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WO2024238872A2 - Conjugués d'anticorps isoindolinone-glutarimide et leurs utilisations - Google Patents

Conjugués d'anticorps isoindolinone-glutarimide et leurs utilisations Download PDF

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Publication number
WO2024238872A2
WO2024238872A2 PCT/US2024/029822 US2024029822W WO2024238872A2 WO 2024238872 A2 WO2024238872 A2 WO 2024238872A2 US 2024029822 W US2024029822 W US 2024029822W WO 2024238872 A2 WO2024238872 A2 WO 2024238872A2
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alkyl
antibody conjugate
conjugate composition
aryldiyl
heteroalkyldiyl
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PCT/US2024/029822
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WO2024238872A3 (fr
Inventor
Luca Arista
John A. Flygare
Freya M. HARVEY
Thomas Ryckmans
Sean Wesley Smith
Christoph C. GROHMANN
Bernhard Geierstanger
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Firefly Bio, Inc.
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Publication of WO2024238872A2 publication Critical patent/WO2024238872A2/fr
Publication of WO2024238872A3 publication Critical patent/WO2024238872A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6863—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from stomach or intestines cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the disclosure relates generally to antibody conjugate compositions, intermediates for their manufacture, and methods of their use.
  • the compositions are useful for facilitating intracellular degradation of target proteins.
  • the ubiquitin proteasome system can be manipulated to conduct targeted degradation of specific proteins. Promoting the targeted degradation of pathogenic proteins using small molecule degraders is a new modality in the treatment of diseases, including redirecting the activity of E3 ligases such as cereblon.
  • Proteolysis targeting chimera compounds called PROTAC (Sakamoto, K. M , et al. (2001) Proc. Natl. Acad. Sci. USA 98:8554-8559; Sun, X. et al. (2019) Signal Transduct. Target. Ther. 4:64; Schapira, M., et al (2019) Nat. Rev. Drug Discov.
  • PROTAC comprise three parts, including a ligand for binding a target protein, another ligand for recruiting an E3 ligase, and a linker to help anchor the target protein to the E3 ubiquitin ligase to promote its ubiquitination and subsequent proteasomal degradation. Similar to PROTAC, molecular glues can also cause ubiquitination and degradation of a target protein.
  • molecular glues are small molecular weight compounds that trigger a compact protein-protein interaction between a target protein and an E3 ubiquitin ligase.
  • Molecular glues are typically smaller than PROTAC and may have better pharmacological properties, higher membrane permeability, better cellular uptake, and better penetration of the blood-brain barrier.
  • Molecular glues promote the poly-ubiquitination and proteasomal degradation of various disease-associated protein targets (Chamberlain, P., et al (2019) Drug Disc. Today. Tech. 31:29-34; WO 2022/152821).
  • the molecular glue molecules bind to both the E3 ligase and the target protein, thereby mediating an alteration of the ligase surface and enabling an interaction with the target protein.
  • Examples include the IMiD (immunomodulatory imide drug) class including thalidomide, lenalidomide and pomalidomide, each approved for use in treating hematological cancers. More efficient targeting strategies are still required.
  • GSPT1 contains a well-defined degron, a peptidic motif that signals for degradation, allowing for the recruitment of the E3 ligase cereblon (CRBN) and subsequent proteasomal degradation in the presence of molecular glue degraders.
  • GSPT1 (G1 to S phase transition 1) is a translation termination factor that recognizes the termination codon by binding eRFl, forcing the proteins to dissociate from the ribosome after translation
  • Downregulation of GSPT1 can cause the abnormal expression of key proteins, inhibit proliferation or induce apoptosis in diverse tumor cells (Chauvin, C., et al. (2007) Mol. Cell. Biol. 27:5619-5629; Matyskiela, M. E., et al. (2016) Nature 535:252-257; Yang, I, et al. J. Med. Chem. (2019) 62:9471-9487).
  • Targeted therapeutic agents to treat hyperproliferative disorders like cancer, and other disease are of interest.
  • the invention is generally directed to an antibody conjugate composition
  • an antibody conjugate composition comprising an isoindolinone-glutarimide moiety covalently attached to an antibody by an antibody linker, wherein the antibody binds to a tumor-associated antigen or cell-surface receptor.
  • the antibody conjugate composition has Formula I: or a pharmaceutically acceptable salt thereof, wherein:
  • L is the antibody linker
  • IG is the isoindolinone-glutarimide moiety; and p is an integer from 1 to 12
  • Another aspect of the invention is an antibody conjugate composition selected from Formulae la and lb:
  • Another aspect of the invention is an isoindolinone-glutarimide linker compound selected from Formulae Ila and lib: lib wherein the substituents are defined herein.
  • Another aspect of the invention is the antibody conjugate composition prepared by conjugation of a cysteine amino acid of an antibody with an isoindolinone-glutarimide linker compound.
  • Another aspect of the invention is a process for preparing the antibody conjugate comprising reacting a cysteine amino acid of an antibody with an isoindolinone-glutarimide linker compound.
  • Another aspect of the invention is a pharmaceutical composition comprising a therapeutically effective amount of the antibody conjugate composition, and one or more pharmaceutically acceptable diluent, vehicle, carrier or excipient
  • Another aspect of the invention is a method for treating cancer comprising administering a therapeutically effective amount of the pharmaceutical composition to a patient in need thereof,
  • Another aspect of the invention is a use of the antibody conjugate composition in the manufacture of a medicament for the treatment of cancer in a mammal.
  • Another aspect of the invention is the antibody conjugate composition for use in a method for treating cancer.
  • Figure 1 shows a graph of in vivo tumor volume over time in the treatment of CD22+ WSU-DLCL2 syngeneic tumor bearing CB17 SCID mice with pinatuzumab, and anti-CD22 conjugates IGAC-59 and IGAC-62 (Table 3).
  • Figure 2 shows a graph of in vivo tumor volume over time in the treatment of CD22+ WSU-DLCL2 syngeneic tumor bearing CB17 SCID mice with pinatuzumab, and anti-CD22 conjugates IGAC-53, IGAC-60 and IGAC-62 (Table 3).
  • Figure 3 shows a graph of in vivo tumor volume over time in the treatment of HER2+ NCI-N87 gastric tumor xenograft bearing female BALB/c nude mice with anti-HER2 conjugates IGAC-58 and IGAC-61 (Table 3).
  • Figure 4 shows a graph of in vivo tumor volume over time in the treatment of HER2+ NCI-N87 gastric tumor xenograft bearing female BALB/c nude mice with anti-HER2 conjugates IGAC-54, IGAC-55, IGAC-56 and IGAC-57 (Table 3).
  • antibody or “antibody construct” refer to a polypeptide comprising an antigen binding region (including the complementarity determining region (CDRs)) from an immunoglobulin gene or fragments thereof.
  • antibody specifically encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa) connected by disulfide bonds.
  • Each chain is composed of structural domains, which are referred to as immunoglobulin domains. These domains are classified into different categories by size and function, e g., variable domains or regions on the light and heavy chains (VL and VH, respectively) and constant domains or regions on the light and heavy chains (CL and CH, respectively).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, referred to as the paratope, primarily responsible for antigen recognition, i.e., the antigen binding domain.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • IgG antibodies are large molecules of about 150 kDa composed of four peptide chains.
  • IgG antibodies contain two identical class y heavy chains of about 50 kDa and two identical light chains of about 25 kDa, thus a tetrameric quaternary structure The two heavy chains are linked to each other and to a light chain each by disulfide bonds. The resulting tetramer has two identical halves, which together form the Y-like shape Each end of the fork contains an identical antigen binding domain.
  • IgGl IgG2
  • IgG3 IgG4
  • IgG4 IgG4
  • IgGl is the most abundant
  • the antigen binding domain of an antibody will be most critical in specificity and affinity of binding to cancer cells.
  • Bispecific antibodies are antibodies that bind two distinct epitopes to cancer (Suurs F.V. et al Pharmacology & Therapeutics 201: 103-119). Bispecific antibodies may engage immune cells to destroy tumor cells, deliver isoindolinone-glutarimide moi eties to tumors, and/or block tumor signaling pathways.
  • An antibody that targets a particular antigen includes a bispecific or multispecific antibody with at least one antigen binding region that targets the particular antigen.
  • the targeted monoclonal antibody is a bispecific antibody with at least one antigen binding region that targets tumor cells.
  • antigens include but are not limited to: mesothelin, prostate specific membrane antigen (PSMA), HER2, TROP2, CEA, CEACAM5, EGFR, 5T4, Nectin4, CCL-1, CCR7, CD19, CD20, CD22, CD30, CD33, CD70, CD79b, CD 123, CDH3, B7H3, B7H4 (also known as 08E), Integrin-beta6, protein tyrosine kinase 7 (PTK7), glypican-3, GPC-1, LIV-1, Folate receptor alpha, Claudinl8.2, RG1, fucosyl-GMl, tissue factor (CD142), cKit (CD117), Axl, , GC-C, CTLA-4, and CD44 (WO 2017/196598).
  • Other antigen binding regions of bispecific antibodies include those in the following section: ANTIBODY TARGETS.
  • the antibody construct is an antigen-binding antibody “fragment,” which comprises at least an antigen-binding region of an antibody, alone or with other components that together constitute the antibody construct.
  • antibody “fragments” are known in the art, including, for instance, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHi domains, (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab’ fragment, which results from breaking the disulfide bridge of an F(ab’)2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a single chain Fv (scFv), which is a monovalent Fab fragment.
  • the antibody or antibody fragment can be part of a larger construct, for example, a conjugate or fusion construct of the antibody fragment to additional regions.
  • the antibody fragment can be fused to an Fc region as described herein
  • the antibody fragment e.g., a Fab or scFv
  • the antibody fragment can be part of a chimeric antigen receptor or chimeric T-cell receptor, for instance, by fusing to a transmembrane domain (optionally with an intervening linker or “stalk” (e.g., hinge region)) and optional intercellular signaling domain.
  • the antibody fragment can be fused to the gamma and/or delta chains of a t-cell receptor, so as to provide a T-cell receptor like construct that binds PD-L1 .
  • the antibody fragment is part of a bispecific T-cell engager (BiTEs) comprising a CD1 or CD3 binding domain and linker.
  • BiTEs bispecific T-cell engager
  • the antibody construct comprises an Fc domain.
  • the antibody construct is a fusion protein.
  • the antigen binding domain can be a single-chain variable region fragment (scFv).
  • scFv single-chain variable region fragment
  • dsFv disulfide-stabilized variable region fragments
  • the antibody construct or antigen binding domain may comprise one or more variable regions (e.g., two variable regions) of an antigen binding domain of an antibody, each variable region comprising a CDR1, a CDR2, and a CDR3.
  • Cysteine-mutant antibody is an antibody in which one or more amino acid residues of an antibody are substituted with cysteine residues.
  • a cysteine-mutant antibody may be prepared from the parent antibody by antibody engineering methods (Junutula, J et al., (2008b) Nature Biotech., 26(8):925-932; Doman et al. (2QQ9) Blood 114(13):2721-2729; US 7521541; US 7723485; US 2012/0121615; WO 2009/052249).
  • Cysteine residues provide for site-specific conjugation of a drug moiety such as a isoindolinone-glutarimide compound to the antibody through the reactive cysteine thiol groups at the engineered cysteine sites but do not perturb immunoglobulin folding and assembly or alter antigen binding and effector functions.
  • Cysteine- mutant antibodies can be conjugated to the isoindolinone-glutarimide linker compound with uniform stoichiometry of the antibody conjugate (e.g., up to two isoindolinone-glutarimide moieties per antibody in an antibody that has a single engineered, mutant cysteine site).
  • the isoindolinone-glutarimide linker compound has a reactive electrophilic group to react specifically with the free cysteine thiol groups of the cysteine-mutant antibody.
  • Epitope means any antigenic determinant or epitopic determinant of an antigen to which an antigen binding domain binds (i.e., at the paratope of the antigen binding domain).
  • Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific three dimensional structural characteristics, as well as specific charge characteristics
  • Fc receptor refers to a receptor that binds to the Fc region of an antibody
  • FcyR which binds to IgG
  • FcaR which binds to IgA
  • FceR which binds to IgE.
  • the FcyR family includes several members, such as Fcyl (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16A), and FcyRIIIB (CD16B).
  • the Fey receptors differ in their affinity for IgG and also have different affinities for the IgG subclasses (e.g., IgGl, IgG2, IgG3, and IgG4).
  • Nucleic acid or amino acid sequence “identity,” as referenced herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence.
  • the percent identity is the number of nucleotides or amino acid residues that are the same (i.e., that are identical) as between the optimally aligned sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). Alignment of sequences and calculation of percent identity can be performed using available software programs.
  • Such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e g., BLAST 2.1, BL2SEQ, BLASTp, BLASTn, and the like) and FASTA programs (e.g., FASTA3x, FASTM, and S SEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106( ⁇ Q ⁇ .
  • Percent (%) identity of sequences can be also calculated, for example, as 100 x [(identical positions)/min(TGA, TGB)], where TGA and TGB are the sum of the number of residues and internal gap positions in peptide sequences A and B in the alignment that minimizes TGA and TGB. See, e.g., Russell et al., J. Mol Biol., 244: 332-350 (1994).
  • the “antibody construct” or “binding agent” comprises Ig heavy and light chain variable region polypeptides that together form the antigen binding site.
  • Each of the heavy and light chain variable regions are polypeptides comprising three complementarity determining regions (CDR1, CDR2, and CDR3) connected by framework regions.
  • the antibody construct can be any of a variety of types of binding agents known in the art that comprise Ig heavy and light chains.
  • the binding agent can be an antibody, an antigen-binding antibody “fragment,” or a T-cell receptor.
  • Amino acid refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein.
  • Amino acids include naturally-occurring a-amino acids and their stereoisomers, as well as unnatural (non-naturally occurring) amino acids and their stereoisomers.
  • “Stereoisomers” of a given amino acid refer to isomers having the same molecular formula and intramolecular bonds but different three-dimensional arrangements of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid).
  • amino acids can be glycosylated (e.g., //-linked glycans, O-linked glycans, phosphoglycans, C-linked glycans, or glypication) or deglycosylated.
  • Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine.
  • Naturally-occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and combinations thereof.
  • Stereoisomers of naturally- occurring a-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.
  • D-alanine D-
  • Naturally-occurring amino acids include those formed in proteins by post-translational modification, such as citrulline (Cit).
  • Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, A-substituted glycines, and A-methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally- occurring amino acids.
  • amino acid analogs can be unnatural amino acids that have the same basic chemical structure as naturally-occurring amino acids (i.e., a carbon that is bonded to a hydrogen, a carboxyl group, an amino group) but have modified side-chain groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally -occurring amino acid.
  • Linker refers to a functional group that covalently bonds two or more moieties in an antibody conjugate compound.
  • the linking moiety can serve to covalently bond a drug isoindolinone-glutarimide moiety to an antibody in an antibody conjugate composition.
  • Useful bonds for connecting linking moieties to proteins and other materials include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas.
  • Divalent refers to a chemical moiety that contains two points of attachment for linking two functional groups; polyvalent linking moieties can have additional points of attachment for linking further functional groups.
  • Divalent radicals may be denoted by the suffix “diyl”.
  • divalent linking moieties include divalent polymer moieties such as divalent poly(ethylene glycol), divalent cycloalkyl, divalent heterocycloalkyl, divalent aryl, and divalent heteroaryl group.
  • a “divalent cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group” refers to a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group having two points of attachment for covalently linking two moieties in a molecule or material. Cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups can be substituted or unsubstituted. Cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, alkoxy, and others.
  • a wavy line (“ ”) and one or more asterisks (*) represents a point of attachment of the specified chemical moiety to another moiety. If the specified chemical moiety has two wavy lines (“ present, it will be understood that the chemical moiety can be used bilaterally, i.e., as read from left to right or from right to left. In some embodiments, a specified moiety having two wavy lines present is considered to be used as read from left to right.
  • Alkyl refers to a straight (linear) or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, for example from one to six, one to eight, one to twelve, one to twenty, or one to forty.
  • alkyl groups include, but are not limited to, methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1 -propyl (n-Pr, n- propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 )2), 1 -butyl (n-Bu, n-butyl, - CH 2 CH 2 CH 2 CH 3 ), 2-m ethyl- 1 -propyl (i-Bu, i-butyl, -CH 2 CH(CH 3 )2), 2 -butyl (s-Bu, s-butyl, - CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1 -pentyl (n-pentyl, - CH 2 CH 2 CH 2 CH 2 CH 3
  • alkyldiyl refers to a divalent alkyl radical.
  • alkyldiyl groups include, but are not limited to, methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (- CH 2 CH 2 CH 2 -), and the like.
  • An alkyldiyl group may also be referred to as an “alkylene” group.
  • Alkynyl refers to a straight (linear) or branched, unsaturated, aliphatic radical having the number of carbon atoms indicated and at least one carbon-carbon triple bond, sp. Alkynyl can include from two to about 12 or more carbons atoms.
  • C 2 -C 6 alkynyl includes, but is not limited to ethynyl (-CACI I), propynyl (propargyl, -CI FCUCI I), butynyl, pentynyl, hexynyl, and isomers thereof Alkynyl groups can be substituted or unsubstituted.
  • alkynylene or “alkynyldiyl” refer to a divalent alkynyl radical.
  • Heteroalkyl or “heteroalkylene” refer to a monovalent, straight or branched chain alkyl group, as defined above, comprising at least one heteroatom including but not limited to Si, N, O, P or S within the alkyl chain or at a terminus of the alkyl chain. In some embodiments, a heteroatom is within the alkyl chain. In other embodiments, a heteroatom is at a terminus of the alkylene and thus serves to join the alkyl to the remainder of the molecule. In some embodiments, a heteroalkyl group may have 1 to 12 carbon atoms (C 1 -C 12 heteroalkyl).
  • a heteroalkyl group may have 1 to 24 carbon atoms (C 1 -C24 heteroalkyl). In some embodiments, a heteroalkyl group may have 1 to 40 carbon atoms (C 1 -C40 heteroalkyl). Unless stated otherwise specifically in the specification, a heteroalkyl group is optionally substituted.
  • heteroalkyl groups can be substituted with 1 -6 fluoro (F) substituents, for example, on the carbon backbone (as -CHF- or -CF 2 -) or on terminal carbons of straight chain or branched heteroalkyls (such as -CHF2 or -CF3).
  • a terminal polyethylene glycol (PEG) moiety is a type of heteroalkyl group.
  • exemplary heteroalkyl groups also include ethylene oxide (e.g., polyethylene oxide), propylene oxide, amino acid chains (i.e., short to medium length peptides such as containing 1-15 amino acids), and alkyl chains connected via a variety of functional groups such as amides, disulfides, ketones, phosphonates, phosphates, sulfates, sulfones, sulfonamides, esters, ethers, -S-, carbamates, ureas, thioureas, anhydrides, or the like (including combinations thereof).
  • a heteroalkyl group includes a poly amino acid having 1-10 amino acids.
  • a heteroalkyl group includes a polyamin
  • Heteroalkyl groups include a solubilizing unit comprising one or more groups of polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside, or combinations thereof.
  • Heteroalkenyl refers to a heteroalkyl group, as defined above, that contains at least one carbon-carbon double bond.
  • Heteroalkynyl refers to a heteroalkyl group, as defined above, that contains at least one carbon-carbon triple bond.
  • Heteroalkyldiyl refers to a divalent form of a heteroalkyl group as defined above.
  • a heteroalkyldiyl group may have 1 to 12 carbon atoms (C 1 - C 12 heteroalkyl diyl).
  • a heteroalkyldiyl group may have 1 to 24 carbon atoms (C 1 -C 24 heteroalkyldiyl).
  • a heteroalkyldiyl group may have 1 to 40 carbon atoms (C 1 -C 40 heteroalkyldiyl).
  • a divalent polyethylene glycol (PEG) moiety with one to about 50 units of -OCH 2 CH 2 - is a type of heteroalkyl diyl group.
  • Heteroalkenyl diyl refers to a divalent form of a heteroalkenyl group.
  • Heteroalkynyldiyl refers to a divalent form of a heteroalkynyl group.
  • carrier refers to a saturated or partially unsaturated, monocyclic, fused bicyclic, or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated.
  • Saturated monocyclic carbocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl.
  • Saturated bicyclic and polycyclic carbocyclic rings include, for example, norbomane, [2.2.2] bicyclooctane, decahydronaphthalene and adamantane.
  • Carbocyclic groups can also be partially unsaturated, having one or more double or triple bonds in the ring.
  • carbocyclic groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1 ,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbomene, and norbomadiene.
  • cycloalkyl diyl refers to a divalent cycloalkyl radical.
  • Aryl refers to a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms (C 6 - C 20 ) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system.
  • Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by a bond to form a biaryl group.
  • Representative aryl groups include phenyl, naphthyl and biphenyl.
  • Other aryl groups include benzyl, having a methylene linking group.
  • Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl.
  • Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl.
  • arylene or “aryldiyl” mean a divalent aromatic hydrocarbon radical of 6-20 carbon atoms (C 6 - C 20 ) derived by the removal of two hydrogen atom from a two carbon atoms of a parent aromatic ring system.
  • Some aryldiyl groups are represented in the exemplary structures as “Ar”.
  • Aryldiyl includes bicyclic radicals comprising an aromatic ring fused to a saturated, partially unsaturated ring, or aromatic carbocyclic ring.
  • Typical aryldiyl groups include, but are not limited to, radicals derived from benzene (phenyldiyl), substituted benzenes, naphthalene, anthracene, biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene, 1, 2,3,4- tetrahydronaphthyl, and the like.
  • Aryldiyl groups are also referred to as “arylene”, and are optionally substituted with one or more substituents described herein.
  • heterocycle refers to a saturated or a partially unsaturated (i.e., having one or more double and/or triple bonds within the ring) carbocyclic radical of 3 to about 20 ring atoms in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur, the remaining ring atoms being C, where one or more ring atoms is optionally substituted independently with one or more substituents described below.
  • a heterocycle may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.
  • Heterocycles are described in Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W A Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. “Heterocyclyl” also includes radicals where heterocycle radicals are fused with a saturated, partially unsaturated ring, or aromatic carbocyclic or heterocyclic ring.
  • heterocyclic rings include, but are not limited to, morpholin-4-yl, piperidin-l-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-l-yl, thiomorpholin-4-yl, S- dioxothiomorpholin-4-yl, azocan-l-yl, azetidin-l-yl, octahydropyrido[l,2-a]pyrazin-2-yl, [l,4]diazepan-l-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperaz
  • heterocyclyl diyl refers to a divalent, saturated or a partially unsaturated (i.e., having one or more double and/or triple bonds within the ring) carbocyclic radical of 3 to about 20 ring atoms in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur, the remaining ring atoms being C, where one or more ring atoms is optionally substituted independently with one or more substituents as described.
  • Examples of 5- membered and 6-membered heterocyclyldiyls include morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl, thiomorpholinyldiyl, and S- dioxothiomorpholinyldiyl.
  • heteroaryl refers to a monovalent aromatic radical of 5-, 6-, or 7-membered rings, and includes fused ring systems (at least one of which is aromatic) of 5-20 atoms, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazol
  • heteroaryldiyl refers to a divalent aromatic radical of 5-, 6-, or 7-membered rings, and includes fused ring systems (at least one of which is aromatic) of 5-20 atoms, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • Examples of 5-membered and 6-membered heteroaryldiyls include pyridyldiyl, imidazolyl diyl, pyrimidinyl diyl, pyrazolyldiyl, triazolyldiyl, pyrazinyldiyl, tetrazolyldiyl, furyldiyl, thienyldiyl, isoxazolyl diyl diyl, thiazolyldiyl, oxadi azolyldiyl, oxazolyldiyl, isothiazolyldiyl, and pyrrolyldiyl.
  • the heterocycle or heteroaryl groups may be carbon (carbon-linked), or nitrogen (nitrogen-linked) bonded where such is possible.
  • carbon bonded heterocycles or heteroaryls are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6,
  • nitrogen bonded heterocycles or heteroaryls are bonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3 -imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3- pyrazoline, piperidine, piperazine, indole, indoline, IH-indazole, position 2 of a isoindole, or isoindolinone, position 4 of a morpholine, and position 9 of a carbazole, or p-carboline.
  • halo and “halogen,” by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.
  • quaternary ammonium salt refers to a tertiary amine that has been quaternized with an alkyl substituent (e g., a C 1 -C4 alkyl such as methyl, ethyl, propyl, or butyl).
  • an alkyl substituent e g., a C 1 -C4 alkyl such as methyl, ethyl, propyl, or butyl.
  • treat refers to any indicia of success in the treatment or amelioration of an injury, pathology, condition (e.g., cancer), or symptom (e.g., cognitive impairment), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology, or condition more tolerable to the patient; reduction in the rate of symptom progression; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom.
  • the treatment or amelioration of symptoms can be based on any objective or subjective parameter, including, for example, the result of a physical examination.
  • cancer refers to cells which exhibit autonomous, unregulated growth, such that the cells exhibit an aberrant growth phenotype characterized by a significant loss of control over cell proliferation.
  • C 6 lls of interest for detection, analysis, and/or treatment in the context of the invention include cancer cells (e.g., cancer cells from an individual with cancer), malignant cancer cells, pre-metastatic cancer cells, metastatic cancer cells, and non-metastatic cancer cells. Cancers of virtually every tissue are known.
  • cancer burden refers to the quantum of cancer cells or cancer volume in a subject. Reducing cancer burden accordingly refers to reducing the number of cancer cells or the cancer cell volume in a subject.
  • cancer cell refers to any cell that is a cancer cell (e.g., from any of the cancers for which an individual can be treated, e.g., isolated from an individual having cancer) or is derived from a cancer cell, e.g., clone of a cancer cell.
  • a cancer cell can be from an established cancer cell line, can be a primary cell isolated from an individual with cancer, can be a progeny cell from a primary cell isolated from an individual with cancer, and the like.
  • the term can also refer to a portion of a cancer cell, such as a sub-cellular portion, a cell membrane portion, or a cell lysate of a cancer cell.
  • cancers are known to those of skill in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, and myelomas, and circulating cancers such as leukemias.
  • solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, and myelomas
  • circulating cancers such as leukemias.
  • cancer includes any form of cancer, including but not limited to, solid tumor cancers (e.g., skin, lung, prostate, breast, gastric, bladder, colon, ovarian, pancreas, kidney, liver, glioblastoma, medulloblastoma, leiomyosarcoma, head & neck squamous cell carcinomas, melanomas, and neuroendocrine) and liquid cancers (e.g., hematological cancers); carcinomas; soft tissue tumors; sarcomas; teratomas; melanomas; leukemias; lymphomas; and brain cancers, including minimal residual disease, and including both primary and metastatic tumors.
  • solid tumor cancers e.g., skin, lung, prostate, breast, gastric, bladder, colon, ovarian
  • pancreas kidney, liver, glioblastoma, medulloblastoma, leiomyosarcoma, head & neck squamous cell carcinomas, melan
  • phrases “effective amount” and “therapeutically effective amount” refer to a dose or amount of a substance such as the antibody conjugate of the invention that produces therapeutic effects for which it is administered.
  • the exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Goodman & Gilman ’s The Pharmacological Basis of Therapeutics, 11 th Edition (McGraw-Hill, 2006); and Remington: The Science and Practice of Pharmacy, 22 nd Edition, (Pharmaceutical Press, London, 2012)).
  • the therapeutically effective amount of the antibody conjugate may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i e , slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • the antibody conjugate may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR)
  • “Recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired (e.g., humans).
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In certain embodiments, the mammal is human.
  • administering refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal, or subcutaneous administration, oral administration, administration as a suppository, topical contact, intrathecal administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to the subject.
  • a slow-release device e.g., a mini-osmotic pump
  • “about X” or “around X” indicates a value of from 0 9X to 1.1X, e g., from 0.95X to 1.05X or from 0.99X to 1.01X.
  • a reference to “about X” or “around X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Accordingly, “about X” and “around X” are intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”
  • the antibody conjugate compositions of the invention comprise an antibody. Included in the scope of the embodiments of the invention are functional variants of the antibody constructs or antigen binding domain described herein.
  • the term “functional variant” as used herein refers to an antibody construct having an antigen binding domain with substantial or significant sequence identity or similarity to a parent antibody construct or antigen binding domain, which functional variant retains the biological activity of the antibody construct or antigen binding domain of which it is a variant.
  • Functional variants encompass, for example, those variants of the antibody constructs or antigen binding domain described herein (the parent antibody construct or antigen binding domain) that retain the ability to recognize target cells expressing a tumor-associated antigen or cell surface receptor to a similar extent, the same extent, or to a higher extent, as the parent antibody construct or antigen binding domain.
  • the functional variant can, for instance, be at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the antibody construct or antigen binding domain.
  • a functional variant can, for example, comprise the amino acid sequence of the parent antibody construct or antigen binding domain with at least one conservative amino acid substitution.
  • the functional variants can comprise the amino acid sequence of the parent antibody construct or antigen binding domain with at least one nonconservative amino acid substitution.
  • the non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent antibody construct or antigen binding domain.
  • a functional variant can, for example, comprise the amino acid sequence of the parent antibody construct or antigen binding with at least one non-canonical amino acid (ncAA) substitution (L Wang, et al, (2001) Science , 292(5516):498-500, CC Liu, PG Schultz, (2010) Annu Rev Biochem. 79:413-44).
  • ncAA non-canonical amino acid
  • the antibodies comprising the antibody conjugate compositions of the invention include Fc engineered variants.
  • the mutations in the Fc region that result in modulated binding to one or more Fc receptors can include one or more of the following mutations: YTE (M252Y/S254T/T256E), LALAPA (L234A/L235A/P329A), SD (S239D), SDIE (S239D/I332E), SE (S267E), SELF (S267E/L328F), SDIE (S239D/I332E), SDIEAL (S239D/I332E/A330L), GA (G236A), ALIE (A330L/I332E), GASDALIE (G236A/S239D/A330L/I332E), V9 (G237D/P238D/P271G/A330R), and VI 1 (G237D/P238D/H 2 68D/P271G, and
  • the antibodies comprising the antibody conjugate compositions of the invention include glycan variants, such as afucosylation
  • the Fc region of the binding agents are modified to have an altered glycosylation pattern of the Fc region compared to the native non-modified Fc region.
  • the antibodies in the antibody conjugate compositions contain a modified Fc region, wherein the modification modulates the binding of the Fc region to one or more Fc receptors.
  • the antibodies in the antibody conjugate contain one or more modifications (e.g., amino acid insertion, deletion, and/or substitution) in the Fc region that results in modulated binding (e.g., increased binding or decreased binding) to one or more Fc receptors (e.g., FcyRI (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD 16a), and/or FcyRIIIB (CD 16b)) as compared to the native antibody lacking the mutation in the Fc region.
  • FcyRI CD64
  • FcyRIIA CD32A
  • FcyRIIB CD32B
  • FcyRIIIA CD 16a
  • FcyRIIIB CD 16b
  • the antibodies in the antibody conjugate compositions contain one or more modifications (e.g., amino acid insertion, deletion, and/or substitution) in the Fc region that reduce the binding of the Fc region of the antibody to FcyRIIB. In some embodiments, the antibodies in the antibody conjugate compositions contain one or more modifications (e.g., amino acid insertion, deletion, and/or substitution) in the Fc region of the antibody that reduce the binding of the antibody to FcyRIIB while maintaining the same binding or having increased binding to FcyRI (CD64), FcyRIIA (CD32A), and/or FcRylllA (CD 16a) as compared to the native antibody lacking the mutation in the Fc region. In some embodiments, the antibodies in the antibody conjugate compositions contain one of more modifications in the Fc region that increase the binding of the Fc region of the antibody to FcyRIIB
  • the modulated binding is provided by mutations in the Fc region of the antibody relative to the native Fc region of the antibody.
  • the mutations can be in a CH 2 domain, a CH 3 domain, or a combination thereof.
  • a “native Fc region” is synonymous with a “wild-type Fc region” and comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature or identical to the amino acid sequence of the Fc region found in the native antibody (e g , cetuximab)
  • Native sequence human Fc regions include a native sequence human IgGl Fc region, native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.
  • Native sequence Fc includes the various allotypes of Fes (Jefferis et al., (2009) mAbs, l(4):332-338).
  • the Fc region of the antibodies of the antibody conjugate compositions are modified to have an altered glycosylation pattern of the Fc region compared to the native non-modified Fc region.
  • Human immunoglobulin is glycosylated at the Asn297 residue in the Cy2 domain of each heavy chain
  • This N-linked oligosaccharide is composed of a core heptasaccharide, N-acetylglucosamine4Mannose3 (GlcNAc4Man3) Removal of the heptasaccharide with endoglycosidase or PNGase F is known to lead to conformational changes in the antibody Fc region, which can significantly reduce antibody-binding affinity to activating FcyR and lead to decreased effector function
  • the core heptasaccharide is often decorated with galactose, bisecting GlcNAc, fucose, or sialic acid, which differentially impacts Fc binding to activating and inhibitory FcyR.
  • the modification to alter the glycosylation pattern is a mutation.
  • Asn297 is mutated to glutamine (N297Q).
  • the antibodies of the antibody conjugate compositions are modified to contain an engineered Fab region with a non-naturally occurring glycosylation pattern.
  • hybridomas can be genetically engineered to secrete afucosylated mAb, desialylated mAb or deglycosylated Fc with specific mutations that enable increased FcRyllla binding and effector function.
  • the antibodies of the antibody conjugate compositions are engineered to be afucosylated or glycosylated.
  • the antibodies in the antibody conjugate compositions are a cysteine-engineered antibody which provides for site-specific conjugation of an adjuvant, label, or drug moiety to the antibody through cysteine substitutions at sites where the engineered cysteines are available for conjugation but do not perturb immunoglobulin folding and assembly or alter antigen binding and effector functions (Junutula, et al., (2008) Nature Biotech , 26(8):925-932; Dornan et al (2009) 5/00 ⁇ 114(13):2721-2729, US 7521541; US 7723485; US 2012/0121615; WO 2009/052249).
  • Cysteine engineered antibody or “cysteine engineered antibody variant” is an antibody in which one or more residues of an antibody are substituted with cysteine residues.
  • Cysteine-engineered antibodies can be conjugated to the isoindolinone- glutarimide (IG) moiety with uniform stoichiometry (e g., up to two IG moieties per antibody in an antibody that has a single engineered cysteine site).
  • IG isoindolinone- glutarimide
  • cysteine-engineered antibodies are used to prepare antibody conjugate compositions with a reactive cysteine thiol residue introduced at a site on the light chain, such as the 149-lysine site (LC K149C), or on the heavy chain such as the 122-serine site (HC S122C), as numbered by Kabat numbering
  • the cysteine-engineered antibodies have a cysteine residue introduced at the 375-serine site (EU numbering) of the heavy chain (HC S375C).
  • the cysteine-engineered antibodies have a cysteine residue introduced at the 118-alanine site (EU numbering) of the heavy chain (HC Al 18C).
  • cysteine-engineered antibodies have a cysteine residue introduced in: (i) the light chain at G64C, R142C, K188C, L201C, T129C, S114C, or E105C according to Kabat numbering; (ii) the heavy chain at DIOIC, V184C, T205C, or S122C according to Kabat numbering; or (iii) other cysteine-mutant antibodies, and as described in Bhakta, S.
  • the antibody is a full-length antibody. In certain embodiments, the antibody is an antigen binding fragment. In some embodiments, the antibody is a humanized antibody
  • the antibody is an anti-CD40 antibody, an antibody selected from an anti-LRRC 1 5 antibody, an anti-CTSK antibody, an anti-ADAM12 antibody, an anti-ITGAl l antibody, an anti-FAP antibody, an anti-NOX4 antibody, an anti-SGCD antibody, an anti- SYNDIG1 antibody, an anti-CDH11 antibody, an anti-PLPP4 antibody, an anti-SLC24A2 antibody, an anti-PDGFRB antibody, an anti-THYl antibody, an anti-ANTXRl antibody, an anti-GASl antibody, an anti-CALHM5 antibody, an anti-SDCl antibody, an anti-HER2 antibody, an anti-TR0P2 antibody, an anti-MSLN antibody, an anti-Nectin4 antibody, an anti- ASGR1 antibody, and an anti-MUC 1 6 antibody.
  • the antibody or Fc fusion protein is selected from: abagovomab, abatacept (also known as ORENCIA®), abciximab (also known as REOPRO®), c7E3 Fab), adalimumab (also known as HUMIRA®), adecatumumab, alemtuzumab (also known as CAMPATH®), MabCampath or Campath- 1H), altumomab, afelimomab, panitumumab, mafenatox, anrukizumab, apolizumab, arcitumomab, aselizumab, atlizumab, atorolimumab, bapineuzumab, basiliximab (also known as SIMULECT®), bavituximab, bectumomab (also known as LYMPHOSCAN®), belimumab (also known as LYMPHO-STAT
  • the antibody conjugate composition of the invention comprises an antibody constmct that comprises an antigen binding domain that specifically recognizes and binds HER2.
  • the antibody conjugate composition comprises an anti-HER2 antibody.
  • an anti-HER2 antibody of an antibody conjugate composition of the invention comprises a humanized anti-HER2 antibody, e.g., huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8, as described in Table 3 of US 5821337, which is specifically incorporated by reference herein.
  • Those antibodies contain human framework regions with the complementarity-determining regions of a murine antibody (4D5) that binds to HER2.
  • the humanized antibody huMAb4D5-8 is also referred to as trastuzumab, commercially available under the tradename HERCEPTINTM (Genentech, Inc.).
  • the antibody construct or antigen binding domain comprises the CDR regions of trastuzumab.
  • the anti-HER2 antibody further comprises the framework regions of the trastuzumab.
  • the anti-HER2 antibody further comprises one or both variable regions of trastuzumab.
  • an anti-HER2 antibody of an antibody conjugate composition of the invention comprises a humanized anti-HER2 antibody, e.g., humanized 2C4, as described in US 7862817.
  • a humanized 2C4 antibody is pertuzumab (CAS Reg. No. 380610-27-5), PERJETATM (Genentech, Inc.).
  • Pertuzumab is a HER dimerization inhibitor (HDI) and functions to inhibit the ability of HER2 to form active heterodimers or homodimers with other HER receptors (such as EGFR/HER1, HER2, HER3 and HER4).
  • HDI HER dimerization inhibitor
  • the antibody construct or antigen binding domain comprises the CDR regions of pertuzumab
  • the anti-HER2 antibody further comprises the framework regions of the pertuzumab.
  • the anti-HER2 antibody further comprises one or both variable regions of pertuzumab.
  • the antibody conjugate composition of the invention comprises an antibody construct that comprises an antigen binding domain that specifically recognizes and binds the B-cell receptor CD22.
  • the antibody conjugate composition comprises an anti-CD22 antibody
  • the anti- CD22 antibody of an antibody conjugate composition is pinatuzumab (CAS Reg. No. 1639820- 81 -7), with complementarity determining regions (CDRs), heavy chain (HC), and light chain (LC) as described in US 8226945 and WO 2007/140371 which are incorporated by reference herein.
  • the antibody of an antibody conjugate composition is capable of binding one or more antigen targets selected from (e.g., specifically binds to a target selected from) 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, AD0RA2A, Aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOCI, AR, aromatase, ATX, AX1, Axl, AZGP1 (zinc-a-glycoprotein), B7.1, B7.2, B7-H1, B7-H3, B7-H4, BAD, BAFF, BAG1, BAH, BCR, BCL2, BCL6, BDNF, BLNK, BLR1 (MDRI5), BlyS, BMP1, BMP2, BMP3B (GDFIO), BMP4, BMP6, BM
  • TNFSF6 FasL
  • TNFSF7 CD27 ligand
  • TNFSF8 CD30 ligand
  • TNFSF9 4-1BB ligand
  • TOLLLP Toll-like receptors
  • TOP2A topoisomerase lia
  • TP53 TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2, TROP2, TRPC6, TSLP, TWEAK, Tyrosinase, uPAR, VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4, Wnt-1, XCL1 (tymphotactin), XCL2 (SCM-Ib), XCRI (GPR5/CCXCR1), YYI, ZFPM2, CLEC4C (BDCA-2, DLEC, CD303, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6
  • CLEC5A MDL-1, CLECSF5), CLEC 1 B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (Dectin-1), PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), NCR1 (CD335, LY94, NKp46), NCR3 (CD335, LY94, NKp46), NCR3 (CD337, NKp30), OSCAR, TARM1, CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD314, NKG2D), NCR2 (CD336, NKp44), PILRB,
  • the antibody binds to an antigen selected from CDH1, CD19, CD20, CD29, CD30, CD38, CD40, CD47, EpCAM, MUC 1 , MUC 1 6, EGFR, HER2, SLAMF7, and gp75.
  • the antibody of an antibody conjugate composition of the invention is capable of binding to one or more tumor-associated antigens (TAA), cell-surface receptors, and immune-specific antigens to confer specificity to the targeting of the conjugate and enable safe and systemic delivery of an active drug moiety.
  • TAA tumor-associated antigens
  • C 6 rtain tumor-associated antigens are known in the art, and can be prepared for use in generating antibodies using methods and information which are well known in the art.
  • researchers In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s).
  • tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells.
  • identification of such tumor-associated cell surface antigen polypeptides allows more specificity in targeting cancer cells for destruction via antibody -based therapies.
  • TAAs include, but are not limited to, those listed below including (l)-(54).
  • information relating to these antigens is listed below and includes names, alternative names, Genbank accession numbers and primary reference(s), following nucleic acid and protein sequence identification conventions of the National C 6 nter for Biotechnology Information (NCBI).
  • Nucleic acid and protein sequences corresponding to TAAs listed below including (l)-(54) are available in public databases such as GenBank.
  • TAAs targeted by antibodies include all amino acid sequence variants and isoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to the sequences identified in the cited references, and/or which exhibit substantially the same biological properties or characteristics as a TAA having a sequence found in the cited references.
  • a TAA having a variant sequence generally is able to bind specifically to an antibody that binds specifically to the TAA with the corresponding sequence listed.
  • BMPR1B bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203
  • BMPR1B bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203
  • W02004063362 Claim 2
  • W02003042661 Claim 12
  • US2003134790-Al Page 38-39
  • W02002102235 Claim 13, Page 296
  • W02003055443 Page 91-92
  • WO200299122 Example 2; Page 528-530
  • W02003029421 (Claim 6); W02003024392 (Claim 2; Fig 112)
  • WO200298358 (Claim 1; Page 183); W0200254940 (Page 100-101); WO200259377(Page 349-350), W0200230268 (Claim 27; Page 376); W0200148204 (Example;
  • MPF MPF
  • MSLN MSLN
  • SMR megakaryocyte potentiating factor
  • mesothelin Genbank accession no. NM_005823
  • Yamaguchi N., et al. Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96 (20): 11531-11536 (1999), Proc. Natl. Acad. Sei. U.S.A. 93 (1): 136- 140 (1996), J. Biol. Chem.
  • Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424) J. Biol. Chem. 277 (22): 19665-19672 (2002), Genomics 62 (2)281-284 (1999), Field, J A , et al (1999) Biochem. Biophys. Res.
  • Serna 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1 -like), transmembrane domain (TM) and short cytoplasmic domain, (30emaphoring) 5B, Genbank accession no AB040878) Nagase T., et al. (2000) DNA Res.
  • PSCA hlg (2700050C 1 2Rik, C530008016Rik, RIKEN cDNA 2700050C 1 2, RIKEN cDNA 2700050C 1 2 gene, Genbank accession no. AY358628); Ross et al. (2002) Cancer Res.
  • ETBR Endothelin type B receptor, Genbank accession no. AY275463
  • Nakamuta M. et al. Biochem. Biophys. Res. Commun. 177, 34-39, 1991
  • Ogawa Y. et al. Biochem. Biophys. Res. Commun. 178, 248-255, 1991
  • Arai H. et al. Jpn. Circ. J. 56, 1303-1307, 1992
  • Arai H. et al. J. Biol. Chem. 268, 3463-3470, 1993
  • Sakamoto A. Yanagisawa M., et al. Biochem. Biophys. Res. Commun.
  • W02004040000 (Claim 151); W02003087768 (Claim 1); W02003016475 (Claim 1); W02003016475 (Claim 1); W0200261087 (Fig 1); W02003016494 (Fig 6); W02003025138 (Claim 12; Page 144), W0200198351 (Claim 1; Page 124-125); EP522868 (Claim 8; Fig 2); W0200177172 (Claim 1; Page 297-299); US2003109676; US6518404 (Fig 3); US5773223 (Claim la; Col 31-34); W02004001004.
  • MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763); W02003104275 (Claim 1); W02004046342 (Example 2); W02003042661 (Claim 12); W02003083074 (Claim 14; Page 61); WG2003018621 (Claim 1); W02003024392 (Claim 2; Fig 93); WO200166689 (Example 6); Cross-references: LocusID:54894; NP_060233.2; NM_017763_l.
  • STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138) Lab. Invest.
  • TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636) Xu, X.Z., et al Proc. Natl. Acad. Set. U.S.A. 98 (19): 10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem.
  • CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212) Ciccodicola, A., et al. EMBO J. 8 (7): 1987-1991 (1989), Am. J. Hum. Genet.
  • CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004) Fujisaku et al. (1989) J. Biol. Chem. 264 (4):2118- 2125); Weis J. J., et al. J. Exp. Med. 167, 1047-1066, 1988; Moore M , et al. Proc. Natl. Acad. Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al. Mol. Immunol. 35, 1025-1031, 1998; Weis J.J., et al. Proc. Natl. Acad. Sci.
  • CD79b (CD79B, CD790, Igb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626 or 11038674) Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126- 4131, Blood (2002) 100 (9):3068-3076, Muller et al. (1992) Eur. J. Immunol.
  • FcRH 2 (IFGP4, IRTA4, SPAP1A (SH 2 domain containing phosphatase anchor protein la), SPAP1B, SPAP1C, Genbank accession no. NM_030764, AY358130) Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54 (2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci. USA. 98 (17):9772-9777 (2001), Xu, M.J., et al. (2001) Biochem. Biophys. Res. Commun.
  • HER2 ErbB2, Genbank accession no. Ml 1730
  • Coussens L. et al. Science (1985) 230(4730):l 132-1139
  • Yamamoto T. et al. Nature 319, 230-234, 1986
  • Semba K. et al. Proc. Natl. Acad. Sci. U.S.A. 82, 6497-6501, 1985
  • Swiercz J.M. et al. J. Cell Biol. 165, 869-880, 2004
  • Kuhns J.J. et al. J. Biol. Chem. 274, 36422-36427, 1999
  • Cho H.-S. et al.
  • NCA NCA (CEACAM6, Genbank accession no. Ml 8728); Barnet T ., et al. Genomics 3, 59-66, 1988; Tawaragi Y., et al. Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R.L., et al. Proc. Natl. Acad. Sci. U.S.A.
  • MDP DPEP1, Genbank accession no. BC017023
  • W02003016475 (Claim 1); WO200264798 (Claim 33; Page 85-87); JP05003790 (Fig 6-8); WO9946284 (Fig 9); Cross-references: MIM:179780; AAH17023.1; BC017023 1.
  • IL20Roc (IL20Ra, ZCYTOR7, Genbank accession no AF184971); Clark H F , et al. Genome Res. 13, 2265-2270, 2003; Mungall A.J., et al. Nature 425, 805-811, 2003; Blumberg H., et al. Cell 104, 9-19, 2001; Dumoutier L., et al. J. Immunol. 167, 3545-3549, 2001; Parrish- Novak J., et al. J. Biol. Chem. 277 , 47517-47523, 2002; Pletnev S., et al. (2003) Biochemistry 42: 12617-12624; Sheikh F., et al.
  • EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no. NM_004442) Chan, J. and Wat, V.M., Oncogene 6 (6), 1057-1061 (1991) Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. Rev. Cytol.
  • W0200206317 (Example 2; Page 320-321, Claim 34; Page 321-322); WO200271928 (Page 468-469); W0200202587 (Example 1; Fig 1); W0200140269 (Example 3; Pages 190-192); W0200036107 (Example 2; Page 205-207); W02004053079 (Claim 12); W02003004989 (Claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318.
  • PSCA Prostate stem cell antigen precursor, Genbank accession no. AJ297436
  • Reiter R.E. et al. Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740, 1998; Gu Z ., et al. Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun.
  • BAFF-R B cell -activating factor receptor, BlyS receptor 3, BR3, Genbank accession No. AF116456
  • BAFF receptor /pi d NP_443177.1 - Homo sapiens Thompson, J.S., et al. Science 293 (5537), 2108-2111 (2001); W02004058309; W02004011611;
  • W02003045422 (Example; Page 32-33); W02003014294 (Claim 35; Fig 6B); W02003035846 (Claim 70; Page 615-616); WO200294852 (Col 136-137); WO200238766 (Claim 3; Page 133); W0200224909 (Example 3; Fig 3); Cross-references: MIM:606269; NP_443177.1;
  • CD22 B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession No. AK026467
  • CD79a (CD79A, CD79oc, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation), pl: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19ql3.2, Genbank accession No NP 001774.10) W02003088808, US20030228319; W02003062401 (claim 9); US2002150573 (claim 4, pages 13-14), WO9958658 (claim 13, Fig 16); WO9207574 (Fig 1); US5644033; Ha et al. (1992) J. Immunol.
  • CXCR5 (Burkitt’s lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pl: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 1 lq23.3, Genbank accession No.
  • NP_001707.1 WO 2004040000; W02004/015426; US2003105292 (Example 2); US6555339 (Example 2); WO 2002/61087 (Fig 1); W0200157188 (Claim 20, page 269); W0200172830 (pages 12-13); WO 2000/22129 (Example 1, pages 152-153, Example 2, pages 254-256); WO 199928468 (claim 1, page 38); US 5440021 (Example 2, col 49-52); WO9428931 (pages 56-58); WO 1992/17497 (claim 7, Fig 5); Dobner et al. (1992) Eur. J. Immunol. 22-.2795-2I99,- Barella et al. ( ⁇ 995) Biochem. J. 3Q9CT3-I79.
  • HLA-DOB Beta subunit of MHC class II molecule (la antigen) that binds peptides and presents them to CD4+ T lymphocytes); 273 aa, pl: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No. NP_002111.1) Tonnelle et al. (1985) EMBO J. 4(11):2839-2847; Jonsson et al. (1989) Immunogenetics 29(6):411-413; Beck et al. (1992) J. Mol. Biol. 228:433-441; Strausberg et al. (2002) Proc. Natl. Acad.
  • P2X5 Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability
  • 422 aa pl: 7.63, MW: 47206 TM: 1
  • Gene Chromosome 17p 13.3, Genbank accession No. NP_002552.2) Le et al. (1991) FEBS Lett. 418(1-2): 195-199, W02004047749, W02003072035 (claim 10); Touchman et al. (2000) Genome Res. 10: 165-173; W0200222660 (claim 20); W02003093444 (claim 1); W02003087768 (claim 1); W02003029277 (page 82).
  • CD72 B-cell differentiation antigen CD72, Lyb-2
  • pl 8.66
  • MW 40225 TM: 1
  • Gene Chromosome 9pl3.3, Genbank accession No. NP 001773.1) W02004042346 (claim 65); WO 2003/026493 (pages 51-52, 57-58); WO 2000/75655 (pages 105-106); Von Hoegen et al. (1990) J. Immunol. 144(12):4870-4877; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99: 16899-16903.
  • LY64 Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosus); 661 aa, pl: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5ql2, Genbank accession No. NP_005573.1) US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al. (1996) Genomics 38(3):299- 304; Miura et al. (1998) Blood 92:2815-2822; W02003083047; WO9744452 (claim 8, pages 57-61); W0200012130 (pages 24-26).
  • RP105 type I membrane protein of the leucine rich repeat
  • FcRHl Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation); 429 aa, pl: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: Iq21-lq22, Genbank accession No NP_443170 1) W02003077836; W0200138490 (claim 6, Fig 18E-1- 18-E-2); Davis et al. (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; W02003089624 (claim 8); EP1347046 (claim 1); W02003089624 (claim 7).
  • IRTA2 Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies
  • TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin); 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession No.
  • PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20; gplOO) BC001414; BT007202; M32295; M77348; NM_006928; McGlinchey, R.P. et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106 (33), 13731-13736; Kummer, M.P. et al. (2009) J Biol. Chem. 284 (4), 2296-2306.
  • TMEFF1 transmembrane protein with EGF-Iike and two follistatin-like domains 1; Tomoregulin-1); H7365; C9orf2; C9ORF2; U19878; X83961; NM 080655; NM_003692; Harms, P.W. (2003) Genes Dev. 17 (21), 2624-2629; Gery, S. et al. (2003) Oncogene 22 (18):2723-2727.
  • GDNF-Ral GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA, RETL1; TRNR1; RET1L; GDNFR-alphal; GFR-ALPHA-1
  • Ly6E lymphocyte antigen 6 complex, locus E; Ly67,RIG-E,SCA-2,TSA-l
  • NP_002337.1 NP_002346.2
  • de Nooij-van Dalen A.G. et al. (2003) Int. J. Cancer 103 (6), 768-774
  • Zammit D J. et al (2002) Mol. Cell. Biol. 22 (3):946-952
  • TMEM46 shisa homolog 2 (Xenopus laevis); SHISA2
  • NP_001007539.1 NM_001007538.1
  • Furushima K. et al. (2007) Bev. Biol. 306 (2), 480-492; Clark, H.F. et al. (2003) Genome Res. 13 (10):2265-2270.
  • Ly6G6D lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1;
  • LGR5 leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67
  • NP_003658.1 NM_003667.2
  • Salanti G. et al. (2009) Am. J. Epidemiol. 170 (5):537- 545; Yamamoto, Y. et al. (2003) Hepatology 37 (3):528-533.
  • RET ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC 1 ; PTC; CDHF12; Hs.168114; RET51; RET-ELE1); NP 066124.1; NM_020975.4; Tsukamoto, H. et al. (2009) Cancer Sci. 100 (10): 1895-1901; Narita, N. et al. (2009) Oncogene 28 (34):3058-3068.
  • LY6K lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226; NP 059997.3; NM 017527.3; Ishikawa, N. et al. (2007) Cancer Res. 67 (24): 11601-11611; de Nooij -van Dalen, A.G. et al (2003) Int. J. Cancer 103 (6):768-774.
  • GPR19 G protein-coupled receptor 19; Mm.4787
  • NP 006134.1 NM_006143.2
  • GPR54 (KISSI receptor; KISS1R; GPR54; HOT7T175; AX0R12); NP_115940.2; NM_032551.4; Navenot, J M. et al. (2009) Mol. Pharmacol. 75 (6): 1300-1306; Hata, K. et al. (2009) Anticancer Res 29 (2):617-623.
  • ASPHD1 aspartate beta-hydroxylase domain containing 1; LOC253982; NP_859069.2; NM_181718.3; Gerhard, D.S. et al. (2004) Genome Res. 14 (10B):2121-2127.
  • Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); NP 000363 1; NM_000372.4; Bishop, D.T. et al. (2009) Nat. Genet. 41 (8):920-925; Nan, H. et al. (2009) Int. J. Cancer 125 (4): 909-917
  • TMEM118 ring finger protein, transmembrane 2, RNFT2; FLJ14627
  • GPR172A G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e); NP 078807.1; NM_024531.3; Ericsson, T.A. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100
  • CD33 a member of the sialic acid binding, immunoglobulin-like lectin family, is a 67-kDa glycosylated transmembrane protein. CD33is expressed on most myeloid and monocytic leukemia cells in addition to committed myelomonocytic and erythroid progenitor cells. It is not seen on the earliest pluripotent stem cells, mature granulocytes, lymphoid cells, or nonhematopoietic cells (Sabbath et al., (1985) J. Clin. Invest. 75:756-56; Andrews et al., (1986) Blood 68: 1030-5). CD33 contains two tyrosine residues on its cytoplasmic tail, each of which is followed by hydrophobic residues similar to the immunoreceptor tyrosine-based inhibitory motif (ITIM) seen in many inhibitory receptors.
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • CLL-1 (CLEC 1 2A, MICE, and DCAL2)
  • CTL/CTLD C-type lectin/C- type lectin-like domain
  • CLL-1 has been shown to be a type II transmembrane receptor comprising a single C-type lectin-like domain (which is not predicted to bind either calcium or sugar), a stalk region, a transmembrane domain and a short cytoplasmic tail containing an ITIM motif.
  • TROP2 tumor-associated calcium signal transducer 2
  • TACSTD2 transmembrane glycoprotein encoded by the TACSTD2 gene
  • TROP2 is an intracellular calcium signal transducer that is differentially expressed in many cancers It signals cells for seif-renewal, proliferation, invasion, and survival. It has stem cell -like qualities. TROP2 is expressed in many normal tissues, though in contrast, it is overexpressed in many cancers (Ohmachi T, et al., (2006) Clin.
  • TROP2 Overexpression of TROP2 is of prognostic significance. Several ligands have been proposed that interact with TROP2. TROP2 signals the cells via different pathways and it is transcriptionally regulated by a complex network of several transcription factors.
  • Human TROP2 (TACSTD2: tumor-associated calcium signal transducer 2, GA733-1, EGP-1, Ml SI; hereinafter, referred to as hTROP2) is a single-pass transmembrane type 1 cell membrane protein consisting of 323 amino acid residues. While the presence of a cell membrane protein involved in immune resistance, which is common to human trophoblasts and cancer cells (Faulk W P, et al. (1978), Proc. Natl. Acad. Sci.
  • TROP2 an antigen molecule recognized by a monoclonal antibody against a cell membrane protein in a human choriocarcinoma cell line was identified and designated as TROP2 as one of the molecules expressed In human trophoblasts (Lipinski M, et al. (1981), Proc. Natl. Acad. Sci. 78(8), 5147-5150). This molecule was also designated as tumor antigen GA733-1 recognized by a mouse monoclonal antibody GA733 (Linnenbach A J, et al., (1989) Proc. Natl. Acad. Sci.
  • the antibody conjugate composition of the invention comprises an isoindolinone- glutarimide moiety (IG).
  • IG isoindolinone- glutarimide moiety
  • the antibody conjugate composition of the present disclosure may be prepared from an isoindolinone-glutarimide (IG) compound selected from Formula III: or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, or isotopic analog thereof, wherein: m is 0, 1 or 2;
  • X 2 is independently selected from the group consisting of F, Cl, Br, I, -CN, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 1 -C 12 heteroalkyl, -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl), -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl), ( C 1 -C 6 alkyldiyl)-(C 6 -C 20 aryl), -(C 1 -C 6 alkyldiyl)-NR a R b , -( C 1 -C 6 alkyldiyl)-OR a , (C 1 -C 6 alkyldiyl)-(Ca-C 20 carbocyclyl), (
  • R a is independently selected from H, C 1 -C 6 alkyl, phenyl, and benzyl, wherein phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, -CN, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, and C 2 -C 12 alkynyl;
  • R b is independently selected from H, OH, C 1 -C 6 alkyl, phenyl, and benzyl, wherein phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, -CN, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, and C 2 -C 12 alkynyl; n is 0, 1, 2, 3, or 4; and
  • An exemplary embodiment of the isoindolinone-glutarimide compound includes wherein m is 0.
  • An exemplary embodiment of the isoindolinone-glutarimide compound includes wherein m is 1.
  • An exemplary embodiment of the isoindolinone-glutarimide compound includes wherein m is 2.
  • An exemplary embodiment of the isoindolinone-glutarimide compound includes wherein X 1 is CH 2 .
  • An exemplary embodiment of the isoindolinone-glutarimide compound includes wherein X 2 is selected from the group consisting of C 1 -C 12 heteroalkyl, -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryl), and -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl).
  • An exemplary embodiment of the isoindolinone-glutarimide compound includes wherein X 2 is — (C 1 — C 12 heteroalkyldiyl)-(C 6 -C 20 aryl).
  • An exemplary embodiment of the isoindolinone-glutarimide compound includes wherein X 2 is: where ** indicates the point of attachment.
  • An exemplary embodiment of the isoindolinone-glutarimide compound includes wherein X 4 is H.
  • An exemplary embodiment of the isoindolinone-glutarimide compound includes wherein X 4 is selected from the group consisting of C 1 -C 12 heteroalkyl, -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryl), -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl), and -(C 1 -C 12 heteroalkyldiyl )-(C 6 -C 20 aryldiyl)-0-(C 2 -C 20 heterocyclyl).
  • An exemplary embodiment of the isoindolinone-glutarimide compound includes wherein X 4 is -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-0-(C 2 -C 20 heterocyclyl), and C 2 -C 20 heterocyclyl is a glucuronide.
  • An exemplary embodiment of the isoindolinone-glutarimide compound includes wherein X 4 of formula la is selected from the formulae: wherein R is selected from H, C 1 -C 6 alkyl, and O-(C 1 -C 6 alkyl); and * indicates the point of attachment.
  • exemplary isoindolinone-glutarimide (IG) compounds of Table la, lb and 1c were prepared and characterized according to the Examples herein. C 6 rtain exemplary IG compounds of Table la were tested for their effects in inhibiting cellular proliferation, including CAL51,; WSU-DLCL2, NCI-N87 and SKBR3.
  • CAL51 is a human breast adenocarcinoma cell line with triple-negative status for expression of estrogen, progesterone and HER2 receptors.
  • WSU- DLCL2 is a human B-C 6 ll non-Hodgkin lymphoma cell line that expresses high levels of CD22.
  • NCI-N87 is a human epithelial cell line established from a gastric carcinoma;
  • SKBR3 is a human epithelial cell line established from a breast adenocarcinoma; both NCI-N87 and SKBR3 cell lines express high levels of HER2 receptor Table la Isoindolinone-glutarimide (IG) compounds
  • the present disclosure provides branched phenyl maleimide compounds (e.g., compounds of Structure (I)) enable the formation of a covalent bond between an isoindolinone-glutarimide linker compound (IG-L) and an antibody (Ab).
  • branched phenyl maleimide compounds e.g., compounds of Structure (I)
  • IG-L isoindolinone-glutarimide linker compound
  • Ab antibody
  • some embodiments provide a compound having the following Structure (I): wherein: one of Y 1 , Y 2 , Y 3 , Y 4 and Y 5 is C-L'-R 1 , another one of Y 1 , Y 2 , Y 3 , Y 4 and Y 5 is C-L 2 - R 2 , and the remaining three of Y 1 , Y 2 , Y 3 , Y 4 and Y 5 are each independently N, C-R 3 , or C-L 3 - R 3a ;
  • R 1 , R 2 , and R 3a each independently comprise one or more moieties selected from an amino acid element, a charged element, a heteroalkylene element, a hydrophilic element, a trigger element, an immolative unit, a polar cap, an isoindolinone-glutarimide moiety, and combinations thereof; provided that at least one of R 1 and R 2 comprises a isoindolinone-glutarimide moiety; each occurrence of R 3 is independently selected from the group consisting of hydrogen, deuterium, alkyl, haloalkyl, halo, alkoxy, haloalkoxy, amino, aminyl, amidyl, aldehyde, hydroxyl, cyano, nitro, thiol, carboxy, carboxyalkyl, alkyl-S(O) 3 H, alkyl-O-P(O)sH, alkyl- P(O) 3 H, -O-carboxyalkyl, -O-alkyl
  • R 4a and R 4b are each independently hydrogen, deuterium, halo, or -S-R 4c wherein R 4c is substituted or unsubstituted C 6 -C 10 aryl or substituted or unsubstituted 5-12 membered heteroaryl; and
  • L 1 , L 2 , and L 3 are each independently a linker comprising an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted heteroalkylene, an optionally substituted heteroalkenylene, an optionally substituted heteroalkynylene, a heteroatomic linker, an optionally substituted cycloalkylene, an optionally substituted arylene, an optionally substituted heterocyclylene, an optionally substituted heteroarylene, or combinations thereof; as a stereoisomer, enantiomer or tautomer thereof or a mixture thereof; or a pharmaceutically acceptable salt, solvate or prodrug thereof.
  • C 6 rtain embodiments provide a compound having the following Structure (la): wherein: one of Y 1 , Y 2 , Y 3 , Y 4 and Y 5 is C-L'-R 1 , another one of Y 1 , Y 2 , Y 3 , Y 4 and Y 5 is C-L 2 - R 2 , and the remaining three of Y 1 , Y 2 , Y 3 , Y 4 and Y 5 are each independently N, C-R 3 , or C-L 3 - R 3a ;
  • R 1 , R 2 , and R 3a each independently comprise one or more moieties selected from an amino acid element, a charged element, a heteroalkylene element, a hydrophilic element, a trigger element, an immolative element, a polar cap, a payload, and combinations thereof; provided that at least one of R 1 and R 2 comprises a payload; each occurrence of R 3 is independently selected from the group consisting of hydrogen, deuterium, alkyl, haloalkyl, halo, alkoxy, haloalkoxy, amino, aminyl, amidyl, aldehyde, hydroxyl, cyano, nitro, thiol, carboxy, carboxyalkyl, alkyl-S(O) 3 H, alkyl-O-P(O) 3 H, alkyl- P(O) 3 H, -O-carboxyalkyl, -O-alkyl-S(O) 3 H, -O-alkyl-O-P(O
  • R 4a and R 4b are each independently hydrogen, deuterium, halo, or -S-R 4c wherein R 4c is substituted or unsubstituted C 6 -C 10 aryl or substituted or unsubstituted 5-12 membered heteroaryl; and
  • L 1 , L 2 , and L 3 are each independently direct bond or a linker comprising an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted heteroalkylene, an optionally substituted heteroalkenylene, an optionally substituted heteroalkynylene, a heteroatomic linker, an optionally substituted cycloalkylene, an optionally substituted arylene, an optionally substituted heterocyclylene, an optionally substituted heteroarylene, or combinations thereof; as a stereoisomer, enantiomer or tautomer thereof or a mixture thereof; or a pharmaceutically acceptable salt, solvate or prodrug thereof.
  • R 4a and R 4b are both hydrogen. In some embodiments, R 4a is halo or R 4b is halo. In some embodiments, R 4a , R 4b , or both have one of the following structures:
  • R 1 R 2 , and/or R 3a comprises elements selected from an amino acid element, a charged element, a heteroalkylene element, a hydrophilic element, a trigger element, an immolative element, a polar cap, a payload, and combinations thereof. It is understood that these elements can be connected in any order and be connected in a linear manner or via a branched connection.
  • R 1 R 2 , and/or R 3a comprises multiple occurrences of an element (e.g, two or more heteroalkylene elements, two or more hydrophilic elements, two or more polar caps, etc.).
  • R 1 , R 2 , or R 3a comprises a branch point as part of an amino acid element (e.g., lysine) wherein additional elements are attached via an epsilon amine of the lysine and other additional elements are linked to the amino acid element via one or more peptide bonds to the alpha carbon of a lysine.
  • an amino acid element comprises one of the following structures:
  • a compound of Structure (I) comprises one of the following structures:
  • R 1 has the following structure: wherein:
  • L la is an amino acid element
  • L lb is a charged element
  • L lc is a heteroalkylene element
  • L ld is a hydrophilic element
  • L le is a trigger element
  • R la is a isoindolinone-glutarimide moiety that is covalently bound to one occurrence of
  • L la , L lb , L lc , L ld , L le , or L lf and the isoindolinone-glutarimide moiety is optionally substituted with a polar cap.
  • n7 is 1, 2, or 3. In some embodiments, n7 is 1 or 2. In some embodiments, n7 is 1 .
  • R 1 has the following structure: wherein:
  • L la is an amino acid element
  • L lb is a charged element
  • L lc is a heteroalkylene element
  • L ld is a hydrophilic element
  • L le is a trigger element
  • R la is a isoindolinone-glutarimide moiety optionally substituted with a polar cap.
  • R 2 has the following structure: wherein:
  • L 2a is an amino acid element
  • L 2b is a charged element
  • L 2C is a heteroalkylene element
  • L 2d is a hydrophilic element
  • R 2a is hydrogen, alkyl, a isoindolinone-glutarimide moiety, or a polar cap
  • m6 is 1, 2, or 3. In some embodiments, m6 is 1 or 2. In some embodiments, m6 is 1.
  • R 3a has the following structure: wherein:
  • L 3a is an amino acid element
  • b is a charged element
  • L 3C is a heteroalkylene element
  • L 3d is a hydrophilic element
  • R 3b is hydrogen, alkyl, or a polar cap.
  • p6 is 1, 2, or 3. In some embodiments, p6 is 1 or 2. In some embodiments, p6 is 1 .
  • n7 is 1 and each of nl through n6 are 1. In some embodiments, n7 is 1 and each of nl through n4 are 0, n5 is 1, and nb is 1. In certain embodiments, n7 is 1 and nl is 0, n2 is 0, n3 is 1, n4 is 0, n5 is 1, and n6 is 1. In certain embodiments, n7 is 1 and nl is 1, n2 is 0, n3 is 1, n4 is 0, n5 is 1, and n6 is 1. In certain embodiments, n7 is 1 and nl is 1, n2 is 1, n3 is 1, n4 is 0, n5 is 1, and n6 is 1. In certain embodiments, n7 is 1 and nl is 1, n2 is 1, n3 is 1, n4 is 0, n5 is 1, and n6 is 1.
  • n7 is 1 and nl is 1, n2 is 1, n3 is 1, n4 is 1, n5 is 1, and n6 is I In certain embodiments, n7 is 1 and nl is 1, n2 is 0, n3 is 1, n4 is 0, n5 is 1, and n6 is 1 In certain embodiments, n7 is 2 In certain embodiments, n7 is 3
  • m6 is 1 and each of ml through m5 are 1. In some embodiments, m6 is 1, ml is 1, m2 is 0, m3 is 0, m4 is 1, and m5 is 0. In some embodiments, m6 is 1, ml is 1, m2 is 1, m3 is 0, m4 is 1, and m5 is 0. In some embodiments, m6 is 1, ml is 1, m2 is 0, m3 is 1, m4 is 1, and m5 is 0. In some embodiments, mb is 1, ml is 1, m2 is 0, m3 is 0, m4 is 1, and m5 is 1. In some embodiments, m6 is 2. In certain embodiments, m6 is 3.
  • p6 is 1 and each of pl through p5 is 1. In certain embodiments, p6 is 1, pl is 1, p2 is 0, p3 is 0, p4 is 1, and p5 is 0. In some embodiments, p6 is 1, pl is 1, p2 is 1, p3 is 0, p4 is 1, and p5 is 0.
  • p6 is 1, pl is 1, p2 is 0, p3 is 1, p4 is 1, and p5 is 0 In some embodiments, p6 is 1, pl is 1, p2 is 0, p3 is 0, p4 is 1, and p5 is 1 In some embodiments, p6 is 2 and at least one occurrence of pl is 1, p2 is 1, p3 is 0, p4 is 1, and p5 is 0. In some embodiments, pb is 2. In certain embodiments, pb is 3.
  • an amino acid element comprises one or more amino acids selected from the group consisting of glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, sarcosine, and beta-alanine.
  • an amino acid element is selected from the group consisting of glycine, sarcosine, beta-alanine, and glutamic acid.
  • an amino acid element comprises a dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide.
  • an amino acid element has one of the following structures: wherein: each occurrence of R 5a is independently hydrogen, alkyl, hydroxyalkyl, or alkoxyalkyl.
  • a charged element comprises moieties with a negative charge at about pH 7.4 (i.e., a range from 6.3 to 8 5).
  • a charged element comprises moieties with a positive charge at about pH 7.4 (i.e., a range from 6.3 to 8 5).
  • a charged element comprises one or more charged amino acid, one or more carboxylic acid, one or more sulfonic acid, one or more sulfonamide, one or more sulfate, one or more phosphate, one or more quaternary amine, one or more sulfamide, one or more sulfinimide, or combinations thereof.
  • a charged amino acid is aspartic acid, glutamic acid, histidine, lysine, or arginine.
  • R 1 , R 2 , or R 3a comprises a non-cleavable linker (e.g, a linker, or segment thereof, that does not include a trigger element or immolative unit).
  • a non-cleavable linker e.g, a linker, or segment thereof, that does not include a trigger element or immolative unit.
  • R 1 , R 2 , or R 3a comprises one of the following structures:
  • each occurrence of R 5b , R 5c R 5d , and R 5e is independently selected from the group consisting of hydrogen, deuterium, alkyl, haloalkyl, halo, alkoxy, haloalkoxy, amino, hydroxyl, cyano, nitro, thiol, carboxyalkyl, alkyl-S(O) 3 H, alkyl-O-P(O) 3 H, alkyl-P(O) 3 H, -O-carboxyalkyl, -O-alkyl-S(O) 3 H, -O-alkyl-O-P(O) 3 H, -O-alkyl-P(O) 3 H, -S(O) 3 H, -OP(O) 3 H, -P(O) 3 H, alkyl-O- P(O) 3 -alkyl, alkyl-P(O) 3 -alkyl, -O-alkyl-S(O) 3 H,
  • a hydrophilic element comprises polyethylene glycol, poly sarcosine, cyclodextrin, c-glycosides, or combinations thereof. In some embodiments, a hydrophilic element comprises one of the following structures:
  • a hydrophilic element comprises one of the following structures: In some embodiments, a hydrophilic element comprises one of the following structures:
  • a hydrophilic element has one of the following structures:
  • a hydrophilic element has the following structure:
  • a hydrophilic element has one of the following structures:
  • a hydrophilic element comprises a polysarcosine. In some embodiments, a hydrophilic element is a polysarcosine comprising the following structure:
  • a hydrophilic element is a polysarcosine with one of the following structures:
  • a hydrophilic element has a molecular weight greater than 150 g/mole, greater than 200 g/mole, greater than 300 g/mole, greater than 400 g/mole, greater than 500 g/mole, greater than 600 g/mole, greater than 700 g/mole, greater than 800 g/mole, greater than 900 g/mole, or greater than 1000 g/mole
  • a hydrophilic element has a molecular weight less than 150 g/mole, less than 200 g/mole, less than 300 g/mole, less than 400 g/mole, less than 500 g/mole, less than 600 g/mole, less than 700 g/mole, less than 800 g/mole, less than 900 g/mole, or less than 1000 g/mole.
  • L 1 is alkylene. In some embodiments, L 1 is C 1 -C 6 alkylene. In certain embodiments, L 2 is alkylene. In some embodiments, L 2 is C 1 -C 6 alkylene. In certain embodiments, I? is alkylene. In some embodiments, L 3 is C 1 -Cg alkylene.
  • L 1 is heteroalkylene. In some embodiments, L 1 is C 1 -C 6 heteroalkylene (i.e., contains from 1-6 carbon atoms and one or more heteroatoms). In certain embodiments, L 2 is heteroalkylene In some embodiments, L 2 is C 1 -Cg heteroalkylene In certain embodiments, I? is heteroalkylene. In some embodiments, L 3 is C 1 -Cg heteroalkylene.
  • L 1 , L 2 , or L 3 are C 1 -Cg heteroalkylene and contain heteroatoms selected form O and N.
  • I? is a direct bond.
  • L 1 , L 2 , or L 3 have one of the following structures:
  • a trigger element comprises a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a glucuronide, a disulfide, a phosphate, a diphosphate, a triphosphate, a hydrazone, or combinations thereof.
  • a trigger element comprises beta-glucuronic acid.
  • a trigger element comprises a dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide
  • a trigger element comprises two or more amino acids selected from the group consisting of valine, citrulline, alanine, glycine, phenylalanine, lysine, or combinations thereof.
  • a trigger element comprises a sequence of amino acids selected from the group consisting of valine-citrulline, valine-alanine, glycine-glycine-phenylalanine-glycine, and combinations thereof.
  • a trigger element comprises one of the following structures, including combinations thereof
  • a trigger element has a molecular weight greater than 150 g/mole, greater than 200 g/mole, greater than 300 g/mole, greater than 400 g/mole, greater than 500 g/mole, greater than 600 g/mole, greater than 700 g/mole, greater than 800 g/mole, greater than 900 g/mole, or greater than 1000 g/mole.
  • a trigger element has a molecular weight less than 150 g/mole, less than 200 g/mole, less than 300 g/mole, less than 400 g/mole, less than 500 g/mole, less than 600 g/mole, less than 700 g/mole, less than 800 g/mole, less than 900 g/mole, or less than 1000 g/mole.
  • a trigger element has the following structure:
  • a trigger element is specifically cleaved by an enzyme.
  • a trigger element can be cleaved by a lysosomal enzyme.
  • a trigger element can be peptide-based or can include peptidic regions that can act as substrates for enzymes.
  • Peptide based trigger elements can be more stable in plasma and extracellular milieu than chemically labile linkers.
  • Exemplary disulfide-containing trigger elements can include the following structures: wherein D is a isoindolinone-glutarimide moiety and R is independently selected at each occurrence from, for example, hydrogen or C 1 -C 6 alkyl. Increasing steric hindrance adjacent to the disulfide bond can increase the stability of the linker.
  • the above structures can result in increased in vivo stability when one or more R groups is selected from a lower alkyl, such as methyl.
  • Peptide bonds can have good serum stability, as lysosomal proteolytic enzymes can have very low activity in blood due to endogenous inhibitors and the unfavorably high pH value of blood compared to lysosomes.
  • Release of a isoindolinone-glutarimide moiety from conjugate of Structure (II) can occur due to the action of lysosomal proteases, e.g., cathepsin and plasmin. These proteases can be present at elevated levels in certain tumor tissues.
  • a trigger element can be cleavable by a lysosomal enzyme.
  • the lysosomal enzyme can be, for example, cathepsin B, P-glucuronidase, or P-galactosidase.
  • a cleavable peptide of a trigger element can be selected from tetrapeptides such as Gly- Phe-Leu-Gly, Ala-Leu-Ala-Leu, tripeptides such as Glu-Val-Cit, or dipeptides such as Val-Cit, Vai-Ala, Ala-Ala, and Phe-Lys. Dipeptides can have lower hydrophobicity compared to longer peptides
  • a trigger element may be a single amino acid residue.
  • the trigger element comprises asparagine, Asn as a legumain cleavable element (Miller, J.T. et al (2021) Bioconjugate Chem. 32(4):842-858
  • Enzymatically cleavable trigger elements be combined with an immolative unit and provide additional spatial separation between a isoindolinone-glutarimide moiety and the site of enzymatic cleavage.
  • the direct attachment of isoindolinone-glutarimide moiety to a peptidic trigger element can result in proteolytic release of a isoindolinone-glutarimide moiety or of an amino acid adduct of a isoindolinone-glutarimide moiety thereby impairing its activity.
  • the use of an immolative unit can allow for the release of the fully active, chemically unmodified isoindolinone-glutarimide moiety upon amide bond hydrolysis.
  • a trigger element can contain a chemically labile group such as hydrazone and/or disulfide groups.
  • a trigger element comprising chemically labile group or groups can exploit differential properties between the plasma and some cytoplasmic compartments.
  • the intracellular conditions that can facilitate release of a isoindolinone-glutarimide moiety for hydrazone containing trigger elements can be the acidic environment of endosomes and lysosomes, while the disulfide containing trigger elements can be reduced in the cytosol, which can contain high thiol concentrations, e.g., glutathione.
  • the plasma stability of a trigger element containing a chemically labile group can be increased by introducing steric hindrance using substituents near the chemically labile group
  • Acid-labile groups such as hydrazone
  • This pH dependent release mechanism can be associated with non-specific release of a isoindolinone- glutarimide moiety.
  • a trigger element can be varied by chemical modification, e.g., substitution, allowing tuning to achieve more efficient release in the lysosome with a minimized loss in circulation.
  • a trigger element comprises a hydrazone moiety having one of the following structures: wherein R is selected from C 1 -C 6 alkyl, aryl, and -O-C 1 -C 6 alkyl
  • Hydrazone-containing trigger elements can contain additional cleavage sites, such as additional acid-labile cleavage sites and/or enzymatically labile cleavage sites (e.g., a disulfide).
  • Conjugates and compounds including exemplary hydrazone-containing trigger elements can include, for example, the following structure: wherein R is selected from C 1 -C 6 alkyl, aryl, and -O-C 1 -C 6 alkyl
  • acid-labile groups that can be included in trigger elements include c/.v-aconityl- containing linkers.
  • rv.s-Aconityl chemistry can use a carboxylic acid juxtaposed to an amide bond to accelerate amide hydrolysis under acidic conditions.
  • Trigger elements can also include a disulfide group.
  • Disulfides can be thermodynamically stable at physiological pH and release a isoindolinone-glutarimide moiety upon internalization of the conjugate of Structure (II) into cells, wherein the cytosol can provide a significantly more reducing environment compared to the extracellular environment. Scission of disulfide bonds can require the presence of a cytoplasmic thiol cofactor, such as (reduced) glutathione (GSH), such that disulfide-containing trigger element can be reasonably stable in circulation, selectively releasing a isoindolinone-glutarimide moiety in the cytosol.
  • GSH cytoplasmic thiol cofactor
  • the intracellular enzyme protein disulfide isomerase can also contribute to the preferential cleavage of disulfide bonds inside cells.
  • GSH can be present in cells in the concentration range of 0.5-10 mM compared with a significantly lower concentration of GSH or cysteine, the most abundant low-molecular weight thiol, in circulation at approximately 5 pM.
  • Tumor cells where irregular blood flow can lead to a hypoxic state, can result in enhanced activity of reductive enzymes and therefore even higher glutathione concentrations.
  • the in vivo stability of a disulfide-containing trigger element can be enhanced by chemical modification of a trigger element, e.g., use of steric hindrance adjacent to the disulfide bond.
  • a trigger element can also be a B-glucuronic acid-based linker. Facile release of a isoindolinone-glutarimide moiety, can be realized through cleavage of the B-glucuronide glycosidic bond by the lysosomal enzyme B-glucuronidase. This enzyme can be abundantly present within lysosomes and can be overexpressed in some tumor types, while the enzyme activity outside cells can be low. B-Glucuronic acid-based linkers can be used to circumvent the tendency of a conjugate to undergo aggregation due to the hydrophilic nature of B-glucuronides.
  • a trigger element comprises a B-glucuronic acid
  • cleavable 0 -glucuronic acid-based linkers useful for linking drugs such as auristatins, camptothecin analogues, doxorubicin analogues, CBI minor-groove binders, and psymberin to antibodies have been described. Accordingly, these P-glucuronic acid-based trigger elements are used in the conjugates of Structure (II).
  • a trigger element comprises a P-galactoside-based linker. 0-Galactoside is present abundantly within lysosomes, while the enzyme activity outside cells is low.
  • a trigger element may include one or more peptides.
  • a peptide can be selected to contain natural amino acids, unnatural amino acids, or any combination thereof.
  • a peptide can be a tripeptide or a dipeptide.
  • a dipeptide comprises L-amino acids, such as Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit, Cit-Lys; Asp- Cit; Cit-Asp; Ala-Vai; Vai-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala; Phe- Cit; Cit-Phe; Leu-Cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; and Trp-Cit, or salts thereof.
  • L-amino acids such as Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit
  • Trigger elements and immolative groups are known (WO 2022/076905) which is hereby incorporated by reference in their entirety.
  • One immolative unit can be a bifunctional para- aminobenzyl alcohol group, which can link to a trigger element through an amino group, forming an amide bond, while an amine containing isoindolinone-glutarimide moiety can be attached through carbamate functionalities to the benzylic hydroxyl group of the para- aminobenzyl alcohol (to give a //-amidobenzyl carbamate.
  • the resulting pro-compound can be activated upon protease-mediated cleavage, leading to a 1,6-elimination reaction releasing the unmodified isoindolinone-glutarimide moiety and remnants of the antibody-linker.
  • an immolative unit comprises paraaminobenzyloxycarbonyl, an aminal, a hydrazine, a disulfide, an amide, an ester, a hydrazine, a phosphotriester, a diester, a P-glucuronide, a double bond, a triple bond, an ether bond, a ketone, a diol, a cyano, a nitro, a quaternary amine, or combinations thereof
  • an immolative unit comprises a paramethoxybenzyl, a dialkyldialkoxysilane, a diaryldialkoxysilane, an orthoester, an acetal, an optionally substituted p-thiopropionate, a ketal, a phosphorami date, a hydrazone, a vinyl ether, an imine, an aconityl, a trityl, a polyketal, a bis
  • an immolative unit comprises one of the following structures:
  • an immolative unit comprises the following structure: wherein:
  • R 6a , R 6b , R 6C , and R 6d are independently hydrogen, an optionally substituted alkyl, an optionally substituted aryl, or optionally substituted heteroaryl, or
  • R 6a and R 6c together with the nitrogen and carbon atoms to which they are attached form azetidinyl, pyrrolodinyl, piperidinyl, or homopiperidinyl and R 6d is hydrogen;
  • Y 1 is -O-, -S-, or -NR 6b -;
  • an immolative unit comprises the following structure: wherein:
  • R 6e , R 6f , R 6g , and R 511 are independently hydrogen, an optionally substituted alkyl, an optionally substituted aryl, or optionally substituted heteroaryl, or
  • R 6a and R 6c together with the nitrogen and carbon atoms to which they are attached form azetidinyl, pyrrolodinyl, piperidinyl, or homopiperidinyl and R 6d is hydrogen;
  • Y 2 is -O-, -S-, or -NR 6f -;
  • an immolative unit comprises the following structure: wherein: each occurrence of R 10 is independently alkyl, alkoxy, or halo;
  • R 11 is hydrogen, alkyl, or -(CH 2 CH 2 O) Z 3-CH 3 ;
  • R 12 is hydrogen or alkyl
  • R 13 is hydrogen or alkyl; zl is 0 or 1; z2 is 0, 1, 2, 3, or 4; and z3 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or
  • an immolative unit comprises one of the following structures: wherein:
  • R 14a , R 14b , R 14C , R 14d , R 14e , and R 14f are each independently hydrogen, alkyl, hydroxyalkyl, or alkoxyalkyl; z4, z5, z6, and z7 are each independently 1, 2, 3, 4, 5, or 6;
  • an immolative unit comprises one of the following structures: wherein: z8 and z9 are each independently 1, 2, 3, 4, 5, or 6; or
  • an immolative unit comprises one of the following structures:
  • each occurrence of R 15 is independently H, methyl, ethyl, isopropyl, tert-butyl, or phenyl;
  • Y 3 is O or CH 2 ; and q5 is an integer ranging from 1-5.
  • an immolative unit has a molecular weight greater than 150 g/mole, greater than 200 g/mole, greater than 300 g/mole, greater than 400 g/mole, greater than 500 g/mole, greater than 600 g/mole, greater than 700 g/mole, greater than 800 g/mole, greater than 900 g/mole, or greater than 1000 g/mole.
  • an immolative unit has a molecular weight less than 150 g/mole, less than 200 g/mole, less than 300 g/mole, less than 400 g/mole, less than 500 g/mole, less than 600 g/mole, less than 700 g/mole, less than 800 g/mole, less than 900 g/mole, or less than 1000 g/mole.
  • an immolative unit and a trigger element together have the following structure: wherein a trigger element is denoted with “peptide” and comprises from one to ten amino acids, and * represents the point of attachment to a isoindolinone-glutarimide moiety.
  • the peptide comprises Val-Cit or Val-Ala.
  • Heterocyclic variants e.g., pyridinyl, pyrimidinyl, etc.
  • an immolative unit contains a phenol group that is covalently bound to the remainder of the molecule through the phenolic oxygen.
  • One such immolative unit relies on a methodology in which a diamino-ethane “Space Link” is used in conjunction with traditional “PABO”-based immolative unit to deliver phenols.
  • a trigger element can include non-cleavable portions or segments.
  • Polyethylene glycol (PEG) and related polymers can be included with cleavable groups such as a disulfide, a hydrazone or a dipeptide to form an immolative group and/or trigger element.
  • esters can be formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a isoindolinone-glutarimide moiety such ester groups can hydrolyze under physiological conditions to release a isoindolinone-glutarimide moiety
  • Other hydrolytically degradable linkages can include carbonate linkages, imine linkages resulting from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; and oligonucleotide linkages formed by a phosphoramidite group, including at the end of a polymer, and a 5’ hydroxyl group of an oligonucleotide.
  • a trigger element, immolative unit (IM), and isoindolinone- glutarimide moiety (IG) together have the following structure:
  • a trigger element is Asn-Cit, Arg-Cit, Val-Glu, Ser-Cit, Lys- Cit, Asp-Cit, Phe-Lys, Glu-Val-Cit, Glu-Val-Cit, Glu-Glu-Val-Cit, or Glu-Glu-Glu-Val-Cit, and an immolative unit is PABC.
  • the phenyl portion of the PABC is substituted with one or more substituents.
  • the substituents have one of the following structures:
  • an immolative group comprises one of the following structures:
  • a trigger element, an immolative unit (IM), and isoindolinone- glutarimide moiety (IG) together have one of the following structures:
  • an immolative unit has a structure selected from the following:
  • a substitution pattern may be 1, 2, 4 (i.e., 1 being a linkage to an IG, 2 being a linkage to the remainder of the molecule and 4 being a linkage to the carbohydrate) or 1, 3, 5 (i.e., 1 being a linkage to an IG, 3 being a linkage to the remainder of the molecule and 4 being a linkage to the carbohydrate).
  • cleavable linkers e.g., linkers with trigger elements or immolative unit
  • linkers need not be cleavable.
  • an IG release may not depend on the differential properties between the plasma and some cytoplasmic compartments.
  • the release of an IG can occur after internalization of the conjugate of Structure (II) via antigen-mediated endocytosis and delivery to lysosomal compartment, where the targeting moiety (or binding fragment thereof) can be degraded to the level of amino acids through intracellular proteolytic degradation. This process can release a isoindolinone- glutarimide moiety or isoindolinone-glutarimide moiety derivative.
  • a isoindolinone-glutarimide moiety or isoindolinone-glutarimide moiety derivative can be more hydrophilic and less membrane permeable, which can lead to less bystander effects and less non-specific toxicities compared to conjugates with a cleavable linker.
  • Conjugates with non-cleavable linkers can have greater stability in circulation than conjugates with cleavable linkers
  • Non-cleavable linkers can include alkylene chains, or can be polymeric, such as, for example, based upon polyalkylene glycol polymers, amide polymers, or can include segments of alkylene chains, poly alkylene glycols and/or amide polymers.
  • the linker can contain a polyethylene glycol segment having from 1 to 6 ethylene glycol units.
  • -L 1 -R. 1 or L 2 -R 2 comprises a linker that is non-cleavable in vivo.
  • a trigger element and an immolative unit (IM) together comprise one of the following structures:
  • a heteroalkylene element comprises polyethylene glycol or polypropylene glycol.
  • a heteroalkylene element comprises one of the following structures: wherein: each occurrence of R 5b , R 5c R 5d , and R 5e is independently selected from the group consisting of hydrogen, deuterium, alkyl, haloalkyl, halo, alkoxy, haloalkoxy, amino, hydroxyl, cyano, nitro, thiol, carboxyalkyl, alkyl-S(O) 3 H, alkyl-O-P(O) 3 H, alkyl-P(O) 3 H, -O-carboxyalkyl, -O-alkyl-S(O) 3 H, -O-alkyl-O-P(O) 3 H, -O-alkyl-P(O) 3 H, -S-alkyl-P(O) 3
  • a polar cap comprises one or more charged amino acid, one or more polyol, or combinations thereof.
  • a polar cap comprises a diol, a triol, a tetraol, or combinations thereof.
  • a polar cap comprises glycerol, trimethylolpropane, pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof.
  • a polar cap comprises one or more natural amino acids.
  • a polar cap comprises one or more non-natural amino acids.
  • a polar cap comprises one or more non-natural amino acids and one or more natural amino acids.
  • a polar cap comprises serine, threonine, cysteine, proline, asparagine, glutamine, lysine, arginine, histidine, aspartate, glutamate, 4- hydroxyproline, 5-hydroxylysine, homoserine, homocysteine, ornithine, beta-alanine, statine, or gamma aminobutyric acid
  • a polar cap comprises aspartic acid, serine, glutamic acid, serine-beta-glucose, or combinations thereof.
  • a polar cap comprises one of the following structures:
  • a polar cap has one of the following structures, including combinations thereof:
  • L 1 , L 2 , or L 3 comprise a linker selected from the group alkylene, alkylene-!/-, alkenylene, alkenylene-I?-, alkynylene, alkynylene-L a -, -L a -, -L a -alkylene-L a -, -L a - alkenylene-L a -, -L a -alkynylene-L a -, and combinations thereof, wherein each alkylene, alkenylene, and alkynylene is optionally substituted and each occurrence of L a is independently selected from -O-, -S-, -N(R 7 )-, -C(O)-, -C(S)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R 7 )-, -N(R 7 )C
  • R 7 is independently selected at each occurrence from hydrogen, -NH 2 , -C(O)OCH 2 C6Hs; and Cnio alkyl, C2 10 alkenyl, C 2-10 alkynyl, C 3-12 cycloalkyl, and 3- to 12-membered heterocycle, each of which
  • each L 1 , L 2 , or L 3 is optionally substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, halo, hydroxyl, cyano, -OR 8 , -SR 8 , amino, aminyl, amido, cycloalkyl, aryl, heterocyclyl, heteroaryl, cycloclkylalkyl, arylalkyl, heterocyclylalkyl, heteroarylalkyl, -C(O)R 8 , -C(O)N(R 8 ) 2 , -N(R 8 )C(O)R 8 , -C(O)OR 8 , -OC(O)R 8 , -S(O)R 8 , -S(O)2R 8 , -P(O)(OR 8 ) 2 , -OP(O)(OR 8 )2, nitro, oxo, thioxo,
  • L 1 , L 2 , or L 3 are independently selected from the following structures: wherein:
  • R a is hydrogen or alkyl; each occurrence of L b is independently a direct bond, an optionally substituted alkylene linker, an optionally substituted heteroalkylene linker, a heteroatomic linker, or a combination thereof; and each occurrence of L c is independently an optionally substituted alkylene linker and provided that at least one of L 1 , L 2 , or L 3 has the following structure:
  • L 1 and L 2 are independently selected from the following structures: wherein:
  • R a is hydrogen or alkyl; each occurrence of L b is independently a direct bond, an optionally substituted alkylene linker, an optionally substituted heteroalkylene linker, a heteroatomic linker, or a combination thereof; each occurrence of L c is independently an optionally substituted alkylene linker; provided that at least one of L 1 or L 2 has the following structure: In more embodiments, L 2 has the following structure:
  • L c is unsubstituted. In some embodiments, L c is a C 1 -Cg alkylene.
  • L c is a C2-C4 alkylene. In some embodiments, L c is a straight C 1 -C 6 alkylene. In more embodiments, L c is a straight, unsubstituted C 1 -C6 alkylene. In more embodiments, L c is a straight, unsubstituted C2-C4 alkylene.
  • the isoindolinone-glutarimide linker compound has one of the following structures (la- 1), (la-2), (la-3), (la-4), (Ia-5),or (la-6): each occurrence of L b is independently a direct bond, an optionally substituted alkylene linker, an optionally substituted heteroalkylene linker, a heteroatomic linker, or combinations thereof; and q6 is 0, 1, or 2. In some embodiments, q6 is 1 and L b is gly-gly.
  • the isoindolinone-glutarimide linker compound has one of the following Structures (Ic-1), (Ic-2), (Ic-3), or (Ic-4):
  • each occurrence of L b is independently a direct bond, an optionally substituted alkylene linker, an optionally substituted heteroalkylene linker, a heteroatomic linker, or combinations thereof; and q7 is 1, 2, or 3.
  • q7 is 2.
  • the isoindolinone-glutarimide linker compound has the following Structure (Id), (le), (If), or (Ig):
  • each occurrence of L b is independently a direct bond, an optionally substituted alkylene linker, an optionally substituted heteroalkylene linker, a heteroatomic linker, or combinations thereof; q8 is 0, 1, or 2; and q9 is 0, 1, or 2.
  • q9 is 0 and q8 is 1.
  • the isoindolinone-glutarimide linker compound has one of the following Structures (Ih) or (li): wherein: each occurrence of L b is independently a direct bond, an optionally substituted alkylene linker, an optionally substituted heteroalkylene linker, a heteroatomic linker, or combinations thereof.
  • L b is a direct bond, an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.
  • L b is a direct bond or has one of the following structures: wherein: each occurrence of R b is independently hydrogen, alkyl, hydroxyalkyl, or alkoxyalkyl.
  • each occurrence of R b is -CH 3 .
  • L 1 or L 2 has the following structure: wherein:
  • X 2 is C-L ⁇ R 1 and X 3 is C-L 2 -R 2 . In some embodiments, X 3 is C-L'-R 1 and X 2 is C-L 2 -R 2 . In some embodiments, X 1 , X 4 , and X 5 are all CR 3 . In some embodiments, X 1 , X 4 , and X 3 are all CH.
  • the conjugates of the invention are prepared by conjugation of an antibody with an isoindolinone-glutarimide linker compound, IG-L.
  • An isoindolinone-glutarimide linker compound is selected from Formulae Ila and Hb : wherein: m is 1 or 2;
  • X 2 is independently selected from the group consisting of F, Cl, Br, I, -CN, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 1 -C 12 heteroalkyl, -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl), -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl), -(C 1 -C 12 heteroalkyldiyl)-(C6-Czo aryl), -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl), (C 1 -C 6 alkyl diyl)-(C 6 -C 20 aryl), -(
  • R a is independently selected from H, C 1 -C 6 alkyl, phenyl, and benzyl, wherein phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, -CN, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, and C 2 -C 12 alkynyl;
  • R b is independently selected from H, OH, C 1 -C 6 alkyl, phenyl, and benzyl, wherein phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, -CN, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, and C 2 -C 12 alkynyl; n is 0, 1, 2, 3, or 4,
  • X 3 is selected from the group consisting of a bond, O, C 1 -C 12 alkyldiyl, C 2 -C 12 alkenyldiyl, C 2 -C 12 alkynyldiyl, C 1 -C 12 heteroalkyldiyl, -(C 1 -C 6 alkyldiyl)-(C 6 -C 20 aryldiyl)-, -(C 1 -C 6 alkyl diyl)NR a -, -(C 1 -C 6 alkyldiyl)O-, -(C 1 -C 6 alkyldiyl)-(C 3 -C 20 carbocyclyldiyl)-, -(C 1 -C 6 alkyl diyl)-(C2-C 20 heterocyclyldiyl)-, -(C 1 -C 6 alkyldiyl)-(C 1 -C 20 heteroaryldiyl
  • X 4 is selected from the group consisting of H, C 1 -C 12 heteroalkyl, -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryl), -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl);
  • X 5 is selected from the group consisting of C 1 -C 12 alkyldiyl, C 2 -C 12 alkenyldiyl, C 2 -C 12 alkynyldiyl, C 1 -C 12 heteroalkyldiyl, -(C 1 -C 6 alkyldiyl)-(C 6 -C 20 aryldiyl), -(C 1 -C 6 alkyldiyl)-(C 6 -C 20 atyldiyl)-O-(C 2 -C 20 heterocyclyl), -(C 1 -C 6 alkyldiyl)NR a -, -(C 1 -C 6 alkyldiyl)O-, -(C 1 -C 6 alkyldiyl)-(C 3 -C 20 carb ocyclyl diyl), -C 1 -C 6 alkyl diyl)-(C 2 -C 20 heterocycly
  • L is the antibody linker
  • Str 1 is a stretcher unit covalently attached to Z
  • PEP is a protease-cleavable, peptide unit covalently attached to Str 1 and IM;
  • IM is an immolative unit covalently attached the isoindolinone-glutarimide moiety; n is 0 or 1; and m is 0 or 1
  • An exemplary embodiment of the isoindolinone-glutarimide linker compound includes wherein PEP-IM is selected from the structures: wherein ** indicates the point of attachment to the isoindolinone-glutarimide moiety.
  • An exemplary embodiment of the isoindolinone-glutarimide linker compound includes wherein Z-Str 1 has the structure: wherein:
  • An exemplary embodiment of the isoindolinone-glutarimide linker compound includes wherein R 1 is selected from -(CH 2 )s-, and -CH 2 CH 2 -.
  • An exemplary embodiment of the isoindolinone-glutarimide linker compound includes wherein R 1 is selected from Cg-C 20 aryldiyl, (C 6 -C 20 aryldiyl)-(C 1 -C 12 alkyldiyl), and (C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl diyl).
  • An exemplary embodiment of the isoindolinone-glutarimide linker compound includes wherein Z-Str 1 has the structure: wherein:
  • L 1 is selected from a bond, C 1 -C 12 alkyldiyl, and C 1 -C 40 heteroalkyl diyl;
  • L la is selected from an amino acid, amino, hydroxyl, halide, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof; o is 0, 1, or 2;
  • L 2 is selected from a bond, C 1 -C 12 alkyldiyl, and C 1 -C 40 heteroalkyl diyl;
  • An exemplary embodiment of the isoindolinone-glutarimide linker compound includes wherein L 1 or L 2 comprise a solubilizing unit selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), C 1 -C 40 heteroalkyl, and a glycoside, or combinations thereof.
  • L 1 or L 2 comprise a solubilizing unit selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), C 1 -C 40 heteroalkyl, and a glycoside, or combinations thereof.
  • the invention includes all reasonable combinations, and permutations of the features, of the Formula II embodiments.
  • the linker units comprise functional groups and subunits which affect stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy properties of the antibody conjugates.
  • the linker unit includes a reactive functional group which reacts, i e. conjugates, with a reactive functional group of the antibody.
  • a nucleophilic group such as a lysine side chain amino of the antibody reacts with an electrophilic reactive functional group of the isoindolinone-glutarimide-linker, IG-L compound to form the antibody conjugate.
  • a cysteine thiol of the antibody reacts with a maleimide or bromoacetamide group of the IG-L compound to form the antibody conjugate.
  • Considerations for the design of the antibody conjugates of the invention include: (1) preventing the premature release of the isoindolinone-glutarimide (IG) moiety during in vivo circulation and (2) ensuring that a biologically active form of the IG moiety is released at the desired site of action at an adequate rate.
  • IG isoindolinone-glutarimide
  • the complex structure of the antibody conjugate together with its functional properties requires careful design and selection of every component of the molecule including antibody, conjugation site, linker structure, and the IG moiety.
  • the linker determines the mechanism and rate of isoindolinone-glutarimide moiety release.
  • the linker unit (L) may be cleavable or non-cleavable.
  • Cleavable linker units may include a peptide sequence which is a substrate for certain proteases such as Cathepsins which recognize and cleave the peptide linker unit, separating the isoindolinone-glutarimide moiety from the antibody (Caculitan NG, et al (2017) Cancer Res. 77(24):7027-7037).
  • Cleavable linker units may include labile functionality such as an acid-sensitive disulfide group (Kellogg, BA et al (2011) Bioconjugate Chem. 22, 717-727; Gört, A. D. et al (2011) Clin. Cancer Res. 17, 6417-6427; Pillow, T., et al (2017) Chem. Sci. 8, 366-370, Zhang D, et al (2016) ACS Med Chem Lett. 7(1 l):988-993).
  • labile functionality such as an acid-sensitive disulfide group
  • the linker is non-cleavable under physiological conditions .
  • physiological conditions refers to a temperature range of 20-40 degrees C 6 lsius , atmospheric pressure (i.e. , 1 atm) , a pH of about 6 to about 8 , and the one or more physiological enzymes, proteases, acids , and bases.
  • the linker comprises a trivalent, branch point as part of an amino acid unit (e.g., lysine) wherein additional linker units are attached via the side chain amine of lysine or linked to other sites of an amino acid unit (US 11, 173,214).
  • an amino acid unit e.g., lysine
  • additional linker units are attached via the side chain amine of lysine or linked to other sites of an amino acid unit (US 11, 173,214).
  • a similar motif could be utilized with a glutamic acid of an amino acid unit.
  • An exemplary additional linker unit is a monovalent solubilizing unit such as one or more units of polyglycine, poly sarcosine, polyethyleneoxy (PEG), C 1 -C 40 heteroalkyl, and a glycoside, or combinations thereof.
  • the solubilizing unit may bear a group at the terminus such as an amino acid, amino, hydroxyl, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof.
  • a group at the terminus such as an amino acid, amino, hydroxyl, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof.
  • a solubilizing unit is a polysarcosine with one of the following structures:
  • an amino acid unit or peptide unit comprises one or more amino acids selected from the group consisting of glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, sarcosine, and beta-alanine.
  • the invention includes an amino acid unit or a peptide linking unit, i.e. L or linker, between the antibody and the IG moiety, comprising a peptide comprising a linear sequence of specific amino acid residues which can be selectively cleaved by a protease such as a cathepsin, caspase, a tumor-associated elastase enzyme or an enzyme with proteaselike or elastase-like activity.
  • the peptide radical may be two to about twelve amino acids. Enzymatic cleavage of a bond within the peptide linker releases an active form of the IG moiety.
  • lysosomal proteases such as cathepsin and plasmin which may be present at elevated levels in certain tumor tissues.
  • the lysosomal enzyme can be, for example, cathepsin B, P-glucuronidase, or P-galactosidase.
  • a cleavable peptide of a peptide linker unit can be selected from tetrapeptides such as Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, and Gly-Gly-Phe-Gly; tripeptides such as Glu-Val-Cit, and Ala- Ala-Ala; and dipeptides such as Val-Cit, Vai-Ala, Ala-Ala, and Phe-Lys.
  • the linker provides sufficient stability of the antibody conjugate in biological media, e.g. culture medium or serum and, at the same time, the desired intracellular action within tumor tissue as a result of its specific enzymatic or hydrolytic cleavability with release of the IG moiety, i.e. “isoindolinone-glutarimide moiety”.
  • the enzymatic activity of a protease, cathepsin, or elastase can catalyze cleavage of a covalent bond of the antibody conjugate under physiological conditions
  • the enzymatic activity being the expression product of cells associated with tumor tissue
  • the enzymatic activity on the cleavage site of the targeting peptide converts the antibody conjugate to an active IG drug free of targeting antibody and linking group.
  • the cleavage site may be specifically recognized by the enzyme Cathepsin or elastase may catalyze the cleavage of a specific peptidic bond between the C-terminal amino acid residue of the specific peptide and the IG moiety of the antibody conjugate.
  • the invention includes a linking unit, i.e. L or linker, between the antibody and the IG moiety, comprising a substrate for glucuronidase (Jeffrey SC, et al (2006) Bioconjug Chem. 17(3):831-40; US11,413,353; US11,173,214), or sulfatase (Bargh ID, et al (2020) Chem Sei. 1 1 (9):2375-2380) cleavage.
  • L includes a Glue unit and comprises a formula selected from:
  • Specific cleavage of the antibody conjugate takes advantage of the presence of tumor infiltrating cells of the immune system and leukocyte- secreted enzymes, to promote the activation of an anticancer drug at the tumor site.
  • Reactive electrophilic reactive functional groups (Q in Formula II) suitable for the isoindolinone-glutarimide linker compound (IG-L) include, but are not limited to, N- hydroxysuccinimidyl (NHS) esters and N-hydroxysulfosuccinimidyl (sulfo-NHS) esters (amine reactive); carbodiimides (amine and carboxyl reactive); hydroxymethyl phosphines (amine reactive); mal eimides (thiol reactive); halogenated acetamides such as A'-iodoacetamides (thiol reactive); aryl azides (primary amine reactive); fluorinated aryl azides (reactive via carbon- hydrogen (C-H) insertion); pentafluorophenyl (PFP) esters (amine reactive); tetrafluorophenyl (TFP) and sulfotetrafluorophenyl (STP) esters (amine reactive); imidoest
  • linkers such as those comprising peptide units and substrates for protease may be labile in the blood stream, thereby releasing unacceptable amounts of the drug prior to internalization in a target cell (Khot, A et al (2015) Bioanafysis 7(13): 1633-1648)
  • Other linkers may provide stability in the bloodstream, but intracellular release effectiveness may be negatively impacted.
  • Linkers that provide for desired intracellular release may have poor stability in the bloodstream.
  • the amount of adjuvant/drug moiety loaded on the antibody i.e. drug loading
  • Aggregate formation may be correlated to the number of equivalents of drug moieties conjugated to the antibody. Under high drug loading, formed aggregates must be removed for therapeutic applications. As a result, drug loading-mediated aggregate formation decreases antibody conjugate yield and can render process scale-up difficult
  • the invention includes all reasonable combinations, and permutations of the features, of the Formula Ila and Hb embodiments.
  • the isoindolinone-glutarimide linker compound has one of the following structures:
  • R lb is an isoindolinone-glutarimide moiety (IG);
  • R 2b has one of the following structures:
  • L lg has one of the following structures:
  • the isoindolinone-glutarimide linker compound has one of the following structures:
  • the isoindolinone-glutarimide linker compound has one of the following structures:
  • the isoindolinone-glutarimide linker compound has one of the following structures:
  • IG-L isoindolinone-glutarimide linker compounds
  • the isoindolinone-glutarimide antibody conjugates (IGAC) of the invention induce target-specific protein degradation. Tumor targeting brings specificity to minimize off-target effects
  • the isoindolinone-glutarimide antibody conjugates (IGAC) of the invention comprise an isoindolinone-glutarimide moiety covalently attached to an antibody by an antibody linker, wherein the antibody binds to a tumor-associated antigen or cell-surface receptor.
  • L is the antibody linker
  • IG is the isoindolinone-glutarimide moiety; and p is an integer from 1 to 12.
  • An exemplary embodiment of the IGAC of Formula I includes wherein a phenolic oxygen of the isoindolinone-glutarimide moiety is attached to the antibody linker.
  • An exemplary embodiment of the IGAC of Formula I includes wherein the nitrogen of the glutarimide group is attached to the antibody linker.
  • An exemplary embodiment of the IGAC of Formula I includes wherein a solubilizing unit selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), C 1 -C 40 heteroalkyl, and a glycoside, or combinations thereof is attached to: (1) a phenolic oxygen, or (2) the nitrogen of the glutarimide group of the isoindolinone-glutarimide moiety.
  • a solubilizing unit selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), C 1 -C 40 heteroalkyl, and a glycoside, or combinations thereof is attached to: (1) a phenolic oxygen, or (2) the nitrogen of the glutarimide group of the isoindolinone-glutarimide moiety.
  • An exemplary embodiment of the IGAC of Formula I includes wherein the terminus of the solubilizing unit is a group selected from an amino acid, amino, hydroxyl, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof.
  • An exemplary embodiment of the IGAC is selected from Formulae la and lb: wherein: m is 0, 1 or 2;
  • X 2 is independently selected from the group consisting of F, Cl, Br, I, -CN, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 1 -C 12 heteroalkyl, -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl), -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl), -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryl), -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryl), -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryl), -(C 1 -C 12 heteroalkyldi
  • R b is independently selected from H, OH, C 1 -C 6 alkyl, phenyl, and benzyl, wherein phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, -CN, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, and C 2 -C 12 alkynyl; n is 0, 1, 2, 3, or 4;
  • X 3 is selected from the group consisting of a bond, O, C 1 -C 12 alkyldiyl, C 2 -C 12 alkenyldiyl, C 2 -C 12 alkynyldiyl, C 1 -C 12 heteroalkyldiyl, -(C 1 -C 6 alkyldiyl)-(C 6 -C 20 aryldiyl)-, -(C 1 -C 6 alkyl diyl)NR a -, -(C 1 -C 6 alkyldiyl)O-, -(C 1 -C 6 alkyldiyl)-(C 3 -C 20 carbocyclyldiyl)-, -(C 1 -C 6 alkyl diyl)-(C2-C 20 heterocyclyldiyl)-, -(C 1 -C 6 alkyldiyl)-(C 1 -C 20 heteroaryldiyl
  • X 4 is selected from the group consisting of H, C 1 -C 12 heteroalkyl, -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryl), -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl);
  • X 5 is selected from the group consisting of a bond, C 1 -C 12 alkyldiyl, C 2 -C 12 alkenyldiyl, C 2 -C 12 alkynyldiyl, C 1 -C 12 heteroalkyldiyl, -(C 1 -C 6 alkyldiyl)-(C 6 -C 20 aryldiyl), -(C 1 -C 6 alkyldiyl)-(C 6 -C 20 aryldiyl)-0-(C 2 -C 20 heterocyclyl), -(C 1 -C 6 alkyldiyl)NR a -, -(C 1 -C 6 alkyldiyl)O-, -(C 1 -C 6 alkyldiyl)-(C 3 -C 20 carbocyclyldiyl), -C 1 -C 6 alkyl diyl)-(C 2 -C
  • An exemplary embodiment of the IGAC of Formula I includes wherein the antibody linker is covalently attached to a cysteine amino acid of the antibody.
  • An exemplary embodiment of the IGAC of Formula I includes wherein the antibody is a cysteine-engineered antibody.
  • An exemplary embodiment of the IGAC of Formulae la and lb includes wherein m is 0.
  • An exemplary embodiment of the IGAC of Formulae la and lb includes wherein m is 1 .
  • An exemplary embodiment of the IGAC of Formulae la and lb includes wherein m is 2.
  • An exemplary embodiment of the IGAC of Formulae la and lb includes wherein X 1 is CH 2 .
  • An exemplary embodiment of the IGAC of Formulae la and lb includes wherein X 2 is selected from the group consisting of C 1 -C 12 heteroalkyl, -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryl), and -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl).
  • An exemplary embodiment of the IGAC of Formulae la and lb includes wherein X 2 is -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryl).
  • An exemplary embodiment of the IGAC of Formulae la and lb includes wherein X 2 is: wherein:
  • An exemplary embodiment of the IGAC of Formulae la includes wherein X 3 is O.
  • An exemplary embodiment of the IGAC of Formulae la includes wherein X 3 is -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyldiyl).
  • An exemplary embodiment of the IGAC of Formulae la includes wherein X 3 is selected from -(C 1 -C 12 heteroalkyl diyl)-(C 6 -C 20 aryldiyl)-O- -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-N(R a )-, and -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyldiyl).
  • An exemplary embodiment of the IGAC of Formulae la includes wherein X 3 is selected from the structures: and wherein:
  • R 6 is independently selected from F, Cl, Br, I, -CN, OH, -O-(C 1 -C 12 alkyl), C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 1 -C 12 heteroalkyl, C 6 -C 20 aryl, C 3 -C 20 carbocyclyl, C 2 -C 20 heterocyclyl, and C 1 -C 20 heteroaryl; nl is 1, 2, 3, or 4;
  • Y 1 is selected from CF2 and NH
  • Y 2 is selected from NH, O, and CH 2 .
  • An exemplary embodiment of the IGAC of Formulae la includes wherein X 3 is selected from: and wherein:
  • R 4 is selected from H and C 1 -C 12 alkyl
  • R 6 is independently selected from F, Cl, Br, I, -CN, OH, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, and C 1 -C 12 heteroalkyl; and q is selected from 0, 1, 2, 3, and 4.
  • An exemplary embodiment of the IGAC of Formulae la includes wherein X 3 is -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryl).
  • An exemplary embodiment of the IGAC of Formulae la includes wherein X 3 is selected from: wherein:
  • R 6 is independently selected from F, Cl, Br, I, -CN, OH, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, and C 1 -C 12 heteroalkyl; and q is selected from 0, 1, 2, 3, and 4.
  • An exemplary embodiment of the IGAC of Formula la includes wherein X 2 is -OH and n is 1 .
  • An exemplary embodiment of the IGAC of Formula la includes wherein X 4 is H.
  • An exemplary embodiment of the IGAC of Formula la includes wherein X 4 is selected from the group consisting of C 1 -C 12 heteroalkyl, -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryl), -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl), and -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-0-(C 2 -C 20 heterocyclyl).
  • An exemplary embodiment of the IGAC of Formula la includes wherein X 4 is -(C 1 -C 12 heteroalkyldiyl)-(C 6 -C 20 aryldiyl)-0-(C 2 -C 20 heterocyclyl), and C 2 -C 20 heterocyclyl is a glucuronide.
  • An exemplary embodiment of the IGAC of Formula la includes wherein X 4 of formula la is selected from the formulae:
  • R is selected from H, C 1 -C 6 alkyl, and O-( C 1 -C 6 alkyl); and * indicates the point of attachment to IG.
  • An exemplary embodiment of the IGAC of Formula I includes Formula lb:
  • An exemplary embodiment of the IGAC of Formula lb includes wherein n is 2, 3 or 4 and one of X 2 is -OH.
  • An exemplary embodiment of the IGAC of Formula lb includes wherein X 5 is selected from -(C 1 -C 12 heteroalkyl diyl )-(C 6 -C 20 aryldiyl) and -(C 1 -C 6 alkyldiyl)-(C 6 -C 20 aryldiyl).
  • Exemplary embodiments of the IGAC of Formula I includes Formulae Ic-g: le;
  • R is independently selected from H, C 1 -C 6 alkyl, and O-( C 1 -C 6 alkyl); n a is an integer from 1 to 5; and Y is CH 2 or O.
  • An exemplary embodiment of the IGAC of Formulas Ic-g includes wherein R is -CH 3 and n a is 1.
  • An exemplary embodiment of the IGAC of Formulas Ic-g includes X 2 is -OH and n is 1.
  • Exemplary embodiments of the IGAC of Formula I include Formulae Ih and li: wherein R is selected from H, C 1 -C 6 alkyl, and O-(C 1 -C 6 alkyl); and X 5a is -(C 1 -C 6 alkyldiyl)-(C 6 -C 20 aryldiyl)-0-(C 2 -C 20 heterocyclyl).
  • An exemplary embodiment of the IGAC of Formulas Ih and li includes wherein C 2 -C 20 heterocyclyl of X 5a is a glucuronide.
  • An exemplary embodiment of the IGAC of Formulas Ih and li includes wherein X 5a is: wherein * indicates the point of attachment.
  • An exemplary embodiment of the IGAC of Formulas Ih and li includes wherein L has the structure: wherein:
  • L 1 is selected from a bond, C 1 -C 12 alkyldiyl, and C 1 -C 40 heteroalkyl diyl;
  • L la is selected from an amino acid, amino, hydroxyl, halide, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof; o is 0, 1, or 2;
  • L 2 is selected from a bond, C 1 -C 12 alkyldiyl, and C 1 -C 40 heteroalkyl diyl; * indicates the point of attachment to a cysteine thiol of Ab; and
  • t is selected from 0, 1, 2, 3, and 4.
  • An exemplary embodiment of the IGAC of Formulae Ij-m includes wherein L 1 is a bond and L la is F.
  • An exemplary embodiment of the IGAC of Formula I includes wherein the antibody linker comprises an immolating group.
  • An exemplary embodiment of the IGAC of Formula I includes wherein the antibody linker comprises a peptide unit.
  • Str is a stretcher unit covalently attached to the antibody
  • PEP is a protease-cleavable, peptide unit covalently attached to Str and IM or IG;
  • IM is an immolative unit covalently attached to IG; n is 0 or 1 ; and m is 0 or 1 .
  • An exemplary embodiment of the IGAC of Formula I includes wherein IG is attached to L by Str.
  • An exemplary embodiment of the IGAC of Formula I includes wherein IG is attached to L by PEP.
  • An exemplary embodiment of the IGAC of Formula I includes wherein IG is attached to L by IM.
  • An exemplary embodiment of the IGAC of Formula I includes wherein Str is a branched linker covalently attached to: (i) the antibody; and (ii) a solubilizing unit comprising a group selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), C 1 -C 40 heteroalkyl, and a glycoside, or combinations thereof, wherein the terminus of the solubilizing unit is a group selected from an amino acid, amino, hydroxyl, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof.
  • An exemplary embodiment of the IGAC of Formula I includes wherein the solubilizing unit and the terminus of the solubilizing unit covalently attached to Str are selected from the structures: wherein * indicates the point of attachment to Str
  • An exemplary embodiment of the IGAC of Formula I includes wherein L is a branched linker and PEP is covalently attached to: (i) Str and IM or IG; and (ii) a solubilizing unit comprises a group selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), C 1 -C 40 heteroalkyl, and a glycoside, or combinations thereof, wherein the terminus of the solubilizing unit is a group selected from an amino acid, amino, hydroxyl, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof.
  • An exemplary embodiment of the IGAC of Formula I includes wherein L is a branched linker and IM is covalently attached to: (i) IG; and (ii) a solubilizing unit comprises a group selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), C 1 -C 40 heteroalkyl, and a glycoside, or combinations thereof, wherein the terminus of the solubilizing unit is a group selected from an amino acid, amino, hydroxyl, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof.
  • An exemplary embodiment of the IGAC of Formula I includes wherein Str has the structure: wherein:
  • An exemplary embodiment of the IGAC of Formula I includes wherein R 1 is selected from -(CH 2 ) 5 -, and -CH 2 CH 2 -.
  • An exemplary embodiment of the IGAC of Formula I includes wherein R 1 is selected from C 6 -C 20 aryldiyl, (C 6 -C 20 aryldiyl)-(C 1 -C 12 alkyldiyl), and (C 6 -C 20 aryldiyl)-(C 1 -C 12 heteroalkyl diyl).
  • An exemplary embodiment of the IGAC of Formula I includes wherein Str is selected from the structure: wherein: one of Y 1 , Y 2 , Y 3 , Y 4 and Y 5 is C-L’-R 1 , another one of Y 1 , Y 2 , Y 3 , Y 4 and Y 5 is C-L 2 - R 2 , and the remaining three of Y 1 , Y 2 , Y 3 , Y 4 and Y 5 are each independently N, C-R 3 , or C-L 3 - R 3a ;
  • R 1 , R 2 , and R 3a each independently comprise one or more moieties selected from an amino acid element, a charged element, a heteroalkylene element, a hydrophilic element, a trigger element, an immolative unit, a polar cap, a isoindolinone-glutarimide moiety, and combinations thereof; provided that at least one of R 1 and R 2 comprises the isoindolinone-glutarimide moiety; each occurrence of R 3 is independently selected from the group consisting of hydrogen, deuterium, alkyl, haloalkyl, halo, alkoxy, haloalkoxy, amino, aminyl, amidyl, aldehyde, hydroxyl, cyano, nitro, thiol, carboxy, carboxyalkyl, alkyl-S(O) 3 H, alkyl-O-P(O) 3 H, alkyl- P(O) 3 H, -O-carboxyalkyl, -O-alkyl
  • R 4a and R 4b are each independently hydrogen, deuterium, halo, or -S-R 4c wherein R 4c is substituted or unsubstituted C 6 -C 10 aryl or substituted or unsubstituted 5-12 membered heteroaryl; and
  • L 1 , L 2 , and L 3 are each independently a linker comprising an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted heteroalkylene, an optionally substituted heteroalkenylene, an optionally substituted heteroalkynylene, a heteroatomic linker, an optionally substituted cycloalkylene, an optionally substituted arylene, an optionally substituted heterocyclylene, an optionally substituted heteroarylene, or combinations thereof; and
  • An exemplary embodiment of the IGAC of Formula I includes wherein Str is selected from the structure: wherein:
  • L 1 is selected from a bond, C 1 -C 12 alkyldiyl, and C 1 -C 40 heteroalkyl diyl;
  • L la is selected from an amino acid, amino, hydroxyl, halide, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof; o is 0, 1, or 2;
  • L 2 is selected from a bond, C 1 -C 12 alkyldiyl, and C 1 -C 40 heteroalkyl diyl;
  • An exemplary embodiment of the IGAC of Formula I includes wherein L 1 or L 2 comprise a solubilizing unit selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), C 1 -C 40 heteroalkyl, and a glycoside, or combinations thereof.
  • L 1 or L 2 comprise a solubilizing unit selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), C 1 -C 40 heteroalkyl, and a glycoside, or combinations thereof.
  • An exemplary embodiment of the IGAC of Formula I includes wherein L 1 or L 2 comprises glucuronic acid having the structure:
  • An exemplary embodiment of the IGAC of Formula I is selected from formulas: wherein * indicates the point of attachment to a cysteine thiol of Ab and ** indicates the point of attachment to IG.
  • An exemplary embodiment of the IGAC of Formula I includes wherein n is 1, m is 1, and PEP-IM has the formula: wherein * indicates the point of attachment to Str and ** indicates the point of attachment to IG;
  • AA is independently selected from a natural or unnatural amino acid side chain, or one or more of AA, and an adjacent nitrogen atom form a 5 -membered ring proline amino acid, and the wavy line indicates a point of attachment;
  • Cyc is selected from C 6 -C 20 aryldiyl and C 1 -C 20 heteroaryldiyl, optionally substituted with one or more groups selected from F, Cl, NO 2 , -OH, -OCH 3 , and glucuronic acid having the structure:
  • R 9 is independently selected from the group consisting of H, C 1 -C 12 alkyl, and - (CH 2 CH 2 O)n-(CH 2 ) m -OH, where m is an integer from 1 to 5, and n is an integer from 2 to 50, or two R 9 groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12, and z is 0 or 1.
  • An exemplary embodiment of the IGAC of Formula I includes wherein AA is independently selected from the side chain of a naturally occurring amino acid.
  • An exemplary embodiment of the IGAC of Formula I includes wherein AA is independently selected from H, -CH 3 , -CH(CH 3 ) 2 , -CFhlGeHs), -CH 2 C(O)NH 2 , -CH 2 CH 2 CO2H, -CH 2 CO2H, -CH 2 CH 2 CH 2 CH 2 NH 2 , -CH 2 CH 2 CH 2 NHC(NH)NH 2 , -CH 2 CH(CH 3 ) 2 , -CH 2 SO 3 H, and -CH 2 CH 2 CH 2 NHC(O)NH 2 ; or two AA form a 5-membered ring proline amino acid.
  • An exemplary embodiment of the IGAC of Formula I includes wherein PEP-IM comprises at least one natural or unnatural amino acid side chain AA substituted with C 1 -C 40 heteroalkyl.
  • An exemplary embodiment of the IGAC of Formula I includes wherein C 1 -C 40 heteroalkyl comprises a solubilizing unit selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside, or combinations thereof
  • An exemplary embodiment of the IGAC of Formula I includes wherein y is 2, PEP is a dipeptide, and PEP-IM has the formula: wherein AA 1 and AA 2 are independently selected from a side chain of a naturally- occurring amino acid.
  • An exemplary embodiment of the IGAC of Formula I includes wherein the dipeptide is selected from ala-ala, val-cit, and phe-ala.
  • An exemplary embodiment of the IGAC of Formula I includes wherein AA 1 is - CH(CH 3 ) 2 , and AA 2 is -CH 2 CH 2 CH 2 NHC(O)NH 2 .
  • An exemplary embodiment of the IGAC of Formula I includes wherein AA 1 and AA 2 are each -CH 3 .
  • An exemplary embodiment of the IGAC of Formula I includes wherein y is 3, PEP is a tripeptide, and PEP-IM has the formula:
  • An exemplary embodiment of the IGAC of Formula I includes wherein y is 4, PEP is a tetrapeptide PEP-IM, and has the formula:
  • An exemplary embodiment of the IGAC of Formula I includes where z is 1 and IM has the formula:
  • ** indicates the point of attachment to IG.
  • An exemplary embodiment of the IGAC of Formula I includes where R 7 has the formula: wherein:
  • R 8 is selected from H and C 1 -C 6 alkyl
  • ** indicates the point of attachment to IG.
  • An exemplary embodiment of the IGAC of Formula I includes where R 8 is H.
  • An exemplary embodiment of the IGAC of Formula I includes where R 8 is -CH 3 .
  • IGAC of Formula I includes wherein IM is selected from the formulae:
  • the isoindolinone-glutarimide antibody conjugates (IGAC) of the invention include those with anti-cancer activity.
  • the IGAC selectively deliver an effective dose of an isoindolinone-glutarimide drug to tumor tissue or hematopoietic cell, whereby greater selectivity (z.e., a lower efficacious dose) may be achieved while increasing the therapeutic index (“therapeutic window”) relative to unconjugated isoindolinone-glutarimide drug.
  • Drug loading in Table 3 is represented as DAR, the number of isoindolinone-glutarimide (IG) moi eties per antibody in an IGAC of Formula I.
  • Drug (IG) loading may range from 1 to about 8 drug moieties (D) per antibody.
  • IGAC of Formula I include mixtures or collections of antibodies conjugated with a range of IG drug moieties, from 1 to about 8.
  • the number of IG drug moieties that can be conjugated to an antibody is limited by the number of reactive or available amino acid side chain residues such as lysine and cysteine.
  • free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein.
  • p may be 1, 2, 3, 4, 5, 6, 7, or 8, and ranges thereof, such as from 1 to 8 or from 2 to 5.
  • Exemplary IGAC of Formula I include, but are not limited to, antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon, R. et al. (2012) Methods in Enzym. 502:123-138).
  • one or more free cysteine residues are already present in an antibody forming intra-chain and inter-chain disulfide bonds (native disulfide groups), without the use of engineering, in which case the existing free, reduced cysteine residues may be used to conjugate the antibody to a drug
  • an antibody is exposed to reducing conditions prior to conjugation of the antibody in order to generate one or more free cysteine residues.
  • p may be limited by the number of attachment sites on the antibody
  • an antibody may have only one or a limited number of cysteine thiol groups, or may have only one or a limited number of sufficiently reactive thiol groups, to which the drug may be attached.
  • one or more lysine amino groups in the antibody may be available and reactive for conjugation with a IG-lmker compound of Formula II.
  • higher drug loading e.g. p >5
  • the average drug loading for an antibody conjugate ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5
  • an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.
  • the loading (drug/antibody ratio) of an antibody conjugate may be controlled in different ways, and for example, by: (i) limiting the molar excess of the IG-linker intermediate compound relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive denaturing conditions for optimized antibody reactivity.
  • the resulting product is a mixture of antibody conjugate compounds with a distribution of one or more IG drug moieties attached to an antibody.
  • the average number of drugs per antibody may be calculated from the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug.
  • Individual IGAC molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, for example hydrophobic interaction chromatography, HIC (McDonagh et al. (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al. (2004) Clin. Cancer Res.
  • a homogeneous antibody conjugate with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.
  • Exemplary embodiments of the isoindolinone-glutarimide antibody conjugates (IGAC) of Formula I are compiled in Table 3. Assessment of IGAC biological activity and other properties may be conducted according to the methods of Example 102.
  • the C 6 ll proliferation dose response IC50 value is an average of repetitive assays C 6 rtain exemplary IGAC were tested for their effects in inhibiting cellular proliferation, including CAL51 ; WSU-DLCL2, NCI-N87 and SKBR3 cell lines.
  • CAL51 is a human breast adenocarcinoma cell line with triple-negative status for expression of estrogen, progesterone and HER2 receptors.
  • WSU-DLCL2 is a human B-C 6 ll non-Hodgkin lymphoma cell line that expresses high levels of CD22.
  • NCI-N87 is a human epithelial cell line established from a gastric carcinoma;
  • SKBR3 is a human epithelial cell line established from a breast adenocarcinoma; both NCI-N87 and SKBR3 cell lines express high levels of HER2 receptor.
  • the invention provides a composition, e g., a pharmaceutically or pharmacologically acceptable composition or formulation, comprising an isoindolinone-glutarimide antibody conjugate (IGAC) composition of the invention as described herein and a pharmaceutically acceptable diluent, vehicle, carrier or excipient.
  • IGAC isoindolinone-glutarimide antibody conjugate
  • the isoindolinone-glutarimide antibody conjugate (IGAC) composition can be the same or different in the pharmaceutical composition, i.e., the composition can comprise IGAC that have the same number of isoindolinone-glutarimide (IG) moieties linked to the same positions on the antibody and/or IGAC that have the same number of (IG) moieties linked to different positions on the antibody, that have different numbers of (IG) moieties linked to the same positions on the antibody, or that have different numbers of (IG) moieties linked to different positions on the antibody.
  • a pharmaceutical composition comprises a mixture of the IGAC, wherein the average drug (IG) loading per antibody in the mixture of antibody conjugate compounds is about 2 to about 8.
  • An IGAC of the invention can have an average IG to antibody ratio (DAR) of about 0.4 to about 10.
  • DAR average IG to antibody ratio
  • the average number of IG moieties per antibody (DAR) in preparations of IGAC from conjugation reactions may be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC.
  • IGAC immunoglobulin-like IGAC
  • separation, purification, and characterization of homogeneous IGAC where p is a certain value from antibody drug conjugates (ADC) with other drug loadings may be achieved by purification means such as reverse phase HPLC or electrophoresis.
  • An isoindolinone-glutarimide antibody conjugate (IGAC) composition can be formulated for parenteral administration, such as intradermal, subcutaneous, intramuscular (IM), or intravenous (IV) injections, infusion, or administration into a body cavity or lumen of an organ.
  • the IGAC as a pharmaceutical composition can be injected into otherwise placed into a specific site of the body, such as a tumor.
  • Compositions for injection will commonly comprise a solution of the IGAC dissolved in a pharmaceutically acceptable carrier Among the acceptable vehicles and solvents that can be employed are water and an isotonic solution of one or more salts such as sodium chloride, e.g., Ringer's solution.
  • sterile fixed oils can conventionally be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed, including synthetic monoglycerides or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of inj ectables.
  • These pharmaceutical compositions desirably are sterile and generally free of undesirable matter.
  • These pharmaceutical compositions can be made sterilized by conventional, well known sterilization techniques.
  • the pharmaceutical compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • the pharmaceutical composition may contain any suitable concentration of the IGAC .
  • concentration of the IGAC in the pharmaceutical composition can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs.
  • concentration of IGAC in a solution formulation for injection will range from about 0.1% (w/w) to about 10% (w/w).
  • the isoindolinone-glutarimide antibody conjugate (IGAC) compositions of the invention may be useful in the treatment of diseases and disorders such as cancer.
  • the IGAC direct a tumor-associated antigen-binding antibody to a cell that expresses the antigen and deliver a cereblon-degrading moiety to the target cell.
  • a target protein such as GSPT1 is ubiquitinated and subsequently degraded
  • the invention provides a method for treating cancer with a pharmaceutical composition of the IGAC.
  • the method includes administering a therapeutically effective amount of an antibody conjugate composition as described herein to a subject in need thereof, such as a patient that has cancer and is in need of treatment for the cancer.
  • the method includes administering a therapeutically effective amount of an IGAC selected from Table 3.
  • the IGAC include those with anticancer activity ( Figures 1-4).
  • the IGAC selectively delivers an effective dose of an active form of the isoindolinone- glutarimide protein target degrader moiety to tumor tissue, whereby greater selectivity (i.e., a lower efficacious dose) may be achieved while increasing the therapeutic index (“therapeutic window”) relative to an unconjugated protein target degrader compound
  • the IGAC may be used to treat various hyperproliferative diseases or disorders, e.g. characterized by the overexpression of a tumor antigen.
  • hyperproliferative disorders include benign or malignant solid tumors and hematological disorders such as leukemia and lymphoid malignancies.
  • cancers to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia or lymphoid malignancies including acute myeloid leukemia, squamous cell cancer, epithelial squamous cell cancer, lung cancer including smallcell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.
  • carcinoma lymphoma, blastoma, sarcoma,
  • the IGAC may be useful in therapy to treat solid tumors such as lung cancer; non-small cell lung cancer, squamous cell lung cancer, small cell lung cancer, breast cancer, and neuroendocrine cancers such as neuroendocrine prostate cancer, castration-resistant neuroendocrine prostate cancer (NEPC) and lung neuroendocrine tumors.
  • the IGAC may be useful in therapy to treat blood-borne hematological cancers such as leukemias; acute myelogenous leukemia (AML) and myelomas; multiple myeloma (MM).
  • an IGAC for use as a medicament is provided.
  • the invention provides an IGAC for use in a method of treating an individual comprising administering to the individual an effective amount of the antibody conjugate composition in a pharmaceutical composition.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein
  • the invention provides for the use of an IGAC in the manufacture or preparation of a medicament.
  • the medicament is for treatment of cancer, the method comprising administering to an individual having cancer an effective amount of the medicament.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein.
  • Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • the IGAC dose can range from about 5 mg/kg (body weight) to about 50 mg/kg, from about 10 pg/kg to about 5 mg/kg, or from about 100 pg/kg to about 1 mg/kg.
  • the IGAC dose can be about 100, 200, 300, 400, or 500 pg/kg.
  • the IGAC dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg.
  • the IGAC dose can also be outside of these ranges, depending on the particular conjugate as well as the type and severity of the cancer or disorder being treated. Frequency of administration can range from a single dose to multiple doses per week, or more frequently. In some embodiments, the IGAC is administered from about once per month to about five times per week. In some embodiments, the IGAC is administered once per week.
  • the IGAC can be used either alone or in combination with other therapeutic agents in a therapy regimen.
  • IGAC may be administered concurrently in a regimen with one or more other drugs during the same treatment cycle, on the same day of treatment as the one or more other drugs, and, optionally, at the same time as the one or more other drugs.
  • the concurrently administered drugs are each administered on day-1 of a 3-week cycle.
  • an IGAC may be co-administered with at least one additional therapeutic agent, such as a chemotherapeutic agent.
  • Such combination therapies encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the IGAC can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent.
  • IGAC can also be used in combination with radiation therapy.
  • the reaction mixture was poured into water (50.0 mL) and stirred for 10 min, then filtered to give a filter cake, the filter cake was concentrated to give a residue.
  • the residue was purified by Prep- HPLC (column: Waters Xbridge 150*25mm* 5um;mobile phase: [water(FA)-ACN];B%: 11%- 41%,10min) to afford l-(3-chloro-4-methylphenyl)-3-((2-(2,6-dioxopiperidin-3-yl)-l- oxoisoindolin-5-yl)methyl)urea (119.12 mg, 267 umol, 29% yield, 99% purity) as a yellow solid.
  • Step B Preparation of Int Is Int 1r
  • Int Ir (1.80 g, 4.86 mmol, 1.00 eq) in tetrahydrofuran (100 mL) and methanol (10.0 mL) was added palladium carbon (180 mg, 10% purity) at nitrogen atmosphere.
  • the mixture was stirred under hydrogen atmosphere at 25 °C for 12 h.
  • the reaction was filtered to give a filter cake.
  • the reaction was diluted with water (50.0 mL) and extracted with ethyl acetate (3 x 50.0 mL). The combined organic layer was washed with brine (50.0 mL) and dried over anhydrous sodium sulfate, filtered and concentrated to give a residue.
  • the mixture was filtered to give a filtrate.
  • the filtrate was purified by reversed phase-HPLC (column: Shim-pack C 1 8 150*25*10um, mobile phase: [water(0.1% formic acid)- acetonitrile]) to afford 2-(2,6- dioxopiperidin-3-yl)-l-oxoisoindoline-5-carbaldehyde, Int Iv, (120 mg, 441 umol, 28% yield) as a yellow solid.
  • Step D Preparation of Compound IG-16
  • 3-(6-(aminomethyl)-l-oxoisoindolin-2-yl)piperidine-2, 6-dione, Int DD, (100 mg, 366 umol, 1.00 eq) in dimethyl formamide (3.00 mL) was added triethylamine (55.5 mg, 549 umol, 76.4 uL, 1.50 eq) and 2-chloro-4-isocyanato-l -methylbenzene (73.6 mg, 439 umol, 1.20 eq) .
  • the mixture was stirred at 25°C for 2 h.
  • the mixture was concentrated in vacuum to give a residue.
  • Step D Preparation of Int 2g and Int 2h
  • the filter cake was diluted with water (10.0 mL).
  • the residue was purified by reversed-phase HPLC(column: Phenom enex luna C 18 150*25mm* 10um;mobile phase: [water(FA)-ACN];B%: 7%- 37%,10min),and the fraction was lyophilized to afford a mixture of 5-amino-2-(2,6-dioxopiperidin- 3-yl)-6-hydroxyisoindoline-l, 3-dione, Int 2g, and 4-amino-2-(2,6-dioxopiperidin-3-yl)-5- hydroxyisoindoline- 1,3 -dione, Int 2h, (200 mg, 318.07 umol,) as a yellow solid.
  • 2-chloro-4-methoxypyrimidine (68.5 mg, 474 umol, 1.00 eq), dicyclohexyl-(2-(2,6-dimethoxyphenyl)phenyl)phosphane (S-Phos) (38.9 mg, 94 7 umol, 0.20 eq) and tris(dibenzylidenethyl acetoacetate)dipalladium(O) (43.38 mg, 47.37 umol, 0.1 eq) were added and the mixture was stirred at 60 °C for 3 h. The mixture was diluted with ethyl acetate (5 mL) and washed with brine (3 > ⁇ 2 mL).
  • the filter cake was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, Eluent of 0-30% ethyl acetate/petroleum ether gradient @ 100 mL/min) to give methyl 4-hydroxy-2-methyl-3- nitrobenzoate, Int 2o, (1 .50 g, 7. 10 mmol, 24% yield) as a yellow solid.
  • the reaction mixture was concentrated under reduced pressure to give a residue
  • the residue was purified by reversed-phase HPLC(column: Waters Xbridge 150 * 25 mm * 5 um; mobile phase: [water( NHiHCChl-ACN]; B%: 32% - 62%, 9 min).
  • the desired fraction was lyophilized to afford 3-(4-amino-6-methoxy-l- oxoisoindolin-2-yl)piperidine-2, 6-dione, Int 2y, (100 mg, 200 umol, 8% yield) as a white solid.
  • Methyl 6-fluoro-2-methyl-3-nitro-benzoate (1.00 g, 4.69 mmol, 1.00 eq) was added into a solution of sodium methoxide (507 mg, 9.38 mmol, 2.00 eq) in methanol (10.0 mL). The mixture was stirred at 25 °C for 12 h. The mixture was concentrated to give a residue. The mixture was concentrated to afford methyl 6-methoxy-2-methyl-3-nitro-benzoate, Int 2z, (980 mg, 4.35 mmol, 93% yield) as a yellow oily substance.

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Abstract

L'invention concerne des conjugués d'anticorps de formule I comprenant un anticorps lié par conjugaison à une ou plusieurs fractions isoindolinone-glutarimide. L'invention concerne également des compositions intermédiaires dérivées d'isoindolinone-glutarimide comprenant un groupe fonctionnel réactif. De telles compositions intermédiaires sont des substrats appropriés pour la formation des conjugués d'anticorps par l'intermédiaire d'un lieur ou d'une fraction de liaison. L'invention concerne en outre des méthodes de traitement de maladies et de troubles tels que le cancer avec les conjugués d'anticorps.
PCT/US2024/029822 2023-05-18 2024-05-17 Conjugués d'anticorps isoindolinone-glutarimide et leurs utilisations WO2024238872A2 (fr)

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US20200199247A1 (en) * 2017-06-07 2020-06-25 Silverback Therapeutics, Inc. Antibody conjugates of immune-modulatory compounds and uses thereof
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