ar or halide, wherein Ar is phenyl, naphthyl, optionally substituted with one or more of C 1 -C 6 alkyl, cyano, nitro or halo; and wherein R 15 , R 16 , R 17 , and R 18 are each independently selected from the group consisting of H, C 1 -C 10 alkyl, C 2 -C 12 alkenyl, aryl; or any two of R 15 , R 16 , R 17 and R 18 together with the carbons they are attached form a cycloalkyl or heteroalkyl ring that is optionally substituted with one or more C 1 -C 10 alkyl or
  • the second phosphorus reagent is selected from (-) S S (S) P Br S S (R) P Br nd p y , p p g e third deprotection reaction on the compound of Formula (L) to produce the compound of Formula (M), wherein this step further includes subjecting the compound of Formula (L) to a specific set of conditions that only removes the fourth protecting group (G 4 ) from the compound of Formula (L).
  • the fourth protecting group (G 4 ) on the compound of Formula (L) is a benzoyl group and the compound of Formula (L) is subjected to a solvent system containing NH 4 OH in methanol to selectively remove the benzoyl group.
  • a solvent system containing NH 4 OH in methanol can be used to selectively remove a benzoyl protecting group from the compounds 113 Attorney Reference: 0080171-000886 disclosed herein.
  • solvent systems and reagents are well-known in the art and therefore are readily applicable by those of ordinary skill in the art.
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (D): Formula (D), G 1 is a first protecting group; G 2 is a second protecting group; G 3 is a third protecting group wherein one of G 1 , G 2 , and G 3 can be selectively removed under conditions that render the others stable; and O N L 2 is selected from the group includin , O N O O O O F d stereoisomers thereof, wherein the terminal nitrogen in 114 Attorney Reference: 0080171-000886 the method including: reacting a compound of Formula (C) with a non-nucleophilic base to produce a first nucleophile; and reacting the first nucleophile with a first electrophile to produce the compound of Formula (D), wherein the compound of Formula (C) has the following structure: Formula (C), G 1 is a first protecting group; and G 2 is a second protecting group, wherein one of G1 and G2 can be selectively removed under conditions that render the other stable.
  • the first nucleophile formed from the reacting of the compound of Formula (C) with the non-nucleophilic base is reacted with a first electrophile having a structure according to Formula (iiia): LG 2 -L 2 -G 3 Formula (iiia) wherein: G 3 is a third protecting group that can be selectively removed under conditions that render G 1 and G 2 stable; LG 2 is a leaving group; and L 2 is selected from the group including: O N O N , Attorney Reference: 0080171-000886 F F F F F F nd O [00141] In exemplary embodiments, the LG 2 unit of Formula (iiia or O .
  • the compound of Formula (C) reacts with lithium bis(trimethylsilyl)amide to produce the first nucleophile.
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (E): wherein: G 1 is a first protecting group; G 2 is a second protecting group; G 3 is a third protecting group, wherein one of G 1 , G 2 , and G 3 can be selectively removed under conditions that render the others stable; 116 Attorney Reference: 0080171-000886 m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); m is selected from 1 and 2; o is selected from 1 and 2; and O N L 2 is selected from the group includin , O N O O O N N N N O d stereoisomers thereof, wherein the terminal nitrogen the method including: reacting a compound of Formula (D) with a diol with
  • the azodicarboxylate that is present during the reacting of the compound of Formula (D) with the diol is selected from, but not limited to, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), potassium azodicarboxylate, di-tert-butyl-azodicarboxylate (Di-tert-butyl-AD), bis(2,2,2- trichloroethyl)azodicarboxylate (TCEAD), dibenzyl-azodicarboxylate (DBAD), di(4- chlorobenzyl)-azodicarboxylate (Di(4-chlorobenzyl)-AD) and/or (1,1′-azodicarbonyl)- azodicarboxylate (ADDP).
  • DEAD diethyl azodicarboxylate
  • DIAD diisopropyl azodicarboxylate
  • DIAD di-tert-butyl-AD
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (F): e G 3 L 2 2a , wherein: G 1 is a first protecting group; G 2 is a second protecting group; G 3 is a third protecting group; G 4 is a fourth protecting group; 120 Attorney Reference: 0080171-000886 G 2a is a fifth protecting group, wherein one of G 1 , G 2 , G 2a , G 3 , and G 4 can be selectively removed under conditions that render the others stable; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); m is selected from 1 and 2; o is selected from 1 and 2; and O N L 2 is selected from the group includin , O N O O O N N N N O n the method including: reacting a compound of Formula (E) with a nucleophile of Formula (vb) in the presence of
  • the azodicarboxylate that is present during the reacting of the compound of Formula (E) with the nucleophile of Formula (vb) is selected from, but not limited to, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), potassium azodicarboxylate, di-tert-butyl-azodicarboxylate (Di-tert-butyl-AD), bis(2,2,2- trichloroethyl)azodicarboxylate (TCEAD), dibenzyl-azodicarboxylate (DBAD), di(4- chlorobenzyl)-azodicarboxylate (Di(4-chlorobenzyl)-AD) and/or (1,1′-azodicarbonyl)- azodicarboxylate (ADDP).
  • DEAD diethyl azodicarboxylate
  • DIAD diisopropyl azodicarboxylate
  • DIAD di-tert-
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (G): e G 3 L P 2 R 2a wherein: P R is a phosphorus group possessing a leaving group; G 1 is a first protecting group; G 2 is a second protecting group; G 3 is a third protecting group,; G 4 is a fourth protecting group; G 2a is a fifth protecting group, wherein one of G 1 , G 2 , G 2a , G 3 , and G 4 can be selectively removed under conditions that render the others stable; 123 Attorney Reference: 0080171-000886 m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); m is selected from 1 and 2; o is selected from 1 and 2; and O N L 2 is selected from the group includin , O N O O O N N N N N O d stereoisomers thereof, wherein the terminal nitrogen the method
  • the first phosphorus reagent that reacts with the compound of Formula (F) is selected from the group including: (-) PSI, (+) PSI, S S Br S S Br S S (S) P R P P NO2 , F .
  • G 1 in the compound of Formula (G) is dimethoxytrityl and the compound of Formula (G) is subjected to dichloroacetic acid and triethylsilane to remove the first protecting group (G 1 ) from the compound.
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (J): e G 3 L 2 a G 2a wherein: P a is phosphorus atom that is either achiral, has a (R)-configuration, or has a (S)-configuration; X a is selected from OH and SH; Y a is selected from O and S; G 2 is a second protecting group; G 3 is a third protecting group; G 4 is a fourth protecting group; 129 Attorney Reference: 0080171-000886 G 2a is a fifth protecting group, wherein one of G 2 , G 2a , G 3 , and G 4 can be selectively removed under conditions that render the others stable; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); m is selected from 1 and 2; o is selected from 1 and 2; and O N L 2 is selected from
  • the subjecting the compound of Formula (H) to the non-nucleophilic base occurs for at least 1 hour.
  • the subjecting the compound of Formula (H) to the non-nucleophilic base occurs at a temperature within the range of 0°C to 25°C.
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (K): wherein: P a is a phosphorus atom that is either achiral, has a (R)-configuration or has a (S)-configuration; X a is selected from OH and SH; Y a is selected from O and S; G 3 is a third protecting group; G 4 is a fourth protecting group, wherein one of G 3 and G 4 can be selectively removed under conditions that render the other stable; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); m is selected from 1 and 2; o is selected from 1 and 2; and O N L 2 is selected from the group includin , O N O O O O O Attorney Reference: 0080171-000886 F F F O N , N N d stereoisomers thereof,
  • the reaction may be conducted in a solvent system containing triethylamine. In one embodiment, the reaction may be conducted in a solvent system containing triethylamine and pyridine.
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (L): 134 Attorney Reference: 0080171-000886 wherein: P a and P b are each independently a phosphorus atom that is either achiral, has a (R)-configuration or has a (S)-configuration; X a and X b are independently selected from OH and SH; Y a and Y b are independently selected from O and S; G 3 is a third protecting group; G 4 is a fourth protecting group, wherein one of G 3 and G 4 can be selectively removed under conditions that render the other stable; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond
  • second phosphorus reagent is selected from the S S S) Br S S ( P (R) P Br , nd und of Formula (M): Attorney Reference: 0080171-000886 wherein: P a and P b are each independently a phosphorus atom that is either achiral, has a (R)-configuration or has a (S)-configuration; X a and X b are independently selected from OH and SH; Y a and Y b are independently selected from O and S; G 3 is a third protecting group; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); m is selected from 1 and 2; o is selected from 1 and 2; and O N L 2 is selected from the group includin , O N O O N N N , the method including: deprotecting a compound of Formula (L) under a specific
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (N): , wherein: P a and P b are each independently a phosphorus atom that is either achiral, has a (R)-configuration or has a (S)-configuration; X a and X b are independently selected from OH and SH; Y a and Y b are independently selected from O and S; 141 Attorney Reference: 0080171-000886 m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); m is selected from 1 and 2; o is selected from 1 and 2; and O N L 2 is selected from the group includin , O N O O N N N , nd stereoisomers thereof, wherein the method including: deprotecting a compound of Formula (M) under a specific set of conditions that removes G 3 from the compound of Formula (M
  • the second protecting group G 2 present in Formula (C), Formula (D), Formula (E), Formula (F), Formula (G), Formula (H), and Formula (J) is a TBS protecting group.
  • the third protecting group G 3 in Formula (D), Formula (E), Formula (F), Formula (G), Formula (H), Formula (J), Formula (K), Formula (L) and Formula (M) is a nitrogen protecting group, for example, tert-butyloxycarbonyl (Boc) or fluorenylmethoxycarbonyl (Fmoc).
  • the fourth protecting group G 4 in Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) and Formula (L) is a benzoyl protecting group.
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (V2): Attorney Reference: 0080171-000886 Formula (V2), an isomer thereof, a deuterated derivative thereof; or a salt thereof, wherein: P a and P b are each independently a phosphorus atom that is either achiral, has a (R)-configuration or has a (S)-configuration; each of X a and X b are independently selected from OH and SH; each of Y a and Y b are independently selected from O and S; Z is selected from CH 2 and O; L 1 is a linker moiety; L 2 is a self-immolative linker unit, where e and d denote attachment points to moieties adjacent to L 2 ; m
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (Va): , wherein: P a and P b are each independently a phosphorus atom that has a (R)- configuration or a (S)-configuration; each of Q a and Q b are independently selected from NH and O; each of V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; and 151 Attorney Reference: 0080171-000886 m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); W is a self-immolative linker; and L 3 is a linker moiety having a structure of: , U
  • the W unit in Formula (Va), Formula (f), and/or O O Attorney Reference: 0080171-000886 S. I , Attorney Reference: 0080171-000886 , Attorney Reference: 0080171-000886 , , Attorney Reference: 0080171-000886 , [00174]
  • the method includes step (a) of installing the first protecting group G 5 onto the compound of Formula (a) to produce the compound of Formula (b), wherein this step further includes reacting the compound of Formula (a) with a first protecting group installation reagent in an organic solvent, optionally in the presence of a base.
  • the first protecting group installation reagent is 2- nitrobenzyl bromide, 2-nitrobenzyl iodide, 2-nitrobenzyl chloride, 4-nitrobenzyl bromide, 4- 160 Attorney Reference: 0080171-000886 nitrobenzyl iodide, or 4-nitrobenzyl chloride
  • the organic solvent is a polar aprotic solvent such as dimethyl formamide, NMP, or DMI.
  • the compound of Formula (a) reacts with the first protecting group installation reagent in the presence of N,N-diisopropylethylamine, TEA, or PMP (1,2,2,6,6-pentamethylpiperidine.
  • the method includes step (b) of installing the second protecting group G 6 onto the compound of Formula (b) to produce the compound of Formula (c), wherein this step further includes reacting the compound of Formula (b) with a second protecting group installation reagent in an organic solvent, optionally in the presence of a base, to produce the compound of Formula (c).
  • the second protecting group installation reagent is 4,4 -dimethoxytrityl chloride and the organic solvent is pyridine.
  • the method includes step (c) of reacting the compound of Formula (c) with the first phosphorus reagent to produce the compound of Formula (d), wherein this step further includes reacting the compound of Formula (c) with the first phosphorus reagent and a non-nucleophilic base to produce the compound of Formula (d).
  • the first phosphorus reagent is selected from the group including (-) PSI, (+) PSI and a phosphorus reagent having the following structure: wherein X is a leaving group uted or unsubstituted –S-Ar, -O-Ar or halide, wherein Ar is phenyl, naphthyl, optionally substituted with one or more of C 1 -C 6 alkyl, cyano, nitro or halo; and wherein R 15 , R 16 , R 17 , and R 18 are each independently selected from the group consisting of H, C 1 -C 10 alkyl, C 2 -C 12 alkenyl, aryl; or any two of R 15 , R 16 , R 17 and R 18 together with the carbons they are attached form a cycloalkyl or heteroalkyl ring that is optionally substituted with one or more C 1 -C 10 alkyl or C 2 -C 12 alkeny
  • the first phosphorus reagent is selected from the S S S S (S) P Br (R) P Br , nd irst phosphorus reagent in the presence of 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU).
  • DBU 1,8-diazabicyclo(5.4.0)undec-7-ene
  • the method includes step (d) of removing the second protecting group G6 from the compound of Formula (d) and subsequently cyclizing the deprotected compound of Formula (d) to produce the compound of Formula (e), wherein this step further includes (i) first subjecting the compound of Formula (d) to an acid and optionally a silicon-containing compound to remove G 6 ; and/or (ii) subsequently subjecting the deprotected compound of Formula (d) to a base in an organic solvent system to produce the compound of Formula (e).
  • the second protecting group G 6 of the compound of Formula (d) is dimethoxytrity (DMT) and the removing of the second protecting group G 6 from the compound of Formula (d) includes subjecting the compound of Formula (d) to dichloroacetic acid and triethylsilane.
  • DMT dimethoxytrity
  • the method includes step (e) of performing the first substitution reaction on the compound of Formula (e) to produce the compound of Formula (f), wherein this step further includes reacting the compound of Formula (e) with a compound of Formula (ei): 162 Attorney Reference: 0080171-000886 wherein: G 7 is a third protecting that can be selectively removed under conditions that render G 5 stable; U is a peptide moiety; W is a self-immolative linker; and LG is a leaving group.
  • the method includes step (f) of performing the second deprotecting reaction on the compound of Formula (f) to produce the compound of Formula (g), wherein this step further includes subjecting the compound of Formula (f) to a metal and a salt in a mixture of organic solvent and water for at least thirty minutes at a temperature of at least 45°C.
  • the first protecting group P 5 is a nitrobenzyl group and the compound of Formula (f) is subjected to ammonium chloride and ethanol to remove P 5 from the compound of Formula (f).
  • the method includes step (g) of performing the third deprotection reaction on the compound of Formula (g) to remove G 6 and, subsequently, performing the second substitution reaction to produce the compound of Formula (Va), wherein this step further includes (i) removing G 6 to produce a deprotected compound of Formula (g); and (ii) reacting the deprotected compound of Formula (g) with an electrophile to produce the compound of Formula (Va).
  • the nucleophile of the deprotected compound of Formula (g) is a compound of formula (gi): 163 Attorney Reference: 0080171-000886 Formula (gi), wherein: P a and P b are each independently a phosphorus atom having either a (R)- configuration or a (S)-configuration; Q a and Q b are independently selected from NH and O; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); W is a self-immolative linker; and Nu is a nucleophilic group.
  • the deprotected compound of Formula (g) reacts with an electrophile having a structure from the group including: 164 Attorney Reference: 0080171-000886 ; Attorney Reference: 0080171-000886 nd wherein: a, b, c and d are independently selected from an integer within the range of 1 to 8; and LG is a leaving group.
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (b): a p p g g configuration; 166 Attorney Reference: 0080171-000886
  • Q a is selected from NH and O
  • Q c is selected from NH 2 and OH
  • V a and V b are independently selected from F and OH
  • Y is selected from H and NH 2
  • each Z a is independently selected from CH 2 , O, and NH
  • m is selected from 1 and 2
  • o is selected from 1 and 2
  • G 5 is a first protecting group; the method including: reacting a compound of Formula (a) with a first protecting group installation reagent, wherein the compound of Formula (a) has the following structure: - configuration; Q a is selected from NH and O; Q
  • the reacting of the compound of Formula (a) with the first protecting group installation reagent occurs in an organic solvent, optionally in the presence of a base.
  • the first protecting group installation reagent is 2- nitrobenzyl bromide, 2-nitrobenzyl iodide, 2-nitrobenzyl chloride, 4-nitrobenzyl bromide, 4- nitrobenzyl iodide, or 4-nitrobenzyl chloride
  • the organic solvent is dimethyl formamide, NMP, or DMI
  • the reacting of the compound of Formula (a) with the first protecting group installation reagent occurs in the presence of the base, the base being N,N- diisopropylethylamine, TEA, or PMP (1,2,2,6,6-pentamethylpiperidine.
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (c): wherein: P a is a phosphorus atom having either a (R)-configuration or (S)- configuration; Q a is selected from NH and O; Q c is selected from NH 2 and OH; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; 168 Attorney Reference: 0080171-000886 each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); G 5 is a first protecting group; and G 6 is a second protecting group, wherein one of G 5 and G 6 can be selectively removed under conditions that render the other stable; the method including: reacting a compound of
  • the reacting of the compound of Formula (b) with the second protecting group installation reagent occurs in an organic solvent, optionally in the presence of a base.
  • the second protecting group installation reagent is 4,4 -dimethoxytrityl chloride and the organic solvent is pyridine.
  • Another aspect of the present disclosure is a method of preparing a compound of Formula (d): wherein: P a is a phosphorus atom having either a (R)-configuration or (S)- configuration; P R is a phosphorus group possessing a leaving group; Q a and Q b are independently selected from NH and O; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); 170 Attorney Reference: 0080171-000886 G 5 is a first protecting group; and G 6 is a second protecting group, wherein one of G 5 and G 6 can be selectively removed under conditions that render the other stable; the method
  • the first phosphorus reagent is selected from (-) PSI, (+) PSI and a phosphorus reagent having a structure according to the following formula: wherein X is a leaving group uted or unsubstituted –S-Ar, -O-Ar or halide, wherein Ar is phenyl, naphthyl, optionally substituted with one or more of C 1 -C 6 alkyl, cyano, nitro or halo; and wherein wherein R 15 , R 16 , R 17 , and R 18 are each independently selected from the group consisting of H, C 1 -C 10 alkyl, C 2 -C 12 alkenyl, aryl; or any two of R 15 , R 16 , R 17 and R 18 together with the carbons they are attached form a cycloalkyl or heteroalkyl ring that is optionally substituted with one or more C 1
  • the reacting of the compound of Formula (c) with the first phosphorus-containing reagent occurs in the presence of 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU).
  • the first phosphorus reagent is selected from the S S P Br S S P Br , nd Attorney Reference: 0080171-000886
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (e): wherein: P a and P b are each independently a phosphorus atom having either a (R)- configuration or a (S)-configuration; Q a and Q b are independently selected from NH and O; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m e
  • the deprotecting of the compound of Formula (d) comprises subjecting the compound of Formula (d) to an acid and optionally a silicon-containing compound.
  • protecting group G 6 in the compound of Formula (d) is a DMT group and the compound of Formula (d) is subjected to dichloroacetic acid and triethylsilane to remove protecting group G 6 .
  • P a and P b are each independently a phosphorus atom having either a (R)- configuration or a (S)-configuration; Q a and Q b are independently selected from NH and O; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); G 5 is a first protecting group; W is a self-immolative unit; U is a peptide moiety; and G 7 is a third protecting group, wherein one of G 5 and G 7 can be
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (g): wherein: P a and P b are each independently a phosphorus atom having either a (R)- configuration or a (S)-configuration; Qa and Qb are independently selected from NH and O; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); and W is a self-immolative unit; U is a peptide moiety; and G7 is a third protecting group; the method including: deprotecting a compound of Formula (f) under a specific set of conditions that only removes G 5 from the compound of Formula (
  • the deprotecting of the compound of Formula (f) comprises subjecting the compound of Formula (f) to a metal and a salt in an organic solvent for at least thirty minutes at a temperature of at least 45°C. 178 Attorney Reference: 0080171-000886 [00207]
  • the protecting group G 5 in the compound of Formula (f) is a nitrobenzyl group and the compound of Formula (f) is subjected to ammonium chloride and ethanol to remove protecting group G 5 .
  • Another aspect of the present disclosure is a method for preparing a compound of Formula (gi): Formula (gi), wherein: P a and P b are each independently a phosphorus atom having either a (R)- configuration or a (S)-configuration; Q a and Q b are independently selected from NH and O; V a and V b are independently selected from F and OH; Y is selected from H and NH 2 ; each Z a is independently selected from CH 2 , O, and NH; m is selected from 1 and 2; o is selected from 1 and 2; m eans that the bond is selected from a single bond ( ), a double bond ( ) of (E)- or (Z)-configuration, or a triple bond ( ); W is a self-immolative linker; U is a peptide moiety; and Nu is a nucleophilic group; 179 Attorney Reference: 0080171-000886 the method including: deprotecting a compound of Formula (gi
  • the present disclosure provides methods for preparing drug-linker combinations that can bind to antibodies or antigen-binding fragments and deliver drugs to specific sites of 180 Attorney Reference: 0080171-000886 interest.
  • the methods disclosed herein can provide drug-linker combinations that can bind specifically to PSMA antibodies and/or antigen-binding fragments and deliver STING agonists to cancerous tissues and/or cells.
  • the ADCs formed from the drug-linker combinations disclosed herein can bind to PSMA with a dissociation constant (KD) of ⁇ 1 mM, ⁇ 100 nM, or ⁇ 10 nM, or any amount in between, as measured by, e.g., BIAcore® analysis.
  • KD dissociation constant
  • the KD is 500 pM to 1 nM, or 1 nM to 10 nM.
  • the KD is ⁇ 10 nM, ⁇ 5 nM, ⁇ 1 nM, or ⁇ 0.5 nM.
  • the drug-linker combinations disclosed herein may be conjugated to antibodies that are internalizing antibodies or internalizing antigen-binding fragments thereof.
  • the internalizing antibodies bind to PSMA expressed on the surface of a cell and enter the cell upon or after binding.
  • the drug moiety of the ADC is released from the antibody moiety of the ADC after the ADC enters and is present in a cell expressing PSMA (i.e., after the ADC has been internalized).
  • the internalizing antibodies bind to PSMA expressed on the cell surface of a cell and the cell is subsequently phagocytosed (e.g., antibody-dependent cellular phagocytosis occurs).
  • the drug moiety of the ADC is released from the antibody moiety of the ADC after the ADC enters and is present in the phagocytic cell (e.g., macrophage, dendritic cell).
  • the drug-linker combinations disclosed herein can also be conjugated to antigen- binding fragments that retain PSMA binding.
  • the antigen binding fragment retains PSMA binding by possessing three heavy chain CDRs (e.g., HCDR1, HCDR2, and HCDR3) and/or three light chain CDRs (e.g., LCDR1, LCDR2, and LCDR3), as defined by the Kabat numbering system.
  • the antigen-binding fragments disclosed herein may retain PSMA binding by comprising a VH domain.
  • Any of the anti-PSMA antibodies and antigen binding fragments disclosed herein may be used as a conjugate, e.g., with a detectable agent and/or another therapeutic agent.
  • the anti-PSMA antibody or antigen binding fragment are used in an antibody-drug conjugate (ADC), e.g., any of the ADCs disclosed herein, preferably to target the drug in the ADC to a cancer cell.
  • ADC antibody-drug conjugate
  • the linker-toxins in the ADCs disclosed herein are surprisingly effective with the anti-PSMA antibodies also disclosed herein.
  • linkers and toxin e.g., a STING agonist
  • 181 Attorney Reference: 0080171-000886
  • Linkers [00214]
  • the drug-linker combinations disclosed herein contain a drug moiety (e.g., a STING agonist) bonded to a linker moiety.
  • the linker moiety has a structure according to the following formula: wherein: L 2 is a first self-immolative linker unit; W is a second self-immolative linker unit; U is a peptide moiety; and K is a spacer unit.
  • the drug moiety is a STING agonist.
  • the linker moiety has a structure according to the following structure: wherein: W is a second self-immolative linker unit; U is a peptide moiety; and 182 Attorney Reference: 0080171-000886 K is a spacer unit.
  • the drug moiety is a STING agonist.
  • the linker moiety is stable extracellularly in a sufficient manner to be therapeutically effective. In some embodiments, the linker moiety is stable outside a cell, such that the ADC remains intact when present in extracellular conditions (e.g., prior to transport or delivery into a cell).
  • intact means that the antibody moiety remains attached to the drug moiety (e.g., a STING agonist).
  • stable in the context of a linker or ADC comprising a linker, means that no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linkers (or any percentage in between) in a sample of ADC are cleaved (or in the case of an overall ADC are otherwise not intact) when the ADC is present in extracellular conditions when evaluated over a set period of time.
  • the linkers in ADCs disclosed herein are chosen to remain stable for more than about 48 hours, more than about 60 hours, more than about 72 hours, more than about 84 hours, or more than about 96 hours.
  • Whether a linker moiety is stable extracellularly can be determined, for example, by including an ADC in plasma for a predetermined time period (e.g., 2, 4, 6, 8, 16, or 24 hours) and then quantifying the amount of free drug moiety present in the plasma. Stability may allow the ADC time to localize to target tumor cells and prevent the premature release of the drug, which could lower the therapeutic index of the ADC by indiscriminately damaging both normal and tumor tissues.
  • the linker is stable outside of a target cell and releases the drug moiety from the ADC once inside of the cell, such that the drug moiety can bind to its target (e.g., to STING).
  • an effective linker moiety will: (i) maintain the specific binding properties of the antibody moiety; (ii) allow delivery, e.g., intracellular delivery, of the drug moiety via stable attachment to the antibody moiety; (iii) remain stable and intact until the ADC has been transported or delivered to its target site; and (iv) allow for the therapeutic effect, e.g., cytotoxic effect, of the drug moiety after cleavage.
  • the linker moiety can impact the physico-chemical properties of an ADC.
  • cytotoxic agents are hydrophobic in nature, linking them to the antibody with an additional hydrophobic moiety may lead to aggregation.
  • ADC aggregates are insoluble and often limit achievable drug loading onto the antibody, which can negatively affect the potency of the ADC.
  • 183 Attorney Reference: 0080171-000886 Protein aggregates of biologics, in general, have also been linked to increased immunogenicity.
  • linker moieties disclosed herein result in ADCs with low aggregation levels and desirable levels of drug loading.
  • a linker moiety is conjugated to the antibody or antigen-binding fragment through a cysteine.
  • a linker moiety is conjugated to the antibody or antigen-binding fragment through a lysine.
  • Suitable methods for conjugating linker moieties of the present disclosure to an antibody include the technologies for directed attachment to a lysine on a heavy chain of an antibody, to a cysteine on the heavy chain of an antibody, and to a cysteine on the light chain of an antibody, e.g., as disclosed in PCT applications WO 2017/213267, WO 2017/106643, and WO 2016/205618, and in Junutula et al. (2008) Journal of Immunological Methods 332:41–52, all of which are herein incorporated by reference in their entireties.
  • a linker moiety is conjugated to the antibody or antigen-binding fragment on the light chain, e.g., at a cysteine on the light chain, e.g., at cysteine-80 on the light chain.
  • a linker moiety is conjugated to the antibody or antigen-binding fragment on the heavy chain, e.g., at a cysteine on the heavy chain, e.g., at cysteine-118 on the heavy chain.
  • a linker moiety disclosed herein can be "cleavable” or “non-cleavable” (Ducry and Stump, Bioconjugate Chem. (2010) 21:5-13).
  • Cleavable linkers are designed to release the drug when subjected to certain environment factors, e.g., when internalized into the target cell, whereas non-cleavable linker moieties generally rely on the degradation of the antibody moiety itself.
  • the linker moiety is a non-cleavable linker.
  • the drug moiety of the ADC is released by degradation of the antibody moiety.
  • the linker moiety is cleavable.
  • Cleavable linkers are designed to release the drug when subjected to certain environmental factors, e.g., when internalized into the target cell.
  • a cleavable linker moiety refers to any linker that comprises a cleavable moiety.
  • the term “cleavable moiety” refers to any chemical bond that can be cleaved. Suitable cleavable chemical bonds are known in the art and include, but are not limited to, acid labile bonds, protease/peptidase labile bonds, photolabile bonds, disulfide bonds, and esterase labile bonds.
  • Linker moieties comprising a cleavable moiety can allow for the release of the drug moiety from the ADC via cleavage at a particular site in the linker.
  • the linker moiety is cleavable under intracellular conditions, such that cleavage of the linker sufficiently releases the drug moiety from the antibody moiety in the intracellular environment to activate the drug and/or render the drug therapeutically effective.
  • the drug moiety is not cleaved from the antibody moiety until the ADC enters a cell that expresses an antigen specific for the antibody moiety of the ADC, and the drug moiety is cleaved from the antibody moiety upon entering the cell.
  • the linker moiety comprises a cleavable moiety that is positioned such that no part of the linker or the antibody moiety remains bound to the drug moiety upon cleavage.
  • cleavable linkers include acid labile linkers, protease/peptidase-sensitive linkers, photolabile linkers, dimethyl-, disulfide-, or sulfonamide-containing linkers.
  • the linker moiety is cleavable by a cleaving agent, e.g., an enzyme, that is present in the intracellular environment (e.g., within a lysosome, endosome, or caveolea).
  • the linker moiety can include, e.g., a peptide moiety (i.e., U) that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
  • the linker moiety includes a cleavable peptide linker or moiety.
  • a cleavable peptide linker or moiety refers to any linker moiety that comprises a cleavable peptide moiety.
  • cleavable peptide moiety refers to any chemical bond linking amino acids (natural or synthetic amino acid derivatives) that can be cleaved by an agent that is present in the intracellular environment.
  • a cleavable peptide linker or moiety is more stably conjugated to an antibody disclosed herein compared to an acid labile linker.
  • the linker moiety is an enzyme-cleavable linker and contains a cleavable peptide moiety (i.e., U) that is cleavable by the enzyme.
  • the cleavable peptide moiety is cleavable by a lysosomal enzyme, e.g., cathepsin or legumain (also known as asparaginyl endopeptidase or vacuolar processing enzyme).
  • the linker moiety contains a cathepsin-cleavable linker.
  • the linker moiety contains a legumain-cleavable linker.
  • the cleavable peptide moiety in the linker is cleavable by a lysosomal cysteine cathepsin, such as cathepsin B, C, F, H, K, L, O, S, V, X, or W.
  • the cleavable peptide moiety is cleavable by cathepsin B.
  • An exemplary dipeptide that may be cleaved by cathepsin B is valine-citrulline (Val-Cit). See, e.g., Dubowchik et al. (2002) Bioconjugate Chem.13:855-69.
  • cleavable peptide moiety i.e., U
  • cysteine endopeptidase such as legumain.
  • An exemplary monopeptide that may be cleaved by legumain is asparagine (Asn).
  • the cleavable peptide moiety (i.e., U) in the linker moiety comprises an amino acid unit.
  • the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug moiety from the ADC upon exposure to one or more intracellular proteases, such as one or more lysosomal enzymes. See, e.g., Doronina et al. (2003) Nat. Biotechnol.21:778-84; and Dubowchik and Walker (1999) Pharm. Therapeutics 83:67-123.
  • Exemplary amino acid units include, but are not limited to, monopeptides, dipeptides, tripeptides, tetrapeptides, and pentapeptides.
  • Exemplary monopeptides include, but are not limited to, asparagine (Asn) and aspartic acid (Asp).
  • Exemplary dipeptides include, but are not limited to, valine-citrulline (Val-Cit), alanine-asparagine (Ala-Asn), alanine- phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys), alanine-lysine (Ala-Lys), alanine- valine (Ala-Val), valine-alanine (Val-Ala), valine-lysine (Val-Lys), lysine-lysine (Lys-Lys), phenylalanine-citrulline (Phe-Cit), leucine-citrulline (Leu-Cit), isoleucine-citrulline (Ile-Cit), tryptophan-citrulline (Trp-Cit), and phenylalanine-alanine (Phe-Ala).
  • Exemplary tripeptides include, but are not limited to, alanine-alanine-asparagine (Ala-Ala-Asn), glycine-valine- citrulline (Gly-Val-Cit), glutamic acid-valine-citrulline, glycine-glycine-glycine (Gly-Gly-Gly), phenylalanine-phenylalanine-lysine (Phe-Phe-Lys), alanine-phenylalanine-lysine (Ala-Phe-Lys), glycine-valine-alanine (Gly-Val-Ala), and glycine-phenylalanine-lysine (Gly-Phe-Lys).
  • Exemplary tetrapeptides include, but are not limited to, glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).
  • Other exemplary amino acid units include, but are not limited to, Gly-Phe- Leu-Gly, Ala-Leu-Ala-Leu, Phe-N9-tosyl-Arg, and Phe-N9-Nitro-Arg, as described in, e.g., U.S. Patent No.6,214,345.
  • an amino acid unit may comprise amino acid residues comprising at least one methyl group, e.g., a monomethyl or dimethyl group.
  • Exemplary amino acid units that comprise amino acid residues comprising at least one methyl group include, but are not limited to, N-methylated alanine ((NMe)Ala), methylated aspartic acid (Asp(OMe)) and dimethylated lysine (Val-Lys(Me) 2 ).
  • the amino acid unit 186 Attorney Reference: 0080171-000886 in the linker moiety comprises Val-Ala.
  • the amino acid unit in the linker moiety comprises Val-Cit.
  • An amino acid unit may comprise amino acid residues that occur naturally and/or minor amino acids and/or non-naturally occurring amino acid analogs, such as citrulline.
  • Amino acid units can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, a lysosomal protease such as legumain or cathepsin B, C, D, or S.
  • the linker moiety comprises an antibody attachment moiety.
  • An antibody attachment moiety may be used, for example, to link the antibody moiety to the linker, which in turn may link to the drug moiety, e.g., indirectly through a cleavable moiety (e.g., a cleavable peptide).
  • the linker moiety comprises an antibody attachment moiety comprising a maleimide moiety (Mal).
  • maleimide moiety means a compound that contains a maleimide group and that is reactive with a sulfhydryl group, e.g., a sulfhydryl group of a cysteine residue on the antibody moiety.
  • linker moiety attaches to the antibody or antigen- binding fragment via a Mal moiety.
  • the Mal moiety is reactive with a cysteine residue on the antibody or antigen-binding fragment.
  • the Mal moiety is joined to the antibody or antigen-binding fragment via the cysteine residue.
  • the Mal moiety is a maleimidocaproyl (MC) moiety.
  • the linker moiety attaches to the antibody or antigen-binding fragment via an MC moiety.
  • the MC moiety is reactive with a cysteine residue on the antibody or antigen-binding fragment.
  • the MC moiety is joined to the antibody or antigen-binding fragment via the cysteine residue.
  • the linker moiety comprises a Mal moiety and a cleavable peptide moiety (i.e., U).
  • the cleavable peptide moiety comprises an amino acid unit.
  • the amino acid unit comprises Val-Cit. In some embodiments, the amino acid unit comprises Val-Ala. In some embodiments, the Mal moiety attaches the antibody moiety to the cleavable peptide moiety in the linker moiety. In some embodiments, the 187 Attorney Reference: 0080171-000886 cleavable peptide moiety comprises an amino acid unit. In some embodiments, the amino acid unit comprises Val-Cit. In some embodiments, the amino acid unit comprises Val-Ala. In some embodiments, the linker moiety comprises Mal-Val-Cit. In some embodiments, the linker comprises Mal-Val-Ala.
  • the linker moiety comprises an MC moiety and a cleavable peptide moiety (i.e., U).
  • the cleavable peptide moiety comprises an amino acid unit.
  • the amino acid unit comprises Val-Cit.
  • the amino acid unit comprises Val-Ala.
  • the MC moiety attaches the antibody moiety to the cleavable peptide moiety in the linker moiety.
  • the cleavable peptide moiety comprises an amino acid unit.
  • the amino acid unit comprises Val-Cit.
  • the amino acid unit comprises Val-Ala.
  • the linker moiety comprises MC-Val-Cit. In some embodiments, the linker moiety comprises MC-Val-Ala. [00236] In some embodiments, the linker moiety comprises at least one spacer unit (i.e., K) joining the antibody moiety to the drug moiety. In some embodiments, the spacer unit joins a cleavage site (e.g., a cleavable peptide moiety) in the linker moiety to the antibody moiety. In some embodiments, the spacer unit joins a cleavage site (e.g., a cleavable peptide moiety) in the linker moiety to the drug moiety.
  • a spacer unit joins a cleavage site (e.g., a cleavable peptide moiety) in the linker moiety to the drug moiety.
  • the linker moiety, and/or spacer unit in the linker moiety is substantially hydrophilic.
  • the linker moiety includes one or more polyethylene glycol (PEG) moieties, e.g., 1, 2, 3, or 4 PEG moieties.
  • the linker moiety includes one or more alkyl moieties, e.g., 1, 2, 3, 4, or 5 alkyl moieties.
  • the spacer unit in the linker moiety comprises one or more PEG moieties.
  • the spacer unit comprises -(PEG) m -, and m is an integer from 1 to 10.
  • m ranges from 1 to 4; or from 2 to 4. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, the spacer unit comprises (PEG) 2 , (PEG) 3 , or (PEG) 4 . In some embodiments, the spacer unit comprises PEG 2 -Lys( ⁇ -PEG 8 -OMe)-PEG 2 . [00238] In some embodiments, the spacer unit in the linker moiety comprises an alkyl moiety.
  • the spacer unit comprises -(CH 2 ) n -, and n is an integer from 1 to 10 (i.e., n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n is 3, 4, or 5. In some 188 Attorney Reference: 0080171-000886 embodiments, the spacer unit comprises (CH 2 ) 3 , or (CH 2 ) 4 , or (CH 2 ) 5 . In some embodiments, the spacer unit comprises CH 2 -CH 2 . [00239] In some embodiments, the spacer unit comprises one or more O . In some embodiments, the spacer unit comprises one or more O and (PEG)2.
  • the spacer unit comprises one or more O conjugated to (PEG) 2 .
  • the O O spacer unit comprises one or more (Formula (II)).
  • a linker moiety comprising a spacer unit may provide benefits for various D moieties, including, e.g., improved conjugation stability, improved plasma stability, and/or in vivo anti-tumor activity compared to other linker moieties comprising alternative spacer units.
  • benefits of using a linker moiety comprising a spacer unit with a STING agonist disclosed herein can include improved conjugation stability, improved plasma stability, and/or in vivo anti-tumor activity.
  • the linker moiety comprises a spacer unity and a payload comprising a STING agonist disclosed herein and demonstrates superior properties when conjugated to an anti-PSMA antibody disclosed herein.
  • a spacer unit can be used, for example, to link the antibody moiety to the drug moiety, either directly or indirectly. In some embodiments, the spacer unit links the antibody moiety to the drug moiety directly.
  • the antibody moiety and the drug moiety are attached via a spacer unit comprising one or more alkyl moieties (e.g., (CH 2 ) 3 , or (CH 2 ) 4 , or (CH 2 ) 5 ).
  • the antibody moiety and the drug moiety are attached via a spacer unit comprising one or more PEG moieties (e.g., (PEG) 2 or (PEG) 3 or (PEG) 4 ).
  • the antibody moiety and the drug moiety are attached via a spacer unit comprising Formula (II).
  • the spacer unit links the antibody moiety to the drug moiety indirectly.
  • the spacer unit links the antibody moiety to the drug moiety indirectly through a cleavable moiety (e.g., a cleavable peptide) and/or an antibody 189 Attorney Reference: 0080171-000886 attachment moiety to join the spacer unit to the antibody moiety, e.g., a maleimide moiety or a carbobenzoxy-L-glutaminyl-glycine moiety.
  • the spacer unit attaches to the antibody moiety (i.e., the antibody or antigen-binding fragment) via a maleimide moiety (Mal).
  • a spacer unit that attaches to the antibody or antigen-binding fragment via a Mal is referred to herein as a “Mal-spacer unit.”
  • the Mal-spacer unit is reactive with a cysteine residue on the antibody or antigen-binding fragment.
  • the Mal-spacer unit is joined to the antibody or antigen-binding fragment via the cysteine residue.
  • the Mal- spacer unit comprises a PEG moiety.
  • the Mal-spacer unit comprises an alkyl moiety.
  • the Mal-spacer unit comprises Formula (II).
  • the spacer unit attaches to the antibody moiety (i.e., the antibody or antigen- binding fragment) via a maleimidocaproyl moiety (MC).
  • a spacer unit that attaches to the antibody or antigen-binding fragment via an MC is referred to herein as an “MC-spacer unit.”
  • the MC-spacer unit is reactive with a cysteine residue on the antibody or antigen-binding fragment.
  • the MC-spacer unit is joined to the antibody or antigen-binding fragment via the cysteine residue.
  • the MC-spacer unit comprises a PEG moiety.
  • the MC-spacer unit comprises an alkyl moiety.
  • the MC-spacer unit comprises Formula (II).
  • the linker comprises the Mal-spacer unit or MC-spacer unit and a cleavable peptide moiety (i.e., U).
  • the cleavable peptide moiety comprises an amino acid unit.
  • the amino acid unit comprises Val-Cit.
  • the amino acid unit comprises Val-Ala.
  • the linker moiety comprises the Mal-spacer unit or MC-spacer unit and an amino acid unit.
  • the linker moiety comprises Mal-(CH 2 ) n and an amino acid unit, where n is 3 to 5, or 3, 4, or 5.
  • the linker moiety comprises MC-(CH 2 ) n and an amino acid unit, where n is 3 to 5, or 3, 4, or 5. [00244] In some embodiments, the linker moiety comprises Mal-(PEG) m and an amino acid unit, where m is 2 to 4, or 2, 3, or 4. In some embodiments, the linker moiety comprises MC-(PEG) m and an amino acid unit, where m is 2 to 4, or 2, 3, or 4. In some embodiments, the linker moiety further comprises a cleavable dipeptide, e.g., Val-Cit or Val-Ala.
  • the linker moiety comprises a cleavable dipeptide, e.g., Val-Cit or Val-Ala. In some embodiments, the linker moiety comprises Mal-Formula (II)-Val- Cit. In some embodiments, the linker moiety comprises Mal-Formula (II)-Val-Ala. [00246] In some embodiments, the Mal-spacer unit or MC-spacer unit attaches the antibody moiety (i.e., the antibody or antigen-binding fragment) to the cleavable moiety in the linker moiety.
  • the antibody moiety i.e., the antibody or antigen-binding fragment
  • the MC-spacer unit comprises an alkyl moiety.
  • the cleavable moiety in the linker moiety is joined directly to the drug moiety and/or to the antibody moiety.
  • a spacer unit is used to attach the cleavable moiety in the linker moiety to the drug moiety and/or to the antibody moiety.
  • the drug moiety may be any STING agonist drug moiety disclosed herein, e.g., a compound of Formula (III), Formula (IV), or a compound disclosed in Table 3, infra.
  • the drug moiety is attached to the cleavable moiety in the linker moiety by a spacer unit.
  • the drug moiety is Compound 1.
  • the Compound 1 moiety is attached to the cleavable moiety in the linker moiety by a spacer unit.
  • the drug moiety, e.g., Compound 1 is attached to the cleavable moiety in the linker moiety by a self-immolative unit.
  • the drug moiety e.g., Compound 1
  • the cleavable moiety comprises an amino acid unit
  • a further spacer unit 191 Attorney Reference: 0080171-000886 e.g., comprising one or more alkyl or PEG moieties or Formula (II), joins the cleavable moiety to the antibody moiety.
  • the drug moiety e.g., Compound 1
  • the drug moiety is joined to an anti-PSMA antibody via a Mal-spacer unit in the linker moiety joined to a cleavable peptide moiety and a pAB or pABC self-immolative unit.
  • the drug moiety, e.g., Compound 1 is joined to an anti-PSMA antibody via an MC-spacer unit in the linker moiety joined to a cleavable peptide moiety and a pAB or pABC self-immolative unit.
  • a spacer unit may be “self-immolative” or “non-self-immolative.”
  • a “non-self- immolative” spacer unit is one in which part or all of the spacer unit remains bound to the drug moiety upon cleavage of the linker moiety.
  • Examples of non-self-immolative units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit.
  • Non-self-immolative units may eventually degrade over time but do not readily release a linked native drug entirely under cellular conditions.
  • a “self-immolative” unit comprises any structure that allows for release of the native drug moiety after administration to a subject, e.g., under intracellular conditions.
  • a “native drug” is one where no part of the spacer unit or other chemical modification remains after cleavage/degradation of the spacer unit.
  • Self-immolation chemistry is known in the art and may be readily selected for the disclosed ADCs.
  • the spacer unit attaching the cleavable moiety in the linker to the drug moiety e.g., Compound 1
  • the linker moiety comprises at least one self-immolative unit (i.e., W and/or L 2 ).
  • linker moieties disclosed herein may comprise a first self- immolative unit (L 2 ).
  • the phrase “linker moiety comprising at least one self-immolative unit” may indicate a linker moiety comprising one self-immolative unit or a linker moiety comprising one or more self-immolative units.
  • the linker moiety disclosed herein comprises a first self-immolative unit (L 2 ) and a second self-immolative unit (W).
  • the second self-immolative unit in the linker moiety comprises a p-aminobenzyl unit.
  • a p-aminobenzyl alcohol is attached to an amino acid unit or other cleavable moiety in the linker moiety via an amide bond, and a carbamate, methylcarbamate, or carbonate is made between the pABOH and the drug moiety.
  • the second self-immolative unit is or comprises p-aminobenzyl (pAB).
  • the second self-immolative unit is or comprises p-aminobenzyloxycarbonyl (pABC).
  • the pAB is substituted with 1-3 substituents chosen from methyl, fluoro, chloro, trifluoromethyl, C 6 -C 10 aryl, and C 5 -C 12 heteroaryl.
  • exemplary substituted pAB units are disclosed in Table 1.
  • a linker moiety disclosed herein comprises a second self-immolative linker unit (i.e., W) selected from the self-immolative units disclosed in Table 1, infra. Table 1.
  • Exemplary Second Self-Immolative Linker (W) Units Substituted pAB Substituted pAB Moiet Structure 193 Attorney Reference: 0080171-000886 Substituted pAB Substituted pAB Moiety Structure Moiety 194 Attorney Reference: 0080171-000886 Substituted pAB Substituted pAB Moiety Structure Moiety 195 Attorney Reference: 0080171-000886 Substituted pAB Substituted pAB Moiety Structure Moiety 196 Attorney Reference: 0080171-000886 Substituted pAB Substituted pAB Moiety Structure Moiety 197 Attorney Reference: 0080171-000886 [00256] Linker moieties may be modified to achieve desirable properties of an ADC, e.g., stability, tolerability, and/or efficacy.
  • a linker moiety comprising a modified pAB or pABC moiety may increase ADC stability and/or in vivo ADC tolerability (as determined by, for example, percent body weight loss) while minimizing reduced ADC efficacy when compared to a linker moiety comprising pAB or pABC.
  • Certain additional modifications to the linker-drug structure e.g., spacer units or modified drug moiety attachment points, may be required to obtain one or more (e.g., all of these) properties. For instance, certain modifications or combinations of modifications may need to be made to enhance ADC stability while avoiding loss of efficacy.
  • an ADC comprising LP1, LP2, LP16, LP20, LP26, or LP28 may achieve desirable properties of an ADC, e.g., stability, tolerability, and/or efficacy when compared to other anti-PSMA ADCs.
  • the linker moieties disclosed herein comprise a first self- immolative unit (i.e., L 2 ).
  • the first self-immolative unit attaches the linker moiety to the drug moiety (e.g., Compound 1).
  • a linker-payload conjugate as disclosed herein may provide superior technical benefits, e.g., superior stability and/or improved activity, compared to other linker- payload conjugates comprising any of the payload compounds disclosed herein.
  • Exemplary first self-immolative units i.e., L 2
  • a linker-payload conjugate comprises a first self-immolative unit listed in Table 2, infra.
  • a linker-payload conjugate comprises Val-Ala-pAB and a first self- immolative unit selected from Table 2.
  • a linker-payload conjugate comprises Val-Ala-pABC and a first self-immolative unit selected from Table 2. In some embodiments, a linker-payload conjugate comprises Val-Cit-pAB and a first self-immolative unit selected from Table 2. In some embodiments, a linker-payload conjugate comprises Val-Cit- pABC and a first self-immolative unit selected from Table 2. Table 2.
  • the first self-immolative unit comprises a Unit 1 (MEC) moiety.
  • MEC Unit 1
  • a MEC moiety attaches the first self-immolative unit to the drug moiety (e.g., Compound 1) (“self-immolative unit-MEC moiety”).
  • the first self-immolative unit comprises a Unit 2 moiety.
  • a Unit 6 moiety attaches the first self-immolative unit to the drug moiety (e.g., Compound 1) (“self-immolative unit-Unit 6 moiety”).
  • the first self-immolative unit comprises a Unit 7 moiety.
  • a Unit 7 moiety attaches the first self-immolative unit to the drug moiety (e.g., Compound 1) (“self-immolative unit-Unit 7 moiety”).
  • the first self-immolative unit comprises a Unit 8 moiety.
  • a Unit 8 moiety attaches the first self-immolative unit to the drug moiety (e.g., Compound 1) (“self-immolative unit-Unit 8 moiety”).
  • the first self-immolative unit comprises a Unit 9 moiety. In some embodiments, a Unit 9 moiety attaches the first self-immolative unit to the drug moiety (e.g., Compound 1) (“self-immolative unit-Unit 9 moiety”). In some embodiments, the first self-immolative unit comprises a Unit 10 moiety. In some embodiments, a Unit 10 moiety attaches the first self-immolative unit to the drug moiety (e.g., Compound 1) (“self-immolative unit-Unit 10 moiety”). In some embodiments, the first self-immolative unit comprises a Unit 11 moiety.
  • a cleavable moiety in a linker attaches directly or indirectly to a sulfur in the drug moiety.
  • the drug moiety may be any suitable drug moiety disclosed herein, e.g., a compound of Formula (III), Formula (IV), or a compound disclosed in Table 3, infra.
  • the drug moiety is or comprises Compound 1.
  • the cleavable moiety in the linker moiety attaches directly or indirectly to the sulfur in a STING agonist drug moiety disclosed herein (e.g., Compound 1).
  • the one or more self-immolative unit(s) comprises pAB.
  • the linker moiety comprises Val-Cit-pAB. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker moiety comprises Val-Ala-pAB.
  • a cleavable moiety in the linker moiety attaches directly or indirectly to a nitrogen in the drug moiety.
  • the drug moiety may be a STING agonist drug moiety disclosed herein, e.g., a compound of Formula (III), Formula (IV), or a compound disclosed in Table 3 , infra. In some embodiments, the drug moiety is or comprises Compound 1. In some embodiments, the one or more self-immolative unit(s) comprises pAB.
  • the one or more self-immolative unit(s) comprises pABC. In some embodiments, the one or more self-immolative unit(s) comprises a MEC moiety. In some embodiments, the one or more self-immolative unit(s) comprises pABC-MEC moiety. In some embodiments, the carboxylate moiety of the pABC is bound to the n-methyl moiety of the MEC to form an N- methylcarbamate moiety. In some embodiments, the one or more self-immolative unit(s) comprises a Unit 8 moiety. In some embodiments, the one or more self-immolative unit(s) comprises pABC-Unit 8 moiety.
  • the one or more self-immolative unit(s) comprises a Unit 9 moiety. In some embodiments, the one or more self-immolative unit(s) comprises pABC-Unit 9 moiety. In some embodiments, the one or more self-immolative unit(s) comprises a Unit 11 moiety. In some embodiments, the one or more self-immolative unit(s) comprises pABC-Unit 11 moiety. [00263] In some embodiments, the linker moiety comprises a third spacer unit between the first spacer unit and the second spacer unit. In some embodiments, the second and/or third spacer unit is selected from a moiety of Table 2, supra.
  • the pABC or pABC-MEC moiety undergoes self- immolation upon cleavage of the cleavable moiety, and Compound 1 is released from the ADC in its native, active form.
  • the release of Compound 1 from the antibody and linker moiety occurs in a stepwise fashion, wherein first the cleavable moiety in the linker moiety is cleaved, then the pABC moiety undergoes self-immolation, and then the MEC moiety undergoes self-immolation.
  • the cleavable moiety comprises an amino acid unit.
  • the linker moiety comprises amino acid unit-pABC.
  • the linker moiety comprises amino acid unit-pABC-MEC moiety. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker moiety comprises Val-Cit-pABC. In some embodiments, the linker moiety comprises Val-Cit-pABC-MEC moiety. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker moiety comprises Val-Ala-pABC. In some embodiments, the linker moiety comprises Val-Ala-pABC- MEC moiety.
  • the pABC or pABC-Unit 8 moiety undergoes self- immolation upon cleavage of the cleavable moiety, and Compound 1 is released from the ADC in its native, active form.
  • the release of Compound 1 from the antibody and linker moiety occurs in a stepwise fashion, wherein first the cleavable moiety in the linker moiety is cleaved, then the pABC moiety undergoes self-immolation, and then the Unit 8 moiety undergoes self-immolation.
  • the cleavable moiety comprises an amino acid unit.
  • the linker moiety comprises amino acid unit-pABC.
  • the linker moiety comprises amino acid unit-pABC-Unit 8 moiety. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker moiety comprises Val-Cit-pABC. In some embodiments, the linker moiety comprises Val-Cit-pABC-Unit 8 moiety. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker moiety comprises Val-Ala-pABC. In some embodiments, the linker moiety comprises Val-Ala- pABC-Unit 8 moiety.
  • the linker moiety comprises amino acid unit-pABC. In some embodiments, the linker moiety comprises amino acid unit-pABC-Unit 9 moiety. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker moiety comprises Val-Cit-pABC. In some embodiments, the linker moiety comprises Val-Cit-pABC-Unit 9 moiety. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker moiety comprises Val-Ala-pABC. In some embodiments, the linker moiety comprises Val-Ala- pABC-Unit 9 moiety.
  • the pABC or pABC-Unit 11 moiety undergoes self- immolation upon cleavage of the cleavable moiety, and Compound 1 is released from the ADC in its native, active form.
  • the release of Compound 1 from the antibody and linker moiety occurs in a stepwise fashion, wherein first the cleavable moiety in the linker moiety is cleaved, then the pABC moiety undergoes self-immolation, and then the Unit 11 moiety undergoes self-immolation.
  • the cleavable moiety comprises an amino acid unit.
  • the linker moiety comprises amino acid unit-pABC.
  • the linker moiety comprises amino acid unit-pABC-Unit 11 moiety. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker moiety comprises Val-Cit-pABC. In some embodiments, the linker moiety comprises Val-Cit-pABC-Unit 11 moiety. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker moiety comprises Val-Ala-pABC. In some embodiments, the linker moiety comprises Val-Ala- pABC-Unit 11 moiety.
  • the at least one self-immolative unit (e.g., pAB, pABC, pABC-MEC moiety, pABC-Unit 8 moiety, pABC-Unit 9 moiety, or pABC-Unit 11 moiety) undergoes self-immolation upon cleavage of a cleavable peptide moiety in the linker moiety.
  • the self-immolation of the at least one self-immolative unit occurs in a stepwise manner after cleavage of a cleavable peptide moiety in the linker moiety, starting from the self-immolative moiety closest to the cleavable peptide moiety.
  • the at least one self-immolative unit (e.g., pAB, pABC, pABC-MEC moiety, 204 Attorney Reference: 0080171-000886 pABC-Unit 8 moiety, pABC-Unit 9 moiety, or pABC-Unit 11 moiety) undergoes self- immolation in a stepwise manner after cleavage of a cleavable peptide moiety in the linker moiety, wherein the first self-immolative unit (e.g., pABC or pAB) undergoes self-immolation prior to self-immolation of the second self-immolative unit (e.g., MEC moiety, Unit 8 moiety, Unit 9 moiety, Unit 11 moiety).
  • the first self-immolative unit e.g., pABC or pAB
  • the second self-immolative unit e.g., MEC moiety, Unit 8 moiety, Unit 9 moiety, Unit 11 mo
  • the cleavable peptide moiety comprises an amino acid unit.
  • the linker moiety comprises amino acid unit-pAB.
  • the linker moiety comprises amino acid unit-pABC.
  • the linker moiety comprises amino acid unit-pABC-MEC moiety.
  • the linker moiety comprises amino acid unit-pABC-Unit 8 moiety.
  • the linker moiety comprises amino acid unit-pABC-Unit 9 moiety.
  • the linker moiety comprises amino acid unit-pABC-Unit 11 moiety.
  • the amino acid unit is Val-Cit.
  • the linker moiety comprises Val-Cit-pAB.
  • the linker moiety comprises Val-Cit-pABC. In some embodiments, the linker moiety comprises Val-Cit-pABC-MEC moiety. In some embodiments, the linker moiety comprises Val-Cit-pABC-Unit 8 moiety. In some embodiments, the linker moiety comprises Val-Cit-pABC-Unit 9 moiety. In some embodiments, the linker moiety comprises Val-Cit- pABC-Unit 11 moiety. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker moiety comprises Val-Ala-pAB. In some embodiments, the linker moiety comprises Val-Ala-pABC.
  • the linker moiety comprises Val-Ala- pABC-MEC moiety. In some embodiments, the linker moiety comprises Val-Ala-pABC-Unit 8 moiety. In some embodiments, the linker moiety comprises Val-Ala-pABC-Unit 9 moiety. In some embodiments, the linker moiety comprises Val-Ala-pABC-Unit 11 moiety. [00269] In various aspects, the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises an MC-spacer unit, a cleavable amino acid unit, and a pAB.
  • the linker moiety comprises MC-Val-Cit- pAB. In some embodiments, the linker moiety comprises MC-Val-Ala-pAB.
  • the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises an MC-spacer unit, a cleavable amino acid unit, and a pABC. In some embodiments, the linker moiety comprises MC-Val-Cit- pABC. In some embodiments, the linker moiety comprises MC-Val-Ala-pABC.
  • the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises an MC unit, a cleavable amino acid unit, a pABC, and a MEC moiety.
  • the linker moiety comprises MC- Val-Cit-pABC-MEC moiety.
  • the linker moiety comprises MC-Val-Ala- pABC-MEC moiety.
  • the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises an MC unit, a cleavable amino acid unit, a pABC, and a Unit 8 moiety.
  • the linker moiety comprises MC- Val-Cit-pABC-Unit 8 moiety.
  • the linker moiety comprises MC-Val-Ala- pABC-Unit 8 moiety.
  • the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises an MC unit, a cleavable amino acid unit, a pABC, and a Unit 9 moiety.
  • the linker moiety comprises MC- Val-Cit-pABC-Unit 9 moiety.
  • the linker moiety comprises MC-Val-Ala- pABC-Unit 9 moiety.
  • the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises an MC unit, a cleavable amino acid unit, a pABC, and a Unit 11 moiety.
  • the linker moiety comprises MC-Val-Cit-pABC-Unit 11 moiety.
  • the linker moiety comprises MC- Val-Ala-pABC-Unit 11 moiety.
  • the antibody moiety is conjugated to the drug moiety via a linker moiety comprising a maleimidicopryl moiety (MC) and an amino acid.
  • the antibody moiety is conjugated to the drug moiety via a linker moiety comprising a maleimidocaproyl moiety (MC), an amino acid, and a pAB. In some embodiments, the antibody moiety is conjugated to the drug moiety via a linker moiety comprising a maleimidocaproyl moiety (MC), an amino acid, and a pABC. In some embodiments, the antibody moiety is conjugated to the drug moiety via a linker moiety comprising a maleimidocaproyl moiety (MC), an amino acid, a pABC, and a MEC moiety.
  • the antibody moiety is conjugated to the drug moiety via a linker moiety comprising a maleimidocaproyl moiety (MC), an amino acid, a pABC, and a Unit 8 moiety.
  • the antibody moiety is conjugated to the drug moiety via a linker moiety 206 Attorney Reference: 0080171-000886 comprising a maleimidocaproyl moiety (MC), an amino acid, a pABC, and a Unit 9 moiety.
  • the antibody moiety is conjugated to the drug moiety via a linker moiety comprising a maleimidocaproyl moiety (MC), an amino acid, a pABC, and a Unit 11 moiety.
  • the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises a Mal-spacer unit, a cleavable amino acid unit, and a pAB.
  • the linker moiety comprises Mal-Formula (II)-Val-Cit-pAB.
  • the linker moiety comprises Mal-Formula (II)-Val-Ala- pAB.
  • the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises a Mal-spacer unit, a cleavable amino acid unit, and a pABC.
  • the linker moiety comprises Mal-Formula (II)-Val-Cit- pABC. In some embodiments, the linker moiety comprises Mal-Formula (II)-Val-Ala-pABC.
  • the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises a Mal unit, a cleavable amino acid unit, a pABC, and a MEC moiety. In some embodiments, the linker moiety comprises Mal- Formula (II)-Val-Cit-pABC-MEC moiety.
  • the linker moiety comprises Mal-Formula (II)-Val-Ala-pABC-MEC moiety.
  • the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises a Mal unit, a cleavable amino acid unit, a pABC, and a Unit 8 moiety.
  • the linker moiety comprises Mal- Formula (II)-Val-Cit-pABC-Unit 8 moiety.
  • the linker moiety comprises Mal-Formula (II)-Val-Ala-pABC-Unit 8 moiety.
  • the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises a Mal unit, a cleavable amino acid unit, a pABC, and a Unit 9 moiety.
  • the linker moiety comprises Mal- Formula (II)-Val-Cit-pABC-Unit 9 moiety.
  • the linker moiety comprises Mal-Formula (II)-Val-Ala-pABC-Unit 9 moiety.
  • the antibody moiety of the ADC is conjugated to the drug moiety via a linker moiety, wherein the linker moiety comprises a Mal unit, a cleavable amino acid unit, a pABC, and a Unit 11 moiety.
  • the linker moiety comprises Mal-Formula (II)-Val-Cit-pABC-Unit 11 moiety.
  • the linker moiety comprises Mal-Formula (II)-Val-Ala-pABC-Unit 11 moiety.
  • the drug moiety (D) of the linker-drug conjugates and ADCs disclosed herein can be any chemotherapeutic agent.
  • the drug moiety is a STING agonist.
  • Exemplary STING agonists are known in the art and include cyclic dinucleotides, e.g., macrocycle-bridged STING agonists, non-cyclic dinucleotides.
  • the drug moiety is a non-cyclic dinucleotide.
  • the drug moiety is a macrocycle- bridged STING agonist.
  • the drug moiety of the linker-drug conjugates and ADCs disclosed herein comprises a compound according to one of the following Formulae: Y b Y b O W Pb Xb O W Pb Xb an isomer thereof, a deuterated derivative of the compound or isomer; or a salt of the compound, isomer, or deuterated derivative; wherein, independently for each occurrence, ⁇ each of Pa and Pb, when not achiral, is independently selected from (R)-configuration and (S)-configuration; ⁇ each of Q a and Q b is independently selected from NH and O; ⁇ each of V a and V b is independently selected from F and OH; ⁇ W 1 is selected from H and NH 2 ; ⁇ each of X a and X b is independently selected from OH and SH; ⁇ each of Y a and Y b is independently selected from O and S; ⁇ each Z a and Z b is independently selected from CH 2 ,
  • each of P a and P b is racemic or achiral. In some embodiments, P a is racemic or achiral and P b is selected from (R)-configuration and (S)- configuration. In some embodiments, P a is selected from (R)-configuration and (S)-configuration and P b is racemic or achiral. In some embodiments, each of P a and P b is selected from (R)- configuration and (S)-configuration. [00286] In some embodiments, P a is (R)-configuration and P b is (R)-configuration. In some embodiments, P a is (R)-configuration and P b is (S)-configuration.
  • P a is (S)-configuration and P b is (R)-configuration. In some embodiments, P a is (S)-configuration and P b is (S)-configuration.
  • Q a is O and Q b is O. In some embodiments, Q a is NH and Q b is O. In some embodiments, Q a is O and Q b is NH. In some embodiments, Q a is NH and Q b is NH.
  • V a is OH and V b is OH. In some embodiments, V a is F and V b is OH. In some embodiments, V a is OH and V b is F.
  • V a is F and V b is F.
  • W 1 is H. In some embodiments, W 1 is NH 2 .
  • X a is OH and X b is OH.
  • X a is SH and X b is OH.
  • X a is OH and X b is SH.
  • X a is SH and X b is SH.
  • Y a is O and Y b is O.
  • Y a is S and Y b is O.
  • Y a is O and Y b is S.
  • Y a is S and Y b is S.
  • Z a is NH and Z b is selected from CH 2 , O, and NH. In some embodiments, Z a is NH and Z b is CH 2 . In some embodiments, Z a is NH and Z b is O. In some embodiments, Z a is NH and Z b is NH. [00293] In some embodiments, Z a is O and Z b is selected from CH 2 , O, and NH. In some embodiments, Z a is O and Z b is CH 2 . In some embodiments, Z a is O and Z b is O.
  • Z a is O and Z b is NH. 209 Attorney Reference: 0080171-000886 [00294] In some embodiments, Z a is CH 2 and Z b is selected from CH 2 , O, and NH. In some embodiments, Z a is CH 2 and Z b is CH 2 . In some embodiments, Z a is CH 2 and Z b is O. In some embodiments, Z a is CH 2 and Z b is NH. [00295] In some embodiments, is a single bond. In some embodiments, is a double bond of (E)-configuration. In some embodiments, is a double bond of (Z)-configuration. In some embodiments, is a triple bond.
  • At least one of X a and X b is SH and each of Z a and Z b is independently selected from CH 2 , O, and NH.
  • X a is SH and each of Z a and Z b is independently selected from CH 2 , O, and NH.
  • X b is SH and each of Z a and Z b is independently selected from CH 2 , O, and NH.
  • each of X a and X b is SH and each of Z a and Z b is independently selected from CH 2 , O, and NH.
  • At least one of Z a and Z b is NH and X a and X b are selected from OH and SH.
  • Z a is NH and X a and X b are selected from OH and SH.
  • Z b is NH and X a and X b are selected from OH and SH.
  • each of Z a and Z b is NH and X a and X b are selected from OH and SH.
  • the bridge of the drug moiety is an aliphatic group in which at least one CH 2 unit has been replaced by an NH group. In some embodiments, the aliphatic group is fully saturated.
  • the aliphatic group contains at least one unit of unsaturation.
  • the bridge is an aliphatic group in which one CH 2 unit has been replaced by an NH group. In some embodiments, the bridge is an aliphatic group in which two CH 2 units have been replaced by an NH group.
  • the bridge H H N N N N atoms comprise H . In some embodiments, the bridge atoms comprise H . H N N In some embodiments, the bridge atoms comprise H . [00299]
  • D compri und of Formula (III) and X a is SH. In some embodiments, D comprises a compound of Formula (III) and X b is SH.
  • D comprises a compound of Formula (IV) and X a is SH. In some embodiments, D comprises a compound of Formula (IV) and X b is SH. [00300] In some embodiments, D comprises a compound of Formula (III) selected from: 210 Attorney Reference: 0080171-000886 O O O (R) S H O (R) P P SH F O F O N H N N N H N N , S H , S H , O H , S H , , Attorney Reference: 0080171-000886 O O (R O O P OH ) P SH F O N N H F O N N N N O , S H H O , an [00301] In some embodiments, the compound of Formula (III) is selected from: (R) O (R O O ) P SH O P SH F O N N N F O N N O , SH O , a [00302] In some embodiments, D comprises a compound of Formula (IV) selected from: O O P SH (For
  • D comprises a compound of Formula (IV) selected from: 212 Attorney Reference: 0080171-000886 O (R) O O (R) P SH O P SH HO O N HO O N H N N H N N O , S H O , an [00304] In some embodiments, D comprises a compound selected from: O O O P SH O P SH F O O OH O , O H O O , SH O , an sa s e eo . [00305] In some embodiments, the STING agonist is Compound 1.
  • Compound 1 also encompasses salts of the structure shown above unless context indicates otherwise.
  • the drug moiety is Compound 1.
  • a linker e.g., the linker of an ADC
  • a linker of an ADC is attached to Compound 1 via the S-14 sulfur on Compound 1.
  • a linker e.g., the linker of an ADC
  • a linker, e.g., the linker of an ADC is attached to Compound 1 via the N-39 nitrogen on Compound 1.
  • the linker of the ADC covalently attaches to the S-14 sulfur on Compound 1 via pAB.
  • the pAB is an analog of pAB as disclosed above.
  • the linker of the ADC covalently attaches to the N-34 nitrogen on Compound 1 via pABC.
  • the linker of the ADC covalently attaches to the N-39 nitrogen on Compound 1 via pABC.
  • the linker of the ADC covalently attaches to the N-34 nitrogen on Compound 1 via a second self immolative unit as disclosed below.
  • the linker of the ADC covalently attaches to the N-39 nitrogen on Compound 1 via a second self immolative unit as disclosed below.
  • the STING agonist is Compound 2.
  • the structure of Compound 2 is shown below: 214 Attorney Reference: 0080171-000886 O O P SH (R) F O
  • the term Compound 2 also encompasses salts of the structure shown above unless context indicates otherwise.
  • the drug moiety is Compound 2.
  • a linker e.g., the linker of an ADC, is attached to Compound 1 via the S-14 sulfur on Compound 2.
  • a linker e.g., the linker of an ADC
  • a linker is attached to Compound 1 via the N-34 nitrogen on Compound 2.
  • a linker e.g., the linker of an ADC
  • the linker of the ADC covalently attaches to the S-14 sulfur on Compound 2 via pAB.
  • the pAB is an analog of pAB as disclosed above.
  • the linker of the ADC covalently attaches to the N-34 nitrogen on Compound 2 via pABC.
  • the linker of the ADC covalently attaches to the N-39 nitrogen on Compound 2 via pABC. In some embodiments, the linker of the ADC covalently attaches to the N-34 nitrogen on Compound 2 via a second self immolative unit as disclosed below. In some embodiments, the linker of the ADC covalently attaches to the N-39 nitrogen on Compound 2 via a second self immolative unit as disclosed below. In some embodiments, the STING agonist is selected from a compound of Table 3, infra. Table 3.
  • Exemplary STING agonist compounds O O O (R) (R) P SH O P SH 215 Attorney Reference: 0080171-000886 O O SH ( O (S) P O R) P SH F O F O 216 Attorney Reference: 0080171-000886 O O O (R) P SH O P SH HO NH HO NH 217 Attorney Reference: 0080171-000886 S S OH OH O P O P F (R) O F (R) O [ ] ur er sc ose are somers o e compoun s o a e , eu era e er va ves of the compounds and isomers; and salts of the compounds, isomers, and deuterated derivatives.
  • an intermediate such as a precursor of a linker disclosed above, is reacted with the drug moiety under appropriate conditions.
  • reactive groups are used on the drug and/or the intermediate or linker.
  • the product of the reaction between the drug and the intermediate, or the derivatized drug is subsequently reacted with the antibody or antigen-binding fragment under appropriate conditions, e.g., according to the methods discussed below.
  • the linker or intermediate may first be reacted with the antibody or a derivatized antibody, and then reacted with the drug or derivatized drug.
  • a number of different reactions are available for covalent attachment of drugs and/or linkers to the antibody moiety.
  • Linker-Drug Conjugates [00312] The present disclosure provides linker-drug conjugates comprising L-D, wherein L is a cleavable linker that covalently attaches to D.
  • linker-drug conjugate and “linker-payload conjugate” are used interchangeably herein.
  • the linker-drug conjugates disclosed herein are suitable for conjugation to a variety of antibodies, including anti-PSMA antibodies disclosed herein.
  • D is a compound that forms a covalent bond with L, which results in the loss of at least one hydrogen radical.
  • D may be any suitable compound that would benefit from a disclosed linker.
  • D is selected from any of the compounds disclosed herein.
  • L may be selected from any linker disclosed herein.
  • D comprises a compound according to one of the following Formulae: an isomer thereof, a deuterated derivative of the compound or isomer; or a salt of the compound, isomer, or deuterated derivative; wherein, independently for each occurrence, ⁇ each of P a and P b , when not racemic or achiral, is independently selected from (R)- configuration and (S)-configuration; ⁇ each of Q a and Q b is independently selected from NH and O; ⁇ each of V a and V b is independently selected from F and OH; ⁇ W 1 is selected from H and NH 2 ; ⁇ each of X a and X b is independently selected from OH and SH; 219 Attorney Reference: 0080171-000886 ⁇ each of Y a and Y b is independently selected from O and S; ⁇ each Z a and Z b is independently selected from CH 2 , O, and NH; and ⁇ means that the bond is selected from a single bond ( ), a double
  • each of P a and P b is racemic. In some embodiments, P a is racemic or achiral and P b is selected from (R)-configuration and (S)-configuration. In some embodiments, P a is selected from (R)-configuration and (S)-configuration and P b is racemic or achiral. In some embodiments, each of P a and P b is selected from (R)-configuration and (S)- configuration. [00314] In some embodiments, P a is (R)-configuration and P b is (R)-configuration. In some embodiments, P a is (R)-configuration and P b is (S)-configuration.
  • P a is (S)-configuration and P b is (R)-configuration. In some embodiments, P a is (S)-configuration and P b is (S)-configuration.
  • Q a is O and Q b is O. In some embodiments, Q a is NH and Q b is O. In some embodiments, Q a is O and Q b is NH. In some embodiments, Q a is NH and Q b is NH.
  • V a is OH and V b is OH. In some embodiments, V a is F and V b is OH. In some embodiments, V a is OH and V b is F.
  • V a is F and V b is F.
  • W 1 is H. In some embodiments, W 1 is NH 2 .
  • X a is OH and X b is OH.
  • X a is SH and X b is OH.
  • X a is OH and X b is SH.
  • X a is SH and X b is SH.
  • Y a is O and Y b is O. In some embodiments, Y a is S and Y b is O. In some embodiments, Y a is O and Y b is S.
  • Y a is S and Y b is S.
  • Z a is NH and Z b is selected from CH 2 , O, and NH.
  • Z a is NH and Z b is CH 2 .
  • Z a is NH and Z b is O.
  • Z a is NH and Z b is NH. 220 Attorney Reference: 0080171-000886 [00321]
  • Z a is O and Z b is selected from CH 2 , O, and NH.
  • Z a is O and Z b is CH 2 .
  • Z a is O and Z b is O.
  • Z a is O and Z b is NH.
  • Z a is CH 2 and Z b is selected from CH 2 , O, and NH.
  • Z a is CH 2 and Z b is CH 2 .
  • Z a is CH 2 and Z b is O.
  • Z a is CH 2 and Z b is NH.
  • At least one of X a and X b is SH and each of Z a and Z b is independently selected from CH 2 , O, and NH.
  • X a is SH and each of Z a and Z b is independently selected from CH 2 , O, and NH.
  • X b is SH and each of Z a and Z b is independently selected from CH 2 , O, and NH.
  • each of X a and X b is SH and each of Z a and Z b is independently selected from CH 2 , O, and NH.
  • Z a and Z b is NH and X a and X b are selected from OH and SH.
  • Z a is NH and X a and X b are selected from OH and SH.
  • Z b is NH and X a and X b are selected from OH and SH.
  • each of Z a and Z b is NH and X a and X b are selected from OH and SH.
  • D comprises a compound of Formula (III) and X a is SH.
  • D comprises a compound of Formula (III) and X b is SH.
  • D comprises a compound of Formula (IV) and X a is SH. In some embodiments, D comprises a compound of Formula (IV) and X b is SH.
  • the bridge of the linker-drug conjugate is an aliphatic group in which at least one CH 2 unit has been replaced by an NH group. In some embodiments, the aliphatic group is fully saturated. In some embodiments, the aliphatic group contains at least one unit of unsaturation. In some embodiments, the bridge is an aliphatic group in which one CH 2 unit has been replaced by an NH group. In some embodiments, the bridge is an aliphatic group in which two CH 2 units have been replaced by an NH group.
  • the H N N bridge atoms comprise H . In some embodiments, the bridge atoms comprise H N H N N N H . In some embodiments, the bridge atoms compris H .In some 221 Attorney Reference: 0080171-000886 embodiments, L is attached to D via a sulfur atom. In some embodiments, L is attached to D at the S-2 sulfur or the S-14 sulfur. In some embodiments, L is attached to D at the S-2 sulfur. In some embodiments, L is attached to D at the S-14 sulfur. [00328] In some embodiments, D comprises a compound of Formula (III) and Z a is NH. In some embodiments, D comprises a compound of Formula (III) and Z b is NH.
  • D comprises a compound of Formula (IV) and Z a is NH. In some embodiments, D comprises a compound of Formula (IV) and Z b is NH. [00329] In some embodiments, L is attached to D via a bridge nitrogen atom. In some embodiments, L is attached to D at the N-34 nitrogen or the N-39 nitrogen. In some embodiments, L is attached to D at the N-34 nitrogen. In some embodiments, L is attached to D at the N-39 nitrogen. [00330] In some embodiments, D comprises a compound of Formula (III). Exemplary compounds of Formula (III) are shown below.
  • D comprises a compound of Formula (III) selected from: O O (R) (R) O P SH O P SH F O F O O , S H O , S H O , Attorney Reference: 0080171-000886 O O O P SH O P OH F O N H N N F O N N N O N H O , S H O , , S H , S H , an .
  • the compound of Formula (III) is selected from: (R O O O ) (R) P SH O P SH O , Attorney Reference: 0080171-000886 O (R) O O P SH O P SH F O (R) N F O H N N N O , an [00332]
  • D comprises Compound 1. In some embodiments, D comprises Compound 2.
  • D comprises a compound of Formula (IV) selected from: O O Pb SH (Formula (VII)) [00334] In some embodiments, D comprises a compound of Formula (IV) selected from: O (R) O S H O (R) O P P SH HO O HO O O , S H O , an . [00335] In some embodiments, X a or X b is SH and L is attached to D via a sulfur atom at the S-2 sulfur or the S-14 sulfur. In some embodiments, Z a or Z b is NH and L is attached to D via a nitrogen atom at the N-34 nitrogen or the N-39 nitrogen.
  • D comprises a compound of Formula (III), X a is SH, and L is attached to D at the S-2 sulfur.
  • D comprises a compound of Formula (III), X b is SH, and L is attached to D at the S-14 sulfur.
  • D comprises a compound of Formula (III), Z a is NH, and L is attached to D at the N-34 nitrogen.
  • D comprises a compound of Formula (III), Z b is NH, and L is attached to D at the N-39 nitrogen.
  • D comprises a compound of Formula (IV) and L is attached to D at the S-2 sulfur.
  • D comprises a compound of Formula (IV) and L is attached to D at the S-14 sulfur. In some embodiments, D comprises a compound of Formula (IV) and L is attached to D at the N-34 nitrogen. In some embodiments, D comprises a compound of Formula (IV) and L is attached to D at the N-39 nitrogen. [00338] In some embodiments, D comprises Compound 1. In some embodiments, D comprises Compound 2.
  • L is attached to D via a sulfur atom at the S-2 sulfur or the S-14 sulfur. In some embodiments, L is attached to D at the S-2 sulfur. In some embodiments, L is attached to D at the S-14 sulfur. [00341] In some embodiments, L is attached to D via a nitrogen atom at the N-34 nitrogen or the N-39 nitrogen.
  • L is attached to D at the N-34 nitrogen. In some embodiments, L is attached to D at the N-39 nitrogen. [00342] In some embodiments of linker-payload conjugates comprising L-D, L is any linker disclosed herein. In some embodiments of linker-payload conjugates comprising L-D, D is any drug moiety disclosed herein. [00343] In some embodiments of linker-payload conjugates comprising L-D, wherein L is a cleavable linker that covalently attaches to D, the cleavable linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety is cleavable by a protease.
  • the protease is legumain or cathepsin.
  • the cleavable peptide moiety comprises an amino acid unit.
  • the amino acid unit comprises Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Asn, Ala-(NMe)Ala-Asn, Asn, Gly-Gly-Phe- Gly, or Gly-Val-Ala.
  • the amino acid unit comprises Val-Ala.
  • the amino acid unit comprises Val-Cit.
  • the linker-payload conjugate comprises Val-Ala
  • D is selected from a compound of Table 3.
  • the linker-payload conjugate comprises Val-Cit, and D is selected from a compound of Table 3. [00345] In some embodiments, the linker-payload conjugate comprises Formula (II), and D is selected from a compound of Table 3. [00346] In some embodiments, the linker-payload conjugate comprises Formula (II)-Val- Ala, and D is selected from a compound of Table 3. In some embodiments, the linker-payload conjugate comprises Formula (II)-Val-Cit, and D is selected from a compound of Table 3. [00347] In some embodiments, the linker-drug conjugate comprises MC-Val-Cit-pABC- MEC-Compound 1.
  • the linker-drug conjugate comprises MC-Val-Ala- pABC-MEC-Compound 1 (e.g., LP1 or LP2). In some embodiments, the linker-drug conjugate comprises MC-Val-Cit-pABC-Unit 8-Compound 1. In some embodiments, the linker-drug conjugate comprises MC-Val-Ala-pABC-Unit 8-Compound 1 (e.g., LP16). In some embodiments, the linker-drug conjugate comprises MC-Val-Cit-pABC-Unit 9-Compound 1.
  • the linker-drug conjugate comprises MC-Val-Ala-pABC-Unit 9-Compound 1 (e.g., LP20). In some embodiments, the linker-drug conjugate comprises MC-Val-Cit-pABC- Unit 11-Compound 1. In some embodiments, the linker-drug conjugate comprises MC-Val-Ala- pABC-Unit 11-Compound 1 (e.g., LP28). [00348] In some embodiments, the linker-drug conjugate comprises Mal-Formula (II)- Val-Cit-pABC-MEC-Compound 1.
  • the linker-drug conjugate comprises Mal-Formula (II)-Val-Cit-pABC-Unit 8-Compound 1. In some embodiments, the linker-drug conjugate comprises Mal-Formula (II)-Val-Cit-pABC-Unit 9-Compound 1. In some embodiments, the linker-drug conjugate comprises Mal-Formula (II)-Val-Cit-pABC-Unit 11- Compound 1. In some embodiments, the linker-drug conjugate comprises Mal-Formula (II)-Val- Ala-pABC-MEC-Compound 1.
  • the linker-drug conjugate comprises Mal- Formula (II)-Val-Ala-pABC-Unit 8-Compound 1. In some embodiments, the linker-drug conjugate comprises Mal-Formula (II)-Val-Ala-pABC-Unit 9-Compound 1. In some embodiments, the linker-drug conjugate comprises Mal-Formula (II)-Val-Ala-pABC-Unit 11- Compound 1. [00349] In some embodiments, the linker-drug conjugate comprises Mal-Formula (II)- Val-Cit-pAB-Unit 9-Compound 1.
  • the linker-drug conjugate comprises Mal-Formula (II)-Val-Ala-pAB-Unit 9-Compound 1. In some embodiments, the linker-drug conjugate comprises LP25. [00350] In some embodiments, the linker-drug conjugate comprises Mal-Formula (II)- Val-Cit-pAB-Unit 11-Compound 1. In some embodiments, the linker-drug conjugate comprises Mal-Formula (II)-Val-Ala-pAB-Unit 11-Compound 1. In some embodiments, the linker-drug conjugate comprises LP26. [00351] Exemplary linker-drug conjugates of the invention are disclosed in Table 4 and 5, infra.
  • the linker-drug conjugate is selected from the linker-drug conjugates shown in Tables 4 and 5. 227 Attorney Reference: 0080171-000886 Table 4. Exemplary S-attached Linker-Drug Conjugates O(R) O P SH F O N H N N 228 Attorney Reference: 0080171-000886 O(R) O P SH N F O N H N H O O O 229 Attorney Reference: 0080171-000886 O O O O O O O O O 230 Attorney Reference: 0080171-000886 Cl O O O O O O O O O O 231 Attorney Reference: 0080171-000886 O O O O O a e .
  • an exemplary linker-drug conjugate or a salt thereof may be referred to as “LP3” and has the structure of
  • an exemplary linker-drug conjugate or a salt thereof may be referred to as “LP1” and has the structure of LP1 shown below: O O P (R) SH N N F O N O [00354]
  • an exemplary linker-drug conjugate or a salt thereof may be referred to as “LP2” and has the structure of LP2 shown below: O O P (S) SH F O O [00355]
  • an exemplary linker-drug conjugate or a salt thereof has the structure of LP16 shown below: O O P SH O Attorney Reference: 0080171-000886 [00356]
  • an exemplary linker-drug conjugate or a salt thereof has the structure of LP20 shown below: O O P SH F O N H N N O [00357]
  • an exemplary linker-drug conjugate or a salt thereof has the structure of
  • a linker-payload disclosed herein e.g., LP1, LP2, LP3, LP16, LP20, LP26, or LP28
  • a linker-payload disclosed herein e.g., LP1, LP2, LP3, LP16, LP20, LP26, or LP28
  • a linker-payload disclosed herein, e.g., LP1, LP2, LP3, LP16, LP20, LP26, or LP28 has superior in vivo anti-tumor activity over prior art linker-STING agonist conjugates.
  • a linker-payload disclosed herein e.g., LP1, LP2, LP3, LP16, LP20, LP26, or LP28, has superior tolerability in vivo over prior art linker-STING agonist conjugates.
  • linker-payload conjugates wherein D is a STING agonist, e.g., a compound of Formula (III), Formula (IV), Compound 1, and L is conjugated to D at the N-34 nitrogen or the N-39 nitrogen (e.g., LP16, LP20, LP26, or LP28), the linker-payload conjugate demonstrates superior properties (e.g., plasma stability, in vitro immune responses, in vivo anti-tumor activity, tolerability, stimulation of an anti-immune response in the tumor microenvironment) compared to other linker-payload conjugates comprising a compound of Formula (III), Formula (IV), that are conjugated to D at alternative attachment points, e.g., at a sulfur, e.g., S-2 or S-14.
  • superior properties e.g., plasma stability, in vitro immune responses, in vivo anti-tumor activity, tolerability, stimulation of an anti-immune response in the tumor microenvironment
  • linker-payload conjugates disclosed herein wherein L comprises a spacer unit comprising Formula (II)
  • the linker-payload conjugate demonstrates superior properties (e.g., improved conjugation stability, improved plasma stability, in vivo anti- tumor activity) compared to other linker-payload conjugates comprising alternative spacer units.
  • benefits of using a linker comprising Formula (II) with a STING agonist disclosed herein, e.g., a compound of Formula (III), Formula (IV), e.g., Compound 1 may include improved conjugation stability, improved plasma stability, and in vivo anti-tumor activity.
  • a linker-payload conjugate comprising a linker comprising Formula (II) and a payload comprising a STING agonist disclosed herein, e.g., a compound of Formula (III), Formula (IV), demonstrates superior properties when conjugated to an anti-PSMA antibody disclosed herein.
  • an anti-PSMA antibody moiety or an antigen-binding fragment thereof as disclosed herein may be conjugated (i.e., covalently attached, e.g., by a linker) to a drug moiety, wherein the drug moiety when not conjugated to an antibody moiety has 245 Attorney Reference: 0080171-000886 a cytotoxic or cytostatic effect.
  • the drug moiety exhibits reduced or no cytotoxicity when bound in a conjugate but resumes cytotoxicity after cleavage from the linker and antibody moiety.
  • an ADC for use as a human therapeutic agent may require more than the identification of an antibody capable of binding to a desired target or targets and attaching to a drug used on its own to treat cancer.
  • Linking the antibody to the drug may have significant and unpredictable effects on the activity of one or both of the antibody and the drug, effects which will vary depending on the type of linker and/or drug chosen.
  • the components of the ADC are selected to (i) retain one or more therapeutic properties exhibited by the antibody and drug moieties in isolation, (ii) maintain the specific binding properties of the antibody moiety; (iii) optimize drug loading and drug-to-antibody ratios; (iv) allow targeted tumor cell delivery, e.g., intracellular delivery, of the drug moiety via stable attachment to the antibody moiety; (v) reduce toxicity compared to non-targeted and/or systemic delivery of the drug moiety; (vi) retain ADC stability as an intact conjugate until transport or delivery to a target site; (vii) minimize aggregation of the ADC prior to or after administration; (viii) exhibit in vivo anti-cancer treatment efficacy comparable to or superior to that of the antibody and drug moieties in isolation; (ix) minimize off-target killing by the drug moiety; (x) exhibit desirable pharmacokinetic and pharmacodynamics properties, formulatability, and toxicologic/immunologic profiles; (xi) maintain
  • the ADC compounds of the present disclosure have superior stability as an intact conjugate until transported or delivered to a target site compared to ADC compounds comprising other antibodies, e.g., J591 or deJ591, and/or other linkers.
  • the ADC compounds of the present disclosure are less immunogenic compared to ADC compounds comprising other antibodies, e.g., J591 or deJ591, and/or other linkers.
  • the ADC compounds of the present disclosure may selectively deliver an effective dose of a cytotoxic or cytostatic agent to cancer cells or to tumor tissue. It has been discovered that the disclosed ADCs have potent cytotoxic and/or cytostatic activity against cells 246 Attorney Reference: 0080171-000886 expressing PSMA. In some embodiments, the cytotoxic and/or cytostatic activity of the ADC is dependent on PSMA expression level in a cell. In some embodiments, the disclosed ADCs are particularly effective at killing cancer cells expressing a high level of PSMA, as compared to cancer cells expressing the same antigen at a low level. In some embodiments, the disclosed ADCs are particularly effective at killing high PSMA-expressing cancers such as prostate cancer.
  • targeted killing of PSMA-expressing cancer cells is improved by the presence or recruitment of myeloid cells (e.g., macrophages and/or dendritic cells).
  • ADCs disclosed herein demonstrate PSMA-specific binding on PSMA-expressing cells, e.g., in PSMA-expressing cancers.
  • the disclosed ADCs upon binding PSMA, the disclosed ADCs are internalized.
  • release of the drug moiety, e.g., Compound 1 results in STING pathway activation and release of proinflammatory cytokines (e.g., IFN ⁇ ).
  • proinflammatory cytokines e.g., IFN ⁇ .
  • release of proinflammatory cytokines promotes myeloid cell activation.
  • the disclosed ADCs activate myeloid cells, e.g., macrophages or dendritic cells. Without being bound by theory, myeloid cell activation may be a result of phagocytosis of PSMA-expressing cancer cells bound by the disclosed ADCs.
  • the disclosed ADCs activate macrophages.
  • the activated macrophages are proinflammatory (M1) macrophages.
  • tumor-associated macrophages or M2 macrophages undergo proinflammatory activation upon administration of the disclosed ADCs.
  • the activated macrophages release pro-inflammatory cytokines and chemokines (e.g., TNF ⁇ , CXCL10, IL-6, IFN ⁇ , and/or IL-1 ⁇ ). In some embodiments, the activated macrophages promote further myeloid cell activation. In some embodiments, the activated macrophages promote the generation of cytotoxic T cells. In some embodiments, the activated macrophages demonstrate increased phagocytosis of cancer cells. In some embodiments, administration of the disclosed ADCs stimulates Type I IFN-dependent anti- tumor activity.
  • an “activated macrophage” is synonymous with a “polarized macrophage.”
  • ADC compounds comprising an antibody or antigen-binding fragment thereof (Ab) which targets a tumor cell, a drug moiety (D), and a linker moiety (L) that covalently attaches Ab to D.
  • the antibody or antigen-binding fragment is able 247 Attorney Reference: 0080171-000886 to bind to a tumor-associated antigen (e.g., PSMA) with high specificity and high affinity.
  • the antibody or antigen-binding fragment is internalized into a target cell upon binding, e.g., into a degradative compartment in the cell.
  • ADCs internalize upon binding to a target cell, undergo degradation, and release the drug moiety.
  • the drug moiety may be released from the antibody and/or the linker moiety of the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.
  • target cells bound by the ADC are phagocytosed by a myeloid cell, e.g., a macrophage or dendritic cell.
  • the ADCs upon phagocytosis the ADCs undergo degradation and release the drug moiety.
  • the drug moiety is released in the phagolysosome of the myeloid cell (e.g., a macrophage or dendritic cell).
  • p is from 1 to 12, or 2 to 11. In some embodiments, p is from 1 to 8. In some embodiments, p is from 4 to 11. In some embodiments, p is from 4 to 8. In some embodiments, p is 2. In some embodiments, p is 4.
  • p is 7. In some embodiments, p is 11. [00372]
  • a linker comprising a thiol- reactive group is used to generate a conjugated antibody or antigen-binding fragment, e.g., by reacting with the antibody or antigen-binding fragment at a cysteine residue.
  • the cysteine residue is at amino acid position 80 on the light chain. In some embodiments, the cysteine residue is at amino acid position 118 on the heavy chain.
  • the starting materials used are either commercially available or can be readily prepared by standard methods from known materials.
  • the disclosed conjugatable linker- payloads can be prepared using the reactions and techniques described herein.
  • all proposed reaction conditions including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment, and workup procedures, can be chosen to be the conditions standard for that reaction, unless otherwise indicated. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule should be compatible with the reagents and reactions proposed. Substituents not compatible with the reaction conditions are apparent to one skilled in the art, and alternate methods are therefore indicated herein.
  • NMR spectra were recorded using a Bruker Ascend 400MHz spectrometer. Chemical shifts ( ⁇ ) are reported in ppm relative to the residual solvent signal (measurement range – 6.4 kHz). 1 H NMR data are reported as follows: chemical shift (multiplicity, coupling constants and number of hydrogens). Multiplicity is abbreviated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad).
  • aqueous layer was extracted with dichloromethane (12.3 ml) and the combined organic layers were washed with 30% aq NaCl (6.2 ml) and dried over MgSO 4 . Filtration followed by concentration in vacuo provided 1.10 g of off-white foam solid.
  • methanol (7.9 ml) and ammonium hydroxide (7.9 mL) were added to the resulting product.
  • the resulting mixture was heated at 52 o C for 5h, cooled to rt, and diluted with water (9.9 ml).
  • the resulting mixture was stirred for 1h at rt and then DIEA (0.075 ml, 0.43 mmol) was added. After 1h stirring, the reaction mixture was kept at 0 o C for 4 days. The reaction mixture was warmed to rt, and treated with MTBE (9 mL) and a sat'd NH 4 Cl (10 mL). The supernatant was removed by decantation and the remaining residue was dried in vacuo to give 245 mg of the target product.
  • the reaction mixture was diluted with EtOAc (10 ml) and treated with a sat'd NaHCO 3 solution (10 mL). The layers were separated and the aqueous layer was extracted two times with EtOAc (10 mL each). The combined organic layers were washed with brine (5 mL), dried over MgSO 4 , filtered and concentrated in vacuo. The resulting residue was dissolved in MeCN (5 ml), treated with MTBE (5 mL) followed by n-heptane (5 mL). The resulting slurry was kept at -20 o C overnight and filtered, rinsing with MTBE followed by n-heptane.
  • the reaction mixture was stirred for 1h at 0 o C and for 1h at rt, and kept for 4 days at -20 o C.
  • the resulting mixture was warmed to rt and treated with additional tert-butyl ((S)-1-(((S)-1-((4-(iodomethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3- methyl-1-oxobutan-2-yl)carbamate (41 mg, 0.082 mmol) over 3h.
  • the resulting mixture was diluted with EtOAc (20 mL), washed with a sat' Na 2 S 2 O 3 solution (5 mL) and brine (5 mL x 2).
  • reaction mixture was treated with a sat'd aqeous NaHCO 3 solution (10 mL) and then extracted with DCM (10 mL each) twice. The combined organic layers were dried over MgSO 4 , filtered,and concentrated in vacuo. The residue was triturated with n-heptane. The resulting slurry was filtered through a syringe filter. The solid was dissolved in DCM and concentrated in vacuo, leading to 3.4 mg of the target product.

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Abstract

L'invention divulgue des composés qui peuvent être utilisés pour préparer des conjugués anticorps-médicament et possèdent les formules suivantes : (i) ou (ii). L'invention divulgue également des procédés de création de ces composés.
PCT/US2025/043044 2024-08-21 2025-08-21 Synthèse de combinaisons médicament-lieur pour conjugués anticorps-médicament Pending WO2026044149A2 (fr)

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