WO2023107674A2 - Stapled peptide-antibody conjugates (spacs) and uses thereof - Google Patents

Stapled peptide-antibody conjugates (spacs) and uses thereof Download PDF

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Publication number
WO2023107674A2
WO2023107674A2 PCT/US2022/052358 US2022052358W WO2023107674A2 WO 2023107674 A2 WO2023107674 A2 WO 2023107674A2 US 2022052358 W US2022052358 W US 2022052358W WO 2023107674 A2 WO2023107674 A2 WO 2023107674A2
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Prior art keywords
spac
certain embodiments
stapled
antibody
peptide
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PCT/US2022/052358
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English (en)
French (fr)
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WO2023107674A3 (en
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Rida MOURTADA
Daniel T. COHEN
John Ernest Vallarta BAJACAN
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Lytica Therapeutics Inc
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Lytica Therapeutics Inc
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Priority to IL312929A priority Critical patent/IL312929A/en
Priority to CA3238784A priority patent/CA3238784A1/en
Priority to EP22905164.4A priority patent/EP4444736A2/en
Priority to AU2022407066A priority patent/AU2022407066A1/en
Priority to JP2024534450A priority patent/JP2024545161A/ja
Priority to KR1020247023088A priority patent/KR20240125945A/ko
Priority to CN202280090496.9A priority patent/CN119110805A/zh
Priority to MX2024007006A priority patent/MX2024007006A/es
Publication of WO2023107674A2 publication Critical patent/WO2023107674A2/en
Publication of WO2023107674A3 publication Critical patent/WO2023107674A3/en
Anticipated expiration legal-status Critical
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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
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • ADCs are described in, e.g., Drago et al., Nature Reviews Clinical Oncology, 2021, vol.18, 327–344; Baah et al., Molecules, 2021, 26(10), 2943; Khongorzul et al., Mol. Cancer Res., 2020, 18(1):3-19; and Beck et al., Nature Reviews Drug Discovery, 2017, vol.16, 315–337.
  • Described herein is a new class of therapeutic agents that resolves many of the issues with current antibody-drug conjugates (ADCs) and expands the payload beyond the current, highly cytotoxic agents. Relying on a unique class of peptides known as stapled peptides, the conjugates described herein have distinct advantages without many of the liabilities of current ADCs.
  • Stapled peptides are synthetic peptides comprising at least two unnatural amino acids connected via a crosslink (i.e., “staple”).
  • a stapled peptide can comprise at least two unnatural olefin-bearing amino acids cyclized via metathesis to form an internal hydrocarbon crosslink (i.e., “hydrocarbon staple”).
  • This process of cyclization, or “stapling,” can help fold the peptide in an alpha-helical confirmation or other secondary structure, recapturing the natural three-dimensional orientation of protein structures to mimic their biological functions.
  • stapled peptide technology can be found in, e.g., International PCT Application Publication Nos. WO 2017/004591, published January 5, 2017; WO 2019/018499, published January 24, 2019; and WO 2021/126827, published June 24, 2021, the entire contents of each of which are incorporated herein by reference.
  • the low cell penetrance of some stapled peptides can be an asset rather than a liability.
  • a stapled peptide to an antibody or antigen-binding fragment thereof, one can take advantage of the conjugate’s internalization to deliver a cargo/payload (i.e., a stapled peptide) into a cell that would otherwise be inactive or have low activity outside of the cell.
  • a cargo/payload i.e., a stapled peptide
  • the ability of antibodies to aggregate payloads at the plasma membrane of receptor-positive cells may allow for targeted delivery of otherwise non-specifically lytic stapled peptides.
  • stapled peptide-antibody conjugates comprising a stapled peptide conjugated to an antibody or antigen-binding fragment thereof.
  • the stapled peptide is conjugated to the antibody or antigen-binding fragment thereof via a linker.
  • the SPACs provided herein can be used to deliver stapled peptides to cells (e.g., cancer cells, bacterial cells) with relatively high selectivity and/or relatively low off-target toxicity.
  • SPACs provided herein comprise an antibody or antigen-binding fragment thereof.
  • Antibodies including intact antibodies and antigen-binding fragments), variants, and derivatives thereof include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), single-domain antibodies (sdAb), epitope-binding fragments (e.g., Fab, Fab', and F(ab')2, Fd, Fvs, single- chain Fvs (scFv), rlgG, single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing either a VL or VH domain, fragments produced by an Fab expression library), and anti-idiotypic (anti-Id) antibodies.
  • Antibody molecules of the conjugates can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • the antibody is a monoclonal antibody (mAb) or antigen- binding fragment thereof.
  • the antibody is an anti-cancer antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof is directed against a target antigen expressed on a cancer cell (e.g., expressed specifically on a cancer cell).
  • the antibody is a homolog of an antibody or antigen-binding fragment thereof described herein.
  • the antibody is an antibody-drug conjugate (ADC), or antigen binding fragment thereof.
  • the SPAC comprises an antibody or antigen binding fragment thereof conjugated to dual payloads: (i) a stapled peptide or pharmaceutically acceptable salt thereof; and (ii) a second agent (i.e., the “drug” component of the antibody-drug conjugate).
  • the stapled peptide component of the SPAC may be any stapled peptide (e.g., any singly stapled, doubly stapled, multiply stapled, or stitched peptide).
  • the stapled peptide is a stapled anti-cancer peptide.
  • the stapled peptide is a stapled antimicrobial peptide (StAMP).
  • the stapled peptide is a stapled Magainin peptide (e.g., stapled Magainin II peptide).
  • the stapled peptide is a stapled Esculentin peptide (e.g., stapled Esculentin-1A peptide).
  • the stapled peptide is an inhibitor of a protein-protein interaction (PPI).
  • the stapled peptide is an inhibitor of a BCL-2 family member protein (e.g., BCL-xL, BCL-2, BCL-W, or MCL1).
  • the stapled peptide is an activator of a BCL-2 family member protein effector (e.g., BAX, BAK, or BOK).
  • the stapled peptide is a stapled BCL-2-interacting mediator of cell death (BIM) peptide.
  • the stapled peptide is a ⁇ - catenin inhibitor (e.g., an inhibitor of Wnt/ ⁇ -catenin signaling).
  • the stapled peptide is an MDM2 and/or MDMX inhibitor (e.g., inhibits the binding of MDM2 and/or MDMX to p53).
  • MDM2 and/or MDMX inhibitor e.g., inhibits the binding of MDM2 and/or MDMX to p53.
  • Other examples of stapled peptides are provided herein.
  • the antibody or antigen-binding fragment thereof is directly conjugated to the stapled peptide (e.g., via a bond).
  • the antibody or antigen-binding fragment thereof is conjugated to the stapled peptide via a linker.
  • the linker is a cleavable linker (e.g., a pH cleavable linker, or a linker cleavable by a protease, esterase, or intracellular disulfide reduction).
  • the linker is a peptidic linker (e.g., a cleavable peptidic linker).
  • pharmaceutical compositions comprising a SPAC provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a therapeutically effective amount of a SPAC provided herein (e.g., for treating cancer or inhibiting tumor growth in a subject).
  • SPACs Stapled-peptide antibody conjugates
  • cells e.g., cancer cells, bacterial cells
  • diseases e.g., proliferative diseases such as cancer, infectious diseases.
  • Methods provided herein include: (i) Methods of treating and/or preventing a disease in a subject comprising administering to the subject a therapeutically and/or prophylactically effective amount of a SPAC provided herein, or a pharmaceutical composition thereof.
  • the disease is a proliferative disease (e.g., cancer); (ii) Methods of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of a SPAC provided herein, or a pharmaceutical composition thereof; (iii) Methods of inhibiting tumor growth in a subject comprising administering to the subject an effective amount of a SPAC provided herein, or a pharmaceutical composition thereof; (iv) Methods of delivering a stapled peptide into a cell comprising contacting the cell with a SPAC provided herein, or a pharmaceutical composition thereof.
  • the cell is a cancer cell.
  • the stapled peptide in delivered to the cell in vitro.
  • the stapled peptide is delivered to the cell in vivo (i.e., in a subject); and (v) Method of triggering cancer cell death comprising contacting the cancer cell with an effective amount of a SPAC provided herein, or a pharmaceutical composition thereof.
  • the stapled peptide is delivered to the cell in vivo (i.e., in a subject).
  • kits comprising a SPAC provided herein, or a pharmaceutical composition thereof.
  • kits described herein may include a single dose or multiple doses of the SPAC or pharmaceutical composition thereof.
  • the kits described herein are useful in any method or use provided herein, and optionally further comprise instructions for using the kit (e.g., instructions for using the SPAC or composition included in the kit).
  • instructions for using the kit e.g., instructions for using the SPAC or composition included in the kit.
  • a methods of preparing a SPAC described herein are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the invention will be apparent from the Definitions, Examples, Figures, and Claims.
  • DEFINITIONS General Definitions [021] The following definitions are general terms used throughout the present application.
  • peptide and “polypeptide” are used interchangeably and refer to a polymer of amino acid residues linked together by peptide bonds.
  • the terms also include proteins, and refer to peptides, polypeptides, and proteins, of any size, structure, or function.
  • a peptide will be at least three amino acids long, or at least the length required by an amino acid sequence provided herein.
  • a peptide may refer to an individual peptide or a collection of peptides.
  • Peptides provided herein can include natural amino acids and/or unnatural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a peptide chain) in any combination.
  • amino acids in a peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification.
  • a peptide may be a fragment or modified version of a naturally occurring peptide or protein.
  • a peptide may be naturally occurring, recombinant, synthetic, or any combination of these.
  • amino acid refers to a molecule containing both an amino group and a carboxyl group.
  • Amino acids include alpha–amino acids, the generic structure of which is depicted below. Each amino acid referred to herein may be denoted by a 1- to 4-letter code (e.g., R and Arg represent L-Arginine, hArg represents L-homoarginine).
  • Suitable amino acids include, without limitation, natural alpha–amino acids such as D– and L–isomers of the 20 common naturally occurring alpha–amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, as provided below), and unnatural alpha–amino acids.
  • natural alpha–amino acids such as D– and L–isomers of the 20 common naturally occurring alpha–amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, as provided below), and unnatural alpha–amino acids.
  • Exemplary natural alpha-amino acids include L–alanine (A), L–arginine (R), L–asparagine (N), L–aspartic acid (D), L–cysteine (C), L–glutamic acid (E), L–glutamine (Q), glycine (G), L–histidine (H), L–isoleucine (I), L– leucine (L), L–lysine (K), L–methionine (M), L–phenylalanine (F), L–proline (P), L–serine (S), L–threonine (T), L–tryptophan (W), L–tyrosine (Y), and L–valine (V).
  • A L–alanine
  • R L–arginine
  • N L–asparagine
  • D L–aspartic acid
  • C L–cysteine
  • E L–glutamic acid
  • Q L–glutamine
  • G L–histidine
  • Exemplary unnatural alpha-amino acids include D–arginine, D–asparagine, D–aspartic acid, D–cysteine, D–glutamic acid, D–glutamine, D–histidine, D–isoleucine, D–leucine, D– lysine, D–methionine, D–phenylalanine, D–proline, D–serine, D–threonine, D–tryptophan, D–tyrosine, D–valine, Di-vinyl, ⁇ -methyl-alanine (Aib), ⁇ -methyl-arginine, ⁇ -methyl- asparagine, ⁇ -methyl-aspartic acid, ⁇ -methyl-cysteine, ⁇ -methyl-glutamic acid, ⁇ -methyl- glutamine, ⁇ -methyl-histidine, ⁇ -methyl-isoleucine, ⁇ -methyl-leucine, ⁇ -methyl-lysine, ⁇ - methyl
  • amino acid substitution when used in reference to an amino acid sequence refers to an amino acid of the amino acid sequence being replaced by a different amino acid (e.g., replaced by a natural or unnatural amino acid).
  • An amino acid sequence provided herein may comprise or include one or more amino acid substitutions. Specific amino acid substitutions are denoted by commonly used colloquial nomenclature in the art of peptide sequencing to denote amino acid sequence variations. For example, when referring to SEQ ID NO: 2 (below), an “amino acid substitution at H7” refers to the histidine (H) at position 7 of the amino acid sequence being replaced by a different amino acid (e.g., a natural or unnatural amino acid other than histidine).
  • amino acid substitution “H7K” refers to replacing the histidine (H) at position 7 of the amino acid sequence of SEQ ID NO: 2 with lysine (K), resulting in an amino acid sequence represented by SEQ ID NO: 3 (below).
  • amino acid addition when used in reference to an amino acid sequence refers to an amino acid (e.g., a natural or unnatural amino acid) being inserted between two amino acids of the amino acid sequence, or added at either end of the sequence.
  • amino acid sequence herein can comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid additions.
  • amino acid deletion when used in reference to an amino acid sequence refers to an amino acid of the amino acid sequence being deleted from the amino acid sequence.
  • an amino acid sequence herein can comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions.
  • the term “salt” refers to any and all salts, and encompasses pharmaceutically acceptable salts. Salts include ionic compounds that result from the neutralization reaction of an acid and a base. A salt is composed of one or more cations (positively charged ions) and one or more anions (negative ions) so that the salt is electrically neutral (without a net charge).
  • Salts of the peptides of this invention include those derived from inorganic and organic acids and bases.
  • acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2–hydroxy–ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2– naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
  • non-hydrogen group refers to any group that is defined for a particular variable that is not hydrogen.
  • aliphatic refers to alkyl, alkenyl, alkynyl, and carbocyclic groups.
  • heteroaliphatic refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.
  • haloalkyl groups include –CHF 2 , ⁇ CH 2 F, ⁇ CF 3 , ⁇ CH 2 CF 3 , ⁇ CF 2 CF 3 , ⁇ CF 2 CF 2 CF 3 , ⁇ CCl 3 , ⁇ CFCl 2 , ⁇ CF 2 Cl, and the like.
  • heteroalkyl refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain.
  • alkylene is the divalent moiety of alkyl
  • alkenylene is the divalent moiety of alkenyl
  • alkynylene is the divalent moiety of alkynyl
  • heteroalkylene is the divalent moiety of heteroalkyl
  • heteroalkenylene is the divalent moiety of heteroalkenyl
  • heteroalkynylene is the divalent moiety of heteroalkynyl
  • carbocyclylene is the divalent moiety of carbocyclyl
  • heterocyclylene is the divalent moiety of heterocyclyl
  • arylene is the divalent moiety of aryl
  • heteroarylene is the divalent moiety of heteroaryl.
  • Antibody molecules of the conjugates can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • the term “antigen-binding fragment,” as used herein, refers to one or more fragments of an immunoglobulin that retain the ability to specifically bind to a target antigen.
  • the antigen-binding function of an immunoglobulin can be performed by fragments of a full- length antibody.
  • binding fragments encompassed by the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L , and C H 1 domains; (ii) a F(ab') 2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and C H 1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb (Ward et al., Nature, 1989, 341, 544-546) including VH and VL domains; (vi) a dAb fragment that consists of a VH domain; (vii) a dAb that consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and
  • anti-cancer antibody refers to an antibody that targets an antigen expressed on a cancer cell (e.g., a cancer-associated antigen or cancer-specific antigen), can inhibit the growth of a cancer or tumor, and/or can trigger cancer cell death.
  • the antibody is an anti-cancer mAb or an antigen-binding fragment thereof.
  • Table 3 below provides examples of anti-cancer mAbs (including generic name, known target antigen(s), and exemplary use(s)). Other non-limiting examples of anti-cancer mAbs are provided above in Table 2.
  • Therapeutic applications of the anti-cancer mAbs listed below are not limited the particular known target antigen(s) and exemplary use(s) provided. Table 3. Examples of Anti-Cancer Monoclonal Antibodies (mAbs)
  • the SPAC comprises an antibody or antigen- binding fragment thereof conjugated to (i) a stapled peptide or pharmaceutically acceptable salt thereof; and (ii) a second agent (i.e., the “drug” component of the ADC).
  • a SPAC provided herein can comprise two or more distinct payloads which can be selected from different classes of agents (e.g., with different mechanisms of action).
  • ADCs Antibody-Drug Conjugates
  • antigen refers to an agent which is targeted by and binds an antibody.
  • antigens trigger the immune system to produce antibodies against the antigens in what is known as an immune response.
  • an antigen is expressed in a cell or on the surface of a cell (e.g., a cancer cell).
  • an antibody described herein is an antibody directed against a cluster of differentiation (CD) antigen (e.g., CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a (LFA-1), CD15, CD18 (ITGB2), CD19, CD20 (MS4A1), CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD37, CD38, CD40, CD41, CD44, CD49b (ITGA2), CD51, CD52, CD54 (ICAM-1), CD56, CD62L, CD70, CD74, CD79B, CD80, CD125, CD140a, CD142, CD147, CD152 (CTLA4), CD154, CD200, CD221, CD240D, CD248, CD257 (BAFF), CD274 (PD-L1), CD276, CD279 (PD-1)).
  • the antibody is an antibody directed against human epidermal growth factor receptor 2 (HER2), or an antigen-binding fragment thereof. In certain embodiments, the antibody is a mAb directed against HER2, or an antigen-binding fragment thereof. In certain embodiments, the antibody is trastuzumab. In certain embodiments, antibody is pertuzumab. In certain embodiments, the antibody is margetuximab. [096] In certain embodiments, the antibody is an antibody directed against CD38, CD33, CD22, TROP2, CD30, CD79b, or Nectin-4, or antigen-binding fragment thereof.
  • HER2 human epidermal growth factor receptor 2
  • the antibody is a mAb directed against HER2, or an antigen-binding fragment thereof.
  • the antibody is trastuzumab. In certain embodiments, antibody is pertuzumab. In certain embodiments, the antibody is margetuximab. [096] In certain embodiments, the antibody is an antibody directed against CD38, CD33, CD
  • the antibody is a mAb directed against CD38, CD33, CD22, TROP2, CD30, CD79b, or Nectin-4, or an antigen-binding fragment thereof.
  • the antibody is an antibody directed against CD38, or an antigen-binding fragment thereof.
  • the antibody is a mAb directed against CD38, or an antigen-binding fragment thereof.
  • the antibody daratumumab.
  • the antibody is isatuximab.
  • TM4SF1-Targeting Antibodies [098] TM4SF1 (Transmembrane-4 L Six Family member 1) is a membrane glycoprotein that was originally discovered on many human epithelial tumor cells.
  • TM4SF1 is known to support growth, invasion, and metastasis and is therefore a therapeutic target in cancer. See, e.g., Hellström et al., Proc. Natl. Acad. Sci. USA, 1986, 83(18), 7059-7063; Kao et al., Clin.
  • Patent Nos.7,192,713; 7,786,072; 8,895,699; 9,505,801; 9,951,099; and 10,487,110 the entire contents of each of which is incorporated herein by reference.
  • Other examples of stapled peptide technology can be found in, e.g., International PCT Application Publication Nos.
  • Stapled peptides of the disclosure include (i) “singly stapled” peptides, meaning peptides including one internal crosslink connecting two crosslinked amino acids; (ii) “doubly stapled” peptides, meaning peptides including two internal crosslinks, each connecting a different pair of crosslinked amino acids; and (iii) “stitched” peptides, meaning peptides including at least two tandem staples, i.e., staples attached to the same crosslinked amino acid.
  • Stapled peptides can include more than two crosslinks (i.e., multiply stapled), with any number of the staples in the stitched configuration.
  • a crosslink is attached to the ⁇ -positions of the crosslinked amino acids.
  • crosslinked amino acids are separated by 3 amino acids in the amino acid sequence, forming an “i+4 crosslink.”
  • crosslinked amino acids are separated by 4 amino acids in the amino acid sequence, forming an “i+5 crosslink.”
  • crosslinked amino acids are separated by 6 amino acids in the amino acid sequence, forming an “i+7 crosslink.”
  • crosslinked amino acids are separated by 7 amino acids in the amino acid sequence, forming an “i+8 crosslink.”
  • Stapling e.g., crosslinking
  • a peptide can stabilize a secondary structure (e.g., ⁇ -helical secondary structure) of the peptide.
  • one or more crosslinks of a stapled peptide provided herein stabilize an ⁇ -helix of the peptide.
  • a peptide has increased ⁇ -helicity as compared to a corresponding unstapled (e.g., uncrosslinked) peptide.
  • a stapled peptide can exhibit ⁇ -helical stability by the maintenance of ⁇ -helical structure as measured by circular dichroism or NMR.
  • the stapled peptide exhibits at least a 1.1, 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, or 2- fold increase in ⁇ -helicity (e.g., as determined by circular dichroism or NMR) compared to a corresponding unstapled peptide.
  • a stapled peptide provided herein can exhibit about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% ⁇ -helicity (e.g., as determined by circular dichroism or NMR) compared to a corresponding unstapled peptide.
  • a stapled peptide can be of any length. In certain embodiments, the stapled peptide is 100 amino acids or fewer in length. In certain embodiments, the stapled peptide is 90 amino acids or fewer in length. In certain embodiments, the stapled peptide is 80 amino acids or fewer in length.
  • the stapled peptide is 70 amino acids or fewer in length. In certain embodiments, the stapled peptide is 60 amino acids or fewer in length. In certain embodiments, the stapled peptide is 50 amino acids or fewer in length. In certain embodiments, the stapled peptide is 45 amino acids or fewer in length. In certain embodiments, the stapled peptide is 40 amino acids or fewer in length. In certain embodiments, the stapled peptide is 35 amino acids or fewer in length. In certain embodiments, the stapled peptide is 30 amino acids or fewer in length. In certain embodiments, the stapled peptide is 25 amino acids or fewer in length. In certain embodiments, the stapled peptide is 20 amino acids or fewer in length.
  • the stapled peptide is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids or fewer in length. In certain embodiments, the stapled peptide is at least the length of an amino acid sequence provided herein. In certain embodiments, the stapled peptide is the length of any amino acid sequence provided herein. [108] In certain embodiments, the stapled peptide is conjugated to one or more stapled peptides (e.g., via a linker, such as a cleavable linker).
  • a linker such as a cleavable linker
  • the stapled peptide is part of a string of two or more stapled peptides linked to one another in a linear or non-linear fashion (e.g., via linkers, such as cleavable linkers).
  • the stapled peptide is a stapled anti-cancer peptide.
  • “Stapled anti-cancer peptide” also referred to as “stapled oncolytic peptide” refers to a stapled variant of a peptide having anti-cancer activity (i.e., a peptide capable of triggering cancer cell death and/or inhibiting the growth of a cancer or tumor).
  • the stapled peptide triggers cancer cell death.
  • stapled anti-cancer peptides i.e., stapled oncolytic peptides
  • stapled peptide component of the SPACs any of which can be used as the stapled peptide component of the SPACs provided herein
  • the stapled peptide is a stapled antimicrobial peptide (StAMP).
  • StAMP stapled antimicrobial peptide
  • StAMPs any of which can be used as the stapled peptide component of the SPACs provided herein, can be found in International PCT Application Publication Nos. WO 2017/004591, published January 5, 2017; and WO 2019/018499, published January 24, 2019, the entire contents of each of which are incorporated herein by reference.
  • a stapled peptide may have both anti-cancer and antimicrobial activity.
  • Stapled Peptide Inhibitors of Protein-Protein Interaction (PPI) [111]
  • the stapled peptide is an inhibitor of a protein-protein interaction (PPI).
  • the PPI is associated with cancer formation and/or proliferation. See, e.g., Wang et al. Chem.
  • the stapled peptide is an inhibitor of a BCL-2 family member protein (e.g., BCL-xL, BCL-2, BCL-W, or MCL1).
  • a BCL-2 family member protein e.g., BCL-xL, BCL-2, BCL-W, or MCL1.
  • the stapled peptide is an MCL-1 inhibitor (e.g., MCL-1 SAHB A , MCL-1 SAHB B , MCL-1 SAHB C , MCL-1 SAHB D , MCL-1 SAHB E ).
  • the stapled peptide is a BFL-1 inhibitor. See, e.g., Huhn et al., Cell Chem. Biol., 2016, 23(9): 1123-1134, the entire contents of which is incorporated herein by reference.
  • the stapled peptide is MCL-1 SAHB D as described in, e.g., Stewart et al., Nat. Chem.
  • MCL-1 SAHBD comprises the following amino acid sequence: wherein both X are connected via the crosslink alk.
  • the stapled peptide is an activator of a BCL-2 family member protein effector (e.g., BAX, BAK, or BOK).
  • the stapled peptide is a stapled BH3 peptide.
  • the stapled peptide is a stapled BCL-2- interacting mediator of cell death (BIM) peptide.
  • the stapled peptide is BIM SAHBA1 or BIM SAHBA2.
  • the stapled peptide is BID SAHB A . See, e.g., Walensky et al., Moelcular Cell, 2006, vol.24, 199-210.
  • the stapled peptide is a stapled PUMA peptide. See, e.g., Edwards et al., Chemistry & Biology, 2013, vol.20, 888-902.
  • the stapled peptide is a ⁇ -catenin inhibitor. In certain embodiments, the stapled peptide is an inhibitor of Wnt/ ⁇ -catenin signaling. In certain embodiments, the stapled peptide is a stapled BCL9 peptide. See, e.g., Takada et al., Sci. Transl. Med.2012, 4(148):148ra117, pp.1-19; Grossman et al., PNAS, 2012, 109(44), 17942-17947; and Liao et al., Cell Discovery, 2020, 6, No.35, the entire contents of each of which is incorporated herein by reference.
  • the stapled peptide is xStAx-34 as described in, e.g., Grossman et al., PNAS, 2012, 109(44), 17942-17947, the entire contents of which is incorporated herein by reference.
  • xStAx-34 comprises the following amino acid sequence: RWPQXILDXHVRRVWR (SEQ ID NO: 149), wherein both X are connected via the crosslink alk.
  • the stapled peptide is SAH-BCL9B as described in, e.g., Takada et al., Sci. Transl. Med.2012, 4(148):148ra117, pp.1-19, the entire contents of which is incorporated herein by reference.
  • SAH-BCL9 B comprises the following amino acid sequence: LSQEQLEHRERSLXTLRXIQRBLF (SEQ ID NO: 150), wherein both X are connected via the crosslink alk.
  • the stapled peptide is an MDM2 and/or MDMX inhibitor. In certain embodiments, the stapled peptide is an MDM2 inhibitor. In certain embodiments, the stapled peptide is an MDMX inhibitor. In certain embodiments, the stapled peptide inhibits the binding of MDM2 to p53. In certain embodiments, the stapled peptide inhibits the binding of MDMX to p53. In certain embodiments, the stapled peptide is a stapled p53 peptide. See, e.g., Verdine et al., J. Am. Chem.
  • the stapled peptide is ALRN-6924 or ATSP-7041. See, e.g., Ye Che, Methods Mol. Biol., 2019, 2001, 97-106; and Chang et al., PNAS, 2013, 110(36), E3445- E3454, the entire contents of each of which are incorporated herein by reference.
  • the stapled peptide comprises one of the following amino acid sequences (wherein both X are amino acids connected via a crosslink): LTFXEYWAQLXSAA (SEQ ID NO: 161), LTFXEYWAQLXSAAGGG (SEQ ID NO: 162), LTFXEYWAQLXSAA-PEG3 (SEQ ID NO: 163), or a pharmaceutically acceptable salt thereof.
  • the stapled peptide comprises one of the following (wherein R 8 and S 5 are joined together to form the crosslink alk2): LTFR 8 EYWAQLS 5 SAA-NH 2 (SEQ ID NO: 164), LTFR 8 EYWAQLS 5 SAAGGG-NH 2 (SEQ ID NO: 165), LTFR 8 EYWAQLS 5 SAA-PEG3-NH 2 (SEQ ID NO: 166), or a pharmaceutically acceptable salt thereof.
  • Stapled Magainin Peptides [122] In certain embodiments, the stapled peptide is based on the amino acid sequence of a Magainin peptide (e.g., Magainin II).
  • the Magainins are a class of antimicrobial peptides (AMPs) originally found in the African clawed frog (Xenopus laevis).
  • the peptides are cationic, generally lack a stable conformation in water but form amphipathic ⁇ -helices in membranes. They are generally known to disrupt the cell membranes of a broad spectrum of cells, including bacteria, protozoa, and fungi. They have also been reported to have anti- cancer activity.
  • the amino acid sequences of the peptides known as “Magainin I” and “Magainin II” are provided below.
  • Magainin I GIGKFLHSAGKFGKAFVGEIMKS (SEQ ID NO: 153)
  • Magainin II GIGKFLHSAKKFGKAFVGEIMNS (SEQ ID NO: 154)
  • stapled Magainin peptides any of which can be used as the stapled peptide component of the SPACs provided herein, can be found in International PCT Application Publication Nos. WO 2017/004591, published January 5, 2017; WO 2019/018499, published January 24, 2019; and WO 2021/126827, published June 24, 2021, the entire contents of each of which are incorporated herein by reference. See also, e.g., U.S.
  • the stapled peptide comprises the amino acid sequence: G X 1 G K F X 2 H S K K K F G K A X 3 V G E X 4 (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof, wherein: X 1 , X 2 , X 3 , and X 4 are amino acids (i.e., crosslinked amino acids); X 1 and X 2 are connected via a crosslink, and X 3 and X 4 are connected via a crosslink; and the amino acid sequence includes 0 to 9 amino acid substitutions, inclusive, at positions other than X 1 , X 2 , X 3 , and X 4 . In certain embodiments, the amino acid sequence comprises 0 amino acid substitutions.
  • the amino acid sequence comprises 1 amino acid substitution. In certain embodiments, the amino acid sequence comprises 2 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 3 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 4 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 5 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 6 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 7 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 8 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 9 amino acid substitutions.
  • the stapled peptide comprises the amino acid sequence: G X 1 G K F X 2 H S K K K F G K A X 3 V G E X 4 A K K (SEQ ID NO: 2), or a pharmaceutically acceptable salt thereof, wherein: X 1 , X 2 , X 3 , and X 4 are amino acids (i.e., crosslinked amino acids); X 1 and X 2 are connected via a crosslink, and X 3 and X 4 are connected via a crosslink; the amino acid sequence includes 0 to 11 amino acid substitutions, inclusive, at positions other than X 1 , X 2 , X 3 , and X 4 .
  • the amino acid sequence comprises 0 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 1 amino acid substitution. In certain embodiments, the amino acid sequence comprises 2 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 3 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 4 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 5 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 6 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 7 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 8 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 9 amino acid substitutions.
  • a stapled peptide or pharmaceutically acceptable salt thereof comprises one of the following amino acid sequences: [128] In certain embodiments, the stapled peptide is of one of SEQ ID NOs: 1-57 and 167- 186, or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide is of one of SEQ ID NOs: 1-57 and 167-186, or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with –NH 2 .
  • the stapled peptide is of one of SEQ ID NOs: 1-57 and 167-186, or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with –NH 2 ; and wherein X 1 and X 2 , and X 3 and X 4 , are each connected via the crosslink (alk).
  • the stapled peptide comprises the amino acid sequence: G X 1 G K F X 2 Dap K K K K F G K A X 3 V G E X 4 A K K (SEQ ID NO: 48), or a pharmaceutically acceptable salt thereof.
  • the stapled peptide or pharmaceutically acceptable salt thereof is of SEQ ID NO: 48, wherein X 1 and X 2 , and X 3 and X 4 , are connected via a crosslink of the formula (alk); and wherein the C-terminus of the stapled peptide is amidated with –NH 2 .
  • the stapled peptide comprises the amino acid sequence: G X 1 G Dap F X 2 Dap Dap Dap Dap Dap Dap F G Dap A X 3 V G E X 4 A Dap Dap (SEQ ID NO: 26), or a pharmaceutically acceptable salt thereof.
  • the stapled peptide or pharmaceutically acceptable salt thereof is of SEQ ID NO: 26, wherein X 1 and X 2 , and X 3 and X 4 , are connected via a crosslink of the formula (alk); and wherein the C-terminus of the stapled peptide is amidated with –NH 2 .
  • the stapled peptide comprises the amino acid sequence: G X 1 G K F X 2 K K K K F G K A X 3 V G E X 4 A K K (SEQ ID NO: 5), or a pharmaceutically acceptable salt thereof.
  • the stapled peptide or pharmaceutically acceptable salt thereof is of SEQ ID NO: 5, wherein X 1 and X 2 , and X 3 and X 4 , are connected via a crosslink of the formula (alk); and wherein the C-terminus of the stapled peptide is amidated with –NH 2 .
  • the stapled peptide comprises the amino acid sequence: G X 1 G K F X 2 H K K K K F G K A X 3 V F E X 4 A K K (SEQ ID NO: 23), or a pharmaceutically acceptable salt thereof.
  • the stapled peptide or pharmaceutically acceptable salt thereof is of SEQ ID NO: 23, wherein X 1 and X 2 , and X 3 and X 4 , are connected via a crosslink of the formula (alk); and wherein the C-terminus of the stapled peptide is amidated with –NH 2 .
  • the stapled peptide comprises the amino acid sequence: [134] or a pharmaceutically acceptable salt thereof.
  • the stapled peptide or pharmaceutically acceptable salt thereof is of SEQ ID NO: 24, wherein X 1 and X 2 , and X 3 and X 4 , are connected via a crosslink of the formula (alk); and wherein the C-terminus of the stapled peptide is amidated with –NH 2 .
  • a stapled peptide or pharmaceutically acceptable salt thereof provided herein comprises one of the following amino acid sequences: [135]
  • the stapled peptide is of one of SEQ ID NOs: 58-71, or a pharmaceutically acceptable salt thereof.
  • the stapled peptide is of one of SEQ ID NOs: 58-71, or a pharmaceutically acceptable salt thereof, wherein the C- terminus is amidated with –NH 2 .
  • the stapled peptide is of one of SEQ ID NOs: 58-71, or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with –NH 2 ; and wherein X 1 and X 2 , and X 3 and X 4 , are each connected via the crosslink (alk).
  • the stapled peptide comprises the following amino acid sequence: G X 1 G K F X 2 K K K K K X 3 V G E X 4 A K K (SEQ ID NO: 72), or a pharmaceutically acceptable salt thereof, wherein: X 1 , X 2 , X 3 , and X 4 are amino acids (i.e., crosslinked amino acids); X 1 and X 2 are connected via a crosslink, and X 3 and X 4 are connected via a crosslink; and the amino acid sequence optionally includes 0 to 5 amino acid substitutions, inclusive, at positions other than X 1 , X 2 , X 3 , and X 4 .
  • the amino acid sequence comprises 0 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 1 amino acid substitution. In certain embodiments, the amino acid sequence comprises 2 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 3 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 4 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 5 amino acid substitutions.
  • the stapled peptide or a pharmaceutically acceptable salt thereof comprises SEQ ID NO: 72. In certain embodiments, the stapled peptide is of SEQ ID NO: 72, or a pharmaceutically acceptable salt thereof.
  • the stapled peptide is of SEQ ID NO: 72, or a pharmaceutically acceptable salt thereof, wherein the C- terminus is amidated with –NH 2 .
  • the stapled peptide is of SEQ ID NO: 72, or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with –NH 2 ; and wherein X 1 and X 2 , and X 3 and X 4 , are each connected via the crosslink (alk).
  • Stapled Esculentin Peptides [138]
  • the stapled peptide is based on the amino acid sequence of an Esculentin peptide (e.g., Esculentin-1A).
  • Esculentins are a class of cytotoxic peptides with antibacterial and antifungal activity that were originally found in the skin secretions of many species of frogs and toads.
  • Esculentin-1A is a particular antimicrobial peptide (AMP) originally isolated from frog skin. Esculentin peptides can also have anti-cancer activity.
  • the amino acid sequence of the peptide known as “Esculentin-1A” is provided below.
  • a stapled peptide is based on amino acids 1-21 of SEQ ID NO: 155.
  • Esculentin-1A [139]
  • the stapled peptide is a stapled Esculentin peptide.
  • the stapled peptide is a stapled Esculentin-1A peptide.
  • stapled Esculentin peptides any of which can be used as the stapled peptide component of the SPACs provided herein, can be found in, e.g., U.S. Provisional Applications, U.S.S.N. 63/185,641, filed May 7, 2021; and 63/185,673, filed May 7, 2021, the entire contents of each of which are incorporated herein by reference. See also, e.g., Mourtada, et al., Nature Biotechnology, 2019, vol.37, 1186–1197, the entire contents of which is incorporated herein by reference.
  • the stapled peptide comprises the amino acid sequence: G X 1 F S K X 2 K G K K I K N L X 3 I S G X 4 K G (SEQ ID NO: 73), or a pharmaceutically acceptable salt thereof, wherein: X 1 , X 2 , X 3 , and X 4 are amino acids (i.e., crosslinked amino acids); X 1 and X 2 are connected via a crosslink, and X 3 and X 4 are connected via a crosslink; and the amino acid sequence includes 0 to 9 amino acid substitutions, inclusive, at positions other than X 1 , X 2 , X 3 , and X 4 .
  • the amino acid sequence comprises 0 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 1 amino acid substitution. In certain embodiments, the amino acid sequence comprises 2 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 3 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 4 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 5 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 6 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 7 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 8 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 9 amino acid substitutions.
  • the stapled peptide or pharmaceutically acceptable salt thereof comprises one of the following amino acid sequences: [141] In certain embodiments, the stapled peptide is of one of SEQ ID NOs: 73-96, or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide is of one of SEQ ID NOs: 73-96, or a pharmaceutically acceptable salt thereof, wherein the C- terminus of the peptide is amidated with –NH 2 .
  • the stapled peptide is of SEQ ID NO: 73, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with –NH 2 .
  • the stapled peptide is of SEQ ID NO: 73, or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with –NH 2 ; and wherein X 1 and X 2 , and X 3 and X 4 , are each connected via the crosslink (alk).
  • the amino acid sequence comprises 1 amino acid substitution. In certain embodiments, the amino acid sequence comprises 2 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 3 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 4 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 5 amino acid substitutions. In certain embodiments, the amino acid sequence comprises 6 amino acid substitutions.
  • X 1 and X 2 are connected via a dithio crosslink (e.g., (mxy), (pxy), (but), (bbn), (bbf), (bbp), (pfb), (hfb)).
  • the stapled peptide is one of the following, or a pharmaceutically acceptable salt thereof: [153] In certain embodiments, the stapled peptide is one of the following, or a pharmaceutically acceptable salt thereof:
  • the stapled peptide is one of the following, or a pharmaceutically acceptable salt thereof:
  • an amino acid sequence (e.g., SEQ ID NOs: 1-147 provided herein) comprises X 3 and X 4
  • X 3 and X 4 are crosslinked amino acids connected via a crosslink.
  • the following certain embodiments describing stapled peptide crosslinks apply to all stapled peptides described herein, including all amino acid sequences (e.g., SEQ ID NOs: 1-147) and variants thereof described herein.
  • the crosslinks are independently attached to the ⁇ -positions of the crosslinked amino acids (e.g., ⁇ -positions of X 1 , X 2 , X 3 , and X 4 ).
  • the crosslinks are independently attached to the ⁇ -positions of the crosslinked amino acids (e.g., X 1 , X 2 , X 3 , and X 4 ), and the crosslinked amino acids are independently ⁇ , ⁇ - disubstituted amino acids.
  • each crosslink is independently from about 5 ⁇ to about 35 ⁇ in length, inclusive. In certain embodiments, each crosslink is independently from about 5 ⁇ to about 25 ⁇ in length, inclusive (e.g., in the case of i+4 crosslinks). In certain embodiments, each crosslink is independently from about 6 ⁇ to about 22 ⁇ in length, inclusive.
  • each crosslink is independently from about 7 ⁇ to about 20 ⁇ in length, inclusive. In certain embodiments, each crosslink is independently from about 8 ⁇ to about 18 ⁇ in length, inclusive. In certain embodiments, each crosslink is independently from about 9 ⁇ to about 17 ⁇ in length, inclusive. each crosslink is independently about 10 ⁇ to about 16 ⁇ in length, inclusive. In certain embodiments, each crosslink is independently from about 11 ⁇ to about 15 ⁇ in length, inclusive. In certain embodiments, each crosslink is independently from about 12 ⁇ to about 14 ⁇ in length, inclusive. In certain embodiments, each crosslink is independently about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 ⁇ in length.
  • each crosslink is independently from about 15 ⁇ to about 35 ⁇ in length, inclusive (e.g., in the case of i+7 crosslinks). In certain embodiments, each crosslink is independently from about 17 ⁇ to about 33 ⁇ in length, inclusive. In certain embodiments, each crosslink is independently from about 19 ⁇ to about 31 ⁇ in length, inclusive. In certain embodiments, each crosslink is independently from about 20 ⁇ to about 30 ⁇ in length, inclusive. In certain embodiments, each crosslink is independently from about 22 ⁇ to about 29 ⁇ in length, inclusive. each crosslink is independently about 24 ⁇ to about 28 ⁇ in length, inclusive. In certain embodiments, each crosslink is independently from about 25 ⁇ to about 27 ⁇ in length, inclusive.
  • each crosslink is independently about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 ⁇ in length.
  • the length of each crosslink is approximately equal to the length of 5 to 25 carbon-carbon and/or carbon-sulfur bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon-carbon and/or carbon- sulfur bonds, inclusive.
  • the length of each crosslink is approximately equal to the length of 5 to 20 carbon-carbon and/or carbon-sulfur bonds, inclusive.
  • the length of each crosslink is approximately equal to the length of 5 to 15 carbon-carbon and/or carbon-sulfur bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 5 to 13 carbon-carbon and/or carbon- sulfur bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 6 to 12 carbon-carbon and/or carbon-sulfur bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 7 to 11 carbon-carbon and/or carbon-sulfur bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 8 to 10 carbon-carbon and/or carbon- sulfur bonds, inclusive.
  • the length of each crosslink is approximately equal to the length of 10 to 20 carbon-carbon and/or carbon-sulfur bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 11 to 19 carbon-carbon and/or carbon-sulfur bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 12 to 18 carbon-carbon and/or carbon- sulfur bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 13 to 17 carbon-carbon and/or carbon-sulfur bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 14 to 16 carbon-carbon and/or carbon-sulfur bonds, inclusive.
  • At least one crosslink spans at least one turn of an ⁇ -helix of the peptide. In certain embodiments, each crosslink spans at least one turn of an ⁇ -helix of the peptide. In certain embodiments, at least one crosslink spans one turn of an ⁇ -helix of the peptide. In certain embodiments, each crosslink spans one turn of an ⁇ -helix of the peptide.
  • each pair of crosslinked amino acids are independently connected by a crosslink to form the following formula: , wherein ⁇ denotes the ⁇ -carbons of the crosslinked amino acids; L 1 is a crosslink; and each instance of R 1 is independently hydrogen or optionally substituted C1-6 alkyl.
  • each crosslink (e.g., L 1 ) is independently optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted acylene, or any combination thereof.
  • each crosslink (e.g., L 1 ) is independently a hydrocarbon crosslink.
  • Hydrocarbon crosslink for the purposes of this disclosure is a crosslink consisting of optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, and combinations thereof.
  • each crosslink e.g., L 1
  • each crosslink is independently optionally substituted alkenylene (e.g., unsubstituted alkenylene).
  • each crosslink is independently of the following formula: ; wherein each n is independently an integer from 1-10, inclusive. In certain embodiments, the sum of two n on the same crosslink is 6.
  • the crosslinked amino acids are independently ⁇ , ⁇ -disubstituted amino acids.
  • each pair of crosslinked amino acids e.g., X 1 and X 2 , and X 3 and X 4
  • ⁇ denotes the ⁇ -carbons of the crosslinked amino acids
  • R 1 is independently optionally substituted C1-6 alkyl.
  • the sum of two n on the same crosslink is 6.
  • a crosslink (e.g., L 1 ) is independently optionally substituted alkylene (e.g., unsubstituted alkylene).
  • each crosslink is independently of the following formula: ; wherein m is an integer from 1-20, inclusive. In certain embodiments, m is 6.
  • a pair of crosslinked amino acids (e.g., X 1 and X 2 , and X 3 and X 4 ) are independently joined by a crosslink to form the following formula: , wherein ⁇ denotes the ⁇ -carbons of the crosslinked amino acids; and wherein each instance of R 1 is independently optionally substituted C 1-6 alkyl.
  • m is 6.
  • a crosslink e.g., L 1
  • a pair of crosslinked amino acids e.g., X 1 and X 2 , and X 3 and X 4
  • ⁇ denotes the ⁇ -carbons of the crosslinked amino acids.
  • a crosslink e.g., L 1
  • a crosslink is independently of the formula: .
  • a pair of crosslinked amino acids are independently connected via a crosslink to form the following formula: wherein ⁇ denotes the ⁇ -carbons of the crosslinked amino acids.
  • a crosslink e.g., L 1
  • a pair of crosslinked amino acids are independently connected via a crosslink to form the following formula: , wherein ⁇ denotes the ⁇ -carbons of the crosslinked amino acids.
  • a crosslink is independently a dithio crosslink of the following formula: , wherein each n is independently an integer from 1-10, inclusive; and L 2 is optionally substituted alkylene, , , optionally substituted arylene, optionally substituted heteroarylene, or –A 1 -A 1 –; wherein each instance of A 1 is independently optionally substituted arylene or optionally substituted heteroarylene.
  • each instance of n is 1.
  • each instance of n is 2.
  • a crosslink is independently a dithio crosslink of one of the following formulae: , , , In certain embodiments, each instance of n is 1.
  • each instance of n is 2.
  • a crosslink is independently a dithio crosslink of one of the following formulae: , Wherein each n is independently an integer from 1-10, inclusive. In certain embodiments, each instance of n is 1. In certain embodiments, each instance of n is 2. [188] For example, in certain embodiments, a crosslink is independently of one of the following formulae:
  • crosslinks (mxy), (pxy), (but), (bbn), (bbf), (bbp), (pfb), and (hfb) referenced herein are formed by crosslinking two cysteine (C) residues of a peptide.
  • a crosslink (e.g., L 1 ) is independently a triazolylene crosslink.
  • a “triazolylene crosslink” is a crosslink interrupted by at least one triazolylene moiety (e.g., [191]
  • a crosslink is independently a triazolylene crosslink of the following formula: , wherein each n is independently an integer from 1-10, inclusive. In certain embodiments, the sum of two n on the same crosslink is 5.
  • a crosslink is independently a triazolylene crosslink of one of the following formulae: .
  • n and R 1 apply to all generic formulae and subgenera provided herein, as well as all stapled and unstapled peptides provided herein.
  • the sum of two n on the same crosslink is an integer from 3-9, inclusive. In certain embodiments, the sum of two n on the same crosslink is an integer from 4-8, inclusive. In certain embodiments, the sum of two n on the same crosslink is an integer from 5-7, inclusive. In certain embodiments, the sum of two n on the same crosslink is 5. In certain embodiments, the sum of two n on the same crosslink is 6. In certain embodiments, the sum of two n on the same crosslink is 7.
  • At least one instance of n is 1. In certain embodiments, at least one instance of n is 2. In certain embodiments, at least one instance of n is 3. In certain embodiments, at least one instance of n is 4. In certain embodiments, at least one instance of n is 5. In certain embodiments, at least one instance of n is 6. In certain embodiments, at least one instance of n is 7. In certain embodiments, at least one instance of n is 8. In certain embodiments, at least one instance of n is 9. In certain embodiments, at least one instance of n is 10. [196] In certain embodiments, m is an integer from 3-9, inclusive. In certain embodiments, m is an integer from 4-8, inclusive.
  • m is an integer from 5-7, inclusive. In certain embodiments, m is 5. In certain embodiments, m is 6. In certain embodiments, m is 7. [197] In certain embodiments, at least one instance of R 1 is hydrogen. In certain embodiments, each instance of R 1 is hydrogen. In certain embodiments, at least one instance of R 1 is unsubstituted C 1-6 alkyl. In certain embodiments, at least one instance of R 1 is unsubstituted C 1-3 alkyl. In certain embodiments, at least one instance of R 1 is methyl. In certain embodiments, each instance of R 1 is methyl.
  • Stapled peptides can comprise modifications to the C-terminus and/or N-terminus of the polypeptide.
  • a stapled peptide comprises a modified C-terminus. Examples of C-terminus modifications are described herein.
  • the stapled peptide comprises an amidated C-terminus.
  • Stapled peptides may also be amidated at the C-terminus with an amino acid, peptide, or protein.
  • the amino acid, peptide, or protein can be natural or unnatural.
  • the stapled peptide comprises a peptide conjugated to the C-terminus.
  • the peptide is from 2 to 6 amino acids in length, inclusive, and comprises amino acids selected from G, E, S, A, and K.
  • the peptide is from 2 to 6 amino acids in length, inclusive, and comprises amino acids selected from G, E, and S.
  • the peptide is from 2 to 6 amino acids in length, inclusive, and comprises amino acids selected from G and E.
  • the peptide is 2 amino acids in length and comprises amino acids selected from G and E. In certain embodiments, the peptide is 3 amino acids in length and comprises amino acids selected from G and E. In certain embodiments, the peptide is 4 amino acids in length and comprises amino acids selected from G and E.
  • Non-limited examples of peptides which can be conjugated to the C-terminus of the stapled peptide are the following: GE, AG, AA, GG, GGE, GGS, GGG, GGK, GGQ, GGGC (SEQ ID NO: 187), GGGE (SEQ ID NO: 156), GGEE (SEQ ID NO: 157), or GGSGGS (SEQ ID NO: 158).
  • the polymer is PEG3. As described herein, PEG3 is of the formula: .
  • the stapled peptide is amidated at the C-terminus with a group of the following formula: –NH-(PEG)-CONH 2 , wherein PEG is polyethylene glycol.
  • the stapled peptide is amidated at the C-terminus with a group of the following formula: –NH(CH 2 CH 2 O) 1-20 CH 2 CH 2 CONH 2 .
  • the stapled peptide is amidated at the C-terminus with a group of one of the following formulae: –NHCH 2 CH 2 OCH 2 CH 2 CONH 2 , –NH(CH 2 CH 2 O) 2 -CH 2 CH 2 CONH 2 , –NH(CH 2 CH 2 O) 3 - CH 2 CH 2 CONH 2 , –NH(CH 2 CH 2 O)4- CH 2 CH 2 CONH 2 , or –NH(CH 2 CH 2 O)5-CH 2 CH 2 CONH 2 .
  • the stapled peptide comprises a small molecule conjugated to the C-terminus of the peptide.
  • the small molecule is an anti-cancer agent.
  • a SPAC comprises a stapled peptide conjugated to an antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof is conjugated to the N-terminus of the stapled peptide.
  • the antibody or antigen-binding fragment thereof is conjugated to the C-terminus of the stapled peptide.
  • the antibody or antigen-binding fragment thereof is conjugated to an internal position on the stapled peptide (e.g., to an amino acid residue or to the crosslink of the stapled peptide).
  • the stapled peptide is conjugated through a thiol of the antibody or antigen-binding fragment thereof. In certain embodiments, the stapled peptide is conjugated through cysteine residue of the antibody or antigen-binding fragment thereof. In certain embodiments, the stapled peptide is conjugated through an amine of the antibody or antigen-binding fragment thereof. In certain embodiments, the stapled peptide is conjugated through a lysine residue of the antibody or antigen-binding fragment thereof. [210] In certain embodiments, the antibody or antigen-binding fragment thereof is conjugated to the stapled peptide directly (e.g., via a bond).
  • the antibody or antigen-binding fragment thereof is conjugated to the stapled peptide via a linker.
  • Linker refers to the moiety linking the antibody or antigen-binding fragment thereof to the stapled peptide, not to be confused with the one or more “crosslinks” connecting amino acids of the stapled peptides.
  • the linker comprises optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted acylene, or any combination thereof.
  • the linker comprises optionally substituted alkylene.
  • the linker comprises optionally substituted alkenylene.
  • the linker comprises optionally substituted alkynylene.
  • the linker comprises optionally substituted heteroalkylene.
  • the linker is pH cleavable or cleavable by a protease, esterase, or intracellular disulfide reduction. In certain embodiments, the linker is cleavable by a protease. See, e.g., Bargh et al., Chem. Soc. Rev.2019, 48(16), 4361-4374; Zheng Su et al., “Antibody–drug conjugates: Recent advances in linker chemistry”, Acta Pharmaceutica Sinica B, 2021; and Leriche et al., Bioorganic & Medicinal Chemistry, 2012, vol.20, 571-582, the entire contents of each of which is incorporated herein by reference.
  • the linker is a peptidic linker. In certain embodiments, the linker is a cleavable peptidic linker. “Peptidic linker” as used herein refers to a linker comprising two or more amino acids linked via peptide bonds.
  • the peptidic linker comprises —Y A Y B Y C Y D – (SEQ ID NO: 159), wherein: Y A is glycine, glutamic acid, or is absent; Y B is valine, phenylalanine, alanine, tyrosine, or glycine; Y C is citrulline, arginine, lysine, alanine, or glycine; and Y D is glycine or is absent. [215] In certain embodiments, the peptidic linker comprises –GGFG– (SEQ ID NO: 160). In certain embodiments, the peptidic linker comprises –GGG–.
  • the peptidic linker comprises —EVC–. See, e.g., Anami et al., Nature Communications, 2018, 9, 2512, the entire contents of which is incorporated herein by reference.
  • the peptidic linker comprises —valine-citrulline— (i.e., –V-C–). Table 5 below shows non-limiting examples of such linkers, represented by linking reagent and the corresponding resulting linker structure. Table 5. Examples of Linkers and Linking Reagents [217]
  • the linker comprises a triazolylene moiety. As described herein, linkers comprising triazolylene moieties may be formed by azide-alkyne cycloaddition reactions. Non-limiting examples of triazole-containing linkers are shown below in Table 9. Table 9. Examples of Trazolylene-Containing Linkers
  • a SPAC provided herein includes 1 stapled peptide conjugated to the antibody or antigen-binding fragment thereof (i.e., a 1:1 stapled peptide to antibody ratio).
  • 2 or more stapled peptides are conjugated to the antibody or antigen-binding fragment thereof (i.e., a 2:1 stapled peptide to antibody ratio or greater).
  • 1 to 20 stapled peptides, inclusive are conjugated to the antibody or antigen-binding fragment thereof (i.e., a 1:1 to 20:1 stapled peptide to antibody ratio, inclusive).
  • 2 to 20 stapled peptides, inclusive are conjugated to the antibody or antigen-binding fragment thereof (i.e., a 2:1 to 20:1 stapled peptide to antibody ratio, inclusive).
  • 1 to 10 stapled peptides, inclusive are conjugated to the antibody or antigen-binding fragment thereof (i.e., a 1:1 to 10:1 stapled peptide to antibody ratio, inclusive).
  • 2 to 10 stapled peptides, inclusive are conjugated to the antibody or antigen-binding fragment thereof (i.e., a 2:1 to 10:1 stapled peptide to antibody ratio, inclusive).
  • 5 to 10 stapled peptides, inclusive are conjugated to the antibody or antigen-binding fragment thereof (i.e., a 1:1 to 10:1 stapled peptide to antibody ratio, inclusive).
  • the antibody or antigen-binding fragment thereof is conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 stapled peptides.
  • about 8 stapled are conjugated to the antibody or antigen-binding fragment thereof (i.e., an 8:1 stapled peptide to antibody ratio).
  • the stapled peptides are the same or different, in any combination. Additional Embodiments [219] Additional embodiments and examples of SPACs are provided below, including in Table 6.
  • Click chemistry is a chemical approach introduced by Sharpless in 2001 and describes chemistry tailored to generate substances quickly and reliably by joining small units together. See, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004-2021; Evans, Australian Journal of Chemistry (2007) 60: 384-395.
  • Exemplary coupling reactions include, but are not limited to, formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems); azidealkyne Huisgen cycloaddition; thiol-yne addition; imine formation; Michael additions (e.g., mal eimide addition); and Diels- Alder reactions (e.g., tetrazine [4 + 2] cycloaddition).
  • nucleophilic displacement reactions e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems
  • azidealkyne Huisgen cycloaddition thiol-yne addition
  • imine formation Michael additions (e.g., mal eimide addition)
  • the first reactive moiety is an azide; and the second reactive moiety is an alkyne. In certain embodiments, the first reactive moiety is an alkyne and the second reactive moiety is an azide.
  • a method for preparing a stapled peptide-antibody conjugate (SPAC) described herein comprises a step of contacting a stapled peptide comprising an azide with an antibody or antigen-binding fragment thereof comprising an alkyne under conditions sufficient to form a triazolylene-containing linker, thereby forming the SPAC.
  • the stapled peptide comprising an azide comprises one of the following formulae (e.g., to form any one of linkers A1-A20):
  • stapled peptides comprising any one of SEQ ID NOs: 161-166, or a pharmaceutically acceptable salt thereof; and further comprising an azide.
  • stapled peptides comprising any one of SEQ ID NOs: 161-166, or a pharmaceutically acceptable salt thereof; and further comprising one of the following formulae at the N-terminus of the amino acid sequence: ,
  • the antibody or antigen binding fragment thereof comprises a terminal alkyne (e.g., for use in copper-promoted cycloaddition with an azide).
  • the antibody or antigen binding fragment thereof comprises cyclic alkyne (e.g., for use in strain-promoted (e.g., copper-free) cycloaddition with an azide).
  • cyclic alkyne moieties include DBCO and sulfo-DBCO: Pharmaceutical Compositions, Kits, and Administration [247] The present disclosure provides pharmaceutical compositions comprising a SPAC disclosed herein.
  • the pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers/excipients.
  • a SPAC described herein is provided in an effective amount in the pharmaceutical composition.
  • the effective amount is a therapeutically effective amount (e.g., for treating cancer in a subject and/or inhibiting tumor growth in a subject).
  • the effective amount is a prophylactically effective amount.
  • Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology.
  • compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.
  • Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross- linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.
  • crospovidone cross-linked poly(vinyl-pyrrolidone)
  • sodium carboxymethyl starch sodium starch glycolate
  • Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cell
  • Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives.
  • the preservative is an antioxidant.
  • the preservative is a chelating agent.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof.
  • EDTA ethylenediaminetetraacetic acid
  • salts and hydrates thereof e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like
  • citric acid and salts and hydrates thereof e.g., citric acid mono
  • antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
  • Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
  • Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
  • Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen- free water, isotonic saline
  • Liquid dosage forms for parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, and suspensions.
  • the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.
  • SPACs provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
  • the SPACs and compositions provided herein can be administered by any route, including, parenteral, enteral (e.g., oral), intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • enteral e.g., oral
  • intravenous intramuscular
  • intra-arterial intramedullary
  • intrathecal subcutaneous
  • intraventricular transdermal
  • interdermal interdermal
  • rectal intravaginal
  • topical as by powders, ointments, creams, and/or drops
  • mucosal nasal, buccal
  • Specifically contemplated routes are intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site.
  • intravenous administration e.g., systemic intravenous injection
  • regional administration via blood and/or lymph supply
  • direct administration to an affected site.
  • the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate a certain route of administration).
  • the exact amount of a SPAC required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular SPAC, mode of administration, and the like.
  • an effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses).
  • any two doses of the multiple doses include different or substantially the same amounts of a SPAC described herein.
  • the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks.
  • the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell.
  • the duration between the first dose and last dose of the multiple doses is three months, six months, or one year.
  • the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell.
  • Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult.
  • the amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
  • a SPAC or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents).
  • a pharmaceutical composition described herein including a SPAC described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the SPAC and the additional pharmaceutical agent, but not both.
  • the additional pharmaceutical agent achieves a desired effect for the same disorder.
  • the additional pharmaceutical agent achieves different effects.
  • the SPAC or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies.
  • Pharmaceutical agents include therapeutically active agents.
  • Pharmaceutical agents also include prophylactically active agents.
  • Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S.
  • SPACs proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic SPACs or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.
  • CFR Code of Federal Regulations
  • the additional pharmaceutical agents include, but are not limited to, anti-proliferative agents, anti-cancer agents, anti-angiogenesis agents, steroidal or non-steroidal anti- inflammatory agents (NSAIDs), immunosuppressants, anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, pain-relieving agents, anesthetics, anti–coagulants, inhibitors of an enzyme, steroidal agents, steroidal or antihistamine, antigens, vaccines, antibodies, decongestant, sedatives, opioids, analgesics, anti–pyretics, hormones, and prostaglandins.
  • NSAIDs steroidal or non-steroidal anti- inflammatory agents
  • immunosuppressants anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, pain-relieving agents, anesthetic
  • the additional pharmaceutical agent is an anti-cancer agent.
  • Anti-cancer agents encompass biotherapeutic anti-cancer agents as well as chemotherapeutic agents.
  • Exemplary biotherapeutic anti-cancer agents include, but are not limited to, interferons, cytokines (e.g., tumor necrosis factor, interferon ⁇ , interferon ⁇ ), vaccines, hematopoietic growth factors, monoclonal serotherapy, immunostimulants and/or immunomodulatory agents (e.g., IL-1, 2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) and antibodies (e.g.
  • chemotherapeutic agents include, but are not limited to, anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g. goscrclin and leuprolide), anti- androgens (e.g. flutamide and bicalutamide), photodynamic therapies (e.g.
  • vertoporfin BPD- MA
  • phthalocyanine phthalocyanine
  • photosensitizer Pc4 demethoxy-hypocrellin A (2BA-2-DMHA)
  • nitrogen mustards e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan
  • nitrosoureas e.g. carmustine (BCNU) and lomustine (CCNU)
  • alkylsulphonates e.g. busulfan and treosulfan
  • triazenes e.g. dacarbazine, temozolomide
  • platinum containing compounds e.g.
  • paclitaxel or a paclitaxel equivalent such as nanoparticle albumin-bound paclitaxel (Abraxane), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel, e.g., 2'-paclitaxel methyl 2-glucopyran
  • etoposide etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mytomycin C
  • anti- metabolites DHFR inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonuclotide reductase inhibitors (e.g.
  • uracil analogs e.g.5-fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine
  • cytosine analogs e.g. cytarabine (ara C), cytosine arabinoside, and fludarabine
  • purine analogs e.g. mercaptopurine and Thioguanine
  • Vitamin D3 analogs e.g. EB 1089, CB 1093, and KH 1060
  • isoprenylation inhibitors e.g.
  • lovastatin dopaminergic neurotoxins (e.g.1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), actinomycin (e.g. actinomycin D, dactinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g.
  • thapsigargin Ca 2+ ATPase inhibitors
  • imatinib thalidomide, lenalidomide
  • tyrosine kinase inhibitors e.g., axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN TM , AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®
  • axitinib
  • Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent.
  • the additional pharmaceutical agents may also be administered together with each other and/or with the SPAC or composition described herein in a single dose or composition or administered separately in different doses or compositions.
  • the particular combination to employ in a regimen will take into account compatibility of the SPAC described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved.
  • it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
  • kits e.g., pharmaceutical packs.
  • the kits provided may comprise a pharmaceutical composition or SPAC described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container).
  • a container e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container.
  • provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or SPAC described herein.
  • the pharmaceutical composition or SPAC described herein provided in the first container and the second container are combined to form one unit dosage form.
  • SPACs for use in treating and/or preventing a disease in a subject.
  • uses of SPACs, and pharmaceutical compositions thereof, for the manufacture of medicaments are provided herein.
  • the disease is a proliferative disease (e.g., cancer), infectious disease (e.g., bacterial infection), inflammatory disease, or autoimmune disease.
  • the disease is a proliferative disease (e.g., cancer).
  • methods of treating a proliferative disease (e.g., cancer) in a subject comprising administering to the subject a therapeutically effective amount of a SPAC provided herein, or a pharmaceutical composition thereof.
  • a proliferative disease may be associated with: (1) the pathological proliferation of normally quiescent cells; (2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); (3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or (4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis.
  • Exemplary proliferative diseases include cancers (i.e., “malignant neoplasms”), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.
  • certain “benign” tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor’s neoplastic cells, and these tumors are referred to as “pre-malignant neoplasms.”
  • An exemplary pre-malignant neoplasm is a teratoma.
  • a “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue.
  • a malignant neoplasm generally has the capacity to metastasize to distant sites.
  • cancer refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located.
  • cancer refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues.
  • the cancer is a solid tumor.
  • the cancer is a hematopoietic cancer (i.e., hematological cancer).
  • the cancer is a hematopoietic cancer (e.g., leukemia (e.g., acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma (e.g., Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL)), non- Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma)), f
  • ALL acute lymphocy
  • the cancer is leukemia.
  • the cancer is acute lymphoblastic leukemia (ALL).
  • the cancer is early T-cell precursor (ETP)-acute lymphoblastic leukemia (ALL).
  • the cancer is selected from endocrine/exocrine cancers (e.g., thyroid cancer (e.g., papillary thyroid carcinoma, follicular thyroid carcinoma; medullary thyroid carcinoma, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma), pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors, ductal adenocarcinoma, insulinoma, glucagonoma, vipoma), adrenal gland cancer, neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP- NET), carcinoid tumor), sebaceous gland carcinoma, sweat gland carcinoma).
  • thyroid cancer e.g., papillary thyroid carcinoma, follicular thyroid
  • the cancer is oral cancer (e.g., buccal cavity cancer, lip cancer, tongue cancer, mouth cancer, pharynx cancer, hypopharynx cancer (e.g., hypopharyngeal carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer), salivary gland cancer).
  • the cancer is esophageal cancer (e.g., esophageal squamous cell carcinoma, esophageal adenocarcinoma, Barrett’s adenocarcinoma, esophageal leiomyosarcoma).
  • the cancer is gastrointestinal cancer (e.g., anal cancer, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), gall bladder cancer, gastric cancer (e.g., stomach cancer (e.g., stomach adenocarcinoma)), gastrointestinal stromal tumor (GIST), small bowel cancer (e.g., appendix cancer, small bowel carcinoma, e.g., small bowel adenocarcinoma), small intestine cancer, large bowel cancer, large intestine cancer).
  • gastrointestinal cancer e.g., anal cancer, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), gall bladder cancer, gastric cancer (e.g., stomach cancer (e.g., stomach adenocarcinoma)), gastrointestinal stromal tumor (GIST), small bowel cancer (e.g., appendix cancer, small bowel carcinoma, e
  • the cancer is lung cancer (e.g., bronchus cancer (e.g., bronchogenic carcinoma, bronchial adenoma), alveolar carcinoma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, chondromatous hamartoma, papillary adenocarcinoma).
  • lung cancer e.g., bronchus cancer (e.g., bronchogenic carcinoma, bronchial adenoma), alveolar carcinoma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, chondromatous hamartoma, papillary adenocarcinoma).
  • the cancer is a genitourinary cancer (e.g., bladder cancer (e.g., urothelial carcinoma), urethral cancer, kidney cancer (e.g
  • the cancer is a gynecological cancer (e.g., endometrial cancer (e.g., uterine cancer (e.g., uterine sarcoma, choriocarcinoma), endometrial carcinoma), cervical cancer (e.g., cervical adenocarcinoma), ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), germ cell cancer, vulvar cancer (e.g., Paget’s disease of the vulva) vaginal cancer, fallopian tube cancer).
  • endometrial cancer e.g., uterine cancer (e.g., uterine sarcoma, choriocarcinoma), endometrial carcinoma)
  • cervical cancer e.g., cervical adenocarcinoma
  • ovarian cancer e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ova
  • the cancer is breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast, triple negative breast cancer, HER-2 positive breast cancer, HER2-negative breast cancer).
  • the cancer is skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC), dermatofribroma).
  • the cancer expresses an antigen selected from CD38, CD33, CD22, TROP2, CD30, CD79b, and Nectin-4.
  • the cancer expresses said antigen and the antibody component of the SPAC is an antibody directed against said antigen, or an antigen-binding fragment thereof.
  • the cancer expresses said antigen; the antibody component of the SPAC is an antibody directed against said antigen, or an antigen-binding fragment thereof; and the stapled peptide is a stapled anti- cancer peptide.
  • the cancer expresses CD38.
  • a SPAC provided herein, or a pharmaceutical composition thereof.
  • SPACs for use in inhibiting tumor growth in a subject.
  • the tumor may express any of the antigens described herein (e.g., HER2, CD38, CD33, CD22, TROP2, CD30, CD79b, Nectin- 4).
  • the term “inhibit” or “inhibition” in the context of tumor growth refers to a reduction in the rate of growth of the tumor (i.e., reduction in the rate of proliferation of the tumor’s cells). In some embodiments, the term refers to a reduction in the rate of tumor growth to a level that is statistically significantly lower than an initial rate (e.g., the rate of tumor growth before administration or application of a SPAC provided herein).
  • tumor size is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) relative to its size prior to treatment.
  • Size of a tumor may be measured by any reproducible means of measurement.
  • the size of a tumor may be measured as a diameter of the tumor or by any reproducible means of measurement.
  • the tumor size is reduced by at least 25% relative to its size prior to treatment.
  • treating cancer and/or inhibiting tumor growth may further result in a decrease in number of tumors.
  • tumor number is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) relative to number prior to treatment.
  • Number of tumors may be measured by any reproducible means of measurement.
  • the number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification (e.g., 2x, 3x, 4x, 5x, 10x, or 50x).
  • treating cancer can result in a decrease in number of metastatic nodules in other tissues or organs distant from the primary tumor site.
  • treating cancer can result in an increase in average survival time of a population of subjects treated according to the present disclosure in comparison to a population of untreated subjects. For example, the average survival time is increased by more than 30 days (more than 60 days, 90 days, or 120 days).
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with the compound of the present disclosure.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with the compound of the present disclosure.
  • treating cancer can also result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. For example, the mortality rate is decreased by more than 2% (e.g., more than 5%, 10%, or 25%).
  • a decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with the compound of the present disclosure.
  • a decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with the compound of the present disclosure.
  • treating cancer can also result in an increased average progression-free survival time of a population of treated subjects in comparison to an untreated population. For example, the average progression-free survival time is increased by more than 30 days (more than 60 days, 90 days, or 120 days).
  • Also provided herein are methods of delivering a stapled peptide into a cell comprising contacting the cell with a SPAC provided herein, or a pharmaceutical composition thereof.
  • the cell is a cancer cell.
  • the stapled peptide has improved cellular uptake relative to a corresponding unconjugated stapled peptide.
  • the stapled peptide in delivered to the cell in vitro.
  • the stapled peptide is delivered to the cell in vivo (i.e., in a subject).
  • the stapled peptide is delivered to the cells of a biological sample.
  • Also provided herein are methods of triggering cancer cell death comprising contacting the cancer cell with an effective amount of a SPAC provided herein, or a pharmaceutical composition thereof.
  • the method is a method for selectively triggering cancer cell death (i.e., selectively killing cancer cells).
  • a SPAC provided herein is selectively cytotoxic to cancer cells.
  • the cell is contacted in vitro.
  • the cell is contacted in vivo (i.e., in a subject).
  • the cell is contacted in a biological sample.
  • the selectivity in any of the foregoing embodiments is at least 1.1-fold, at least 1.5-fold, 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 30-fold, at least 50- fold, at least 100-fold, at least 300-fold, at least 500-fold, at least 1,000-fold, at least 3,000- fold, at least 5,000-fold, at least 10,000-fold, at least 30,000-fold, at least 50,000-fold, or at least 100,000-fold.
  • the selectivity is not more than 100,000-fold, not more than 10,000-fold, not more than 1,000-fold, not more than 100-fold, not more than 10- fold, or not more than 2-fold.
  • the cells may be cancer cells.
  • the cells are breast cancer (e.g., BT-474, SK-BR-3, MCF-7), ovarian cancer (e.g., SK-OV-3), esophageal cancer (e.g., OE19, KYSE-410), stomach cancer (e.g., NCI-N87), multiple myeloma (e.g., NCIH929, RMPI 8226), colorectal cancer (e.g., HCT-116, COLO-678), lymphoma: (e.g., DAUDI) or ALL (e.g., DND41) cells.
  • breast cancer e.g., BT-474, SK-BR-3, MCF-7
  • ovarian cancer e.g., SK-OV-3
  • esophageal cancer e.g., OE19, KYSE-410
  • stomach cancer e.g., NCI-N87
  • multiple myeloma e.
  • bacterial infections include, but are not limited to, skin infections (e.g., bacterial cellulitis, wound infections), gastrointestinal infections, throat infections (e.g., strep throat), urinary tract infections (UTIs), genito-urinary infections, sexually-transmitted diseases (e.g., gonorrhea, chlamydia, syphilis), pulmonary infections (e.g., pneumonia, pneumococcal pneumonia, tuberculosis, whooping cough), food poisoning, sepsis, bacterial meningitis, Lyme disease, cholera, botulism, tetanus, anthrax, blood infections, and systemic infections.
  • skin infections e.g., bacterial cellulitis, wound infections
  • gastrointestinal infections e.g., strep throat
  • urinary tract infections e.g., genito-urinary infections
  • sexually-transmitted diseases e.g., gonorrhea, ch
  • the microbial infection is an infection with bacteria, i.e., a “bacterial infection.”
  • the bacteria are Gram-negative bacteria.
  • the bacteria are Gram-positive bacteria.
  • Gram-negative bacteria were first defined by their ability not to retain Gram staining; however, since then Gram-negative bacteria have been further defined as bacteria generally having a cell wall with a thin peptidoglycan layer and have an outer lipid membrane.
  • Gram- positive bacteria are bacteria that take up the crystal violet color in the Gram staining test, and generally have cell walls comprising a thick peptidoglycan layer and no outer lipid membrane.
  • an “antibiotic-resistant bacterial infection” is a bacterial infection caused by antibiotic-resistant bacteria. “Antibiotic resistance” occurs when bacteria evolve mechanisms that protect them from the effects of antibiotics. Microbes resistant to multiple antimicrobials are referred to as “multidrug resistant” (MDR). In certain embodiments, methods herein are for treating multidrug resistant bacterial infections. In certain embodiments, methods herein are for killing and/or inhibiting the growth of multidrug resistant bacteria.
  • Solid phase peptide synthesis Fmoc-based solid-phase peptide synthesis was used to synthesize the antimicrobial peptides and their stapled derivatives.
  • Cell culture Cell lines were maintained in appropriate medium supplemented with fetal bovine serum to a final concentration of 10%.
  • 72 hour cytotoxicity assay Cells were plated in a 96-well format, and after 24 hour incubation, serial dilutions of SPACs from a 1 mg/mL stock, or vehicle, were then added to the cells in a final volume of 100 ⁇ l. After incubating at 37 °C for 72 hours, 100 ⁇ l of CellTiter-Glo ® reagent was added to the cells, and the plates were incubated 15 minutes at room temperature. Luminescence was then measured on a microplate reader.
  • Example 1 Preparation of SPACs [331] Peptide conjugation protocol: (1) Prepare stapled peptide using solid-phase synthesis; (2) On resin, to an N-terminally deprotected stapled peptide, add a linking reagent. For example, 6-maleimidohexanoyl-Val-Cit-p-aminobenzoylcarbonate-4-nitrophenyl ester (Mc- Val-Cit-PABC-PNP) can be used; (3) Cleave from resin and purify by HPLC; (4) In a separate batch, reduce the antibody using TCEP (see protocol below); (5) Conjugate the stapled peptide to the antibody; (6) Quench unreacted peptide using N-acetylcysteine; and (7) Purify by salt exchange.
  • a linking reagent for example, 6-maleimidohexanoyl-Val-Cit-p-aminobenzoylcarbonate-4-nitrophenyl ester (Mc- Val-C
  • Antibody conjugation protocol (1) Buffer exchange the antibody into PBS-E, pH 6-8, by the following protocol: (a) Dilute a 21 mg/mL solution of antibody provided by the manufacturer to 1 mg/mL in PBS-E, pH 6-8, (b) Remove the loading buffer by centrifugating using a spin column.
  • Anti-CD38 SPACs Comprising PPI Inhibitors
  • PPI peptide protein-protein interaction
  • FIGS.3-5 SPACs 8, 11, and 16(see Table 7), each comprising the anti-CD38 antibody daratumumab, inhibit the proliferation of multiple myeloma cells.
  • Example 5 Selectivity of Anti-HER2 SPACs Comprising PPI inhibitors
  • SPACs can be highly selective, which is beneficial when trying to avoid on-target toxicity associated with traditional ADCs. SPACs can also overcome low receptor expression by taking advantage of oncogene addiction.
  • FIG.6A shows results in a HER2-low cell line, MCF7, which has wild-type (wt) p53 status and is thus sensitive to MDM2 inhibition.
  • Trastuzumab emtansine a traditional ADC, shows no activity in this cell line, while SPAC 7 provided herein (see Table 7) comprising an MDM2 inhibitor shows anti- proliferative activity.
  • FIG.6B shows results in a cell line that highly expresses HER2 but has a mutated p53 and thus is insensitive to MDM2 inhibition.
  • the traditional ADC trastuzumab emtansine shows efficacy at relatively low concentration while SPAC 7 (see Table 7) requires a relatively high concentration.
  • a stapled peptide-antibody conjugate comprising a stapled peptide conjugated to an antibody or antigen-binding fragment thereof.
  • the SPAC of paragraph 1 wherein the antibody is a monoclonal antibody (mAb) or antigen-binding fragment thereof.
  • the SPAC of paragraph 1 or 2 wherein the antibody is an anti-cancer antibody or antigen-binding fragment thereof.
  • the SPAC of any one of paragraphs 1-3 wherein the antibody or antigen- binding fragment thereof is directed to a target antigen expressed on a cancer cell.
  • HER2 human epidermal growth factor receptor 2
  • the SPAC of paragraph 5 wherein the antibody is trastuzumab. 7. The SPAC of paragraph 5, wherein the antibody is pertuzumab. 8. The SPAC of any one of paragraphs 1-4, wherein the antibody is an antibody directed against CD38, CD33, CD22, TROP2, CD30, CD79b, Nectin-4, or TM4SF1, or antigen-binding fragment thereof. 9. The SPAC of any one of paragraphs 1-4, wherein the antibody is an antibody directed against TM4SF1, or an antigen-binding fragment thereof. 10. The SPAC of any one of paragraphs 1-4, wherein the antibody is an antibody directed against CD38, or an antigen-binding fragment thereof. 11. The SPAC of paragraph 10, wherein the antibody daratumumab. 12.
  • ADC antibody- drug conjugate
  • the antibody is trastuzumab emtansine.
  • the stapled peptide is a stapled anti-cancer peptide.
  • the SPAC of any one of paragraphs 1-14, wherein the stapled peptide is a stapled antimicrobial peptide (StAMP).
  • StAMP stapled antimicrobial peptide
  • PPI protein-protein interaction
  • BIM cell death
  • the stapled peptide comprises the amino acid sequence: G X 1 G K F X 2 H S K K K F G K A X 3 V G E X 4 (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof, wherein: X 1 , X 2 , X 3 , and X 4 are amino acids; X 1 and X 2 are connected via a crosslink, and X 3 and X 4 are connected via a crosslink; and the amino acid sequence includes 0 to 9 amino acid substitutions, inclusive, at positions other than X 1 , X 2 , X 3 , and X 4 . 35.
  • the stapled peptide comprises the amino acid sequence: G X 1 G K F X 2 H S K K K F G K A X 3 V G E X 4 A K K (SEQ ID NO: 2), or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 0 to 11 amino acid substitutions, inclusive, at positions other than X 1 , X 2 , X 3 , and X 4 .
  • the stapled peptide comprises the amino acid sequence: G X 1 G K F X 2 Dap K K K K F G K A X 3 V G E X 4 A K K (SEQ ID NO: 48), or a pharmaceutically acceptable salt thereof.
  • the stapled peptide comprises the amino acid sequence: G X 1 G Dap F X 2 Dap Dap Dap Dap Dap F G Dap A X 3 V G E X 4 A Dap Dap (SEQ ID NO: 26), G X 1 G K F X 2 K K K K K F G K A X 3 V G E X 4 A K K (SEQ ID NO: 5), G X 1 G K F X 2 H K K K K F G K A X 3 V F E X 4 A K K (SEQ ID NO: 23), G X 1 G Dab F X 2 Dab Dab Dab Dab F G Dab A X 3 V G E X 4 A Dab Dab (SEQ ID NO: 24), or a pharmaceutically acceptable salt thereof.
  • the stapled peptide comprises the amino acid sequence: G X 1 F S K X 2 K G K K I K N L X 3 I S G X 4 K G (SEQ ID NO: 73), and pharmaceutically acceptable salts thereof, wherein: X 1 , X 2 , X 3 , and X 4 are amino acids; X 1 and X 2 are connected via a crosslink, and X 3 and X 4 are connected via a crosslink; and the amino acid sequence includes 0 to 9 amino acid substitutions, inclusive, at positions other than X 1 , X 2 , X 3 , and X 4 . 41.
  • the SPAC of paragraph 40 wherein the stapled peptide comprises SEQ ID NO: 73, or a pharmaceutically acceptable salt thereof.
  • the stapled peptide comprises SEQ ID NO: 105, or a pharmaceutically acceptable salt thereof.
  • the stapled peptide comprises the amino acid sequence: G X 1 F S K X 2 K G K K I K N L L L X 3 S G L X 4 K G G E (SEQ ID NO: 113), or a pharmaceutically acceptable salt thereof.
  • the SPAC of paragraph 36 wherein the stapled peptide is of SEQ ID NO: 48, wherein X 1 and X 2 , and X 3 and X 4 , are connected via a crosslink of the following formula: wherein ⁇ denotes the ⁇ -carbons of the crosslinked amino acids; and wherein the C-terminus of the stapled peptide is amidated with –NH 2 . 50.
  • the SPAC of paragraph 37 wherein the stapled peptide is of SEQ ID NO: 26, wherein X 1 and X 2 , and X 3 and X 4 , are connected via a crosslink of the following formula (alk); and wherein the C-terminus of the stapled peptide is amidated with –NH 2 . 51.
  • the SPAC of paragraph 42 wherein the stapled peptide is of SEQ ID NO: 73, wherein X 1 and X 2 , and X 3 and X 4 , are connected via a crosslink of the following formula (alk); and wherein the C-terminus of the stapled peptide is amidated with –NH 2 .
  • the SPAC of paragraph 43 wherein the stapled peptide is of SEQ ID NO: 105, wherein X 1 and X 2 , and X 3 and X 4 , are connected via a crosslink of the following formula (alk); and wherein the C-terminus of the stapled peptide is amidated with –NH 2 . 53.
  • a pharmaceutical composition comprising an SPAC of any one of paragraphs 1-70 and a pharmaceutically acceptable carrier.
  • a method of inhibiting tumor growth in a subject comprising administering to the subject an SPAC of any one of paragraphs 1-70, or a pharmaceutical composition thereof.
  • a method of delivering a stapled peptide to a cell comprising contacting the cell with an SPAC of any one of paragraphs 1-70, or a pharmaceutical composition thereof.

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IL312929A IL312929A (en) 2021-12-10 2022-12-09 Paired peptide-antibody conjugates and their uses
CA3238784A CA3238784A1 (en) 2021-12-10 2022-12-09 Stapled peptide-antibody conjugates (spacs) and uses thereof
EP22905164.4A EP4444736A2 (en) 2021-12-10 2022-12-09 Stapled peptide-antibody conjugates (spacs) and uses thereof
AU2022407066A AU2022407066A1 (en) 2021-12-10 2022-12-09 Stapled peptide-antibody conjugates (spacs) and uses thereof
JP2024534450A JP2024545161A (ja) 2021-12-10 2022-12-09 ステープルドペプチド－抗体抱合体（ＳＰＡＣｓ）およびそれらの使用
KR1020247023088A KR20240125945A (ko) 2021-12-10 2022-12-09 스테이플링된 펩티드-항체 접합체 (spac) 및 그의 용도
CN202280090496.9A CN119110805A (zh) 2021-12-10 2022-12-09 钉合肽-抗体偶联物(spac)及其用途
MX2024007006A MX2024007006A (es) 2021-12-10 2022-12-09 Conjugados de anticuerpo-peptido engrapado (spac) y usos de los mismos.

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