Nailing peptide-antibody conjugates (SPACs) and uses thereof

Abstract

Provided herein are stapled peptide-antibody conjugates (SPACs) comprising a stapled peptide conjugated to an antibody or an antigen-binding fragment thereof. In certain embodiments, the stapling peptide is coupled to the antibody or antigen-binding fragment thereof via a linker. In certain embodiments, SPACs provided herein can be used to deliver stapling peptides to cells (e.g., cancer cells) with relatively high selectivity and relatively low off-target toxicity. Also provided herein are pharmaceutical compositions and kits comprising the SPACs described herein, methods of using the same (e.g., to treat cancer and / or inhibit tumor growth in a subject), and methods of making the same.

Description

[0001] Related Applications

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Applications USSN 63 / 288,498 filed on December 10, 2021, USSN 63 / 321,968 filed on March 21, 2022, and USSN 63 / 353,275 filed on June 17, 2022, the entire contents of which are incorporated herein by reference.

[0003] background

[0004] Antibody-drug conjugates (ADCs) are a class of unique oncology drugs that can carry highly cytotoxic payloads directly into cells (e.g., cancer cells). This advance in precision medicine has made possible the use of very effective chemotherapeutics that previously had very narrow therapeutic windows. ADCs are described in, for example, 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.

[0005] Interestingly, due to the high potency of its cargo, ADC can be used to treat cancer patients who have been heavily pre-treated with other chemotherapeutic agents (including chemotherapeutic agents of the same therapeutic class) and even the same unconjugated antibodies. However, despite significant progress in this field, some key liabilities of ADC still need to be addressed. One of these problems is that ADC cargo has non-specific toxicity due to, for example, linker instability. Since these cargoes tend to be highly cytotoxic, they can damage healthy tissues when they are inadvertently released outside tumor cells. Efforts to address this problem have focused on creating new linkers with higher stability and release selectivity. SUMMARY OF THE INVENTION

[0007] A new class of therapeutic agents is described herein that solves many problems of current antibody-drug conjugates (ADCs) and expands the payload beyond current highly cytotoxic agents. Relying on a class of unique peptides known as stapled peptides, the conjugates described herein have unique advantages without many of the drawbacks of current ADCs.

[0008] Stapled peptides are synthetic peptides comprising at least two non-natural amino acids connected by cross-linking (ie, "stapled"). For example, stapled peptides may include at least two non-natural olefin-containing amino acids that are cyclized to form internal hydrocarbon cross-links (ie, "hydrocarbon stapling") by translocation. This cyclization or "stapled" process can help the peptide fold into an alpha helical configuration or other secondary structure, thereby regaining the natural three-dimensional orientation of the protein structure to simulate its biological function. Examples of stapled peptide technology can be found in, for example, International PCT Application Publication No. WO 2017 / 004591, published on January 5, 2017, WO 2019 / 018499, published on January 24, 2019, and WO 2021 / 126827, published on June 24, 2021, the entire contents of each of which are incorporated herein by reference. See also, for example, Mourtada et al., Nature Biotechnology, 2019, Vol. 37, 1186-1197, the entire contents of which are incorporated herein by reference.

[0009] Since their development in the early 2000s, stapled peptides have been shown to disrupt protein-protein interactions (PPIs) both outside and inside cells. Currently, Aileron Therapeutics has a stapled peptide drug candidate, ALRN-6924, in a Phase 2 clinical trial as a prophylactic treatment for chemotherapy-induced neutropenia. Although stapled peptides themselves are promising therapeutic agents, a challenge facing some development efforts is the inability to deliver stapled peptides intracellularly without triggering lysis of off-target cells. This is because many stapled peptides already exhibit low cell permeability.

[0010] However, in the context of antibody conjugates, the low cell permeability of certain stapled peptides can be an asset rather than a defect. By coupling the stapled peptide to an antibody or its antigen-binding fragment, the internalization of the conjugate can be utilized to deliver the load / payload (i.e., the stapled peptide) into the cell, which would otherwise be inactive or have low activity outside the cell. Similarly, the ability of antibodies to aggregate payloads at the plasma membrane of receptor-positive cells may allow targeted delivery of stapled peptides that would otherwise be nonspecifically cleaved. The unique properties of the stapled peptides utilized in these systems allow for the delivery of highly effective therapeutic agents, but without the off-target toxicity of previous ADCs.

[0011] Provided herein is a stapled peptide-antibody conjugate (SPAC), comprising a stapled peptide coupled to an antibody or its antigen-binding fragment. In certain embodiments, the stapled peptide is coupled to an antibody or its antigen-binding fragment via a linker. In certain embodiments, provided herein is a SPAC that can be used to deliver a stapled peptide to a cell (e.g., cancer cell, bacterial cell) with relatively high selectivity and / or relatively low off-target toxicity.

[0012] SPACs provided herein include antibodies or antigen-binding fragments thereof. Antibodies (including complete 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., two, three and four specific antibodies, diabodies, tribodies and tetrabodies), single domain antibodies (sdAb), epitope binding fragments (e.g., Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), rIgG, single-chain antibodies, disulfide-linked Fv (sdFv), fragments containing VL or VH domains, fragments produced by Fab expression libraries) and anti-idiotype (anti-Id) antibodies. Fab and F(ab')2 fragments, for example, lack the Fc fragment of a complete antibody. The antibody molecule of the conjugate 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.

[0013] In certain embodiments, the antibody is a monoclonal antibody (mAb) or an antigen binding fragment thereof. In certain embodiments, the antibody is an anti-cancer antibody or an antigen binding fragment thereof. In certain embodiments, the antibody or its antigen binding fragment is directed to a target antigen expressed on a cancer cell (e.g., specifically expressed on a cancer cell). In certain embodiments, the antibody is a homologue of an antibody or its antigen binding fragment described herein.

[0014] In certain embodiments, the antibody is an antibody-drug conjugate (ADC) or an antigen-binding fragment thereof. In such embodiments, the SPAC comprises an antibody or an antigen-binding fragment thereof coupled to a dual payload: (i) a stapled peptide or a pharmaceutically acceptable salt thereof; and (ii) a second agent (i.e., the "drug" component of the antibody-drug conjugate).

[0015] The stapled peptide component of the SPAC can be any stapled peptide (e.g., any single stapled peptide, double stapled peptide, multi-stapled peptide, or stitched peptide). In certain embodiments, the stapled peptide is a stapled anticancer peptide. In certain embodiments, the stapled peptide is a stapled antimicrobial peptide (StAMP). In certain embodiments, the stapled peptide is a stapled magainin (e.g., a stapled magainin II peptide). In certain embodiments, the stapled peptide is a stapled Esculentin peptide (e.g., a stapled Esculentin-1A peptide).

[0016] In certain embodiments, the stapled peptide is an inhibitor of protein-protein interaction (PPI). In certain embodiments, the stapled peptide is a BCL-2 family member protein (e.g., BCL-x L , BCL-2, BCL-W or MCL1). In certain embodiments, the stapled peptide is an activator of a BCL-2 family member protein effector (e.g., BAX, BAK or BOK). In certain embodiments, the stapled peptide is a stapled BCL-2-interacting mediator of cell death (BIM) peptide. In certain embodiments, the stapled peptide is a β-catenin inhibitor (e.g., an inhibitor of Wnt / β-catenin signaling). In certain embodiments, the stapled peptide is an MDM2 and / or MDMX inhibitor (e.g., inhibiting the binding of MDM2 and / or MDMX to p53). Other examples of stapled peptides are provided herein.

[0017] In certain embodiments, the antibody or its antigen-binding fragment is directly coupled to the stapled peptide (e.g., via a bond). In certain embodiments, the antibody or its antigen-binding fragment is coupled to the stapled peptide via a linker. In certain embodiments, the linker is a cleavable linker (e.g., a pH cleavable linker, or a linker cleavable by protease, esterase, or intracellular disulfide reduction). In certain embodiments, the linker is a peptide linker (e.g., a cleavable peptide linker).

[0018] Also provided herein is a pharmaceutical composition comprising a SPAC provided herein and a pharmaceutically acceptable carrier. In certain embodiments, 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).

[0019] The stapled-peptide antibody conjugates (SPACs) provided herein can deliver biologically active stapled peptides to cells (e.g., cancer cells, bacterial cells), and thus can be used to treat and / or prevent various diseases (e.g., proliferative diseases such as cancer, infectious diseases). The methods provided herein include:

[0020] (i) a method for treating and / or preventing a disease in a subject, comprising administering to the subject a therapeutically and / or prophylactically effective amount of a SPAC or a pharmaceutical composition thereof provided herein. In certain embodiments, the disease is a proliferative disease (e.g., cancer);

[0021] (ii) a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a SPAC provided herein or a pharmaceutical composition thereof;

[0022] (iii) a method 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;

[0023] (iv) a method of delivering a stapled peptide into a cell, comprising contacting the cell with a SPAC or a pharmaceutical composition thereof provided herein. In certain embodiments, the cell is a cancer cell. In certain embodiments, the stapled peptide is delivered to a cell in vitro. In certain embodiments, the stapled peptide is delivered to a cell in vivo (i.e., in a subject); and

[0024] (v) a method of triggering cancer cell death, comprising contacting the cancer cell with an effective amount of a SPAC or a pharmaceutical composition thereof provided herein. In certain embodiments, the stapled peptide is delivered to cells in vitro. In certain embodiments, the stapled peptide is delivered to cells in vivo (ie, in a subject).

[0025] (vi) a method for treating and / or preventing an infectious disease (e.g., a bacterial infection) in a subject, comprising administering to the subject an effective amount of a SPAC provided herein or a pharmaceutical composition thereof;

[0026] (vii) a method of killing bacteria and / or inhibiting bacterial growth, comprising contacting the bacteria with an effective amount of a SPAC or a pharmaceutical composition thereof provided herein;

[0027] Also provided herein are SPACs described herein and pharmaceutical compositions thereof for use in any of the methods described herein. In another aspect, provided herein are uses of SPACs described herein and pharmaceutical compositions thereof for the preparation of a medicament.

[0028] In another aspect, provided herein is a kit comprising a SPAC or a pharmaceutical composition thereof provided herein. The kit described herein may include a single dose or multiple doses of a SPAC or a pharmaceutical composition thereof. The kit described herein may be used for any method or use provided herein, and optionally further comprises instructions for using the kit (e.g., instructions for using the SPAC or composition contained in the kit).

[0029] Also provided herein are methods of making the SPACs described herein.

[0030] Details of certain embodiments of the invention 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, drawings and claims.

[0031] definition

[0032] General Definition

[0033] The following definitions are general terms used in this application.

[0034] The terms "peptide" and "polypeptide" are used interchangeably and represent a polymer of amino acid residues linked together by peptide bonds. The term also includes proteins and represents peptides, polypeptides and proteins of any size, structure or function. Typically, a peptide will be at least three amino acids in length, or at least the length required for the amino acid sequence provided herein. A peptide can represent a single peptide or a collection of peptides. The peptides provided herein can include any combination of natural amino acids and / or non-natural amino acids (i.e., compounds that do not exist in nature but can be incorporated into a peptide chain). One or more amino acids in a peptide can be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, joints for coupling or functionalization, or other modifications. 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.

[0035] The term "amino acid" refers to a molecule containing an amino group and a carboxyl group. Amino acids include α-amino acids, the general structure of which is shown below. Each amino acid mentioned herein can be represented by a 1 to 4 letter code (e.g., R and Arg represent L-arginine, and hArg represents L-homoarginine).

[0036]

[0037] Suitable amino acids include, but are not limited to, natural α-amino acids such as the D- and L-isomers of the 20 common naturally occurring α-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 α-amino acids.

[0038] Exemplary natural α-amino acids (single letter codes are provided in parentheses) 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).

[0039] Exemplary non-natural α-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, α-methyl-alanine (Aib), α-methyl-arginine, α-methyl-asparagine, , α-methyl-aspartic acid, α-methyl-cysteine, α-methyl-glutamic acid, α-methyl-glutamine, α-methyl-histidine, α-methyl-isoleucine, α-methyl-leucine, α-methyl-lysine, α-methyl-methionine, α-methyl-phenylalanine, α-methyl-proline, α-methyl-serine, α-methyl-threonine, α-methyl-tryptophan, α-methyl-tyrosine, α-methyl-valine, norleucine and terminally unsaturated α-amino acids. There are many known non-natural amino acids, any of which can be included in the peptides of the present disclosure. See, for example, S. Hunt, The Non-Protein Amino Acids: In Chemistry and Biochemistry of the Amino Acids, GC Barrett, Chapman and Hall, 1985. Non-natural amino acids also include amino acids containing nitrogen substituents.

[0040] Table 1 below provides certain amino acids referred to herein (by name, structure and 1 to 4 letter code).

[0041] Table 1. Certain amino acids

[0042]

[0043]

[0044]

[0045] When used with reference to an amino acid sequence, the term "amino acid replacement" means that an amino acid in the amino acid sequence is replaced by a different amino acid (e.g., replaced by a natural or non-natural amino acid). The amino acid sequences provided herein may include or include one or more amino acid replacements. Specific amino acid replacements are represented by colloquial nomenclature commonly used in the field of peptide sequencing to represent amino acid sequence changes. For example, when referring to SEQ ID NO: 2 (hereinafter), "amino acid replacement at H7" means that the histidine (H) at position 7 of the amino acid sequence is replaced by a different amino acid (e.g., a natural or non-natural amino acid other than histidine). And, for example, when referring to SEQ ID NO: 2, the amino acid replacement "H7K" means that the histidine (H) at position 7 in the amino acid sequence of SEQ ID NO: 2 is replaced by lysine (K), resulting in an amino acid sequence represented by SEQ ID NO: 3 (hereinafter).

[0046] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Position number G <![CDATA[X 1 ]]> G K F <![CDATA[X 2 ]]> H S K K K F G K A <![CDATA[X 3 ]]> V G E <![CDATA[X 4 ]]> A K K SEQ ID NO:2 G <![CDATA[X 1 ]]> G K F <![CDATA[X 2 ]]> K S K K K F G K A <![CDATA[X 3 ]]> V G E <![CDATA[X 4 ]]> A K K SEQ ID NO:3

[0047] When used in reference to an amino acid sequence, the term "amino acid addition" means that an amino acid (e.g., a natural or unnatural amino acid) is inserted between two amino acids in the amino acid sequence, or is added at either end of the sequence. Standard colloquial nomenclature is used to represent specific amino acid additions (e.g., when referring to SEQ ID NO: 2, "G3_K4insX" indicates that the putative amino acid X is inserted between amino acids G3 and K4 of the amino acid sequence). In certain embodiments, the amino acid sequences herein may comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid additions.

[0048] When used with reference to an amino acid sequence, the term "amino acid deletion" means that the amino acid of the amino acid sequence is deleted from the amino acid sequence. Standard colloquial nomenclature is used to represent specific amino acid deletions (e.g., when referring to SEQ ID NO: 2, "G1del" means that the amino acid G1 is deleted from the sequence). In certain embodiments, the amino acid sequences herein may comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid deletions.

[0049] The term "salt" as used herein means any and all salts, and encompasses pharmaceutically acceptable salts. Salts include ionic compounds produced by the neutralization reaction of an acid and a base. Salts are composed of one or more cations (positively charged ions) and one or more anions (negative ions), so that the salt is electrically neutral (no net charge). Salts of the peptides of the present invention include salts derived from inorganic and organic acids and bases. Examples of acid addition salts are salts formed of an amino group with an inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or an organic acid 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. Other 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, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, hippurate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Other salts include ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates.

[0050] The term "pharmaceutically acceptable salt" means a salt that is suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., within the scope of sound medical judgment, and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19 (incorporated herein by reference). Pharmaceutically acceptable salts of the peptides of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed of an amino group with an inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or an organic acid 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. Other pharmaceutically acceptable 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, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates.

[0051] Throughout this disclosure, references to "stapled peptides" are intended to encompass peptides comprising any amino acid sequence provided herein (including any disclosed amino acid substitutions, additions, deletions and / or modifications), as well as pharmaceutically acceptable salts, stereoisomers, tautomers, isotopically labeled derivatives, solvates, hydrates, polymorphs, cocrystals and prodrugs thereof.

[0052] The terms "composition" and "formulation" are used interchangeably.

[0053] A "subject" to which administration is contemplated means a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young, middle-aged, or elderly person)) or a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog), or a poultry (e.g., a commercially relevant poultry such as a chicken, duck, goose, or turkey)). In certain embodiments, the non-human animal is a fish, a reptile, or an amphibian. The non-human animal may be male or female at any stage of development. The non-human animal may be a transgenic animal or a genetically engineered animal. The term "patient" means a human subject in need of treatment for a disease.

[0054] The term "biological sample" means any sample, including tissue samples (such as tissue sections and needle biopsies of tissue); cell samples (e.g., cytological smears (such as Pap smears or blood smears) or cell samples obtained by microdissection); samples of whole organisms (such as samples of yeast or bacteria); or cell fractions, fragments, or organelles (such as obtained by lysing cells and separating their components by centrifugation or other means). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules from a first biological sample.

[0055] The term "administering" means implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a peptide described herein or a composition thereof into or onto the body of a subject.

[0056] The term "treatment" means reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease described herein. In certain embodiments, treatment may be administered after one or more signs or symptoms of the disease have occurred or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to susceptible subjects before symptoms occur (e.g., in view of symptom history and / or in view of exposure to pathogens). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0057] The terms "condition," "disease," and "disorder" are used interchangeably.

[0058] An "effective amount" of a SPAC described herein means an amount sufficient to elicit a desired biological response. An effective amount of a SPAC described herein may vary with factors such as the desired biological endpoint, the severity of the side effects, disease or disorder, the identity, pharmacokinetics and pharmacodynamics of the specific peptide, the condition being treated, the mode, route and desired or required frequency of administration, the species, age and health or general condition of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a SPAC described herein in a single dose. In certain embodiments, an effective amount is the total amount of a SPAC described herein in multiple doses.

[0059] A "therapeutically effective amount" of a SPAC described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or alleviate one or more symptoms associated with the condition. A therapeutically effective amount of a SPAC refers to an amount of a therapeutic agent that provides a therapeutic benefit in the treatment of a condition when used alone or in combination with other therapies. The term "therapeutically effective amount" may include an amount that improves overall treatment, reduces or avoids symptoms, signs, or causes of a condition, and / or enhances the therapeutic effect of another therapeutic agent.

[0060] A "prophylactically effective amount" of a SPAC described herein is an amount sufficient to prevent a disorder or one or more symptoms associated with a disorder or to prevent its recurrence. A prophylactically effective amount of a SPAC refers to an amount of a therapeutic agent that provides a prophylactic benefit in the prevention of a disorder when used alone or in combination with other agents. The term "prophylactically effective amount" may include an amount that improves all prevention, or enhances the prophylactic efficacy of other prophylactic agents.

[0061] The term "prevention" refers to the preventive treatment of a subject who is not currently suffering from a disease and has not suffered from a disease in the past but has a risk of developing a disease, or a subject who has suffered from a disease in the past and does not currently suffer from a disease but has a risk of disease recurrence (regression). In certain embodiments, the subject has a higher risk of developing a disease or a higher risk of disease recurrence than the average healthy member of the population.

[0062] Chemical Definition

[0063] The definition of specific functional groups and chemical terms is described in more detail below. Chemical elements are identified according to the periodic table (Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition, inner cover), and specific functional groups are generally defined as described herein. In addition, the general principles of organic chemistry, as well as specific functional moieties and reactivity are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Michael B. Smith, March's Advanced Organic Chemistry, 7th edition, John Wiley & Sons, Inc., New York, 2013; Richard C. Larock, Comprehensive Organic Transformations, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd edition, Cambridge University Press, Cambridge, 1987.

[0064] The peptides described herein may contain one or more asymmetric centers and may therefore exist in various stereoisomeric forms, e.g., enantiomers and / or diastereomers. For example, the peptides described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of a mixture of stereoisomers, including a racemic mixture and a mixture enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis. See, e.g., Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, EL Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions, p. 268 (EL Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present invention further encompasses peptides as each isomer substantially free of other isomers, and alternatively as a mixture of various isomers.

[0065] In the formula, the key is a single bond, a hyphen is a single bond or does not exist, and the bond or is a single bond or a double bond. or A double or triple bond.

[0066] Unless otherwise provided, the formulas and structures depicted herein include peptides that do not include isotopically enriched atoms, and also include peptides that include isotopically enriched atoms ("isotopically labeled derivatives"). For example, a peptide having a structure of the invention but with hydrogen replaced by deuterium or tritium, 19 F 18 F is replaced, or carbon is 13 C- or 14C-enriched carbon-substituted peptides are within the scope of the present disclosure. Such peptides can be used as analytical tools or probes in, for example, biological assays. The term "isotope" refers to a variant of a particular chemical element such that although all isotopes of a given element have the same number of protons in each atom of the element, those isotopes differ in the number of neutrons.

[0067] When a range of values ​​("range") is listed, it includes every value and subrange within the range. Unless otherwise provided, a range includes values ​​at both ends of the range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl.

[0068] Use of the phrase "at least one instance" means 1, 2, 3, 4 or more instances, but also encompasses ranges, for example, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 4, from 2 to 3, or from 3 to 4 instances, inclusive.

[0069] "Non-hydrogen group" means any group as defined for a particular variable that is not hydrogen.

[0070] The term "aliphatic" refers to alkyl, alkenyl, alkynyl, and carbocyclic groups. Likewise, the term "heteroaliphatic" refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.

[0071] The term "alkyl" refers to the residue of a straight-chain or branched saturated hydrocarbon group having 1 to 20 carbon atoms ("C 1-20 In certain embodiments, an alkyl group has 1 to 6 carbon atoms (“C 1-6 C 1-6 Examples of alkyl groups include methyl (C1), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentyl, pentyl, neopentyl, 3-methyl-2-butanyl, tert-pentyl) and hexyl (C6) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), n-dodecyl (C9), n-octyl (C10), n-dodecyl (C11), n-octyl (C12), n-dodecyl (C13), n-dodecyl (C14), n-dodecyl (C15), n-dodecyl (C16), n-dodecyl (C17), n-dodecyl (C18), n-dodecyl (C19), n-dodecyl (C20), n-dodecyl (C21), n-dodecyl (C22), n-dodecyl (C23), n-dodecyl (C24), n-dodecyl (C25), n-dodecyl (C26), n-dodecyl (C27), n-dodecyl (C28), n-dodecyl (C29), n-dodecyl (C30), n-dodecyl (C31), n-dodecyl (C32), n-dodecyl (C33), n-dodecyl (C34), n-dodecyl (C35), n-dodecyl (C36), n-dodecyl (C37), n-dodecyl (C38), n-dodecyl (C39), n-dodecyl (C41), n-dodecyl (C42), n-dodecyl (C43), n-dodecyl (C412 )wait.

[0072] The term "haloalkyl" is a substituted alkyl group in which one or more hydrogen atoms are independently replaced by halogen (e.g., fluorine, bromine, chlorine, or iodine). "Perhaloalkyl" is a subset of haloalkyl and refers to an alkyl group in which all hydrogen atoms are independently replaced by halogen (e.g., fluorine, bromine, chlorine, or iodine). In certain embodiments, the haloalkyl moiety has 1 to 20 carbon atoms ("C 1-20 In some embodiments, all haloalkyl hydrogen atoms are independently replaced by fluorine to provide a "perfluoroalkyl" group. In some embodiments, all haloalkyl hydrogen atoms are independently replaced by chlorine to provide a "perchloroalkyl" group. Examples of haloalkyl groups include -CHF2, -CH2F, -CF3, -CH2CF3, -CF2CF3, -CF2CF2CF3, -CCl3, -CFCl2, -CF2Cl, etc.

[0073] The term "heteroalkyl" refers to an alkyl group that further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur, which is located within the parent chain (e.g., inserted between adjacent carbon atoms of the parent chain) and / or at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group ("heteroalkyl"). 1-20 alkyl").

[0074] The term "alkenyl" refers to the residue of a straight or branched chain hydrocarbon group having 1 to 20 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In certain embodiments, an alkenyl group has 1 to 20 carbon atoms ("C 1-20 The carbon-carbon double bond(s) may be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). In an alkenyl group, a C=C double bond whose stereochemistry is not specified (e.g., -CH=CHCH3 or ) can be in the (E)- or (Z)-configuration.

[0075] The term "heteroalkenyl" refers to an alkenyl group that further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur, which is located within the parent chain (e.g., inserted between adjacent carbon atoms of the parent chain) and / or at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having 1 to 20 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain ("heteroalkenyl"). 1-20 ("alkenyl").

[0076] The term "alkynyl" refers to the residue of a straight or branched chain hydrocarbon group ("C 1-20 The one or more carbon-carbon triple bonds may be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).

[0077] The term "heteroalkynyl" refers to an alkynyl group that further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur, which is located within the parent chain (e.g., inserted between adjacent carbon atoms of the parent chain) and / or at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having 1 to 20 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain ("heteroalkynyl"). 1-20 ("alkynyl").

[0078] The term "carbocyclyl" or "carbocyclic" refers to a non-aromatic ring system having 3 to 14 ring carbon atoms ("C 3-14 In certain embodiments, the carbocyclyl group has 3 to 6 ring carbon atoms ("C 3-6 "Carbocyclyl"). Exemplary C 3-6 Carbocyclyl groups include cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. As shown in the foregoing examples, in certain embodiments, the carbocyclyl group is monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing a fused, bridged, or spiro ring system such as a bicyclic system ("bicyclic carbocyclyl") or a tricyclic system ("tricyclic carbocyclyl")), and may be saturated or may contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes ring systems in which a carbocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the carbocyclyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the carbocyclic ring system.

[0079] The term "heterocyclyl" or "heterocyclic" refers to the residue of a 3-14 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur ("3-14 membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as long as valence permits. In certain embodiments, the heterocyclyl is a substituted or unsubstituted 3- to 7 membered monocyclic heterocyclyl, wherein 1, 2 or 3 atoms in the heterocyclic system are independently oxygen, nitrogen or sulfur, as long as valence permits. The heterocyclyl group may be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., fused, bridged or spirocyclic systems such as bicyclic systems ("bicyclic heterocyclyl") or tricyclic systems ("tricyclic heterocyclyl")), and may be saturated or may contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems may include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring as defined above is fused to one or more carbocyclyl groups, wherein the point of attachment is on the carbocyclyl or heterocyclyl ring; or ring systems in which a heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such cases the number of ring members continues to refer to the number of ring members in the heterocyclyl ring system.

[0080] The term "aryl" refers to the residue of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., sharing 6, 10 or 14 π electrons in the cyclic array) having 6-14 ring carbon atoms and zero heteroatoms ("C 6-14 In certain embodiments, an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl). In certain embodiments, an aryl group has 10 ring carbon atoms (“C 10 In certain embodiments, an aryl group has 14 ring carbon atoms ("C 14 "Aryl" also includes ring systems in which an aryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, wherein the residue or point of attachment is on the aryl ring, and in such cases the number of carbon atoms continues to refer to the number of carbon atoms in the aryl ring system.

[0081] The term "heteroaryl" refers to the residue of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., sharing 6, 10 or 14 π electrons in a cyclic array), wherein there are ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur ("5-14 membered heteroaryl"). In certain embodiments, the heteroaryl is a substituted or unsubstituted 5 or 6 membered monocyclic heteroaryl, wherein 1, 2, 3 or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen or sulfur. In certain embodiments, the heteroaryl is a substituted or unsubstituted 9 or 10 membered bicyclic heteroaryl, wherein 1, 2, 3 or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen or sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as long as valence permits. Heteroaryl polycyclic ring systems may include one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment is on the heteroaryl ring, and in such cases, the number of ring members continues to represent the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in such cases, the number of ring members represents the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. For polycyclic heteroaryl groups in which one ring does not contain heteroatoms (e.g., indolyl, quinolyl, carbazolyl, etc.), the point of attachment may be on either ring (e.g., the ring with heteroatoms or the ring without heteroatoms).

[0082] The addition of the suffix "-ene" to a group indicates that the group is a divalent moiety, for example, alkylene is a divalent moiety of alkyl, alkenylene is a divalent moiety of alkenyl, alkynylene is a divalent moiety of alkynyl, heteroalkylene is a divalent moiety of heteroalkyl, heteroalkenylene is a divalent moiety of heteroalkenyl, heteroalkynylene is a divalent moiety of heteroalkynyl, carbocyclylene is a divalent moiety of carbocyclyl, heterocyclylene is a divalent moiety of heterocyclyl, arylene is a divalent moiety of aryl, and heteroarylene is a divalent moiety of heteroaryl.

[0083] Unless otherwise clearly provided, chemical moieties are optionally substituted.The term "optionally substituted" means substituted or unsubstituted.In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic radical, heterocyclic radical, aryl, heteroaryl, acyl groups are optionally substituted.In general, when referring to chemical groups, the term "substituted" means that at least one hydrogen present in the group is replaced by an allowable substituent, for example, a substituent producing a stable compound after substitution, for example, a compound that will not spontaneously undergo conversion (such as by rearrangement, cyclization, elimination or other reactions).Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is the same or different at each position.The present invention is not limited in any way by the exemplary substituents described herein.

[0084] Exemplary substituents include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa 、-ON(R bb )2、-N(R bb )2、-N(R bb )3 + X - 、-N(OR cc )R bb , -SH, -SR aa 、-SCN、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc 2. -CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2, -OC(=O)N(R bb )2.-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2, -C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb)N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)(R aa )2、-P(=O)(OR cc )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)(N(R bb )2)2、-OP(=O)(N(R bb )2)2、-NR bb P(=O)(R aa )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(N(R bb )2)2、-P(R cc )2、-P(OR cc )2、-P(R cc )3 + X - 、-P(OR cc )3 + X - 、-P(R cc )4、-P(OR cc )4、-OP(Rcc )2.-OP(R cc )3 + X - 、-OP(OR cc )2.-OP(OR cc )3 + X - 、-OP(R cc )4.-OP(OR cc )4, -B(R aa )2、-B(OR cc )2. -BR aa (OR cc ), C 1-20 Alkyl, C 1-20 Perhaloalkyl, C 1-20 Alkenyl, C 1-20 Alkynyl, hetero C 1-20 Alkyl, hetero C 1-20 Alkenyl, hetero C 1-20 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 Aryl and 5-14 membered heteroaryl; wherein X - is the counter ion;

[0085] Or two geminal hydrogen atoms on a carbon atom are replaced by groups =O, =S, =NN (R bb )2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O)2R aa , =NR bb or = NOR cc replace;

[0086] in:

[0087] R aa Each instance of is independently selected from C 1-20 Alkyl, C 1-20 Perhaloalkyl, C 1-20 Alkenyl, C 1-20 Alkynyl, hetero C 1-20 Alkyl, hetero C 1-20 Alkenyl, hetero C 1-20 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 Aryl and 5-14 membered heteroaryl, or two R aa The groups are connected to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring;

[0088] Rbb Each instance of is independently selected from hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc 2. -CO2R aa 、-SO2R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc 2. -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)(R aa )2.-P(=O)(OR cc )2、-P(=O)(N(R cc )2)2、C 1-20 Alkyl, C 1-20 Perhaloalkyl, C 1-20 Alkenyl, C 1-20 Alkynyl, hetero C 1-20 Alkyl, hetero C 1-20 Alkenyl, hetero C 1-20 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 Aryl and 5-14 membered heteroaryl, or two R bb The groups are connected to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring;

[0089] R cc Each instance of is independently selected from hydrogen, C 1-20 Alkyl, C 1-20 Perhaloalkyl, C 1-20 Alkenyl, C 1-20 Alkynyl, hetero C 1-20 Alkyl, hetero C 1-20 Alkenyl, hetero C 1-20 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 Aryl and 5-14 membered heteroaryl, or two R cc The groups are connected to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each X - It is a counter ion.

[0090] In certain embodiments, each substituent is independently halogen, substituted (eg, substituted with one or more halogens), or unsubstituted C 1-6 Alkyl, -OR aa 、-SR aa 、-N(R bb )2, -CN, -SCN, -NO2, -N3, -C(=O)R aa 、-CO2R aa 、-C(=O)N(R bb )2, -OC(=O)R aa 、-OCO2R aa 、-OC(=O)N(R bb )2.-NR bb C(=O)R aa 、-NR bb CO2R aa or -NR bb C(=O)N(R bb )2.

[0091] The term "halo" or "halogen" refers to fluorine (F, -F), chlorine (Cl, -Cl), bromine (Br, -Br), or iodine (I, -I).

[0092] The term "hydroxyl" or "hydroxy" refers to the group -OH. The term "substituted hydroxy" by extension refers to a hydroxy group in which the oxygen atom directly attached to the parent molecule is replaced by a group other than hydrogen, and includes groups selected from: -OR aa 、-ON(R bb )2, -OC(=O)SR aa 、-OC(=O)R aa 、-OCO2R aa 、-OC(=O)N(R bb )2, -OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-OC(=NR bb )N(R bb )2. -OS(=O)R aa 、-OSO2R aa 、-OSi(R aa )3.-OP(R cc )2.-OP(R cc )3 + X - 、-OP(OR cc )2.-OP(OR cc )3 +X - ,-OP(=O)(R aa )2.-OP(=O)(OR cc )2 and -OP(=O)(N(R bb ))2, where X - , R aa , R bb and R cc As defined herein.

[0093] The term "thiol" or "thio" refers to the group -SH. The term "substituted thiol" or "substituted thio" by extension refers to a thiol group in which the sulfur atom directly attached to the parent molecule is replaced by a group other than hydrogen, and includes groups selected from: -SR aa 、-S-SR cc 、-SC(=S)SR aa 、-SC(=S)OR aa 、-SC(=S)N(R bb )2, -SC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)N(R bb )2 and -SC(=O)R aa , where R aa , R bb and R cc As defined herein.

[0094] The term "amino" refers to the group -NH2. The term "substituted amino" refers by extension to monosubstituted amino, disubstituted amino or trisubstituted amino. In certain embodiments, the "substituted amino" is a monosubstituted amino or disubstituted amino group. The term "monosubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is replaced by one hydrogen and one non-hydrogen group, and includes groups selected from the group consisting of: -NH(R bb )、-NHC(=O)R aa 、-NHCO2R aa 、-NHC(=O)N(R bb )2, -NHC(=NR bb )N(R bb )2. -NHSO2R aa 、-NHP(=O)(OR cc )2 and -NHP(=O)(N(R bb )2)2, where R aa , R bb and R cc As defined herein, and wherein the group -NH(R bb) bb The term "disubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is replaced by two non-hydrogen groups, and includes groups selected from the group consisting of: -N(R bb )2.-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2.-NR bb C(=NR bb )N(R bb )2.-NR bb S02R aa 、-NR bb P(=O)(OR cc )2 and -NR bb P(=O)(N(R bb )2)2, where R aa , R bb and R cc As defined herein, the proviso is that the nitrogen atom directly attached to the parent molecule is not replaced by hydrogen. The term "trisubstituted amino" means an amino group in which the nitrogen atom directly attached to the parent molecule is replaced by three groups, and includes groups selected from the following: -N(R bb )3 and -N(R bb )3 + X - , where R bb and X - As defined herein.

[0095] The term "acyl" refers to an acyl group having the general formula -C(=O)R aa 、-C(=O)OR aa 、-C(=O)-OC(=O)R aa 、-C(=O)SR aa 、-C(=O)N(R bb )2, -C(=S)R aa 、-C(=S)N(R bb )2 and -C(=S)S(R aa ),-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-C(=NR bb )SR aa and -C(=NR bb )N(R bb )2, wherein R aaand R bb As defined herein. Exemplary acyl groups include aldehydes (-CHO), carboxylic acids (-CO2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas.

[0096] "Counterions" or "anionic counterions" are negatively charged groups that combine with positively charged groups to maintain electronic neutrality. Anionic counterions can be monovalent (e.g., include one formal negative charge). Anionic counterions can also be multivalent (e.g., include more than one formal negative charge), such as divalent or trivalent. Exemplary counterions include halides (e.g., F - , Cl - Br - ,I - )、NO3 - 、ClO4 - OH - 、H2PO4 - 、HCO3 - 、HSO4 - , sulfonate ions (e.g., methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethane-1-sulfonic acid-2-sulfonate, etc.), carboxylate ions (e.g., acetate, propionate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, etc.), BF4 - PF4 - PF6 - 、AsF6 - 、SbF6 - 、B[3,5-(CF3)2C6H3]4] - 、B(C6F5)4 - ,BPh4 - 、Al(OC(CF3)3)4 - and carborane anions (e.g., CB 11 H 12 - or (HCB 11 Me5Br6) - ). Exemplary counterions that may be multivalent include CO3 2- 、HPO4 2- PO4 3- 、B4O7 2- 、SO4 2- 、S2O3 2-, carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelaate, sebacate, salicylate, phthalate, aspartate, glutamate, etc.), and carborane.

[0097] These and other exemplary substituents are described in more detail in the detailed description, examples, figures, and claims.The present invention is not limited in any way to the foregoing exemplary list of substituents. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, provide non-limiting examples of the invention.

[0100] Figure 1A-1B The cytotoxicity of anti-HER2 SPACs in breast cancer cell lines was demonstrated. Figure 1A The cytotoxicity of anti-HER2 SPACs (SPAC 1 and SPAC 2) in the BT-474 cell line with high HER2 expression (HER2+++) was shown. Figure 1B The cytotoxicity of anti-HER2 SPACs (SPAC 1 and SPAC 2) in the MCF7 cell line with low HER2 expression (HER2+) was shown.

[0101] Figure 2 The cytotoxicity of anti-CD38 SPAC (SPAC 3) in the CD38+ multiple myeloma cell line RPMI 8226 is shown.

[0102] Figure 3 Inhibition of cell proliferation in two CD38+ multiple myeloma cell lines (NCI-H929 and RPMI 8226) with an anti-CD38 SPAC (SPAC 11) containing a stapled MCL-1 inhibitor peptide was shown.

[0103] Figure 4 Inhibition of cell proliferation in a CD38+ multiple myeloma cell line (RPMI 8226) using an anti-CD38 SPAC (SPAC 8) containing a stapled MDM2 inhibitor peptide is shown.

[0104] Figure 5 Inhibition of cell proliferation in a CD38+ multiple myeloma cell line (RPMI 8226) with an anti-CD38 SPAC (SPAC 16) containing a stapled β-catenin inhibitor peptide is shown.

[0105] Figure 6A-6Bshowed that in HER2-low p53 WT breast cancer cell line (MCF7) ( Fig. 6A ) and HER2+++p53 mut breast cancer cell line (SK-BR-3) ( Figure 6B ) compared with the traditional ADC (trastuzumab emtansine).

[0106] Fig. 7A The cytotoxic activity of the stapled peptide comprising SEQ ID NO: 73 in breast cancer cell lines is shown. Figure 7B The cytotoxic activity of the stapled peptide comprising SEQ ID NO: 24 in breast cancer cell lines is shown.

[0107] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0108] Provided herein is a stapled peptide-antibody conjugate (SPAC), comprising a stapled peptide coupled to an antibody or an antigen-binding fragment thereof. In certain embodiments, the stapled peptide is coupled to an antibody or an antigen-binding fragment thereof via a linker. Also provided herein are pharmaceutical compositions and kits comprising a SPAC provided herein, methods for preparing a SPAC provided herein, and methods for using a SPAC provided herein in a subject (e.g., for treating a disease (e.g., cancer), methods for delivering a stapled peptide to a subject's cells (e.g., cancer cells), etc.).

[0109] Stapled Peptide-Antibody Conjugate (SPAC)

[0110] The present disclosure provides a stapled peptide-antibody conjugate (SPAC), which comprises a stapled peptide coupled to an antibody or its antigen-binding fragment. In certain embodiments, the stapled peptide is coupled to an antibody or its antigen-binding fragment via a linker (e.g., a cleavable linker). In certain embodiments, the antibody or its antigen-binding fragment is an anti-cancer antibody or its antigen-binding fragment; and the stapled peptide is a stapled anti-cancer peptide.

[0111] Antibodies and antigen-binding fragments

[0112] The stapled peptide-antibody conjugates (SPACs) provided herein comprise antibodies or antigen-binding fragments thereof. The term "antibody" as used herein refers to a molecule that specifically binds to or reacts immunologically with a specific antigen and includes at least the variable domain of the heavy chain, and typically includes at least the variable domain of the heavy and light chains of an immunoglobulin. Unless otherwise indicated, the term "antibody" (Ab) is intended to include complete (whole) molecules and antibody fragments (e.g., Fab and F(ab')2 fragments) that are capable of specifically binding to a target antigen. Antibodies (including intact antibodies and antigen-binding fragments), variants and derivatives thereof, including, but not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, heteroconjugate antibodies (e.g., bi-, tri- and tetra-specific antibodies, diabodies, tribodies and tetrabodies), single domain antibodies (sdAbs), epitope-binding fragments (e.g., Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), rIgG, single-chain antibodies, disulfide-linked Fv (sdFv), V-containing L or V H The antibody molecule of the conjugate can belong to 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 molecules.

[0113] 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 achieved by a fragment of a full-length antibody. The antibody fragment can be, for example, a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, consisting of V L 、V H , C L and C H 1 domain; (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bond in the hinge region; (iii) H and C H (iv) a single-arm V fragment of the antibody; L and V H Fv fragment composed of structural domains; (v) includes V H and VL domain dAb (Ward et al., Nature, 1989, 341, 544-546); (vi) V H (vii) a dAb fragment consisting of a V H or V L (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be connected by a linker (e.g., a synthetic linker). In addition, although the two domains (V L and V H ) are encoded by separate genes, but they can be joined using recombinant methods via a linker that enables them to form a single protein chain, where V L and V H The regions are paired to form monovalent molecules (referred to as single-chain Fv (scFv)). These antibody fragments (i.e., "antigen-binding fragments") can be obtained using conventional techniques known to those skilled in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis procedures known in the art.

[0114] The antibodies described herein can be of murine, rat, human or any other origin (including chimeric or humanized antibodies and fragments thereof). Any antibody described herein can be monoclonal or polyclonal. "Monoclonal antibody" means a homogeneous antibody population, and "polyclonal antibody" means a heterogeneous antibody population. These two terms do not limit the source of the antibody or how it is made.

[0115] In certain embodiments, the antibody is a monoclonal antibody (mAb) or its antigen-binding fragment. In certain embodiments, the antibody is a complete (i.e., entire) mAb. In certain embodiments, the antibody is an antigen-binding fragment of mAb. Examples of monoclonal antibodies (mAb) (including common names, commercial names, known target antigens, and exemplary uses) are provided in Table 2 below. The therapeutic applications of the mAb listed below are not limited to the specific known target antigens and exemplary uses provided.

[0116] Table 2. Examples of monoclonal antibodies (mAb)

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143] In certain embodiments, the antibody is an anticancer antibody or an antigen-binding fragment thereof. "Anticancer antibody" used herein means an antibody that targets an antigen (e.g., a cancer-associated antigen or a cancer-specific antigen) expressed on a cancer cell, can inhibit the growth of a cancer or tumor, and / or can trigger cancer cell death. In certain embodiments, the antibody is an anticancer mAb or an antigen-binding fragment thereof. Table 3 below provides examples of anticancer mAbs (including common names, known target antigens, and exemplary uses). Table 2 above provides other non-limiting examples of anticancer mAbs. The therapeutic uses of the anticancer mAbs listed below are not limited to specific known target antigens and the exemplary uses provided.

[0144] Table 3. Examples of anti-cancer monoclonal antibodies (mAbs)

[0145]

[0146]

[0147] In certain embodiments, the antibody is a homologue of an antibody provided herein, or an antigen-binding fragment thereof. The term "homologue" used herein means such an antibody: it has an amino acid composition or sequence similar to the disclosed antibody, so as to allow changes that will not adversely affect the ability of the antibody to perform its normal function (e.g., in conjunction with the target antigen). Homologues can have the same, shorter or longer length as disclosed antibodies. Homologues can have at least about 60% (e.g., at least about 60%, at least about 62%, at least about 64%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98% or at least about 99%) sequence identity (i.e., homology) with the amino acid sequence of the disclosed antibody. A homologue may be an antibody sequence modified, for example, by deletion, addition, mutation or substitution of one or more amino acid residues.

[0148] In certain embodiments, the antibody is a homologue of trastuzumab or an antigen-binding fragment thereof. In certain embodiments, the antibody is a homologue of daratumumab or an antigen-binding fragment thereof.

[0149] Antibodies not disclosed herein and / or not yet known in the art can be used in the SPACs provided herein.

[0150] In certain embodiments, the antibody is an antibody-drug conjugate (ADC) or its antigen-binding fragment. In such an embodiment, the SPAC includes an antibody or its antigen-binding fragment coupled to (i) a stapled peptide or its pharmaceutically acceptable salt and (ii) a second agent (i.e., the "drug" component of ADC). In other words, the SPAC provided herein may include two or more unique payloads, which may be selected from different classes of agents (e.g., having different mechanisms of action). For example, the SPAC provided herein may include an anti-cancer stapled peptide or its pharmaceutically acceptable salt coupled to emtansine trastuzumab (i.e., trastuzumab coupled to (i) an anti-cancer stapled peptide or its pharmaceutically acceptable salt and (ii) emtansine). Table 8 below provides examples of antibody-drug conjugates (ADC) (including common names, commercial names, and exemplary uses). Any of these ADCs may be coupled to a stapled peptide or its pharmaceutically acceptable salt to form a SPAC provided herein. The therapeutic uses of the following ADCs are not limited to specific known target antigens and the exemplary uses provided.

[0151] Table 8. Examples of Antibody-Drug Conjugates (ADCs)

[0152]

[0153] As used herein, "antigen" refers to an agent that is targeted by an antibody and binds to an antibody. In some cases, an antigen triggers the immune system to produce antibodies against the antigen, which is a so-called immune response. In certain embodiments, the antigen is expressed in a cell or on the surface of a cell (e.g., a cancer cell). In certain embodiments, the antibodies described herein are directed against a differentiation group (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 (IT GA2), 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)). Other examples of antigens (i.e., antigens to which the antibodies described herein can be directed) include, but are not limited to, glycoproteins (e.g., TROP2, TPBG, EpCAM, CEA, gpA33, mucin, TAG-72, CA-IX, CA-125 (MUC16), PSMA, endoglin, fibronectin, MUC1, mucin CanAg, rabies virus glycoprotein), glycolipids (e.g., gangliosides (e.g., GD2, GD3, GM2), myelin-associated glycoprotein, TAG-72, TN-C, TYRP1), carbohydrates (e.g., Lewis-Y 2), folate binding proteins (e.g., folate receptor 1, folate receptor α), vascular targets (e.g., VEGF, VEGFR, αVβ3, α5β1, VAP-1, VEGF-A, VEGFR-1, VEGFR-2), growth factors (e.g., HGF, IGF-1, NGF, HNGF, TGF-β, TGF-β1, TGF-β2, EGFL7, GDF-8), growth factor receptors (e.g., EGFR / ERBB1 / HER1, ERBB2 / HER2, ERBB3 / HER3, HGFR / c-Me t, HGFR, HHGFR, IGF-1 receptor, PDGF-Rα, PDGF-Rβ, EphA3, TRAIL-R1, TRAIL-R2 (DR5), RANKL), stromal and extracellular matrix antigens (e.g., FAP, Tensacin), activin receptors (e.g., ACVR2B), activin receptor-like kinases (e.g., activin receptor-like kinase 2), angiogenin (e.g., angiopoietin-2, angiopoietin-3), interferons (e.g., INF-α, INF-β, INF-γ), interleukins (e.g., IL 17A, IL 17F, IL20, IL-12, IL-23, IL-13, IL-17, IL-1β, IL-22, IL-4, IL-5, IL-6, IL-6 receptor, IL-2, IL-23A, IL-31RA, IL-4, IL-6, IL-9, ILGF2), integrins (e.g., α4β7, α5β1, α7β7, α IIb β3, α vβ3), complement components (e.g., C5, CFD), chemokines (e.g., CCL11, CCL2 (MCP-1)), chemokine receptors (e.g., CCR2, CCR4, CCR5), Notch receptors (e.g., Notch1, NRP1), virulence factors (e.g., ClfA), colony stimulating factors (e.g., CSF2), colony stimulating factor receptors (e.g., CSF1R), delta-like ligands (e.g., DLL3, DLL4), lipopolysaccharides (e.g., endotoxin), human leukocyte antigens (e.g., HLA-DR), heat shock proteins (e.g., Hsp90), SLAM proteins (e.g., SLAMF7), tissue factor pathway inhibitors, tumor necrosis factor (e.g., TNF-α), tumor necrosis factor receptor (TNFR superfamily member 4), macrophage migration inhibitory factor (e.g., Factor), rhesus monkey factor, neurite outgrowth inhibitor, alpha-fetoprotein, beta-amyloid protein, carcinoembryonic antigen (CEA), proteinase 1 that regulates neuronal apoptosis, Ch4D5, CLDN18.2, LOXL2, MSLN, NCA-90, PCSK9, sclerostin, syndecan 1, STEAP1, TSLP, TWEAK receptor, tumor antigen CTAA16.88, nectin-4 and TM4SF1. Other examples of known antigens can be found in, for example, Tables 2 and 3 above.

[0154] In certain embodiments, the antibody is directed against an antigen expressed on a cancer cell. Different classes of antigens expressed on cancer cells include: (i) "cancer-associated antigens" (CAAs), i.e., antigens expressed on cancer cells that may also be present on normal cells; (ii) "cancer-specific antigens" (CSAs), i.e., antigens expressed on cancer cells but not on normal cells; (iii) "tumor-associated antigens" (TAAs), i.e., antigens expressed on solid tumor cells that may also be present on normal cells; and (iv) "tumor-specific antigens" (TSAs), i.e., antigens expressed on solid tumor cells but not on normal cells. In certain embodiments, the antibody is directed against cancer-associated antigens. In certain embodiments, the antibody is directed against cancer-specific antigens. In certain embodiments, the antibody is directed against tumor-associated antigens. In certain embodiments, the antibody is directed against tumor-specific antigens.

[0155] In certain embodiments, the antibody is directed against a growth factor receptor (e.g., EGFR / ERBB1 / HER1, ERBB2 / HER2, ERBB3 / HER3, HGFR / c-Met, HGFR, HHGFR, IGF-1 receptor, PDGF-Rα, PDGF-Rβ, EphA3, TRAIL-R1, TRAIL-R2 (DR5), RANKL). In certain embodiments, the antibody is an antibody or antigen-binding fragment thereof directed against human epidermal growth factor receptor 2 (HER2). In certain embodiments, the antibody is a mAb or antigen-binding fragment thereof directed against HER2. In certain embodiments, the antibody is trastuzumab. In certain embodiments, the antibody is pertuzumab. In certain embodiments, the antibody is magetuximab.

[0156] In certain embodiments, the antibody is an antibody or antigen binding fragment thereof to CD38, CD33, CD22, TROP2, CD30, CD79b or Nectin-4. In certain embodiments, the antibody is a mAb or antigen binding fragment thereof to CD38, CD33, CD22, TROP2, CD30, CD79b or Nectin-4.

[0157] In certain embodiments, the antibody is an antibody or antigen binding fragment thereof to CD38. In certain embodiments, the antibody is a mAb or antigen binding fragment thereof to CD38. In certain embodiments, the antibody is daratumumab. In certain embodiments, the antibody is isatuximab.

[0158] TM4SF1-targeting antibodies

[0159] TM4SF1 (transmembrane-4L Six family member 1) is a membrane glycoprotein originally discovered on many human epithelial tumor cells. In cancer cells, TM4SF1 is known to support growth, invasion and metastasis, and is therefore a therapeutic target for cancer. See, e.g., et al., Proc. Natl. Acad. Sci. USA, 1986, 83(18), 7059-7063; Kao et al., Clin. Cancer Res., 2003, 9(7), 2807-2816; Gao et al., Cell, 2016, 166(1), 47-62; Shih et al., Cancer Res., 2009, 69(8), 3272-3277; Lin et al., Angiogenesis, 2014, 17(4), 897-907; and Zukauskas et al., Angiogenesis, 2011, 14(3), 345-354, the entire contents of each of which are incorporated herein by reference.

[0160] In certain embodiments, the SPAC provided herein comprises an antibody or antigen binding fragment thereof to TM4SF1. In certain embodiments, the antibody is a mAb or antigen binding fragment thereof to TM4SF1. In certain embodiments, the antibody or antigen binding fragment thereof targets the nucleus of a cancer cell (e.g., targets an antigen in the nucleus of a cancer cell). In certain embodiments, the antibody is capable of being internalized into the nucleus of a cancer cell.

[0161] Antibodies against TM4SF1 are described in, for example, Sciuto et al., Biochem Biophys Res. Commun., 2015, 465(3), 338-43; and Visintin et al., Mol. Cancer. Ther., 2015, 14(8), 1868-1876. In certain embodiments, the antibody is an antibody disclosed in WO 2020 / 176794, published on September 3, 2020; WO 2021 / 222783, published on November 4, 2021; WO 2021 / 195598, published on September 30, 2021; WO 2019 / 046338, published on March 7, 2019; or WO 2019 / 241430, published on December 19, 2019, the entire contents of each of which are incorporated herein by reference.

[0162] Stapled Peptides

[0163] The stapled peptide-antibody conjugates (SPACs) described herein comprise stapled peptides coupled to antibodies. The terms "staple" and "cross-linking" are used interchangeably and refer to a peptide in which two amino acids (i.e., "cross-linked amino acids") are connected via internal cross-links (i.e., "staples") to form a macrocycle. The terms "cross-linking" and "staple" are used interchangeably and refer to a covalently linked portion other than the peptide backbone that connects a pair of cross-linked amino acids to form a macrocycle.

[0164] Stapled peptide technology is described, for example, in U.S. Pat. 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 are incorporated herein by reference. Other examples of stapled peptide technology can be found in, for example, International PCT Application Publication Nos. WO 2017 / 004591, published on January 5, 2017, WO 2019 / 018499, published on January 24, 2019, WO 2021 / 126827, published on June 24, 2021, WO 2014 / 052647, published on April 3, 2014, WO 2014 / 159969, published on October 2, 2014, WO 2011 / 008260, published on January 20, 2011, WO 2009 / 126292, published on October 15, 2009, WO 2013 / 123266, published on August 22, 2013, and WO 2021 / 126827, published on September 23, 2021. 2021 / 188659, 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.

[0165] The stapled peptides of the present disclosure include (i) "single stapled" peptides, i.e., peptides comprising one internal crosslink connecting two crosslinked amino acids; (ii) "double stapled" peptides, i.e., peptides comprising two internal crosslinks, each internal crosslink connecting a different crosslinked amino acid pair; and (iii) "stitched" peptides, i.e., peptides comprising 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., multiple staples), wherein the stitched configuration has any number of staples.

[0166] In certain embodiments, the crosslink is attached to the alpha position of the crosslinked amino acid. In certain embodiments, the crosslinked amino acids are separated by 3 amino acids in the amino acid sequence, thereby forming an "i+4 crosslink". In certain embodiments, the crosslinked amino acids are separated by 4 amino acids in the amino acid sequence, thereby forming an "i+5 crosslink". In certain embodiments, the crosslinked amino acids are separated by 6 amino acids in the amino acid sequence, thereby forming an "i+7 crosslink". In certain embodiments, the crosslinked amino acids are separated by 7 amino acids in the amino acid sequence, thereby forming an "i+8 crosslink".

[0167] Staple (e.g., cross-link) the peptide can stabilize the secondary structure (e.g., alpha helical secondary structure) of the peptide. In certain embodiments, one or more cross-links of the stapled peptides provided herein stabilize the alpha helix of the peptide. In certain embodiments, the peptide has increased alpha helicity compared to a corresponding unstapled (e.g., uncross-linked) peptide.

[0168] Stapled peptides can exhibit alpha helical stability (measured by circular dichroism or NMR) by maintaining an alpha helical structure. For example, in certain embodiments, the stapled peptide exhibits an increase in alpha helicity of at least 1.1, 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, or 2 times compared to the corresponding unstapled peptide (e.g., determined by circular dichroism or NMR). In certain embodiments, the stapled peptides 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% alpha helicity (e.g., determined by circular dichroism or NMR) compared to the corresponding unstapled peptide.

[0169] The stapled peptides may be of any length. In certain embodiments, the stapled peptide is 100 amino acids or less in length. In certain embodiments, the stapled peptide is 90 amino acids or less in length. In certain embodiments, the stapled peptide is 80 amino acids or less in length. In certain embodiments, the stapled peptide is 70 amino acids or less in length. In certain embodiments, the stapled peptide is 60 amino acids or less in length. In certain embodiments, the stapled peptide is 50 amino acids or less in length. In certain embodiments, the stapled peptide is 45 amino acids or less in length. In certain embodiments, the stapled peptide is 40 amino acids or less in length. In certain embodiments, the stapled peptide is 35 amino acids or less in length. In certain embodiments, the stapled peptide is 30 amino acids or less in length. In certain embodiments, the stapled peptide is 25 amino acids or less in length. In certain embodiments, the stapled peptide is 20 amino acids or less in length. In certain embodiments, 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 in length or less. In certain embodiments, the stapled peptide is at least the length of the amino acid sequence provided herein. In certain embodiments, the stapled peptide is the length of any amino acid sequence provided herein.

[0170] In certain embodiments, the stapled peptide is coupled to one or more stapled peptides (e.g., via a linker, such as a cleavable linker). In certain embodiments, the stapled peptide is part of a string of two or more stapled peptides, which are connected to each other in a linear or non-linear manner (e.g., via a linker, such as a cleavable linker).

[0171] In certain embodiments, the stapled peptide is a stapled anticancer peptide. As used herein, a "stapled anticancer peptide" (also referred to as a "stapled oncolytic peptide") represents a stapled variant of a peptide with anticancer activity (i.e., a peptide capable of inducing cancer cell death and / or inhibiting cancer or tumor growth). In certain embodiments, the stapled peptide induces cancer cell death. Examples of stapled anticancer peptides (i.e., stapled oncolytic peptides) can be found in International PCT Application Publication No. WO 2021 / 126827, published on June 24, 2021 (the entire contents of which are incorporated herein by reference), any of which can be used as a stapled peptide component of a SPAC provided herein.

[0172] In certain embodiments, the stapled peptide is a stapled antimicrobial peptide (StAMP). "Stapled antimicrobial peptide" used herein means a stapled variant of a peptide having antimicrobial (e.g., antibacterial) activity. Examples of StAMP can be found in International PCT Application Publication No. WO 2017 / 004591 published on January 5, 2017 and WO2019 / 018499 published on January 24, 2019 (the entire contents of each of which are incorporated herein by reference), any of which can be used as a stapled peptide component of a SPAC provided herein. Stapled peptides can have anticancer and antimicrobial activity.

[0173] Stapled peptide inhibitors of protein-protein interactions (PPIs)

[0174] In certain embodiments, the stapled peptide is an inhibitor of protein-protein interaction (PPI). In certain embodiments, the PPI is associated with cancer formation and / or proliferation. See, for example, Wang et al. Chem. Sci., 2021, 12, 5977; Ali et al., Computational and Structural Biotechnology, Journal, 2019, Vol. 17, 263-281; ​​Iyer, Current Medicinal Chemistry, 2016, Vol. 23, No. 27, pp. 3025-3043; and Jamieson et al., Reports in Organic Chemistry, 2015, 5, 65-74, the entire contents of each of which are incorporated herein by reference.

[0175] Stapled BCL-2 family member protein inhibitors

[0176] For example, in certain embodiments, the stapled peptide is an inhibitor of a BCL-2 family member protein (e.g., BCL-xL, BCL-2, BCL-W, or MCL1). In certain embodiments, the stapled peptide is an MCL-1 inhibitor (e.g., MCL-1SAHBA 、MCL-1SAHB B 、MCL-1SAHB C 、MCL-1SAHB D 、MCL-1SAHB E ). In certain embodiments, 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 are incorporated herein by reference.

[0177] In certain embodiments, the stapled peptide is MCL-1SAHB D , which is described in, for example, Stewart et al., Nat. Chem. Biol., 2010, 6(8): 595-601, the entire contents of which are incorporated herein by reference. MCL-1 SAHB D Contains the following amino acid sequence:

[0178] KALETLRRVGDGVXRNHXTAF (SEQ ID NO: 148),

[0179] The two X's are linked via a crosslink alk.

[0180] In certain embodiments, the stapled peptide is an activator of a BCL-2 family member protein effector (e.g., BAX, BAK, or BOK). In certain embodiments, the stapled peptide is a stapled BH3 peptide. In certain embodiments, the stapled peptide is a stapled BCL-2 interacting cell death mediator (BIM) peptide. In certain embodiments, the stapled peptide is BIM SAHB A1 or BIM SAHB A2 See, e.g., Walensky et al., Science, 2004, 305(5689): 1466-1470; Walensky et al., Mol. Cell., 2006, 24(2): 199-210; Gavathiotis et al., Nature, 2008, 455(7216): 1076-1081; Araghi et al., PNAS, 2018, 115(5), E886-E895, the entire contents of each of which are incorporated herein by reference. See also, e.g., International PCT Application Publication Nos. WO 2010 / 068684, published on June 17, 2010, WO 2016 / 149613, published on September 22, 2016, and WO 2017 / 075349, published on May 4, 2017, the entire contents of each of which are incorporated herein by reference. In certain embodiments, the stapled peptide is BID SAHBA See, e.g., Walensky et al., Molecular Cell, 2006, Vol. 24, 199-210. In certain embodiments, the stapled peptide is a stapled PUMA peptide. See, e.g., Edwards et al., Chemistry & Biology, 2013, Vol. 20, 888-902.

[0181] Stapled β-catenin inhibitors

[0182] In certain embodiments, the stapled peptide is a β-catenin inhibitor. In certain embodiments, the stapled peptide is an inhibitor of Wnt / β-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 are incorporated herein by reference. See also, for example, International PCT Application Publication Nos. WO 2012 / 040459, published on March 29, 2012, WO 2012 / 142604, published on October 18, 2012, WO 2019 / 051327, published on March 14, 2019, and WO 2020 / 041270, published on February 7, 2020, the entire contents of each of which are incorporated herein by reference.

[0183] For example, in certain embodiments, the stapled peptide is xStAx-34, which is described in, e.g., Grossman et al., PNAS, 2012, 109(44), 17942-17947, the entire contents of which are incorporated herein by reference. xStAx-34 comprises the following amino acid sequence: RWPQXILDXHVRRVWR (SEQ ID NO: 149), wherein the two Xs are linked via a crosslink alk.

[0184] For example, in certain embodiments, the stapled peptide is SAH-BCL9 B , which is described in, for example, Takada et al., Sci. Transl. Med. 2012, 4(148):148ra117, pp. 1-19, the entire contents of which are incorporated herein by reference. SAH-BCL9 B It comprises the following amino acid sequence: LSQEQLEHRERSLXTLRXIQRBLF (SEQ ID NO: 150), wherein the two Xs are connected via a cross-linking alk.

[0185] Staple MDM2 / MDMX inhibitors

[0186] In certain embodiments, 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. Soc. 2007, 129, 2456-2457; Bernal et al., Cancer Cell, 2010, 18(5): 411-422; Chang et al., PNAS, 2013, 110(36), E3445-E3454. See, e.g., International PCT Application Publication Nos. WO 2021 / 222243, published on November 4, 2021, WO 2017 / 165617, published on September 28, 2017, WO 2008 / 095063, published on August 7, 2008, WO 2009 / 126292, published on October 15, 2009, and WO 2013 / 123266, published on August 22, 2013, the entire contents of each of which are incorporated herein by reference. See, e.g., U.S. 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 are incorporated herein by reference. In certain embodiments, 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.

[0187] The structures of ALRN-6924 and ATSP-7041 are shown below:

[0188]

[0189] In certain embodiments, the stapled peptide is ATSP-7041, wherein the cyclobutylalanine amino acid (Cba) is replaced with leucine (L) (i.e., SEQ ID NO: 152 with a Cba10L amino acid replacement) (referred to herein as "ATSP-7041Cba10L"). In certain embodiments, the stapled peptide comprises one of the following amino acid sequences (wherein two Xs are amino acids linked via cross-links):

[0190] LTFXEYWAQLXSAA (SEQ ID NO:161),

[0191] LTFXEYWAQLXSAAGGG (SEQ ID NO:162),

[0192] LTFXEYWAQLXSAA-PEG3 (SEQ ID NO:163),

[0193] or a pharmaceutically acceptable salt thereof.

[0194] In certain embodiments, the stapled peptide comprises one of the following amino acid sequences (wherein R 8 and S 5 linked together to form crosslinks (alk2):

[0195] LTFR 8 EYWAQLS 5 SAA-NH2 (SEQ ID NO: 164),

[0196] LTFR 8 EYWAQLS 5 SAAGGG-NH2 (SEQ ID NO: 165),

[0197] LTFR 8 EYWAQLS 5 SAA-PEG3-NH2 (SEQ ID NO: 166),

[0198] or a pharmaceutically acceptable salt thereof.

[0199] nailed magainin

[0200] In certain embodiments, the stapled peptide is based on the amino acid sequence of magainin (e.g., magainin II). The magainin is a class of antimicrobial peptides (AMPs) originally found in African clawed frogs (Xenopus laevis). The peptides are cationic, usually lacking a stable conformation in water, but forming amphipathic alpha helices in the membrane. It is well known that they can destroy the cell membranes of various cells (including bacteria, protozoa and fungi). It has been reported that they also have anticancer activity. The amino acid sequences of the peptides known as "magainin I" and "magainin II" are provided below.

[0201] Magnapin I: GIGKFLHSAGKFGKAFVGEIMKS (SEQ ID NO: 153) Magnapin II: GIGKFLHSAKKFGKAFVGEIMNS (SEQ ID NO: 154)

[0202] Examples of stapled magainin can be found in International PCT Application Publication Nos. WO2017 / 004591, published on January 5, 2017, WO 2019 / 018499, published on January 24, 2019, and WO 2021 / 126827, published on June 24, 2021 (the entire contents of each of which are incorporated herein by reference), any of which can be used as a stapled peptide component of a SPAC provided herein. See also, for example, U.S. Provisional Application USSN 63 / 160,245, filed on March 12, 2021, the entire contents of which are incorporated herein by reference. See also, for example, Mourtada et al., Nature Biotechnology, 2019, Vol. 37, 1186-1197, the entire contents of which are incorporated herein by reference.

[0203] In certain embodiments, the stapled peptide is a stapled magainin. In certain embodiments, the stapled peptide is a stapled magainin II peptide.

[0204] In certain embodiments, the stapled peptide comprises the amino acid sequence: GX 1 GKFX 2 HSKKKF GK AX 3 VGEX 4 (SEQ ID NO:1),

[0205] or a pharmaceutically acceptable salt thereof, wherein:

[0206] X 1 , X 2 , X 3 and X 4 are amino acids (i.e., cross-linked amino acids);

[0207] X 1 and X 2 Connected via crosslinks, and X 3 and X 4 connected via cross-links; and

[0208] The amino acid sequence includes except X 1 , X 2 , X 3 and X 4 In certain embodiments, the amino acid sequence comprises 0 to 9 amino acid replacements (including end values) at positions other than . In certain embodiments, the amino acid sequence comprises 0 amino acid replacements. In certain embodiments, the amino acid sequence comprises 1 amino acid replacement. In certain embodiments, the amino acid sequence comprises 2 amino acid replacements. In certain embodiments, the amino acid sequence comprises 3 amino acid replacements. In certain embodiments, the amino acid sequence comprises 4 amino acid replacements. In certain embodiments, the amino acid sequence comprises 5 amino acid replacements. In certain embodiments, the amino acid sequence comprises 6 amino acid replacements. In certain embodiments, the amino acid sequence comprises 7 amino acid replacements. In certain embodiments, the amino acid sequence comprises 8 amino acid replacements. In certain embodiments, the amino acid sequence comprises 9 amino acid replacements.

[0209] In certain embodiments, the stapled peptide comprises the amino acid sequence: GX 1 GKFX 2 HSKKKF GK AX 3 VGEX 4 AKK (SEQ ID NO: 2),

[0210] or a pharmaceutically acceptable salt thereof, wherein:

[0211] X 1 , X 2 , X 3 and X 4 are amino acids (i.e., cross-linked amino acids);

[0212] X 1 and X 2 Connected via crosslinks, and X 3 and X 4 The amino acid sequence includes X 1 , X 2 , X 3 and X 40 to 11 amino acid replacements (including end values) at positions other than . In certain embodiments, the amino acid sequence comprises 0 amino acid replacements. In certain embodiments, the amino acid sequence comprises 1 amino acid replacement. In certain embodiments, the amino acid sequence comprises 2 amino acid replacements. In certain embodiments, the amino acid sequence comprises 3 amino acid replacements. In certain embodiments, the amino acid sequence comprises 4 amino acid replacements. In certain embodiments, the amino acid sequence comprises 5 amino acid replacements. In certain embodiments, the amino acid sequence comprises 6 amino acid replacements. In certain embodiments, the amino acid sequence comprises 7 amino acid replacements. In certain embodiments, the amino acid sequence comprises 8 amino acid replacements. In certain embodiments, the amino acid sequence comprises 9 amino acid replacements.

[0213] In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof comprises one of the following amino acid sequences:

[0214]

[0215]

[0216]

[0217] In certain embodiments, the stapled peptide has one of SEQ ID NOs: 1-57 and 167-186 or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide has 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 -NH2. In certain embodiments, the stapled peptide has 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 -NH2; and wherein X 1 and X 2 , and X 3 and X 4 Connected to each other via cross-links (alk).

[0218] In certain embodiments, the stapled peptide comprises the amino acid sequence: GX 1 GKFX 2 Dap KKKK FG KAX 3 VGEX 4 AKK (SEQ ID NO:48),

[0219] or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof has SEQ ID NO: 48, wherein X1 and X 2 , and X 3 and X 4 via a cross-link of formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

[0220] In certain embodiments, the stapled peptide comprises the amino acid sequence: GX 1 G Dap FX 2 Dap Dap Dap Dap Dap FG Dap AX 3 VGEX 4 A Dap Dap (SEQ ID NO: 26),

[0221] or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof has SEQ ID NO: 26, wherein X 1 and X 2 , and X 3 and X 4 via a cross-link of formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

[0222] In certain embodiments, the stapled peptide comprises the amino acid sequence: GX 1 GKFX 2 KKKKKF GK AX 3 VGEX 4 AKK (SEQ ID NO:5),

[0223] or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof has SEQ ID NO: 5, wherein X 1 and X 2 , and X 3 and X 4 via a cross-link of formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

[0224] In certain embodiments, the stapled peptide comprises the amino acid sequence: GX 1 GKFX 2 HKKKKF GK AX 3 VFEX 4 AKK (SEQ ID NO: 23),

[0225] or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof has SEQ ID NO: 23, wherein X 1 and X 2 , and X 3 and X 4 via a cross-link of formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

[0226] In certain embodiments, the stapled peptide comprises the amino acid sequence: GX 1 G Dab FX 2 Dab Dab DabDab Dab FG Dab AX 3 VGEX 4 A Dab Dab (SEQ ID NO: 24),

[0227] or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof has SEQ ID NO: 24, wherein X 1 and X 2 , and X 3 and X 4 Via cross-linking of formula (alk); and wherein the C-terminus of the stapled peptide is amidated by -NH2. In certain embodiments, the stapled peptide provided herein or a pharmaceutically acceptable salt thereof comprises one of the following amino acid sequences:

[0228]

[0229]

[0230] In certain embodiments, the stapled peptide has one of SEQ ID NOs: 58-71 or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide has one of SEQ ID NOs: 58-71 or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with -NH2. In certain embodiments, the stapled peptide has one of SEQ ID NOs: 58-71 or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with -NH2; and wherein X 1 and X 2 , and X 3 and X 4 Connected to each other via cross-links (alk).

[0231] In certain embodiments, the stapled peptide comprises the following amino acid sequence: GX 1 GKFX 2 KKKK KX 3VGEX 4 AKK (SEQ ID NO:72),

[0232] or a pharmaceutically acceptable salt thereof, wherein:

[0233] X 1 , X 2 , X 3 and X 4 are amino acids (i.e., cross-linked amino acids);

[0234] X 1 and X 2 Connected via crosslinks, and X 3 and X 4 connected via cross-links; and

[0235] The amino acid sequence optionally includes in addition to X 1 , X 2 , X 3 and X 4 In certain embodiments, the amino acid sequence comprises 0 to 5 amino acid substitutions (including end values) at positions other than . In certain embodiments, 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.

[0236] In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof comprises SEQ ID NO: 72. In certain embodiments, the stapled peptide has SEQ ID NO: 72 or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide has SEQ ID NO: 72 or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with -NH2. In certain embodiments, the stapled peptide has SEQ ID NO: 72 or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with -NH2; and wherein X 1 and X 2 , and X 3 and X 4 Connected to each other via cross-links (alk).

[0237] Stapled Esculent in Peptide

[0238] In certain embodiments, 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 originally found in the skin secretions of various frogs and toads. Esculentin-1A is a special antimicrobial peptide (AMP) originally isolated from frog skin. Esculentin peptides can also have anticancer activity. The amino acid sequence of a peptide referred to as "Esculentin-1A" is provided below. In certain embodiments, the stapled peptide is based on amino acids 1-21 of SEQ ID NO:155.

[0239] Esculentin-1A: (SEQ ID NO:155)

[0240] In certain embodiments, the stapled peptide is a stapled Esculentin peptide. In certain embodiments, the stapled peptide is a stapled Esculentin-1A peptide. Examples of stapled Esculentin peptides can be found in, for example, U.S. Provisional Application USSN 63 / 185,641 filed on May 7, 2021 and 63 / 185,673 filed on May 7, 2021 (the entire contents of each of which are incorporated herein by reference), any of which can be used as a stapled peptide component of a SPAC provided herein. See also, for example, Mourtada, et al., Nature Biotechnology, 2019, Vol. 37, 1186-1197, the entire contents of which are incorporated herein by reference.

[0241] In certain embodiments, the stapled peptide comprises the amino acid sequence:

[0242] GX 1 FSKX 2 KGKKIKNLX 3 ISGX 4 KG (SEQ ID NO:73),

[0243] or a pharmaceutically acceptable salt thereof, wherein:

[0244] X 1 , X 2 , X 3 and X 4 are amino acids (i.e., cross-linked amino acids);

[0245] X 1 and X 2 Connected via crosslinks, and X 3 and X4 connected via cross-links; and

[0246] The amino acid sequence includes except X 1 , X 2 , X 3 and X 4 In certain embodiments, the amino acid sequence comprises 0 to 9 amino acid replacements (including end values) at positions other than . In certain embodiments, the amino acid sequence comprises 0 amino acid replacements. In certain embodiments, the amino acid sequence comprises 1 amino acid replacement. In certain embodiments, the amino acid sequence comprises 2 amino acid replacements. In certain embodiments, the amino acid sequence comprises 3 amino acid replacements. In certain embodiments, the amino acid sequence comprises 4 amino acid replacements. In certain embodiments, the amino acid sequence comprises 5 amino acid replacements. In certain embodiments, the amino acid sequence comprises 6 amino acid replacements. In certain embodiments, the amino acid sequence comprises 7 amino acid replacements. In certain embodiments, the amino acid sequence comprises 8 amino acid replacements. In certain embodiments, the amino acid sequence comprises 9 amino acid replacements.

[0247] In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof comprises one of the following amino acid sequences:

[0248]

[0249]

[0250] In certain embodiments, the stapled peptide has one of SEQ ID NOs: 73-96 or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide has one of SEQ ID NOs: 73-96 or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with -NH2. In certain embodiments, the stapled peptide has one of SEQ ID NOs: 73-96 or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with -NH2; and wherein X 1 and X 2 , and X 3 and X 4 Connected to each other via cross-links (alk).

[0251] In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof comprises SEQ ID NO: 73. In certain embodiments, the stapled peptide has SEQ ID NO: 73 or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide has SEQ ID NO: 73 or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with -NH2. In certain embodiments, the stapled peptide has SEQ ID NO: 73 or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with -NH2; and wherein X 1 and X 2 , and X 3 and X 4 Connected to each other via cross-links (alk).

[0252] In certain embodiments, the stapled peptides provided herein, or pharmaceutically acceptable salts thereof, comprise one of the following amino acid sequences:

[0253]

[0254]

[0255] In certain embodiments, the stapled peptide has one of SEQ ID NOs: 97-104 or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide has one of SEQ ID NOs: 97-104 or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with -NH2. In certain embodiments, the stapled peptide has one of SEQ ID NOs: 97-104 or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with -NH2; and wherein X 1 and X 2 , and X 3 and X 4 Connected to each other via cross-links (alk).

[0256] In certain embodiments, the stapled peptide comprises the amino acid sequence:

[0257] GX 1 FSKX 2 KGKKIKNLLX 3 SGLX 4 G (SEQ ID NO: 105),

[0258] and pharmaceutically acceptable salts thereof, wherein:

[0259] X 1 , X 2 , X 3 and X 4are amino acids (i.e., cross-linked amino acids);

[0260] X 1 and X 2 Connected via crosslinks, and X 3 and X 4 connected via cross-links; and

[0261] The amino acid sequence optionally includes in addition to X 1 , X 2 , X 3 and X 4 In certain embodiments, the amino acid sequence comprises 0 to 8 amino acid replacements (including end values) at positions other than . In certain embodiments, the amino acid sequence comprises 0 amino acid replacements. In certain embodiments, the amino acid sequence comprises 1 amino acid replacement. In certain embodiments, the amino acid sequence comprises 2 amino acid replacements. In certain embodiments, the amino acid sequence comprises 3 amino acid replacements. In certain embodiments, the amino acid sequence comprises 4 amino acid replacements. In certain embodiments, the amino acid sequence comprises 5 amino acid replacements. In certain embodiments, the amino acid sequence comprises 6 amino acid replacements. In certain embodiments, the amino acid sequence comprises 7 amino acid replacements. In certain embodiments, the amino acid sequence comprises 8 amino acid replacements.

[0262] In certain embodiments, the stapled peptide comprises one of the following amino acid sequences:

[0263]

[0264] or a pharmaceutically acceptable salt thereof, wherein:

[0265] X 1 , X 2 , X 3 and X 4 are amino acids (i.e., cross-linked amino acids);

[0266] X 1 and X 2 Connected via crosslinks, and X 3 and X 4 connected via cross-links; and

[0267] The amino acid sequence optionally includes in addition to X 1 , X 2 , X 3 and X 4In certain embodiments, the amino acid sequence comprises 0 to 8 amino acid replacements (including end values) at positions other than . In certain embodiments, the amino acid sequence comprises 0 amino acid replacements. In certain embodiments, the amino acid sequence comprises 1 amino acid replacement. In certain embodiments, the amino acid sequence comprises 2 amino acid replacements. In certain embodiments, the amino acid sequence comprises 3 amino acid replacements. In certain embodiments, the amino acid sequence comprises 4 amino acid replacements. In certain embodiments, the amino acid sequence comprises 5 amino acid replacements. In certain embodiments, the amino acid sequence comprises 6 amino acid replacements. In certain embodiments, the amino acid sequence comprises 7 amino acid replacements. In certain embodiments, the amino acid sequence comprises 8 amino acid replacements.

[0268] In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof comprises one of the following amino acid sequences:

[0269]

[0270] In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof has one of SEQ ID NOs: 105-114. In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof has one of SEQ ID NOs: 105-114, wherein the C-terminus is amidated with -NH2. In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof has one of SEQ ID NOs: 105-114, wherein the C-terminus is amidated with -NH2; and wherein X 1 and X 2 , and X 3 and X 4 Connected to each other via cross-links (alk).

[0271] In certain embodiments, the stapled peptide or a pharmaceutically acceptable salt thereof comprises SEQ ID NO: 105. In certain embodiments, the stapled peptide has SEQ ID NO: 105 or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide has SEQ ID NO: 105 or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with -NH2. In certain embodiments, the stapled peptide has SEQ ID NO: 105 or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with -NH2; and wherein X 1 and X 2 , and X 3 and X 4 Connected to each other via cross-links (alk).

[0272] In certain embodiments, the stapled peptide comprises the amino acid sequence:

[0273] GX 1 FSKX 2 KGKKIKNLLX 3 SGLX 4 KGGE (SEQ ID NO: 113),

[0274] or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide has SEQ ID NO: 113 or a pharmaceutically acceptable salt thereof. In certain embodiments, the stapled peptide has SEQ ID NO: 113 or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with -NH2. In certain embodiments, the stapled peptide has SEQ ID NO: 113 or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with -NH2; and wherein X 1 and X 2 , and X 3 and X 4 Connected to each other via cross-links (alk).

[0275] In certain embodiments, the stapled peptide comprises one of the following amino acid sequences:

[0276]

[0277]

[0278] or a pharmaceutically acceptable salt thereof, wherein:

[0279] X 1 and X 2 are amino acids (i.e., cross-linked amino acids);

[0280] wherein the amino acid sequence comprises except X 1 and X 2 In certain embodiments, the amino acid sequence comprises 0 to 6 amino acid substitutions (inclusive) at positions other than . In certain embodiments, 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, X 1 and X 2 Linked via disulfide cross-links (e.g., (mxy), (pxy), (but), (bbn), (bbf), (bbp), (pfb), (hfb)).

[0281] In certain embodiments, the stapled peptide is one of the following or a pharmaceutically acceptable salt thereof:

[0282]

[0283] In certain embodiments, the stapled peptide is one of the following or a pharmaceutically acceptable salt thereof:

[0284]

[0285]

[0286]

[0287] In certain embodiments, the stapled peptide is one of the following or a pharmaceutically acceptable salt thereof:

[0288]

[0289]

[0290] In certain embodiments, the stapled peptide is the following or a pharmaceutically acceptable salt thereof:

[0291]

[0292]

[0293] Stapled peptide cross-linking

[0294] As described herein, stapled peptides comprise crosslinks (e.g., staples), wherein each crosslink connects two amino acids (i.e., crosslinked amino acids) to form a macrocycle. In certain embodiments, when the amino acid sequence (e.g., SEQ ID NO: 1-147 provided herein) comprises X 1 and X 2 When X 1 and X 2 Likewise, in certain embodiments, when an amino acid sequence (e.g., SEQ ID NOs: 1-147 provided herein) comprises X 3 and X 4 When X 3 and X 4 are cross-linked amino acids connected via cross-links. Certain embodiments describing cross-linking of stapled peptides below apply to all stapled peptides described herein, including all amino acid sequences described herein (eg, SEQ ID NOs: 1-147) and variants thereof.

[0295] In certain embodiments, the crosslinks are independently attached to the alpha position (e.g., X 1 , X 2 , X 3 and X 4 In certain embodiments, the crosslinks are independently attached to the alpha position of the crosslinked amino acid (e.g., X 1 , X 2 , X 3 and X 4 ), and the cross-linking amino acids are independently α,α-disubstituted amino acids.

[0296] In certain embodiments, the length of each cross-link is independently about to about In certain embodiments, the length of each crosslink is independently about to about (inclusive of the end values) (e.g., in the case of i+4 crosslinks). In certain embodiments, the length of each crosslink is independently about to about In certain embodiments, the length of each crosslink is independently about to about In certain embodiments, the length of each crosslink is independently about to about In certain embodiments, the length of each crosslink is independently about to about (inclusive of the end values). The length of each cross-link is independently about to about In certain embodiments, the length of each crosslink is independently about to about In certain embodiments, the length of each crosslink is independently about to about In certain embodiments, the length of 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

[0297] In certain embodiments, the length of each cross-link is independently about to about (inclusive of the end values) (e.g., in the case of i+7 crosslinks). In certain embodiments, the length of each crosslink is independently about to about In certain embodiments, the length of each crosslink is independently about to about In certain embodiments, the length of each crosslink is independently about to about In certain embodiments, the length of each crosslink is independently about to about (inclusive of the end values). The length of each cross-link is independently about to about In certain embodiments, the length of each crosslink is independently about to about In certain embodiments, the length of 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

[0298] In certain embodiments, 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).

[0299] In certain embodiments, each cross-linked length is approximately equal to the length (including end values) of 5 to 20 carbon-carbon and / or carbon-sulfur bonds. In certain embodiments, each cross-linked length is approximately equal to the length (including end values) of 5 to 15 carbon-carbon and / or carbon-sulfur bonds. In certain embodiments, each cross-linked length is approximately equal to the length (including end values) of 5 to 13 carbon-carbon and / or carbon-sulfur bonds. In certain embodiments, each cross-linked length is approximately equal to the length (including end values) of 6 to 12 carbon-carbon and / or carbon-sulfur bonds. In certain embodiments, each cross-linked length is approximately equal to the length (including end values) of 7 to 11 carbon-carbon and / or carbon-sulfur bonds. In certain embodiments, each cross-linked length is approximately equal to the length (including end values) of 8 to 10 carbon-carbon and / or carbon-sulfur bonds.

[0300] In certain embodiments, each cross-linked length is approximately equal to the length (including end values) of 10 to 20 carbon-carbon and / or carbon-sulfur bonds. In certain embodiments, each cross-linked length is approximately equal to the length (including end values) of 11 to 19 carbon-carbon and / or carbon-sulfur bonds. In certain embodiments, each cross-linked length is approximately equal to the length (including end values) of 12 to 18 carbon-carbon and / or carbon-sulfur bonds. In certain embodiments, each cross-linked length is approximately equal to the length (including end values) of 13 to 17 carbon-carbon and / or carbon-sulfur bonds. In certain embodiments, each cross-linked length is approximately equal to the length (including end values) of 14 to 16 carbon-carbon and / or carbon-sulfur bonds.

[0301] In certain embodiments, at least one cross-link spans at least one turn of an alpha helix of the peptide. In certain embodiments, each cross-link spans at least one turn of an alpha helix of the peptide. In certain embodiments, at least one cross-link spans one turn of an alpha helix of the peptide. In certain embodiments, each cross-link spans one turn of an alpha helix of the peptide.

[0302] In certain embodiments, each pair of cross-linked amino acids (e.g., X 1 and X 2 , and X 3 and X 4 ) are independently linked by cross-linking to form the following formula:

[0303]

[0304] Where α represents the α-carbon of the cross-linked amino acid; L 1 is cross-linked; and R 1 Each instance of is independently hydrogen or optionally substituted C 1-6 alkyl.

[0305] In certain embodiments, 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.

[0306] In certain embodiments, each crosslink (e.g., L 1 ) are independently hydrocarbon crosslinks. For the purposes of this disclosure, a "hydrocarbon crosslink" is a crosslink consisting of optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, and combinations thereof.

[0307] In certain embodiments, each crosslink (e.g., L 1 ) is independently an optionally substituted alkenylene (e.g., unsubstituted alkenylene). In certain embodiments, each cross-link independently has the formula: wherein each n is independently an integer from 1 to 10 (inclusive). In certain embodiments, the sum of two n's on the same crosslink is 6.

[0308] In certain embodiments, the cross-linked amino acids (e.g., X 1 , X 2 , X 3 and X 4 ) are independently α,α-disubstituted amino acids. For example, in certain embodiments, each pair of cross-linked amino acids (e.g., X 1 and X 2 , and X 3 and X 4 ) are independently linked by cross-linking to form the following formula:

[0309]

[0310] wherein α represents the α-carbon of the cross-linked amino acid; and wherein R 1 Each instance of is independently optionally substituted C 1-6 In certain embodiments, the sum of two n's on the same crosslink is 6.

[0311] For example, in certain embodiments, cross-linking (e.g., L 1 ) independently have the following formula:

[0312]

[0313] For example, in certain embodiments, a pair of cross-linked amino acids (e.g., X 1 and X 2 , and X 3 and X 4 ) are independently linked via cross-linking to form the following formula:

[0314]

[0315] where α represents the α-carbon of the cross-linked amino acids.

[0316] In certain embodiments, X 1 and X 2 Connect to form the formula (alk).

[0317] In certain embodiments, X 3 and X 4 Connect to form the formula (alk).

[0318] In certain embodiments, crosslinking (e.g., L 1 ) is independently an optionally substituted alkylene (e.g., an unsubstituted alkylene). In certain embodiments, each cross-link independently has the formula: wherein m is an integer from 1 to 20 (inclusive). In certain embodiments, m is 6.

[0319] In certain embodiments, a pair of cross-linked amino acids (e.g., X 1 and X 2 , and X 3 and X 4 ) are independently linked by cross-linking to form the following formula:

[0320]

[0321] wherein α represents the α-carbon of the cross-linked amino acid; and wherein R 1 Each instance of is independently optionally substituted C 1-6 In certain embodiments, m is 6.

[0322] For example, in certain embodiments, cross-linking (e.g., L 1 ) independently have the following formula:

[0323]

[0324] For example, in certain embodiments, a pair of cross-linked amino acids (e.g., X 1 and X 2 , and X 3 and X 4 ) are linked via cross-linking to form the following formula:

[0325]

[0326] where α represents the α-carbon of the cross-linked amino acids.

[0327] In certain embodiments, crosslinking (e.g., L 1 ) independently have the following formula:

[0328]

[0329] For example, in certain embodiments, a pair of cross-linked amino acids (e.g., X 1 and X 2 , and X 3 and X 4 ) are independently linked via cross-linking to form the following formula:

[0330]

[0331] where α represents the α-carbon of the cross-linked amino acids.

[0332] In certain embodiments, crosslinking (e.g., L 1 ) independently have the following formula:

[0333]

[0334] For example, in certain embodiments, a pair of cross-linked amino acids (e.g., X 1 and X 2 , and X 3 and X 4 ) are independently linked via cross-linking to form the following formula:

[0335]

[0336] where α represents the α-carbon of the cross-linked amino acids.

[0337] In certain embodiments, crosslinking (e.g., L 1 ) is independently optionally substituted alkynylene (eg, unsubstituted alkynylene).

[0338] In certain embodiments, crosslinking (e.g., L 1 ) are independently disulfide crosslinks. For the purposes of this disclosure, a "disulfide crosslink" (i.e., "disulfide stapled") is a crosslink comprising two thioethers (i.e., two -S- groups). In certain embodiments, the crosslinks are independently disulfide crosslinks of the formula:

[0339]

[0340] wherein each n is independently an integer from 1 to 10 (inclusive); and

[0341] L is 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. In certain embodiments, each instance of n is 1. In certain embodiments, each instance of n is 2.

[0342] In certain embodiments, the crosslinks are independently disulfide crosslinks of the formula:

[0343]

[0344] wherein each n is independently an integer from 1 to 10 (inclusive); and

[0345] L 2 is an optionally substituted alkylene group, optionally substituted arylene, optionally substituted heteroarylene or -A 1 -A 1 -; where A 1 Each instance of n is independently optionally substituted arylene or optionally substituted heteroarylene. In certain embodiments, each instance of n is 1. In certain embodiments, each instance of n is 2.

[0346] In certain embodiments, the crosslinks are independently disulfide crosslinks of one of the following formulae:

[0347]

[0348] In certain embodiments, each instance of n is 1. In certain embodiments, each instance of n is 2.

[0349] For example, in certain embodiments, the crosslinks are independently disulfide crosslinks of one of the following formulae:

[0350]

[0351] In other embodiments, the crosslinks are independently disulfide crosslinks of the formula:

[0352]

[0353] wherein each n is independently an integer from 1 to 10 (inclusive);

[0354] L 2 is an optionally substituted aromatic ring (eg, a polyhalogenated aryl or heteroaryl ring) or -A 1 -A 1 -; where A 1 Each instance of n is independently an optionally substituted aromatic ring (e.g., a polyhalogenated aryl or heteroaryl ring). In certain embodiments, each instance of n is 1. In certain embodiments, each instance of n is 2.

[0355] In certain embodiments, the crosslinks are independently disulfide crosslinks of one of the following formulae:

[0356]

[0357] wherein each n is independently an integer from 1 to 10 (inclusive). In certain embodiments, each instance of n is 1. In certain embodiments, each instance of n is 2.

[0358] For example, in certain embodiments, the crosslinks independently have one of the following formulae:

[0359]

[0360] For purposes of the present disclosure, the specific crosslinks (mxy), (pxy), (but), (bbn), (bbf), (bbp), (pfb), and (hfb) cited herein are formed by crosslinking two cysteine ​​(C) residues of the peptide. In other words, the peptides provided herein comprising (mxy), (pxy), (but), (bbn), (bbf), (bbp), (pfb), and / or (hfb) crosslinks, or pharmaceutically acceptable salts thereof, include each pair of crosslinked amino acids (e.g., X) linked via disulfide crosslinks to form the following formula: 1 and X 2 , X 3 and X 4 ):

[0361]

[0362] where each α represents a disulfide-linked amino acid (e.g., X 1 , X 2 , X 3 , X 4 ) at the α position, and L 2 As shown in Table 4 below.

[0363] Table 4. Certain disulfide crosslinks

[0364]

[0365] In certain embodiments, crosslinking (e.g., L 1 ) are independently triazole crosslinks. For the purposes of this disclosure, a "triazole crosslink" is a crosslinked structure formed by at least one triazole moiety (e.g., )Interrupted cross-linking.

[0366] In certain embodiments, the crosslinks are independently triazolyl crosslinks of the formula:

[0367]

[0368] wherein each n is independently an integer from 1 to 10 (inclusive). In certain embodiments, the sum of two n's on the same crosslink is 5.

[0369] For example, in certain embodiments, the crosslinks are independently triazolyl crosslinks of one of the following formulae:

[0370] n and R 1 The following embodiments of apply to all formulae and subgenera provided herein, and to all stapled and unstapled peptides provided herein.

[0371] In certain embodiments, the sum of the two n on the same crosslink is an integer from 3 to 9 (inclusive). In certain embodiments, the sum of the two n on the same crosslink is an integer from 4 to 8 (inclusive). In certain embodiments, the sum of the two n on the same crosslink is an integer from 5 to 7 (inclusive). In certain embodiments, the sum of the two n on the same crosslink is 5. In certain embodiments, the sum of the two n on the same crosslink is 6. In certain embodiments, the sum of the two n on the same crosslink is 7.

[0372] In certain embodiments, 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.

[0373] In certain embodiments, m is an integer from 3 to 9 (inclusive). In certain embodiments, m is an integer from 4 to 8 (inclusive). In certain embodiments, m is an integer from 5 to 7 (inclusive). In certain embodiments, m is 5. In certain embodiments, m is 6. In certain embodiments, m is 7.

[0374] In certain embodiments, R 1 At least one example of is hydrogen. In certain embodiments, R 1 Each instance of is hydrogen. In certain embodiments, R 1 At least one instance of unsubstituted C 1-6 In certain embodiments, R 1 At least one instance of unsubstituted C 1-3 In certain embodiments, R 1 At least one example of is methyl. In certain embodiments, R 1 Each instance of is methyl.

[0375] The listing of chemical groups listed in any definition of a variable herein includes the definition of that variable as any single group or combination of the groups listed. An embodiment of a variable listed herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. The enumeration of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0376] Peptide C-terminal modification

[0377] The stapled peptide may comprise one or more additional modifications at any position of the peptide (e.g., on the side chain of an amino acid, on the alpha carbon of an amino acid, on a peptide nitrogen, at the C-terminus, at the N-terminus, etc.). The stapled peptide may comprise modifications to the C-terminus and / or N-terminus of the polypeptide. In certain embodiments, the stapled peptide comprises a modified C-terminus. Examples of C-terminal modifications are described herein.

[0378] In certain embodiments, the stapled peptide comprises an amidated C-terminus. Traditionally, peptides comprise a carboxyl group (-C(=O)OH) at the C-terminus. The stapled peptide may comprise an amide (e.g., -C(=O)NR2, wherein the group NR2 is NH2, a monosubstituted amino, a disubstituted amino, or a trisubstituted amino) at the C-terminus, referred to as an "amidated C-terminus". For example, a peptide "amidated by -NH2 at the C-terminus comprises a group -C(=O)NH2 instead of a carboxyl group (-C(=O)OH). The amidated C-terminus may also be represented by including -NR2 (e.g., -NH2) at the end of the amino acid sequence.

[0379] The stapled peptides can also be amidated with amino acids, peptides or proteins at the C-terminus. The amino acids, peptides or proteins can be natural or non-natural. In certain embodiments, the stapled peptides comprise a peptide coupled to the C-terminus. In certain embodiments, the peptides are 2 to 6 amino acids in length (inclusive) and comprise an amino acid selected from G, E, S, A and K.

[0380] In certain embodiments, the peptide is 2 to 6 amino acids in length (inclusive) and comprises amino acids selected from G, E, and S. In certain embodiments, the peptide is 2 to 6 amino acids in length (inclusive) and comprises amino acids selected from G and E. In certain embodiments, 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-limiting examples of peptides that can be coupled to the C-terminus of the stapled peptide are as follows:

[0381] GE,

[0382] AG,

[0383] AA,

[0384] GG,

[0385] GGE,

[0386] GGS,

[0387] GGG,

[0388] GGK,

[0389] GGQ,

[0390] GGGC (SEQ ID NO: 187),

[0391] GGGE (SEQ ID NO: 156),

[0392] GGEE (SEQ ID NO: 157), or

[0393] GGSGGS (SEQ ID NO: 158).

[0394] The stapled peptide may also comprise a small molecule, lipophilic group or polymer coupled to the C-terminus of the peptide.

[0395] In certain embodiments, the stapled peptide comprises a lipophilic group coupled to the C-terminus of the peptide. In certain embodiments, the lipophilic group is a lipid or fatty acid. In certain embodiments, the lipophilic group is a hydrocarbon chain.

[0396] In certain embodiments, the stapled peptide comprises a polymer coupled to the C-terminus of the peptide. In certain embodiments, the polymer is a polyether, for example, polyethylene glycol (PEG). In certain embodiments, the polymer is PEG. In certain embodiments, the polymer is PEG3. As described herein, PEG3 has the formula:

[0397] In certain embodiments, the stapled peptide is amidated at the C-terminus with a group of the formula: -NH-(PEG)-CONH2, wherein PEG is polyethylene glycol. In certain embodiments, the stapled peptide is amidated at the C-terminus with a group of the formula: -NH(CH2CH2O) 1-20 In certain embodiments, the stapled peptide is amidated at the C-terminus with a group of one of the following formulae: -NHCH2CH2OCH2CH2CONH2, -NH(CH2CH2O)2-CH2CH2CONH2, -NH(CH2CH2O)3-CH2CH2CONH2, -NH(CH2CH2O)4-CH2CH2CONH2, or -NH(CH2CH2O)5-CH2CH2CONH2.

[0398] In certain embodiments, the stapled peptide comprises a small molecule coupled to the C-terminus of the peptide. In certain embodiments, the small molecule is an anticancer agent.

[0399] Linkers and conjugation

[0400] As described herein, the SPAC provided herein comprises a stapled peptide coupled to an antibody or its antigen-binding fragment. In certain embodiments, the antibody or its antigen-binding fragment is coupled to the N-terminus of the stapled peptide. In certain embodiments, the antibody or its antigen-binding fragment is coupled to the C-terminus of the stapled peptide. In certain embodiments, the antibody or its antigen-binding fragment is coupled to an internal position on the stapled peptide (e.g., an amino acid residue or cross-link of the stapled peptide).

[0401] In certain embodiments, the stapled peptide is coupled via a thiol of the antibody or AF thereof. In certain embodiments, the stapled peptide is coupled via a cysteine ​​residue of the antibody or AF thereof. In certain embodiments, the stapled peptide is coupled via an amine of the antibody or AF thereof. In certain embodiments, the stapled peptide is coupled via a lysine residue of the antibody or AF thereof.

[0402] In certain embodiments, the antibody or its antigen binding fragment is directly (e.g., via a bond) coupled to the stapled peptide. In certain embodiments, the antibody or its antigen binding fragment is coupled to the stapled peptide via a linker. "Linker" as used herein means the portion by which the antibody or its antigen binding fragment is connected to the stapled peptide, not to be confused with one or more "crosslinks" connecting the amino acids of the stapled peptide.

[0403] In certain embodiments, 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.

[0404] In certain embodiments, the joint comprises an optionally substituted alkylene. In certain embodiments, the joint comprises an optionally substituted alkenylene. In certain embodiments, the joint comprises an optionally substituted alkynylene. In certain embodiments, the joint comprises an optionally substituted heteroalkylene. In certain embodiments, the joint comprises an optionally substituted heteroalkenylene. In certain embodiments, the joint comprises an optionally substituted heteroalkynylene. In certain embodiments, the joint comprises an optionally substituted carbocyclylene. In certain embodiments, the joint comprises an optionally substituted heterocyclylene. In certain embodiments, the joint comprises an optionally substituted arylene. In certain embodiments, the joint comprises an optionally substituted heteroarylene. In certain embodiments, the joint comprises an optionally substituted acylene.

[0405] In certain embodiments, the linker is a cleavable linker. "Cleavable linker" as used herein means a linker that can be cleaved under physiological conditions. In certain embodiments, the linker can be cleaved by pH cleavage or by protease, esterase or intracellular disulfide reduction. In certain embodiments, the linker can be cleaved by protease. See, for example, 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 are incorporated herein by reference.

[0406] In certain embodiments, the linker is a peptide linker. In certain embodiments, the linker is a cleavable peptide linker. As used herein, "peptide linker" means a linker comprising two or more amino acids connected via a peptide bond. In certain embodiments, the peptide linker comprises -Y A Y B Y C Y D -(SEQ ID NO: 159), wherein:

[0407] Y A is glycine, glutamate, or absent;

[0408] Y B is valine, phenylalanine, alanine, tyrosine, or glycine;

[0409] Y C is citrulline, arginine, lysine, alanine, or glycine; and

[0410] Y D It's glycine, or it's not there.

[0411] In certain embodiments, the peptide linker comprises -GGFG- (SEQ ID NO: 160). In certain embodiments, the peptide linker comprises -GGG-. In certain embodiments, the peptide linker comprises -EVC-. See, e.g., Anami et al., Nature Communications, 2018, 9, 2512, the entire contents of which are incorporated herein by reference.

[0412] In certain embodiments, the peptide linker comprises -valine-citrulline- (ie, -VC-). Table 5 below shows non-limiting examples of such linkers, expressed as linking agents and the corresponding resulting linker structures.

[0413] Table 5. Examples of linkers and ligation reagents

[0414]

[0415]

[0416]

[0417] *In the structure, S represents the connection point to the antibody or antigen-binding fragment thereof; N represents the connection point to the N-terminus of the stapled peptide.

[0418] In certain embodiments, the linker comprises a triazolylene moiety. As described herein, a linker comprising a triazolylene moiety can be formed by an azide-alkyne cycloaddition reaction. Non-limiting examples of triazole-containing linkers are shown in Table 9 below.

[0419] Table 9. Examples of triazole-containing linkers

[0420]

[0421]

[0422]

[0423]

[0424]

[0425] *In the structure, A represents the connection point with the antibody or antigen-binding fragment thereof; N represents the connection point with the N-terminus of the stapled peptide

[0426] In certain embodiments, the SPAC provided herein includes 1 stapled peptide coupled to the antibody or its antigen-binding fragment (i.e., 1: 1 stapled peptide: antibody ratio). In certain embodiments, 2 or more stapled peptides are coupled to the antibody or its antigen-binding fragment (i.e., 2: 1 stapled peptide: antibody ratio or greater). In certain embodiments, 1 to 20 stapled peptides (including end values) are coupled to the antibody or its antigen-binding fragment (i.e., 1: 1 to 20: 1 stapled peptide: antibody ratio (including end values)). In certain embodiments, 2 to 20 stapled peptides (including end values) are coupled to the antibody or its antigen-binding fragment (i.e., 2: 1 to 20: 1 stapled peptide: antibody ratio (including end values)). In certain embodiments, 1 to 10 stapled peptides (including end values) are coupled to the antibody or its antigen-binding fragment (i.e., 1: 1 to 10: 1 stapled peptide: antibody ratio (including end values)). In certain embodiments, 2 to 10 stapled peptides (including end values) are coupled to the antibody or its antigen-binding fragment (i.e., 2: 1 to 10: 1 stapled peptide: antibody ratio (including end values)). In certain embodiments, 5 to 10 stapled peptides (including end values) are coupled to the antibody or its antigen-binding fragment (i.e., 1: 1 to 10: 1 stapled peptide: antibody ratio (including end values)). In certain embodiments, the antibody or its antigen-binding fragment is coupled to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 stapled peptides. In certain embodiments, about 8 stapled peptides are coupled to the antibody or its antigen-binding fragment (i.e., 8: 1 stapled peptide: antibody ratio). In certain embodiments, the stapled peptides are the same or different, in any combination.

[0427] Additional embodiments

[0428] Additional embodiments and examples of SPACs are provided below (including in Table 6).

[0429] In certain embodiments, the stapled peptide is a stapled anti-cancer peptide; and the antibody is a mAb antibody or antigen-binding fragment thereof to HER2. In certain embodiments, the stapled peptide is a stapled anti-cancer peptide; and the antibody is a mAb antibody or antigen-binding fragment thereof to CD38. In certain embodiments, the stapled peptide is a stapled anti-cancer peptide; and the antibody is trastuzumab. In certain embodiments, the stapled peptide is a stapled anti-cancer peptide; and the antibody is trastuzumab enmetuzumab. In certain embodiments, the stapled peptide is a stapled anti-cancer peptide; and the antibody is daratumumab.

[0430] In certain embodiments, the stapled peptide has SEQ ID NO: 48 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against HER2. In certain embodiments, the stapled peptide has SEQ ID NO: 26 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against HER2. In certain embodiments, the stapled peptide has SEQ ID NO: 5 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against HER2.

[0431] In certain embodiments, the stapled peptide has SEQ ID NO: 48 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against CD38. In certain embodiments, the stapled peptide has SEQ ID NO: 26 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against CD38. In certain embodiments, the stapled peptide has SEQ ID NO: 5 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against CD38.

[0432] In certain embodiments, the stapled peptide has SEQ ID NO: 26 or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab (e.g., SPAC 1 in Table 6 below). In certain embodiments, the stapled peptide has SEQ ID NO: 26 or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab enmetuzumab (e.g., SPAC 17 and SPAC 18 in Table 6 below). In certain embodiments, the stapled peptide has SEQ ID NO: 48 or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab (e.g., SPAC 2 in Table 6 below). In certain embodiments, the stapled peptide has SEQ ID NO: 26 or a pharmaceutically acceptable salt thereof; and the antibody is daratumumab (e.g., SPAC 3 in Table 6 below). In certain embodiments, the stapled peptide has SEQ ID NO: 48 or a pharmaceutically acceptable salt thereof; and the antibody is daratumumab (e.g., SPAC 4 in Table 6 below). In certain embodiments, the stapled peptide has SEQ ID NO: 5 or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab (e.g., SPAC 5 in Table 6 below). In certain embodiments, the stapled peptide has SEQ ID NO: 5 or a pharmaceutically acceptable salt thereof; and the antibody is daratumumab (e.g., SPAC 6 in Table 6 below).

[0433] Table 6. Examples of SPACs

[0434]

[0435]

[0436] Additional embodiments and examples of SPACs are provided below (including in Table 7).

[0437] In certain embodiments, the stapled peptide is an MDM2 inhibitor or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against HER2. In certain embodiments, the stapled peptide is ATSP-7041 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against HER2. In certain embodiments, the stapled peptide is ATSP-7041 or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab. In certain embodiments, the stapled peptide is ATSP-7041 or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab enmetuzumab.

[0438] In certain embodiments, the stapled peptide comprises any one of SEQ ID NO: 161-166 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof to HER2. In certain embodiments, the stapled peptide comprises any one of SEQ ID NO: 161-166 or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab. In certain embodiments, the stapled peptide comprises any one of SEQ ID NO: 161-166 or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab enmi. In certain embodiments, the stapled peptide is ATSP-7041Cba10L or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof to HER2. In certain embodiments, the stapled peptide is ATSP-7041Cba10L or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab (e.g., SPAC 7 in Table 7 below). In certain embodiments, the stapled peptide is ATSP-7041Cbal0L or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab emtansine (eg, SPAC 9 in Table 7 below).

[0439] In certain embodiments, the stapled peptide is an MDM2 inhibitor or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against CD38. In certain embodiments, the stapled peptide is ATSP-7041 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against CD38. In certain embodiments, the stapled peptide is ATSP-7041 or a pharmaceutically acceptable salt thereof; and the antibody is daratumumab.

[0440] In certain embodiments, the stapled peptide comprises any one of SEQ ID NOs: 161-166 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof to CD38. In certain embodiments, the stapled peptide comprises any one of SEQ ID NOs: 161-166 or a pharmaceutically acceptable salt thereof; and the antibody is daratumumab. In certain embodiments, the stapled peptide is ATSP-7041Cba10L or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof to CD38. In certain embodiments, the stapled peptide is ATSP-7041Cba10L or a pharmaceutically acceptable salt thereof; and the antibody is daratumumab (e.g., SPAC 8 in Table 7 below.

[0441] In certain embodiments, the stapled peptide is an MCL-1 inhibitor; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against HER2. In certain embodiments, the stapled peptide is MCL-1 SAHB D or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against HER2. In certain embodiments, the stapled peptide is MCL-1SAHB D or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab (eg, SPAC10 in Table 7 below). In certain embodiments, the stapled peptide is MCL-1SAHB D or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab emtansine (eg, SPAC 12 in Table 7 below).

[0442] In certain embodiments, the stapled peptide is an MCL-1 inhibitor; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against CD38. In certain embodiments, the stapled peptide is MCL-1 SAHB D or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against CD38. In certain embodiments, the stapled peptide is MCL-1SAHB D or a pharmaceutically acceptable salt thereof; and the antibody is daratumumab (eg, SPAC11 in Table 7 below).

[0443] In certain embodiments, the stapled peptide is a β-catenin inhibitor; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against HER2. In certain embodiments, the stapled peptide is SAH-BCL9 B or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against HER2. In certain embodiments, the stapled peptide is SAH-BCL9 Bor a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab (eg, SPAC13 in Table 7 below).

[0444] In certain embodiments, the stapled peptide is a β-catenin inhibitor; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against CD38. In certain embodiments, the stapled peptide is SAH-BCL9 B or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against CD38. In certain embodiments, the stapled peptide is SAH-BCL9 B or a pharmaceutically acceptable salt thereof; and the antibody is daratumumab (eg, SPAC 14 in Table 7 below).

[0445] In certain embodiments, the stapled peptide is xStAx-34 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against HER2. In certain embodiments, the stapled peptide is xStAx-34 or a pharmaceutically acceptable salt thereof; and the antibody is trastuzumab (e.g., SPAC 15 in Table 7 below).

[0446] In certain embodiments, the stapled peptide is xStAx-34 or a pharmaceutically acceptable salt thereof; and the antibody is a mAb antibody or an antigen-binding fragment thereof directed against CD38. In certain embodiments, the stapled peptide is xStAx-34 or a pharmaceutically acceptable salt thereof; and the antibody is daratumumab (e.g., SPAC 16 in Table 7 below).

[0447] Table 7. Additional Examples of SPACs

[0448] SPAC Number Stapled Peptides Connector No. Antibody 7 ATSP-7041Cba10L L3 Trastuzumab 8 ATSP-7041Cba10L L3 Daratumumab 9 ATSP-7041Cba10L L3 Trastuzumab Enbrel 10 <![CDATA[MCL-1SAHB D ]]> L2 Trastuzumab 11 <![CDATA[MCL-1SAHB D ]]> L2 Daratumumab 12 <![CDATA[MCL-1SAHB D ]]> L2 Trastuzumab Enbrel 13 <![CDATA[SAH-BCL9 B ]]> L2 Trastuzumab 14 <![CDATA[SAH-BCL9 B ]]> L2 Daratumumab 15 xStAx-34 L2 Trastuzumab 16 xStAx-34 L2 Daratumumab

[0449] In certain embodiments, the stapled peptide is an MDM2 inhibitor; and the antibody is an antibody or antigen-binding fragment thereof to TM4SF1. In certain embodiments, the stapled peptide comprises any one of SEQ ID NOs: 161-166 or a pharmaceutically acceptable salt thereof; and the antibody is an antibody or antigen-binding fragment thereof to TM4SF1. In certain embodiments, the stapled peptide has any one of SEQ ID NOs: 164-166 or a pharmaceutically acceptable salt thereof; and the antibody is an antibody or antigen-binding fragment thereof to TM4SF1. In certain embodiments, the stapled peptide has any one of SEQ ID NOs: 164-166 or a pharmaceutically acceptable salt thereof; and the antibody is an antibody or antigen-binding fragment thereof to TM4SF1; and the linker comprises any one of A1-A20.

[0450] Methods for preparing stapled peptide-antibody conjugates (SPACs)

[0451] Also provided herein are methods for preparing stapled peptide-antibody conjugates (SPACs) described herein. In certain embodiments, a linking agent is used to couple the stapled peptide to an antibody or an antigen-binding fragment thereof. As used herein, a "linking agent" refers to a molecule comprising two reactive moieties, one of which is capable of reacting with a reactive moiety on an antibody to form at least one covalent bond, and the other is capable of reacting with a reactive moiety on a stapled peptide to form at least one covalent bond.

[0452] For example, the linking agent may comprise (i) a portion capable of reacting with a thiol (e.g., a cysteine ​​residue) or an amine (e.g., a lysine residue) of an antibody; and (ii) a portion capable of reacting with the N-terminal amine of a stapled peptide. For example, the linking agent may comprise (i) a maleimide or iodoacetamide (e.g., capable of reacting with a thiol (e.g., a cysteine ​​residue) or an amine (e.g., a lysine residue) of an antibody); and (ii) a carboxylic acid or ester (e.g., capable of reacting with the N-terminal amine of a stapled peptide). Table 5 provides non-limiting examples of linking agents.

[0453] In certain embodiments, a method for preparing a stapled peptide-antibody conjugate (SPAC) described herein comprises the following steps:

[0454] (a) contacting a stapled peptide with a linking agent under conditions sufficient to couple the linking agent to the stapled peptide, thereby forming a stapled peptide-linking agent intermediate; and

[0455] (b) contacting the stapled peptide-linker intermediate with the antibody or antigen-binding fragment thereof under conditions sufficient to couple the stapled peptide-linker intermediate to the antibody or antigen-binding fragment thereof, thereby forming a stapled peptide-antibody conjugate (SPAC).

[0456] In certain embodiments, a method for preparing a stapled peptide-antibody conjugate (SPAC) described herein comprises the following steps:

[0457] (a) contacting the antibody or antigen-binding fragment thereof with a linking agent under conditions sufficient to couple the linking agent to the antibody or antigen-binding fragment thereof, thereby forming an antibody-linking agent intermediate; and

[0458] (b) contacting the antibody-linking agent intermediate with the stapled peptide under conditions sufficient to couple the antibody-linking agent intermediate to the stapled peptide, thereby forming a stapled peptide-antibody conjugate (SPAC).

[0459] In other embodiments, a method for preparing a stapled peptide-antibody conjugate (SPAC) described herein comprises the steps of contacting a stapled peptide comprising a first reactive moiety with an antibody or antigen-binding fragment thereof comprising a second reactive moiety under conditions sufficient to form at least one covalent bond between the first reactive moiety and the second reactive moiety, thereby forming a SPAC. In certain embodiments, the first reactive moiety and the second reactive moiety are "click chemistry" handles that are capable of reacting with each other to form one or more covalent bonds therebetween.

[0460] "Click chemistry" is a chemical scheme introduced by Sharpless in 2001, and describes a chemical method that is tailored to quickly and reliably generate substances by connecting small units together. See, for example, Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004-2021; Evans, Australian Journal of Chemistry (2007) 60: 384-395. Exemplary coupling reactions (some of which can be classified as "click chemistry") include, but are not limited to, the formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g., nucleophilic displacement of halides or the opening of strained ring systems); azide-alkyne Huisgen cycloaddition; thiol-alkyne addition; imine formation; Michael addition (e.g., maleimide addition); and Diels-Alder reaction (e.g., tetrazine [4+2] cycloaddition). Examples of alkyne-azide reactions can be found in, for example, Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40:2004-2021; Kolb and Sharpless, Drug Discov Today (2003) 24:1128-1137; and Evans, Australian Journal of Chemistry (2007) 60:384-395.

[0461] In certain embodiments, 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.

[0462] For example, a method for preparing a stapled peptide-antibody conjugate (SPAC) described herein comprises the steps of contacting an azide-containing stapled peptide with an alkyne-containing antibody or antigen-binding fragment thereof under conditions sufficient to form a triazole-containing linker, thereby forming a SPAC.

[0463] In certain embodiments, the azide-containing stapled peptide comprises one of the following formulae (e.g., to form any of linkers A1-A20):

[0464]

[0465]

[0466] Provided herein are stapled peptides comprising any one of SEQ ID NOs: 161-166 or a pharmaceutically acceptable salt thereof; and further comprising an azide. For example, provided herein are 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:

[0467]

[0468]

[0469] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a terminal alkyne (e.g., for copper-promoted cycloaddition with azide). In certain embodiments, the antibody or antigen-binding fragment thereof comprises a cyclic alkyne (e.g., for strain-promoted (e.g., copper-free) cycloaddition with azide). Non-limiting examples of cyclic alkyne moieties include DBCO and sulfo-DBCO:

[0470]

[0471]

[0472] Pharmaceutical compositions, kits and administration

[0473] The present disclosure provides pharmaceutical compositions comprising the SPAC disclosed herein. The pharmaceutical compositions may comprise one or more pharmaceutically acceptable carriers / excipients. In certain embodiments, the SPAC described herein is provided in a pharmaceutical composition in an effective amount. In certain embodiments, the effective amount is a therapeutically effective amount (e.g., for treating cancer in a subject and / or inhibiting tumor growth in a subject). In certain embodiments, the effective amount is a prophylactic effective amount.

[0474] The pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparation methods include combining the SPAC described herein (i.e., the "active ingredient") with a carrier or excipient and / or one or more other accessory ingredients, and then, if necessary and / or desired, forming and / or packaging the product into a desired single or multiple dose unit.

[0475] Pharmaceutical compositions can be prepared, packaged and / or sold in bulk, as a single unit dose and / or as a plurality of single unit doses. A "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or a convenient fraction of such a dose, such as one-half or one-third of such a dose.

[0476] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in the pharmaceutical compositions described herein will vary, depending on the identity, size, and / or condition of the subject being treated, and further on the route by which the composition is to be administered. The composition may contain 0.1% to 100% (w / w) active ingredient. The composition may contain 0.1% to 50% (w / w) active ingredient.

[0477] Pharmaceutically acceptable excipients for preparing the provided pharmaceutical compositions include inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavoring agents and fragrances may also be present in the compositions.

[0478] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, and mixtures thereof.

[0479] 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 sponges, 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), methyl cellulose, 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.

[0480] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, 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, esters, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers and carboxyvinyl polymers), carrageenan, cellulosic derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate ( 20) Polyoxyethylene sorbitan ( 60), polyoxyethylene sorbitan monooleate ( 80), Sorbitan monopalmitate ( 40) Sorbitan monostearate ( 60), Sorbitan tristearate ( 65), monoolein, sorbitan monooleate ( 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate ( 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., ), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether ( 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, F-68, poloxamer P-188, cetrimide bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium and / or mixtures thereof.

[0481] Exemplary binders include starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, mucilage of isapol husks), carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate, and larch arabogalactan), alginate, polyethylene oxide, polyethylene glycol, inorganic calcium salt, silicic acid, polymethacrylate, waxes, water, alcohol and / or mixtures thereof.

[0482] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoal preservatives, alcohol preservatives, acidic preservatives and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0483] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0484] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium EDTA, disodium EDTA, trisodium EDTA, calcium disodium EDTA, dipotassium EDTA, etc.), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0485] Exemplary antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0486] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and phenylethanol.

[0487] Exemplary acid preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0488] Other preservatives include tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Plus, Methylparaben, 115. II. and

[0489] Exemplary buffers include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium glucoheptonate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium ketovalerate, valeric acid, dibasic calcium phosphate, phosphoric acid, tricalcium 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, disodium hydrogen phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and mixtures thereof.

[0490] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0491] Exemplary natural oils include almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, black currant seed oil, borage oil, juniper oil, chamomile oil, canola oil, caraway oil, palm wax oil, castor oil, cinnamon oil, cocoa butter oil, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, gourd oil, grape seed oil, hazelnut oil, hyssop oil, isopropyl myristate oil, jojoba oil, macadamia nut oil, lavender oil, lavender oil, Lemon oil, Litsea cubeba oil, macadamia oil, mallow oil, mango seed oil, white pondweed seed oil, mink oil, nutmeg oil, olive oil, orange oil, orange roughy oil, palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, sasquana oil, savory oil, sea buckthorn oil, sesame oil, shea butter oil, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, tsubaki oil, vetiver oil, walnut oil, and wheat germ oil. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

[0492] In certain embodiments, the formulation comprises a polymer excipient. In certain embodiments, the formulation comprises a polyether. In certain embodiments, the formulation comprises polyethylene glycol (PEG) (e.g., PEG200, PEG300, PEG400, etc.).

[0493] Liquid dosage forms for parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions and suspensions. In addition to the active ingredient, the liquid dosage form may also contain an inert diluent commonly used in the art, for example, water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In certain embodiments for parenteral administration, the conjugate described herein is mixed with a solubilizer such as Alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[0494] According to known techniques, injectable preparations, for example, sterile injectable aqueous or oily suspensions, can be prepared using suitable dispersants or wetting agents and suspending agents. Sterile injectable products can be sterile injectable solutions, suspensions or emulsions in nontoxic parenteral acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution (U.S. Pharmacopeia) and isotonic sodium chloride solutions. In addition, sterile fixed oils are generally used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectable preparations. In certain embodiments, the carrier is a buffered aqueous solution.

[0495] The injectable formulations can be sterilized, for example, by filtration through a bacteria-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.

[0496] Although the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions suitable for administration to humans, the skilled artisan will appreciate that such compositions are generally suitable for administration to animals of all kinds. Modifications to pharmaceutical compositions suitable for administration to humans so as to make the compositions suitable for administration to various animals are well understood, and a veterinary pharmacologist with ordinary skills can design and / or perform such modifications by ordinary experimentation.

[0497] The SPACs provided herein are generally formulated in dosage unit form for ease of administration and uniformity of dosage. However, it should be understood that the total daily dosage of the compositions described herein will be determined by the physician within the scope of reasonable medical judgment. For any particular subject or organism, the specific therapeutically effective dosage level will depend on a variety of factors, including the severity of the disease and disorder being treated; the activity of the specific active ingredient employed; the specific composition employed; the age, weight, general health, sex and diet of the subject; the time of administration, route of administration and excretion rate of the specific active ingredient employed; the duration of treatment; drugs used in combination or concomitantly with the specific active ingredient employed; and similar factors well known in the medical field.

[0498] The SPACs and compositions provided herein can be administered by any route, including parenteral, enteral (e.g., oral), intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g., by powder, ointment, cream 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. Specific considerations are intravenous administration (e.g., systemic intravenous injection), regional administration through the blood and / or lymphatic supply, and / or direct administration to the affected site. In general, the most appropriate route of administration will depend on a variety of factors, including the nature of the agent (e.g., its stability in the gastrointestinal environment) and / or the condition of the subject (e.g., whether the subject can tolerate a certain route of administration).

[0499] The exact amount of SPAC required to achieve an effective amount will vary from subject to subject, depending on, for example, the species, age, and general condition of the subject, the severity of the side effect or disorder, the identity of the specific SPAC, the mode of administration, etc. The effective amount can be included in a single dose (e.g., a single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amount of the SPAC described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering multiple doses to a subject or applying multiple doses to a tissue or cell is three doses per day, two doses per day, one dose per day, one dose every other day, one dose every three days, one dose per week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first 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. In certain embodiments, the duration between the first and last dose of the multiple doses is three months, six months or one year. In certain embodiments, the duration between the first and last dose of the multiple doses is the lifetime of the subject, tissue or cell.

[0500] The dosage ranges described herein provide guidance for administering provided pharmaceutical compositions to adults. The amount to be administered to, for example, a child or adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or equal to the amount administered to an adult.

[0501] A SPAC or composition as described herein may be co-administered with one or more additional pharmaceutical agents (e.g., therapeutic and / or preventive active agents). The SPAC or composition may be co-administered with another pharmaceutical agent that improves its activity (e.g., treating a disease in a subject in need thereof, preventing a disease in a subject in need thereof, reducing the risk of developing a disease in a subject in need thereof (e.g., efficacy and / or effectiveness)), improves bioavailability, improves safety, reduces drug resistance, reduces and / or changes metabolism, inhibits excretion, and / or changes distribution in a subject or cell. It should also be understood that the therapy used may achieve the desired effect for the same condition, and / or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein comprising a SPAC described herein and another pharmaceutical agent exhibits a synergistic effect that is not present in a pharmaceutical composition comprising one of the SPAC and another pharmaceutical agent but not both. In certain embodiments, the other pharmaceutical agent achieves the desired effect for the same disorder. In certain embodiments, the other pharmaceutical agent achieves different effects.

[0502] The SPAC or composition can be administered simultaneously with, before or after one or more additional pharmaceutical agents, which can be used as, for example, a combination therapy. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as pharmaceutical compounds (e.g., compounds approved by the U.S. Food and Drug Administration for human or veterinary use as specified in the Code of Federal Regulations (CFR), SPACs, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucins, lipoproteins, synthetic SPACs or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNA, RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.

[0503] Such additional pharmaceutical agents include, but are not limited to, antiproliferative agents, anticancer agents, antiangiogenic agents, steroidal or nonsteroidal anti-inflammatory agents (NSAIDs), immunosuppressants, antibacterial agents, antiviral agents, cardiovascular agents, cholesterol-lowering agents, antidiabetic agents, antiallergic agents, contraceptives, analgesics, anesthetics, anticoagulants, enzyme inhibitors, steroidal agents, steroidal or antihistamines, antigens, vaccines, antibodies, decongestants, sedatives, opioids, analgesics, antipyretics, hormones, and prostaglandins.

[0504] In certain embodiments, the additional pharmaceutical agent is an anticancer agent.Anticancer agent"encompasses biotherapeutic anticancer agents as well as chemotherapeutic agents.

[0505] Exemplary biotherapeutic anti-cancer agents include, but are not limited to, interferons, cytokines (e.g., tumor necrosis factor, interferon alpha, interferon gamma), vaccines, hematopoietic growth factors, monoclonal serum therapy, immunostimulants and / or immunomodulators (e.g., IL-1, 2, 4, 6 or 12), immune cell growth factors (e.g., GM-CSF), and antibodies (e.g., Herceptin (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), Vectibix (panitumumab), Rituxan (rituximab), Bexxar (tositumomab), and those listed in Table 2).

[0506] Exemplary chemotherapeutic agents include, but are not limited to, antiestrogens (e.g., tamoxifen, raloxifene, and megestrol acetate), LHRH agonists (e.g., goserelin and leuprolide), antiandrogens (e.g., flutamide and bicalutamide), photodynamic therapy (e.g., vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, and desmethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g., cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g., carmustine (BCNU) and lomustine (CCNU)), alkyl sulfonates (e.g., busulfan and treoxazole), triazenes (e.g., dacarbazine, temozolomide), platinum-containing compounds (e.g., cisplatin, carboplatin, oxaliplatin), Vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), taxanes (e.g., paclitaxel or paclitaxel equivalents such as nanoparticle albumin-bound paclitaxel (Abraxane), docosahexaenoic acid-bound paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamic acid-bound paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three paclitaxel molecules), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognition peptide EC-1), and glucose-conjugated paclitaxel such as 2'-paclitaxel methyl 2-glucopyranosyl succinate;docetaxel, taxol), epipodophyllins (e.g., etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mitomycin C), antimetabolites, DHFR inhibitors (e.g., methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g., mycophenolic acid, tiazofurine), rin), ribavirin and EICAR), ribonucleotide reductase inhibitors (e.g., hydroxyurea and deferoxamine), uracil analogs (e.g., 5-fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g., arabinoside (araC), cytosine arabinoside and fludarabine), purine analogs (e.g., mercaptopurine and thioguanine), vitamin D3 analogs (e.g., EB 1089, CB 1093 and KH 1060), prenylation inhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g. 1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), actinomycins (e.g. actinomycin D, dactinomycin), bleomycins (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracyclines (e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, doxyrubicin, mitoxantrone), MDR inhibitors (e.g. verapamil), Ca; 2+ ATPase inhibitors (e.g., thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN TM , AZD2171), dasatinib ( BMS-354825), erlotinib Gefitinib Imatinib ( CGP57148B, STI-571), lapatinib Letotinib (CEP-701), Neratinib (HKI-272), Nilotinib Simatinib (Simasanib, SU5416), Sunitinib ( SU11248), Tocinib Fantanib ZD6474), varanib (PTK787, PTK / ZK), trastuzumab Bevacizumab Rituximab Cetuximab Panitumumab Ranibizumab Nilotinib Sorafenib Everolimus Alemtuzumab Gemtuzumab ozogamicin Temsirolimus ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK TM ), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647 and / or XL228), proteasome inhibitors (e.g., bortezomib (Velcade)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), rifolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe), and OSI-027 (OSI)), oblimersen, gemcitabine, carminomycin, folinic acid, pemetrexed, cyclophosphamide, dacarbazine, procarbazine, prednisolone, dexamethasone, camptothecin, plicamycin, asparaginase, aminopterin, methotrexate, porfibrinocin, melphalan, isovinblastine, vinblastine oxide, chlorambucil, trabectedin, procarbazine, scutellariae, carminomycin, aminopterin, and hexamethylmelamine.

[0507] Each additional pharmaceutical agent can be administered according to a dose and / or schedule determined for the pharmaceutical agent. The additional pharmaceutical agents can also be administered together with each other and / or with the SPAC or composition described herein in a single dose or composition, or separately in different doses or compositions. The specific combination to be used in the regimen will take into account the compatibility of the SPAC described herein with the additional pharmaceutical agent and / or the desired therapeutic and / or preventive effect to be achieved. In general, it is expected that the level of use of the additional pharmaceutical agent in the combination will not exceed the level at which it is used alone. In certain embodiments, the level used in combination will be lower than the level used alone.

[0508] In certain embodiments, SPACs are used in combination with one or more different treatment modalities, such as radiation therapy or surgery.

[0509] The present disclosure also encompasses kits (e.g., pharmaceutical packages). The kits provided may include a pharmaceutical composition or SPAC described herein and a container (e.g., a vial, an ampoule, a bottle, a syringe, and / or a dispenser bag or other suitable container). In certain embodiments, the kit provided may optionally further include a second container comprising a pharmaceutical excipient for diluting or suspending the pharmaceutical composition or SPAC described herein. In certain embodiments, the pharmaceutical composition or SPAC described herein provided in the first container and the second container are combined to form a unit dosage form. Therefore, in one aspect, a kit comprising a first container is provided, wherein the first container comprises a SPAC or pharmaceutical composition described herein. In certain embodiments, the kit can be used to treat a disease (e.g., cancer) in a subject in need thereof. In certain embodiments, the kit can be used to prevent a disease in a subject in need thereof.

[0510] In certain embodiments, the kit described herein further includes instructions for using the kit. The kit described herein may also include information required by regulatory agencies such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information contained in the kit is prescription information. In certain embodiments, the kit provides instructions for treating a disease (e.g., cancer) in a subject in need thereof. In certain embodiments, the kit provides instructions for preventing a disease (e.g., cancer) in a subject in need thereof. The kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.

[0511] Treatments and uses

[0512] The stapled-peptide antibody conjugates (SPACs) provided herein can deliver biologically active stapled peptides to cells and thus can be used to treat and / or prevent diseases (eg, proliferative diseases (eg, cancer), infectious diseases).

[0513] Provided herein is a method for treating and / or preventing a disease in a subject, the method comprising administering to the subject a therapeutic and / or preventive effective amount of a SPAC or a pharmaceutical composition thereof provided herein. Also provided herein is a SPAC and a pharmaceutical composition thereof for treating and / or preventing a disease in a subject. Also provided herein is the use of a SPAC and a pharmaceutical composition thereof for preparing a drug. In certain embodiments, the disease is a proliferative disease (e.g., cancer), an infectious disease (e.g., a bacterial infection), an inflammatory disease, or an autoimmune disease.

[0514] In certain embodiments, the disease is a proliferative disease (e.g., cancer). Provided herein is a method for treating a proliferative disease (e.g., cancer) in a subject, the method comprising administering to the subject a therapeutically effective amount of a SPAC provided herein or a pharmaceutical composition thereof. Also provided herein is a SPAC and a pharmaceutical composition thereof for treating a proliferative disease (e.g., cancer) in a subject. Also provided herein is the use of a SPAC and a pharmaceutical composition thereof for preparing a drug for treating a proliferative disease (e.g., cancer). In certain embodiments, the proliferative disease is cancer.

[0515] "Proliferative disease" means a disease that occurs due to abnormal growth or extension of cellular proliferation (see, e.g., Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990). Proliferative diseases may be associated with: (1) pathological proliferation of normally quiescent cells; (2) pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); (3) pathological expression of proteolytic enzymes such as matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or (4) pathological angiogenesis in proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancer (i.e., "malignant neoplasms"), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.

[0516] The term "angiogenesis" refers to the physiological process of forming new blood vessels from existing blood vessels. Angiogenesis is different from vasculogenesis, which is the process of forming endothelial cells from mesodermal cell precursors. The first blood vessel in the developing embryo is formed by angiogenesis, after which angiogenesis is responsible for most of the blood vessel growth in normal or abnormal development. Angiogenesis is an important process in growth and development, as well as in wound healing and in granulation tissue formation. However, angiogenesis is also a basic step in the transformation of tumors from a benign state to a malignant state, so angiogenesis inhibitors need to be used in the treatment of cancer. Angiogenesis may be chemically stimulated by angiogenic proteins such as growth factors (e.g., VEGF). "Pathological angiogenesis" refers to abnormal (e.g., excessive or insufficient) angiogenesis, which causes disease and / or is associated with disease.

[0517] The terms "vegetation" and "tumor" are used interchangeably herein and represent an abnormal tissue mass, wherein the growth of the mass exceeds the growth of normal tissue and is not coordinated with the growth of normal tissue. Vegetation or tumor may be "benign" or "malignant" according to the following characteristics: degree of cell differentiation (including morphology and function), growth rate, local invasion and metastasis. "Benign neoplasms" are usually well differentiated, have a characteristic growth slower than malignant neoplasms, and remain confined to the site of origin. In addition, benign neoplasms do not have the ability to infiltrate, invade or metastasize to distant sites. Exemplary benign neoplasms include, but are not limited to lipomas, chondromas, adenomas, soft droops, senile hemangiomas, seborrheic keratosis, freckles and sebaceous hyperplasia. In some cases, some "benign" tumors may later develop into malignant neoplasms, which may be due to additional genetic changes in the neoplastic cell subpopulation of the tumor, and these tumors are referred to as "pre-malignant neoplasms". An exemplary pre-malignant neoplasm is a teratoma. In contrast, "malignant neoplasms" are usually poorly differentiated (anaplastic) and have a characteristic rapid growth with progressive infiltration, invasion and destruction of surrounding tissues. In addition, malignant neoplasms usually have the ability to metastasize to distant sites. The terms "metastasis," "metastatic," or "metastasis" refer to the spread or migration of cancer cells from a primary or original tumor to another organ or tissue, and can usually be identified by the presence of "secondary tumors" or "secondary cell clusters" of the tissue type of the primary or original tumor, but not by the presence of "secondary tumors" or "secondary cell clusters" of the tissue type of the organ or tissue in which the secondary (metastatic) tumor is located.

[0518] The term "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. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a hematopoietic cancer (i.e., a hematological cancer).

[0519] In certain embodiments, the cancer is a hematopoietic cancer (e.g., a leukemia (e.g., acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myeloid 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)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulin lymphoma, hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma, T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome)), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma In some embodiments, the cancer is a leukemia. In some embodiments, the cancer is an acute lymphoblastic leukemia (ALL). In some embodiments, the cancer is an early T-cell precursor (ETP)-acute lymphoblastic leukemia (ALL).

[0520] In certain embodiments, the cancer is a musculoskeletal cancer (e.g., bone cancer (e.g., osteosarcoma, osteoid osteoma, malignant fibrous histiocytoma, Ewing sarcoma, chordoma, malignant giant cell tumor chordoma, chondrosarcoma, osteochondroma, benign enchondroma, chondroblastoma, chondromyxofibroma, myelodysplastic syndrome (MDS)), myoma (e.g., rhabdomyosarcoma, rhabdoid tumor), connective tissue cancer, synovial tumor).

[0521] In certain embodiments, the cancer is a nervous system cancer (e.g., brain cancer (e.g., astrocytoma, medulloblastoma, glioma (e.g., astrocytoma, oligodendroglioma), glioblastoma, glioblastoma multiforme, medulloblastoma, ependymoma, germ cell tumor (i.e., pinealoma), oligodendroglioma, schwannoma, retinoblastoma, congenital tumors, craniopharyngioma), spinal cord cancer, neurofibroma (e.g., neurofibromatosis type 1 or type 2 (NF), schwannomatosis), neuroblastoma, primary neuroectodermal tumor (PNT), meningeal cancer (e.g., meningioma, meningiosarcoma, gliomatosis), skull cancer, acoustic neuroma, ependymoma, hemangioblastoma, eye cancer (e.g., intraocular melanoma, retinoblastoma)). In certain embodiments, the disease to be treated is a brain tumor. In certain embodiments, the disease is pleomorphic xenoanthrocytoma (PXA). In certain embodiments, the disease is pediatric pleomorphic xenoanthrocytoma (PXA).

[0522] In certain embodiments, the cancer is selected from endocrine / exocrine cancer (e.g., thyroid cancer (e.g., papillary thyroid cancer, follicular thyroid cancer; medullary thyroid cancer, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma), pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasms (IPMN), islet cell tumors, ductal adenocarcinoma, insulinoma, glucagonoma, vasodilatory peptide tumor), adrenal cancer, neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), sebaceous gland cancer, sweat gland cancer). In certain embodiments, the cancer is a sweat gland cancer (e.g., sweat gland cancer).

[0523] In certain embodiments, the cancer is liver cancer (e.g., hepatocellular carcinoma (HCC) (e.g., hepatocellular carcinoma, hepatoblastoma, hepatocellular adenoma), malignant hepatoma, hemangioma, biliary cancer (e.g., cholangiocarcinoma)).

[0524] In certain embodiments, the cancer is head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN), adenoid cystic carcinoma). In certain embodiments, the cancer is oral cancer (e.g., oral cancer, lip cancer, tongue cancer, oral cancer, pharyngeal cancer, hypopharyngeal cancer (e.g., hypopharyngeal cancer), laryngeal cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer), salivary gland cancer). In certain embodiments, the cancer is esophageal cancer (e.g., esophageal squamous cell carcinoma, esophageal adenocarcinoma, Barrett's gland carcinoma, esophageal leiomyosarcoma).

[0525] In certain embodiments, the cancer is gastrointestinal cancer (e.g., anal cancer, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), gallbladder cancer, gastric cancer (e.g., gastric cancer (e.g., gastric adenocarcinoma)), gastrointestinal stromal tumor (GIST), small intestine cancer (e.g., appendix cancer, small intestine cancer, e.g., small intestine adenocarcinoma), small intestine cancer, large intestine cancer, large intestine cancer).

[0526] In certain embodiments, the cancer is a cardiovascular cancer (e.g., a primary cardiac tumor, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelial sarcoma, angiosarcoma), endothelial sarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcomas), cardiac myxoma, cardiac rhabdomyoma).

[0527] In certain embodiments, the cancer is lung cancer (e.g., bronchial carcinoma (e.g., bronchial protocarcinoma, bronchial adenoma), alveolar carcinoma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, cartilaginous hamartoma, papillary adenocarcinoma).

[0528] In certain embodiments, the cancer is a genitourinary tract cancer (e.g., bladder cancer (e.g., urothelial carcinoma), urethral cancer, kidney cancer (e.g., Wilms' tumor, also known as Wilman's tumor, renal cell carcinoma), testicular cancer (e.g., seminoma, testicular embryonal carcinoma), germ cell cancer, prostate cancer (e.g., prostate adenocarcinoma), penile cancer (e.g., Paget's disease of the penis and scrotum)).

[0529] In certain embodiments, 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).

[0530] In certain embodiments, the cancer is breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, breast cancer, medullary carcinoma of the breast, triple-negative breast cancer, HER-2 positive breast cancer, HER2-negative breast cancer).

[0531] In certain embodiments, the cancer is skin cancer (eg, squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC), dermatofibroma).

[0532] In certain embodiments, the cancer is a soft tissue cancer (e.g., intraepithelial neoplasm, epithelial carcinoma, epithelial sarcoma, adenocarcinoma, adenoma, fibrosarcoma, fibroma, liposarcoma, lipoma, myxoma, teratoma).

[0533] In certain embodiments, the cancer is a rare cancer. The term "rare cancer" refers to a cancer that occurs in a relatively small number of patients. Rare cancers include, but are not limited to, sarcomas (e.g., soft tissue sarcomas, liposarcoma, uterine sarcoma, leiomyosarcoma, myxofibrosarcoma, osteosarcoma, angiosarcoma, Ewing's sarcoma, synovial sarcoma, rhabdomyosarcoma, endometrial sarcoma), malignant lymphoma, thymic cancer (e.g., thymoma), mesothelioma, gastrointestinal stromal tumor (GIST), neuroendocrine cancer, eye cancer, brain tumor, bone and soft tissue tumor, skin cancer, and germ cell tumor.

[0534] In certain embodiments, the cancer is breast cancer, gastric cancer, ovarian cancer, or esophageal cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is gastric cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is esophageal cancer. In certain embodiments, the cancer is multiple myeloma, leukemia, lymphoma, or colorectal cancer. In certain embodiments, the cancer is multiple myeloma. In certain embodiments, the cancer is leukemia. In certain embodiments, the cancer is lymphoma. In certain embodiments, the cancer is colorectal cancer.

[0535] In certain embodiments, the cancer is a HER2-positive cancer. In certain embodiments, the cancer is a HER2-positive cancer, and the antibody component of the SPAC is an antibody or antigen binding fragment thereof to HER2. In certain embodiments, the cancer is a HER2-positive cancer; the antibody component of the SPAC is an antibody or antigen binding fragment thereof to HER2; and the stapled peptide is a stapled anti-cancer peptide. In certain embodiments, the HER2-positive cancer is breast cancer, gastric cancer, ovarian cancer, or esophageal cancer. In certain embodiments, the HER2-positive cancer is a HER2-positive breast cancer. In certain embodiments, the antibody to HER2 is trastuzumab or pertuzumab. Other examples of antibodies to HER2 are provided herein.

[0536] In certain embodiments, the cancer expresses an antigen selected from CD38, CD33, CD22, TROP2, CD30, CD79b, and Nectin-4. In certain embodiments, the cancer expresses the antigen and the antibody component of the SPAC is an antibody or antigen binding fragment thereof directed against the antigen. In certain embodiments, the cancer expresses the antigen; the antibody component of the SPAC is an antibody or antigen binding fragment thereof directed against the antigen; and the stapled peptide is a stapled anti-cancer peptide.

[0537] In certain embodiments, the cancer expresses CD38. In certain embodiments, the cancer expresses CD38 and the antibody component of the SPAC is an antibody or antigen binding fragment thereof to CD38. In certain embodiments, the cancer expresses CD38; the antibody component of the SPAC is an antibody or antigen binding fragment thereof to CD38; and the stapled peptide is a stapled anti-cancer peptide. In certain embodiments, the cancer expressing CD38 is multiple myeloma, leukemia, lymphoma, or colorectal cancer. In certain embodiments, the antibody to HER2 is daratumumab. Other examples of antibodies to CD38 are provided herein.

[0538] In addition, provided herein is a method for inhibiting tumor growth in a subject, the method comprising administering to the subject an effective amount of a SPAC provided herein or a pharmaceutical composition thereof. Also provided herein is a SPAC and a pharmaceutical composition thereof for inhibiting tumor growth in a subject. Also provided herein is the use of a SPAC and a pharmaceutical composition thereof provided herein for preparing a drug, the drug being used to inhibit tumor growth. The tumor may express any antigen described herein (e.g., HER2, CD38, CD33, CD22, TROP2, CD30, CD79b, Nectin-4).

[0539] The term "inhibit" as used herein in the context of tumor growth, for example, means reducing the growth rate of a tumor (i.e., reducing the proliferation rate of tumor cells). In certain embodiments, the term means that the tumor growth rate is reduced to a level that is statistically significantly lower than the initial rate (e.g., the tumor growth rate before administering or applying the SPAC provided herein). In certain embodiments, the term means that the tumor growth rate is reduced to a rate that is less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001% or less than 0.0001% of the initial rate (e.g., the tumor growth rate before administering or applying the SPAC provided herein).

[0540] In certain embodiments, treating cancer and / or inhibiting tumor growth can result in a decrease in the size or volume of a tumor. For example, after treatment, the size of the tumor is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) relative to its size before treatment. The size of the tumor can be measured by any reproducible measurement method. The size of the tumor can be measured as the diameter of the tumor or by any reproducible measurement method. In certain embodiments, the size of the tumor is reduced by at least 25% relative to its size before treatment.

[0541] In certain embodiments, treating cancer and / or inhibiting tumor growth can further result in a reduction in the number of tumors. For example, after treatment, the number of tumors is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) compared to the number before treatment. The number of tumors can be measured by any reproducible measurement method. The number of tumors can be measured by counting tumors visible to the naked eye or at a specific magnification (e.g., 2x, 3x, 4x, 5x, 10x or 50x).

[0542] In certain embodiments, treatment of cancer can result in a reduction in the number of metastatic nodules in other tissues or organs away from the primary tumor site. For example, after treatment, the number of metastatic nodules is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) relative to the number before treatment. By any reproducible measurement method, the number of metastatic nodules can be measured. By counting macroscopic metastatic nodules or with a specific magnification (e.g., 2x, 10x or 50x), the number of metastatic nodules can be measured.

[0543] In certain embodiments, treating cancer can result in an increase in the mean survival time of a subject population treated according to the present disclosure, compared to an untreated subject population. For example, the mean survival time increases by more than 30 days (more than 60 days, 90 days, or 120 days). By any reproducible means, the increase in the mean survival time of a population can be measured. The increase in the mean survival time of a population can be measured, for example, by calculating the average survival length of a population after starting treatment with a compound of the present disclosure. The increase in the mean survival time of a population can also be measured, for example, by calculating the average survival length of a population after the first round of treatment with a compound of the present disclosure is completed.

[0544] In certain embodiments, treating cancer can also result in a reduction in the mortality rate of the treated subject population compared to an untreated population. For example, the mortality rate is reduced by more than 2% (e.g., more than 5%, 10%, or 25%). The reduction in the mortality rate of the treated subject population can be measured in any reproducible manner, for example, by calculating the average number of disease-related deaths per unit time of the population after starting treatment with a compound of the disclosure. The reduction in the mortality rate of the population can also be measured, for example, by calculating the average number of disease-related deaths per unit time of the population after completing the first round of treatment with a compound of the disclosure.

[0545] In certain embodiments, treating cancer can also result in an increase in the average progression-free survival time of a treated subject population compared to an untreated population. For example, the average progression-free survival time increases by more than 30 days (more than 60 days, 90 days, or 120 days). By any reproducible means, the increase in the average progression-free survival time of a population can be measured. The increase in the average progression-free survival time of a population can be measured, for example, by calculating the average progression-free survival length of a population after starting treatment with a compound of the present disclosure. The increase in the average progression-free survival time of a population can also be measured, for example, by calculating the average progression-free survival length of a population after completing the first round of treatment with a compound of the present disclosure. "Progression-free survival" used herein means the length of time during and after medication or treatment that a disease being treated (e.g., cancer) does not deteriorate.

[0546] Also provided herein is a method for delivering a stapled peptide into a cell, the method comprising contacting a cell with a SPAC or a pharmaceutical composition thereof provided herein. In certain embodiments, the cell is a cancer cell. In certain embodiments, the stapled peptide has improved cellular uptake compared to a corresponding uncoupled stapled peptide. In certain embodiments, the stapled peptide is delivered to in vitro cells. In certain embodiments, the stapled peptide is delivered to in vivo cells (ie, in a subject). In certain embodiments, the stapled peptide is delivered to cells of a biological sample.

[0547] Also provided herein is a method for triggering cancer cell death, the method comprising contacting a cancer cell with an effective amount of a SPAC or a pharmaceutical composition thereof provided herein. In certain embodiments, the method is a method for selectively triggering cancer cell death (i.e., selectively killing cancer cells). In certain embodiments, the SPAC provided herein has selective cytotoxicity to cancer cells. In certain embodiments, the cell is contacted in vitro. In certain embodiments, the cell is contacted in vivo (i.e., in a subject). In certain embodiments, the cell is contacted in a biological sample.

[0548] If a SPAC described herein triggers cell death of one type of cell to a greater extent than another type of cell, it "selectively" triggers the death of one type of cell over another type of cell (e.g., selectively triggers the death of cancer cells over non-cancerous cells). If a SPAC described herein triggers the death of cancer cells to a greater extent than the death of non-cancerous cells, it "selectively" triggers the death of cancer cells. If a peptide described herein is toxic to cancer cells (e.g., by lysing, killing, promoting apoptosis, or otherwise damaging) more than it is to non-cancerous cells, the peptide is "selectively" cytotoxic to cancer cells relative to non-cancerous cells. In certain embodiments, the selectivity in any of the foregoing embodiments is at least 1.1 times, at least 1.5 times, 2 times, at least 3 times, at least 5 times, at least 10 times, at least 30 times, at least 50 times, at least 100 times, at least 300 times, at least 500 times, at least 1,000 times, at least 3,000 times, at least 5,000 times, at least 10,000 times, at least 30,000 times, at least 50,000 times, or at least 100,000 times. In certain embodiments, the selectivity is no more than 100,000 times, no more than 10,000 times, no more than 1,000 times, no more than 100 times, no more than 10 times, or no more than 2 times. Combinations of the above ranges (e.g., at least 2 times and no more than 10,000 times) are also within the scope of the present disclosure.

[0549] As described herein, the cell can be a cancer cell. In certain embodiments, the cell is a 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), gastric 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) cell.

[0550] Also provided herein is a method for treating and / or preventing an infectious disease (e.g., a bacterial infection) in a subject, the method comprising administering to the subject an effective amount of a SPAC provided herein or a pharmaceutical composition thereof. Also provided herein is a SPAC and a pharmaceutical composition thereof for treating and / or preventing an infectious disease (e.g., a bacterial infection) in a subject. Also provided herein is the use of a SPAC and a pharmaceutical composition thereof for preparing a drug, the drug being used to treat and / or prevent an infectious disease (e.g., a bacterial infection). In certain embodiments, the infectious disease is a bacterial infection. In certain embodiments, the bacterial infection is an antibiotic-resistant bacterial infection.

[0551] Also provided herein are methods of killing and / or inhibiting the growth of bacteria (e.g., Gram-negative and / or Gram-positive bacteria), comprising contacting the bacteria (e.g., in vitro or in vivo) with a SPAC provided herein, or a pharmaceutical composition thereof.

[0552] "Infectious disease" means any disease caused by a pathogen (i.e., a pathogenic microorganism). An infectious disease can be caused by bacteria, viruses, parasites, or fungi. An infectious disease can be a microbial infection. "Microbial infection" means being infected by a microorganism (such as a fungus, bacteria, or virus). Various bacterial infections include, but are not limited to, skin infections (e.g., bacterial cellulitis, wound infections), gastrointestinal infections, throat infections (e.g., streptococcal pharyngitis (strep throat)), urinary tract infections (UTI), genitourinary system infections, sexually transmitted diseases (e.g., gonorrhea, chlamydia, syphilis), lung 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. In certain embodiments, the microbial infection is infected by bacteria, i.e., "bacterial infection". In certain embodiments, the bacterium is a gram-negative bacterium. In certain embodiments, the bacterium is a gram-positive bacterium.

[0553] "Gram-negative bacteria" were originally defined by their inability to retain the Gram stain; however, Gram-negative bacteria have since been further defined as bacteria that typically have a cell wall containing a thin peptidoglycan layer and have an outer lipid membrane. "Gram-positive bacteria" are bacteria that appear crystalline purple in the Gram stain test and typically have a cell wall containing a thick peptidoglycan layer and no outer lipid membrane.

[0554] As used herein, "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. Microorganisms that are resistant to multiple antimicrobial agents are referred to as "multidrug-resistant" (MDR). In certain embodiments, the methods herein are used to treat multidrug-resistant bacterial infections. In certain embodiments, the methods herein are used to kill multidrug-resistant bacteria and / or inhibit their growth. Example

[0555] General approach

[0556] Solid Phase Peptide Synthesis: Antimicrobial peptides and their stapled derivatives were synthesized using Fmoc-based solid phase peptide synthesis. To achieve i+4 staple length, α-methyl, α-alkenyl amino acids were used on both sides of three residues (flanking three residues). For the staple reaction, Grubbs first generation ruthenium catalyst dissolved in dichloroethane was added to the peptide still on the resin. To ensure maximum conversion, three to five rounds of staple were performed. After staple, an appropriate maleimide linker was coupled to the N-terminus and the linker-peptide was cleaved from the resin using trifluoroacetic acid, followed by precipitation using a hexane: ether (1:1) mixture and subsequent purification using preparative HPLC. The purity characterization of the final linker-peptide was evaluated using a UHPLC / MS system.

[0557] Cell culture: Cell lines were maintained in appropriate culture medium supplemented with fetal bovine serum to a final concentration of 10%.

[0558] 72-hour cytotoxicity assay: Cells were plated in a 96-well format and after 24 hours of incubation, serial dilutions of SPAC from a 1 mg / mL stock or vehicle were added to the cells in a final volume of 100 μl. After 72 hours of incubation at 37°C, 100 μl of The reagents were added to the cells and the plates were incubated at room temperature for 15 minutes. Luminescence was then measured on a microplate reader.

[0559] Example 1: Preparation of SPAC

[0560] Peptide coupling protocol: (1) Prepare the stapled peptide using solid phase synthesis; (2) Add a linking agent to the N-terminally deprotected stapled peptide on the resin. For example, 6-maleimidocaproyl-Val-Cit-p-aminobenzoyl carbonate-4-nitrophenyl ester (Mc-Val-Cit-PABC-PNP) can be used; (3) Cleave from the resin and purify by HPLC; (4) In a separate batch, reduce the antibody using TCEP (see protocol below); (5) Couple the stapled peptide to the antibody; (6) Quench unreacted peptide using N-acetylcysteine; and (7) Purify by salt exchange.

[0561] Antibody coupling protocol: (1) Antibody buffer exchange into PBS-E (pH 6-8) according to the following protocol: (a) dilute the 21 mg / mL antibody solution provided by the manufacturer to 1 mg / mL in PBS-E (pH 6-8), (b) remove the loaded buffer by centrifugation using a spin column. Add more PBS-E and equilibrate by further centrifugation, (c) add antibody solution (1 mg / mL), centrifuge, collect the effluent, (d) measure the antibody concentration in the eluate, and (e) use an ultrafiltration device according to the manufacturer's instructions to increase the antibody concentration to 10 mg / mL; (2) reduce the antibody with TCEP; (3) couple the peptide by co-incubation at room temperature; (4) quench the reaction with n-acetylcysteine; and (5) purify.

[0562] Example 2: Cytotoxicity of anti-HER2 SPAC

[0563] Anti-HER2 SPAC 1 and SPAC 2 (see Table 6) are cytotoxic to breast cancer cells with relatively low and relatively high HER2 expression. Figure 1A-1B SPAC 1 and SPAC 2 showed cytotoxicity in the BT-474 cell line with high HER2 expression (HER2+++) ( Figure 1A SPAC 1 and SPAC 2 also showed cytotoxicity in the MCF7 cell line with low HER2 expression (HER2+) ( Figure 1B ).

[0564] Example 3: Cytotoxicity of anti-CD38 SPAC

[0565] Anti-CD38 SPAC 3 (see Table 6) is cytotoxic to CD38+ multiple myeloma cells. Figure 2 SPAC 3 showed cytotoxicity in the CD38+ multiple myeloma cell line RPMI 8226.

[0566] Example 4: Anti-proliferative activity of anti-CD38 SPACs containing PPI inhibitors

[0567] Anti-CD38 SPACs are used to deliver a variety of stapled peptide protein-protein interaction (PPI) inhibitors (e.g., MCL-1 inhibitors, MDM2 inhibitors, β-catenin inhibitors) to inhibit the proliferation of cancer cells. Figure 3-5 As shown in , SPACs 8, 11 and 16 (see Table 7), each comprising the anti-CD38 antibody daratumumab, inhibited the proliferation of multiple myeloma cells.

[0568] Example 5: Selectivity of anti-HER2 SPACs containing PPI inhibitors

[0569] As in Figure 6A-6B As shown in , 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 exploiting oncogene addiction. Fig. 6A Results are shown in the HER2-low cell line MCF7, which has a wild-type (wt) p53 state and is therefore sensitive to MDM2 inhibition. Enmetuzumab (a traditional ADC) did not show activity in this cell line, while SPAC 7 (see Table 7) comprising an MDM2 inhibitor provided herein showed antiproliferative activity. Figure 6B Results are shown in cell lines that highly express HER2 but have mutated p53 and are therefore insensitive to MDM2 inhibition. In this case, the traditional ADC trastuzumab shows efficacy at relatively low concentrations, while SPAC 7 (see Table 7) requires relatively high concentrations. These data confirm the selectivity of SPAC. These data also confirm that on-target toxicity is unlikely to occur because high antibody concentrations and receptor expression above that in normal tissues may be required for toxicity.

[0570] Additional embodiments

[0571] Additional embodiments of the present disclosure are indicated by the following numbered paragraphs:

[0572] 1. A stapled peptide-antibody conjugate (SPAC) comprising a stapled peptide conjugated to an antibody or an antigen-binding fragment thereof.

[0573] 2. The SPAC of paragraph 1, wherein the antibody is a monoclonal antibody (mAb) or an antigen-binding fragment thereof.

[0574] 3. The SPAC of paragraph 1 or 2, wherein the antibody is an anti-cancer antibody or an antigen-binding fragment thereof.

[0575] 4. The SPAC of any of paragraphs 1-3, wherein the antibody or antigen-binding fragment thereof is directed against a target antigen expressed on cancer cells.

[0576] 5. The SPAC of any of paragraphs 1-4, wherein the antibody is an antibody or an antigen-binding fragment thereof against human epidermal growth factor receptor 2 (HER2).

[0577] 6. The SPAC of paragraph 5, wherein the antibody is trastuzumab.

[0578] 7. The SPAC of paragraph 5, wherein the antibody is pertuzumab.

[0579] 8. The SPAC of any of paragraphs 1-4, wherein the antibody is an antibody or antigen-binding fragment thereof against CD38, CD33, CD22, TROP2, CD30, CD79b, Nectin-4 or TM4SF1.

[0580] 9. The SPAC of any of paragraphs 1-4, wherein the antibody is an antibody or an antigen-binding fragment thereof directed against TM4SF1.

[0581] 10. The SPAC of any of paragraphs 1-4, wherein the antibody is an antibody or antigen-binding fragment thereof directed against CD38.

[0582] 11. The SPAC of paragraph 10, wherein the antibody is daratumumab.

[0583] 12. The SPAC of any of paragraphs 1-11, wherein the antibody is an antibody-drug conjugate (ADC) or an antigen-binding fragment thereof.

[0584] 13. The SPAC of paragraph 12, wherein the antibody is trastuzumab emtansine.

[0585] 14. The SPAC of any of paragraphs 1-13, wherein the stapled peptide is a stapled anti-cancer peptide.

[0586] 15. The SPAC of any of paragraphs 1-14, wherein the stapled peptide is a stapled antimicrobial peptide (StAMP).

[0587] 16. The SPAC of any of paragraphs 1-15, wherein the stapled peptide is a single stapled, double stapled, or stitched peptide.

[0588] 17. The SPAC of any of paragraphs 1-16, wherein the stapled peptide is an inhibitor of protein-protein interaction (PPI).

[0589] 18. The SPAC of any of paragraphs 1-17, wherein the stapled peptide is an inhibitor of a BCL-2 family member protein.

[0590] 19. The SPAC of paragraph 18, wherein the stapled peptide is BCL-x L , BCL-2, BCL-W and / or MCL1 inhibitors.

[0591] 20. The SPAC of paragraph 19, wherein the stapled peptide is a MCL1 inhibitor.

[0592] 21. The SPAC of any of paragraphs 1-20, wherein the stapled peptide is an activator of a BCL-2 family member protein effector.

[0593] 22. The SPAC of paragraph 21, wherein the stapled peptide is an activator of BAX, BAK or BOK.

[0594] 23. The SPAC of any of paragraphs 1-22, wherein the stapled peptide is a stapled BCL-2 interacting mediator of cell death (BIM) peptide.

[0595] 24. The SPAC of any of paragraphs 1-23, wherein the stapled peptide is SAHB A1 or BIM SAHB A2 .

[0596] 25. The SPAC of any of paragraphs 1-17, wherein the stapled peptide is a β-catenin inhibitor.

[0597] 26. The SPAC of paragraph 25, wherein the stapled peptide is an inhibitor of Wnt / β-catenin signaling.

[0598] 27. The SPAC of any of paragraphs 1-17, wherein the stapled peptide is an MDM2 inhibitor.

[0599] 28. The SPAC of paragraph 27, wherein the stapled peptide inhibits the binding of MDM2 to p53.

[0600] 29. The SPAC of paragraph 27 or 28, wherein the stapled peptide is ALRN-6924, ATSP-7041, or ATSP7041Cba10L.

[0601] 30. The SPAC of paragraph 27 or 28, wherein the stapled peptide comprises any one of SEQ ID NOs: 161-166 or a pharmaceutically acceptable salt thereof.

[0602] 31. The SPAC of any of paragraphs 1-30, wherein the stapled peptide triggers cancer cell death.

[0603] 32. The SPAC of any of paragraphs 1-17, wherein the stapled peptide is a stapled magainin.

[0604] 33. The SPAC of paragraph 32, wherein the stapled peptide is a stapled magainin II peptide.

[0605] 34. The SPAC of paragraph 33, wherein the stapled peptide comprises the amino acid sequence: GX 1 GKFX 2 HSKKK FG KAX 3 VGEX 4 (SEQ ID NO:1),

[0606] or a pharmaceutically acceptable salt thereof, wherein:

[0607] X 1 , X 2 , X 3 and X 4 It is an amino acid;

[0608] X 1 and X 2 Connected via crosslinks, and X 3 and X 4 connected via cross-links; and

[0609] The amino acid sequence includes except X 1 , X 2 , X 3 and X 4 0 to 9 amino acid substitutions (including the end values) at positions other than

[0610] 35. The SPAC of paragraph 34, wherein the stapled peptide comprises the amino acid sequence: GX 1 GKFX 2 HSKKK FG KAX 3 VGEX 4 AKK (SEQ ID NO: 2),

[0611] or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes in addition to X 1 , X 2 , X 3 and X 4 0 to 11 amino acid substitutions (including the end values) at positions other than

[0612] 36. The SPAC of paragraph 35, wherein the stapled peptide comprises the amino acid sequence: GX 1 GKFX 2 Dap KKK KF GKAX 3 VGEX 4 AKK (SEQ ID NO:48),

[0613] or a pharmaceutically acceptable salt thereof.

[0614] 37. The SPAC of paragraph 35, wherein the stapled peptide comprises the amino acid sequence:

[0615]

[0616] or a pharmaceutically acceptable salt thereof.

[0617] 38. The SPAC of any of paragraphs 1-17, wherein the stapled peptide is a stapled Esculentin peptide.

[0618] 39. The SPAC of paragraph 38, wherein the stapled peptide is a stapled esculentin-1A peptide.

[0619] 40. The SPAC of paragraph 39, wherein the stapled peptide comprises the amino acid sequence:

[0620] GX 1 FSKX 2 KGKKIKNLX 3 ISGX 4 KG (SEQ ID NO:73),

[0621] and pharmaceutically acceptable salts thereof, wherein:

[0622] X 1 , X 2 , X 3 and X 4 It is an amino acid;

[0623] X 1 and X 2 Connected via crosslinks, and X 3 and X 4 connected via cross-links; and

[0624] The amino acid sequence includes except X 1 , X 2 , X 3 and X 4 0 to 9 amino acid substitutions (including the end values) at positions other than

[0625] 41. The SPAC of paragraph 40, wherein the stapled peptide comprises the amino acid sequence:

[0626] GX 1 FSKX 2 KGKKIKNLLX 3 SGLX 4 G (SEQ ID NO: 105),

[0627] and pharmaceutically acceptable salts thereof, wherein:

[0628] X 1 , X 2 , X 3 and X 4 It is an amino acid;

[0629] X 1and X 2 Connected via crosslinks, and X 3 and X 4 connected via cross-links; and

[0630] The amino acid sequence optionally includes in addition to X 1 , X 2 , X 3 and X 4 0 to 8 amino acid substitutions (including the end values) at positions other than

[0631] 42. The SPAC of paragraph 40, wherein the stapled peptide comprises SEQ ID NO: 73 or a pharmaceutically acceptable salt thereof.

[0632] 43. The SPAC of paragraph 41, wherein the stapled peptide comprises SEQ ID NO: 105 or a pharmaceutically acceptable salt thereof.

[0633] 44. The SPAC of paragraph 41, wherein the stapled peptide comprises the amino acid sequence:

[0634] GX 1 FSKX 2 KGKKIKNLLX 3 SGLX 4 KGGE (SEQ ID NO: 113),

[0635] or a pharmaceutically acceptable salt thereof.

[0636] 45. The SPAC of any of paragraphs 1-44, wherein the stapled peptide is an alpha helical peptide.

[0637] 46. ​​The SPAC of any of paragraphs 1-45, wherein the stapled peptide is 100 amino acids or less in length.

[0638] 47. The SPAC of any of paragraphs 1-46, wherein the stapled peptide is 30 amino acids or less in length.

[0639] 48. The SPAC of any of paragraphs 1-47, wherein the stapled peptide is coupled to one or more stapled peptides.

[0640] 49. The SPAC of paragraph 36, wherein the stapled peptide has SEQ ID NO: 48, wherein X 1 and X 2 , and X 3 and X 4 The cross-linking is carried out via the following formula:

[0641]

[0642] wherein α represents the α-carbon of the cross-linked amino acids; and wherein the C-terminus of the stapled peptide is amidated with -NH2.

[0643] 50. The SPAC of paragraph 37, wherein the stapled peptide has SEQ ID NO: 26, wherein X 1 and X 2 , and X 3 and X 4 The cross-linking is via the following formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

[0644] 51. The SPAC of paragraph 42, wherein the stapled peptide has SEQ ID NO: 73, wherein X 1 and X 2 , and X 3 and X 4 The cross-linking is via the following formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

[0645] 52. The SPAC of paragraph 43, wherein the stapled peptide has SEQ ID NO: 105, wherein X 1 and X 2 , and X 3 and X 4 The cross-linking is via the following formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

[0646] 53. The SPAC of paragraph 44, wherein the stapled peptide has SEQ ID NO: 113, wherein X 1 and X 2 , and X 3 and X 4 The cross-linking is via the following formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

[0647] 54. The SPAC of any of paragraphs 1-53, wherein the antibody or antigen-binding fragment thereof is coupled to the N-terminus of the stapled peptide.

[0648] 55. The SPAC of any of paragraphs 1-53, wherein the antibody or antigen-binding fragment thereof is coupled to the C-terminus of the stapled peptide via a lysine residue.

[0649] 56. The SPAC of any of paragraphs 1-55, wherein the stapled peptide is coupled to a cysteine ​​residue of the antibody or antigen-binding fragment thereof.

[0650] 57. The SPAC of any of paragraphs 1-55, wherein the stapled peptide is conjugated via a lysine residue of the antibody or antigen-binding fragment thereof.

[0651] 58. The SPAC of any of paragraphs 1-57, wherein the antibody or antigen-binding fragment thereof is coupled to the stapled peptide via a linker.

[0652] 59. The conjugate of paragraph 58, wherein 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.

[0653] 60. The SPAC of paragraph 58 or 59, wherein the linker is a cleavable linker.

[0654] 61. The SPAC of any of paragraphs 58-60, wherein the linker is pH cleavable, or cleavable by a protease, esterase, or intracellular disulfide reduction.

[0655] 62. The SPAC of paragraph 61, wherein the linker is cleavable by a protease.

[0656] 63. The SPAC of any of paragraphs 58-62, wherein the linker is a peptide linker.

[0657] 64. The SPAC of paragraph 63, wherein the peptide linker comprises -Y A Y B Y C Y D -(SEQ ID NO: 159), wherein Y A is glycine, glutamate, or absent; Y B is valine, phenylalanine, alanine, tyrosine or glycine; Y C is citrulline, arginine, lysine, alanine or glycine; and Y D It's glycine, or it's not there.

[0658] 65. The SPAC of paragraph 64, wherein the peptide linker comprises -valine-citrulline-.

[0659] 66. The SPAC of any of paragraphs 58-65, wherein the linker has one of the following formulae:

[0660]

[0661] (Maleimide-hexanoic acid-valine-citrulline-p-aminobenzyl),

[0662]

[0663] (maleimide-hexanoic acid-valine-citrulline-glycine),

[0664]

[0665] (iodoacetamide-(PEG3)-valine-citrulline),

[0666]

[0667] (iodoacetamide-(PEG3)-valine-citrulline-p-aminobenzyl),

[0668]

[0669] (Maleimide-2,3-diaminopropionate-valine-citrulline-p-aminobenzyl),

[0670]

[0671] (Maleimido-hexanoic acid-valine-citrulline),

[0672]

[0673] (maleimide-hexanoic acid-lysine-valine-citrulline), or

[0674]

[0675] (Maleimido-hexanoic acid-(PEG3)-(PEG3)-valine-citrulline);

[0676] Wherein S represents the connection point with the antibody or antigen-binding fragment thereof; N represents the connection point with the N-terminus of the stapled peptide.

[0677] 67. The SPAC of any of paragraphs 58-65, wherein the linker comprises one of the following formulae:

[0678]

[0679]

[0680]

[0681]

[0682]

[0683] Wherein A represents the connection point with the antibody or antigen-binding fragment thereof; N represents the connection point with the N-terminus of the stapled peptide

[0684] 68. The SPAC of any of paragraphs 1-67, wherein the antibody or antigen-binding fragment thereof is conjugated to two or more stapled peptides.

[0685] 69. The SPAC of any of paragraphs 1-68, wherein the antibody or antigen-binding fragment thereof is conjugated to 2-10 stapled peptides, inclusive.

[0686] 70. The SPAC of any of paragraphs 1-69, comprising an antibody or antigen-binding fragment thereof:stapled peptide ratio of about 1:8.

[0687] 71. A pharmaceutical composition comprising the SPAC of any of paragraphs 1-70 and a pharmaceutically acceptable carrier.

[0688] 72. A method of treating a proliferative disease in a subject, the method comprising administering to the subject a SPAC of any of paragraphs 1-70 or a pharmaceutical composition thereof.

[0689] 73. The method of paragraph 72, wherein the proliferative disease is cancer.

[0690] 74. The method of paragraph 73, wherein the cancer is a HER2-positive cancer.

[0691] 75. The method of paragraph 73 or 74, wherein the cancer is colorectal cancer, breast cancer, gastric cancer, ovarian cancer or esophageal cancer.

[0692] 76. The method of any of paragraphs 73-75, wherein the cancer is HER2-positive breast cancer.

[0693] 77. The method of paragraph 73, wherein the cancer expresses CD38.

[0694] 78. The method of paragraph 73 or 77, wherein the cancer is multiple myeloma, leukemia or lymphoma.

[0695] 79. A method of inhibiting tumor growth in a subject, the method comprising administering to the subject a SPAC of any of paragraphs 1-70 or a pharmaceutical composition thereof.

[0696] 80. A method of delivering a stapled peptide to a cell, the method comprising contacting the cell with the SPAC of any of paragraphs 1-70 or a pharmaceutical composition thereof.

[0697] 81. The method of paragraph 80, wherein the cell is a cancer cell.

[0698] 82. The method of paragraph 80 or 81, wherein the stapled peptide has improved cellular uptake compared to a corresponding unconjugated stapled peptide.

[0699] 83. A method of triggering cancer cell death, the method comprising contacting the cancer cell with the SPAC of any of paragraphs 1-70 or a pharmaceutical composition thereof.

[0700] 84. The method of paragraph 83, which is used to selectively kill cancer cells in the presence of non-cancerous cells.

[0701] 85. The method of any of paragraphs 80-84, wherein the cell is in vitro.

[0702] 86. The method of any of paragraphs 80-84, wherein the cell is in a subject.

[0703] 87. The method of any of the preceding paragraphs, wherein the SPAC is administered intravenously.

[0704] 88. The method of any of the preceding paragraphs, wherein the subject is a human.

[0705] 89. The SPAC of any of paragraphs 1-70, for use in the method of any of the preceding paragraphs.

[0706] 90. Use of the SPAC of any of paragraphs 1-70 for the preparation of a medicament.

[0707] 91. A kit comprising the SPAC of any of paragraphs 1-70 or a pharmaceutical composition thereof and optionally instructions for use.

[0708] Equivalent programs and scope

[0709] In the claims, articles such as "a, an," and "the" may refer to one or more than one, unless indicated to the contrary or otherwise apparent from the context. Claims or descriptions including "or" between one or more members of a group are deemed satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise associated with a specified product or process, unless indicated to the contrary or otherwise apparent from the context. The present disclosure includes embodiments wherein exactly one member of the group is present in, used in, or otherwise associated with a given product or process. The present disclosure includes embodiments wherein more than one or all of the members of the group are present in, used in, or otherwise associated with a given product or process.

[0710] In addition, the present disclosure encompasses all variations, combinations and arrangements in which one or more limitations, elements, clauses and descriptive terms from one or more listed claims are introduced into another claim. For example, any claim that is subordinate to another claim can be modified to include one or more limitations found in any other claim that is subordinate to the same basic claim. In the case where the elements are presented as a list, for example, in Markush group format, each subgroup of the elements is also disclosed, and any element can be removed from the group. It should be understood that, in general, in the case where the present disclosure or aspects of the present disclosure are referred to as including specific elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure are composed of such elements and / or features, or are substantially composed of such elements and / or features. For simplicity, those embodiments are not specifically described in this article with such words.

[0711] It should also be noted that the terms "comprising" and "containing" are intended to be open ended and allow for the inclusion of additional elements or steps. Where ranges are given, the endpoints are included. In addition, unless otherwise indicated or otherwise apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges may assume any specific value or sub-range within the range in different embodiments of the present disclosure, accurate to one tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise.

[0712] The present application relates to various granted patents, published patent applications, journal articles and other publications, which are all incorporated herein by reference. If there is a conflict between any incorporated reference and this specification, the specification shall prevail. In addition, any specific embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more claims. Because such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if they are not explicitly set forth herein for exclusion. Any specific embodiment of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of the prior art.

[0713] Those skilled in the art will recognize or be able to determine many equivalents to the specific embodiments described herein using only routine experimentation. The scope of the embodiments described herein is not intended to be limited to the above description, but is as described in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this specification may be made without departing from the spirit or scope of the present disclosure as defined in the following claims.

Claims

1. A stapled peptide-antibody conjugate (SPAC) comprising a stapled peptide conjugated to an antibody or an antigen-binding fragment thereof.

2. The SPAC of claim 1, wherein the antibody is a monoclonal antibody (mAb) or an antigen-binding fragment thereof.

3. The SPAC of claim 1 or 2, wherein the antibody is an anti-cancer antibody or an antigen-binding fragment thereof.

4. The SPAC of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof is directed against a target antigen expressed on cancer cells.

5. The SPAC of any one of claims 1-4, wherein the antibody is an antibody or an antigen-binding fragment thereof against human epidermal growth factor receptor 2 (HER2).

6. The SPAC of claim 5, wherein the antibody is trastuzumab.

7. The SPAC of claim 5, wherein the antibody is Pertuzumab.

8. The SPAC of any one of claims 1-4, wherein the antibody is an antibody or an antigen-binding fragment thereof against CD38, CD33, CD22, TROP2, CD30, CD79b, Nectin-4 or TM4SF1.

9. The SPAC of any one of claims 1-4, wherein the antibody is an antibody or an antigen-binding fragment thereof against TM4SF1.

10. The SPAC of any one of claims 1-4, wherein the antibody is an antibody or an antigen-binding fragment thereof directed against CD38.

11. The SPAC of claim 10, wherein the antibody is daratumumab.

12. The SPAC of any one of claims 1-11, wherein the antibody is an antibody-drug conjugate (ADC) or an antigen-binding fragment thereof.

13. The SPAC of claim 12, wherein the antibody is trastuzumab emtansine.

14. The SPAC of any one of claims 1-13, wherein the stapled peptide is a stapled anti-cancer peptide.

15. The SPAC of any one of claims 1-14, wherein the stapled peptide is a stapled antimicrobial peptide (StAMP).

16. The SPAC of any one of claims 1-15, wherein the stapled peptide is a single stapled, double stapled, or stitched peptide.

17. The SPAC of any one of claims 1-16, wherein the stapled peptide is an inhibitor of a protein-protein interaction (PPI).

18. The SPAC of any one of claims 1-17, wherein the stapled peptide is an inhibitor of a BCL-2 family member protein.

19. The SPAC of claim 18, wherein the stapled peptide is BCL-x L , BCL-2, BCL-W and / or MCL1 inhibitors.

20. The SPAC of claim 19, wherein the stapled peptide is a MCL1 inhibitor.

21. The SPAC of any one of claims 1-20, wherein the stapled peptide is an activator of a BCL-2 family member protein effector.

22. The SPAC of claim 21, wherein the stapled peptide is an activator of BAX, BAK, or BOK.

23. The SPAC of any one of claims 1-22, wherein the stapled peptide is a stapled BCL-2 interacting mediator of cell death (BIM) peptide.

24. The SPAC of any one of claims 1-23, wherein the stapled peptide is SAHB A1 or BIM SAHB A2 .

25. The SPAC of any one of claims 1-17, wherein the stapled peptide is a β-catenin inhibitor.

26. The SPAC of claim 25, wherein the stapled peptide is an inhibitor of Wnt / β-catenin signaling.

27. The SPAC of any one of claims 1-17, wherein the stapled peptide is an MDM2 inhibitor.

28. The SPAC of claim 27, wherein the stapled peptide inhibits binding of MDM2 to p53.

29. The SPAC of claim 27 or 28, wherein the stapled peptide is ALRN-6924, ATSP-7041, or ATSP7041Cba10L.

30. The SPAC of claim 27 or 28, wherein the stapled peptide comprises any one of SEQ ID NOs: 161-166, or a pharmaceutically acceptable salt thereof.

31. The SPAC of any one of claims 1-30, wherein the stapled peptide triggers cancer cell death.

32. The SPAC of any one of claims 1-17, wherein the stapled peptide is stapled magainin.

33. The SPAC of claim 32, wherein the stapled peptide is a stapled magainin II peptide.

34. The SPAC of claim 33, wherein the stapled peptide comprises the amino acid sequence: GX 1 GKFX 2 HSK KK FGKAX 3 VGEX 4 (SEQ ID NO:1), or a pharmaceutically acceptable salt thereof, wherein: X 1 , X 2 , X 3 and X 4 It is an amino acid; X 1 and X 2 Connected via crosslinks, and X 3 and X 4 connected via cross-links; and The amino acid sequence includes except X 1 , X 2 , X 3 and X 4 0 to 9 amino acid substitutions, inclusive, at positions other than .

35. The SPAC of claim 34, wherein the stapled peptide comprises the amino acid sequence: GX 1 GKFX 2 HSK KK FGKAX 3 VGEX 4 AKK (SEQ ID NO: 2), or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes in addition to X 1 , X 2 , X 3 and X 4 0 to 11 amino acid substitutions, inclusive, at positions other than .

36. The SPAC of claim 35, wherein the stapled peptide comprises the amino acid sequence: GX 1 GKFX 2 Dap K KK KFGKAX 3 VGEX 4 AKK (SEQ ID NO:48), or a pharmaceutically acceptable salt thereof.

37. The SPAC of claim 35, wherein the stapled peptide comprises the amino acid sequence: GX 1 G Dap FX 2 DapDap Dap Dap Dap FG Dap AX 3 VGEX 4 A Dap Dap (SEQ ID NO: 26), GX 1 GKFX 2 KK KKKFGKAX 3 VGEX 4 AKK (SEQ ID NO: 5), GX 1 GKFX 2 HKKKKFGK AX 3 VFEX 4 AKK (SEQ ID NO: 23), GX 1 G Dab FX 2 Dab Dab Dab Dab Dab FG Dab AX 3 VGEX 4 A Dab Dab (SEQ ID NO: 24), or a pharmaceutically acceptable salt thereof.

38. The SPAC of any one of claims 1-17, wherein the stapled peptide is a stapled Esculentin peptide.

39. The SPAC of claim 38, wherein the stapled peptide is a stapled esculentin-1A peptide.

40. The SPAC of claim 39, wherein the stapled peptide comprises the amino acid sequence: GX 1 FSKX 2 KGK KI KNLX 3 ISGX 4 KG (SEQ ID NO:73), and pharmaceutically acceptable salts thereof, wherein: X 1 , X 2 , X 3 and X 4 It is an amino acid; X 1 and X 2 Connected via crosslinks, and X 3 and X 4 connected via cross-links; and The amino acid sequence includes except X 1 , X 2 , X 3 and X 4 0 to 9 amino acid substitutions, inclusive, at positions other than .

41. The SPAC of claim 40, wherein the stapled peptide comprises the amino acid sequence: GX 1 FSKX 2 KGK KI KNLLX 3 SGLX 4 G (SEQ ID NO: 105), and pharmaceutically acceptable salts thereof, wherein: X 1 , X 2 , X 3 and X 4 It is an amino acid; X 1 and X 2 Connected via crosslinks, and X 3 and X 4 connected via cross-links; and The amino acid sequence optionally includes in addition to X 1 , X 2 , X 3 and X 4 0 to 8 amino acid substitutions, inclusive, at positions other than .

42. The SPAC of claim 40, wherein the stapled peptide comprises SEQ ID NO: 73 or a pharmaceutically acceptable salt thereof.

43. The SPAC of claim 41, wherein the stapled peptide comprises SEQ ID NO: 105 or a pharmaceutically acceptable salt thereof.

44. The SPAC of claim 41, wherein the stapled peptide comprises the amino acid sequence: GX 1 FSKX 2 KG KKI KNLLX 3 SGLX 4 KGGE (SEQ ID NO: 113), or a pharmaceutically acceptable salt thereof.

45. The SPAC of any one of claims 1-44, wherein the stapled peptide is an alpha helical peptide.

46. ​​The SPAC of any one of claims 1-45, wherein the stapled peptide is 100 amino acids or less in length.

47. The SPAC of any one of claims 1-46, wherein the stapled peptide is 30 amino acids or less in length.

48. The SPAC of any one of claims 1-47, wherein the stapled peptide is coupled to one or more stapled peptides.

49. The SPAC of claim 36, wherein the stapled peptide has SEQ ID NO: 48, wherein X 1 and X 2 , and X 3 and X 4 The cross-linking is carried out via the following formula: wherein α represents the α-carbon of the cross-linked amino acids; and wherein the C-terminus of the stapled peptide is amidated with -NH2.

50. The SPAC of claim 37, wherein the stapled peptide has SEQ ID NO: 26, wherein X 1 and X 2 , and X 3 and X 4 The cross-linking is via the following formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

51. The SPAC of claim 42, wherein the stapled peptide has SEQ ID NO: 73, wherein X 1 and X 2 , and X 3 and X 4 The cross-linking is via the following formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

52. The SPAC of claim 43, wherein the stapled peptide has SEQ ID NO: 105, wherein X 1 and X 2 , and X 3 and X 4 The cross-linking is via the following formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

53. The SPAC of claim 44, wherein the stapled peptide has SEQ ID NO: 113, wherein X 1 and X 2 , and X 3 and X 4 The cross-linking is via the following formula (alk); and wherein the C-terminus of the stapled peptide is amidated with -NH2.

54. The SPAC of any one of claims 1-53, wherein the antibody or antigen-binding fragment thereof is coupled to the N-terminus of the stapled peptide.

55. The SPAC of any one of claims 1-53, wherein the antibody or antigen-binding fragment thereof is coupled to the C-terminus of the stapled peptide via a lysine residue.

56. The SPAC of any one of claims 1-55, wherein the stapled peptide is coupled to a cysteine ​​residue of the antibody or antigen-binding fragment thereof.

57. The SPAC of any one of claims 1-55, wherein the stapled peptide is conjugated through a lysine residue of the antibody or antigen-binding fragment thereof.

58. The SPAC of any one of claims 1-57, wherein the antibody or antigen-binding fragment thereof is coupled to the stapled peptide via a linker.

59. The conjugate of claim 58, wherein 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.

60. The SPAC of claim 58 or 59, wherein the linker is a cleavable linker.

61. The SPAC of any one of claims 58-60, wherein the linker is pH cleavable or cleavable by a protease, an esterase, or intracellular disulfide reduction.

62. The SPAC of claim 61, wherein the linker is cleavable by a protease.

63. The SPAC of any one of claims 58-62, wherein the linker is a peptide linker.

64. The SPAC of claim 63, wherein the peptide linker comprises -Y A Y B Y C Y D -(SEQ ID NO: 159), wherein Y A is glycine, glutamate, or absent; Y B is valine, phenylalanine, alanine, tyrosine or glycine; Y C is citrulline, arginine, lysine, alanine or glycine; and Y D It's glycine, or it's not there.

65. The SPAC of claim 64, wherein the peptide linker comprises -valine-citrulline-.

66. The SPAC of any one of claims 58-65, wherein the linker has one of the following formulae: (Maleimide-hexanoic acid-valine-citrulline-p-aminobenzyl), (maleimide-hexanoic acid-valine-citrulline-glycine), (iodoacetamide-(PEG3)-valine-citrulline), (iodoacetamide-(PEG3)-valine-citrulline-p-aminobenzyl), (Maleimide-2,3-diaminopropionate-valine-citrulline-p-aminobenzyl), (Maleimido-hexanoic acid-valine-citrulline), (maleimide-hexanoic acid-lysine-valine-citrulline), or (Maleimido-hexanoic acid-(PEG3)-(PEG3)-valine-citrulline); Wherein S represents the connection point with the antibody or antigen-binding fragment thereof; N represents the connection point with the N-terminus of the stapled peptide.

67. The SPAC of any one of claims 58-65, wherein the linker comprises one of the following formulae: Wherein A represents the connection point with the antibody or antigen-binding fragment thereof; N represents the connection point with the N-terminus of the stapled peptide.

68. The SPAC of any one of claims 1-67, wherein the antibody or antigen-binding fragment thereof is conjugated to two or more stapled peptides.

69. The SPAC of any one of claims 1-68, wherein the antibody or antigen-binding fragment thereof is conjugated to 2-10 stapled peptides, inclusive.

70. The SPAC of any one of claims 1-69, comprising an antibody or antigen-binding fragment thereof:stapled peptide ratio of about 1:

8.

71. A pharmaceutical composition comprising the SPAC of any one of claims 1-70 and a pharmaceutically acceptable carrier.

72. A method of treating a proliferative disease in a subject, the method comprising administering to the subject the SPAC of any one of claims 1-70 or a pharmaceutical composition thereof.

73. The method of claim 72, wherein the proliferative disease is cancer.

74. The method of claim 73, wherein the cancer is a HER2-positive cancer.

75. The method of claim 73 or 74, wherein the cancer is colorectal cancer, breast cancer, gastric cancer, ovarian cancer, or esophageal cancer.

76. The method of any one of claims 73-75, wherein the cancer is HER2-positive breast cancer.

77. The method of claim 73, wherein the cancer expresses CD38.

78. The method of claim 73 or 77, wherein the cancer is multiple myeloma, leukemia, or lymphoma.

79. A method of inhibiting tumor growth in a subject, the method comprising administering to the subject a SPAC of any one of claims 1-70 or a pharmaceutical composition thereof.

80. A method of delivering a stapled peptide to a cell, the method comprising contacting the cell with the SPAC of any one of claims 1-70 or a pharmaceutical composition thereof.

81. The method of claim 80, wherein the cell is a cancer cell.

82. The method of claim 80 or 81, wherein the stapled peptide has improved cellular uptake compared to a corresponding unconjugated stapled peptide.

83. A method of triggering cancer cell death, the method comprising contacting the cancer cell with a SPAC or a pharmaceutical composition thereof as described in any one of claims 1-70.

84. The method of claim 83, used to selectively kill cancer cells in the presence of non-cancerous cells.

85. The method of any one of claims 80-84, wherein the cell is in vitro.

86. The method of any one of claims 80-84, wherein the cell is in a subject.

87. The method of any of the preceding claims, wherein the SPAC is administered intravenously.

88. The method of any of the preceding claims, wherein the subject is a human.

89. The SPAC of any one of claims 1-70 for use in the method of any one of the preceding claims.

90. Use of the SPAC of any one of claims 1-70 for the preparation of a medicament.

91. A kit comprising the SPAC or pharmaceutical composition thereof of any one of claims 1-70 and optional instructions for use.

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