Two-step drug delivery system

Abstract

Disclosed herein is a two-component two-stop (TCTS) method of delivering a payload (e.g., a dye or therapeutic agent) to a target in vitro or in the body of a subject. The invention also encompasses components — compounds and compositions — useful in the method, pharmaceutical compositions comprising them, kits comprising them, and methods of their use to treat diseases and disorders.

Description

[0001] TWO-STEP DRUG DELIVERY SYSTEM

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. provisional patent application no. 63 / 665,572, filed June 28, 2024, the entirety of which is incorporated herein by reference.

[0004] STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0005] This invention was made with government support under HT9425-23-1-0358 awarded by the Defense Health Agency, Medical Research and Development Branch, and CA247685 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] 1. BACKGROUND

[0007] Despite recent developments in cancer therapy, cancer is the first or second leading cause of death before the age of 70 years in 112 of 183 countries and ranks third or fourth in a further 23 countries. Sung H. et al. “Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries” CA Cancer J Clin. (2021) 71: 209- 249. New approaches to treating cancer, and solid tumor cancers in particular, are greatly needed.

[0008] One approach has been to develop new small molecule drugs that that can kill cancer cells by affecting previously unaddressed mechanisms of action. Another approach has focused on ways of targeting the delivery of chemotherapeutic agents so that their effect on healthy cells is minimized. A popular embodiment of this approach is the antibody-drug conjugate (ADC), which uses a monoclonal antibody covalently attached to a cytotoxic drug to deliver the drug to cancer cells. See, e.g., Fu Z., et al. “Antibody drug conjugate: the "biological missile" for targeted cancer therapy” Signal Transduct Target Ther. ICIT) 7( 1) :93. ADCs combine both the advantages of highly specific targeting ability and highly potent killing effect to achieve accurate and efficient elimination of cancer cells.

[0009] While ADCs have proven effective — over 14 have been approved in the United States since 2020 — they can be extraordinarily difficult to engineer and manufacture at scale. Challenges include drug-to-antibody ratio variability, conjugation variability, and stability during storage. Additional challenges can include low blood residency time, low penetration capacity to tumor microenvironment, low payload potency, immunogenicity, unusual off-target toxicity, drug resistance, and the lack of stable linkage in blood circulation. Nejadmoghaddam M.R., et al. “Antibody-Drug Conjugates: Possibilities and Challenges” Avicenna J Med Biotechnol. (2019) 1 l(l):3-23. For these reasons, a need remains for new approaches to targeted drug delivery. 2. SUMMARY

[0010] This invention provides a method of selectively delivering a payload (e.g. , a dye or therapeutic agent) to a target, such as a cell or extracellular environment. Particular embodiments of the invention provide methods of treating diseases and conditions (e.g., cancer) and imaging targets (e.g., by delivering a dye to a cancerous cell).

[0011] One embodiment is composition comprising a reactive transformable peptide monomer (TPM) of formula:

[0012] A^L^-B^L^-X or X-L^-B^L^-A1wherein: A1is a hydrophobic moiety; B1is a peptide that forms a p-sheet; each of L1Aand L1Bis independently absent or is a linker; and X is a reactive moiety capable of undergoing a bioorthogonal reaction; which composition forms a micelle in water.

[0013] In particular embodiments, the composition further comprises a targeting TPM of formula:

[0014] A2-L2A-B2-L2B-C or C-L2A-B2-L2B-A2wherein: A2is a hydrophobic moiety; B2is a peptide that forms a p-sheet; each of L2Aand L2Bis independently absent or is a linker; and C is a hydrophilic targeting agent that binds to a receptor, which receptor is expressed within or on the surface of the target.

[0015] Preferred TPM-based compositions of the invention form micelles in water, and are referred to herein as “nanoparticles”.

[0016] Another embodiment of the invention provides a method of delivering a chemical moiety D to a target, comprising: contacting the target with a first component, which first component is a TPM-based composition of the invention (e.g., a nanoparticle); and contacting the target with a second component, which second component is a compound of formula D-L3-Y; wherein: the target is a cell or an extracellular microenvironment; D is a dye or a therapeutic agent; L3is a cleavable linker; Y is a reactive moiety capable of undergoing a bioorthogonal reaction with X; and the second component is contacted with the target at a time T after the first component is contacted with the target. In preferred embodiments, T is at least 1 hour.

[0017] Another embodiment of the invention provides pharmaceutical formulations and kits comprising the compositions (e.g., nanoparticles) disclosed herein.

[0018] Another embodiment of the invention is a method of treating a disease or condition, comprising administering to a patient in need thereof a first component, which first component is a TPM-based composition of the invention (e.g., a nanoparticle); and contacting the target with a second component, which second component is a compound of formula D-L3-Y ; wherein the second component is contacted with the target at a time T after the first component is contacted with the target.

[0019] Another embodiment of the invention is a method of imaging a target in a subject, comprising administering to the subject a first component, which first component is a TPM-based composition of the invention (e.g., a nanoparticle); and contacting the target with a second component, which second component is a compound of formula D-L3-Y ; wherein the second component is contacted with the target at a time T after the first component is contacted with the target.

[0020] 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Certain aspects of the invention may be understood with reference to the accompanying figures.

[0022] FIGS. 1A and IB show the general concept of TCTS drug administration using some specific embodiments described herein. In the first step, a first component is administered to a subject, which forms fibrils at the target site. The second component is then administered to the subject, where it undergoes bioorthogonal click chemistry to provide an intermediate. The active agent is released from the intermediate by cleavage of the linker attaching it to the rest of the molecule.

[0023] FIG. 2 shows a synthetic route to obtain TPM(P22).

[0024] FIG. 3 shows an electrospray ionization mass spectrometry (ESI-MS) spectrum of TMP(P22).

[0025] FIG. 4 shows a synthetic route to obtain TPM(LXY30).

[0026] FIG. 5 shows an ESI-MS of TPM(LXY30).

[0027] FIG. 6 shows a synthetic route to obtain TPM(TCO) I.

[0028] FIG. 7 shows an ESI-MS of TPM(TCO) I.

[0029] FIG. 8 shows an ESI-MS of TPM(TCO) II.

[0030] FIGS. 9A and 9B provide synthetic routes to obtain CBZ-based TPMs. FIG. 9A shows a synthetic route to obtain CBZ-PEG4-mTz; FIG. 9B shows a synthetic route to obtain CBZ-SS-PEG4- mTz.

[0031] FIG. 10 shows an ESI-MS of CBZ-SS-PEG4-mTz.

[0032] FIG. 11 shows a synthetic route to obtain PTX-PEG-mTz.

[0033] FIG. 12 shows an ESI-MS of PTX-PEG-mTz.

[0034] FIG. 13 shows a synthetic route to obtain 0XA-PEG4-mTz.

[0035] FIG. 14 shows an ESI-MS of 0XA-PEG4-mTz.

[0036] FIGS. 15A, 15B, and 15C show synthetic approaches to obtain DMl-PEG4-mTz, Resiqimod- PEG4-mTz, and MMAE-PEG4-mTz, respectively.

[0037] FIG. 16 shows a synthetic route to obtain DOTAGA-d-(AeeA)4-mTz.

[0038] FIG. 17 shows an ESI-MS of DOTAGA-d-(AeeA)4-mTz.

[0039] FIG. 18 shows a synthetic route to obtain D0TAGA-PEG8-mTz.

[0040] FIG. 19 shows an ESI-MS of D0TAGA-PEG8-mTz.

[0041] FIG. 20A shows the particle size and morphology of TNPs I.

[0042] FIG. 20B shows the particle size and morphology of TNPs II.

[0043] FIG. 21 provides data showing the in vitro stability of TNPs in human plasma.

[0044] FIG. 22 shows morphological transformation of TNPs I upon binding to SKOV-3 and A549 cells.

[0045] FIGS. 23A and 23B show morphological transformation of TNPs II upon binding to SKOV-3 and A549 cells under confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. FIG. 24 provides data obtained from the administration of TNPs to mice: Table 1 (complete blood count (CBC) of TNPs) and Table 2 (comprehensive metabolic panel (CMP) of TNPs).

[0046] FIG. 25 provides data obtained from the administration of a second component to mice: Table 3 (Complete blood count of PTX-PEG4-mTz) and Table 4 (comprehensive metabolic panel (CMP) of PTX- PEG4-mTz).

[0047] FIG. 26 shows the body weight changes of mice after treatment with PTX-PEG4-mTz (N=6).

[0048] 4. DETAILED DESCRIPTION

[0049] This invention is directed, in part, to a two-component two-stop (TCTS) method of delivering a payload (e.g., a dye or therapeutic agent) to a target in vitro or in the body of a subject. The invention also encompasses components — compounds and compositions — useful in the method, pharmaceutical compositions comprising them, kits comprising them, and methods of their use to treat diseases and disorders.

[0050] This invention is based, in part, on the discovery that nanoparticles comprising the transformable peptide monomers (TPMs) disclosed herein can provide a means to delivering a compound to a target, which compound is capable of undergoing bioorthogonal coupling with a second compound delivered sometime later. That second compound contains a linker that is cleaved after coupling at the target to release its payload.

[0051] Aspects of this invention build on discoveries reported in the UC Publications and in: Zhang, L., et al. , “Transformable peptide nanoparticles arrest HER2 signaling and cause cancer cell death in vivo” Nature Nanotechnology (2020) 15: 145-153; Zhang, L., et al., “Tumor Receptor-Mediated In Vivo Modulation of the Morphology, Phototherapeutic Properties, and Pharmacokinetics of Smart Nanomaterials” ACS Nano (2021) 15(l):468-479; and Zhang, L., et al., “Programmable Bispecific Nano- immunoengager That Captures T Cells and Reprograms Tumor Microenvironment” Nano Lett. (2022) 22:6866-6876

[0052] 4.1. DEFINITIONS

[0053] Unless otherwise indicated, the term “about” means ± 10% of the indicated range.

[0054] Unless otherwise indicated, the term “combination”, when used to describe the administration of two or more drugs to a patient, means that the two or more drugs are administered in a way such that their effects overlap or that one drug will affect the efficacy or safety of a second drug. The two or more drugs need not be administered at the same time or by the same route of administration.

[0055] The term “hydrophobic moiety” refers to a compound or part of the compound that is substantially insoluble in water.

[0056] The term “include” — like “includes” and “including” — has the same meaning as “include, but are not limited to,” and the term “includes” has the same meaning as “includes, but is not limited to.” Similarly, the term “such as” has the same meaning as the term “such as, but not limited to.” The terms “manage,” “managing” and “management” mean preventing the recurrence of the specified disease or disorder in a patient who has already suffered from the disease or disorder, and / or lengthening the time that a patient who has suffered from the disease or disorder remains in remission. The terms encompass modulating the threshold, development and / or duration of the disease or disorder or changing the way that a patient responds to the disease or disorder.

[0057] When used herein to describe the assembly of compounds of the invention, the terms “nanoparticle” and “micellar nanoparticle” refer to a micelle.

[0058] As used herein, the term “patient” means a subject having a disease or health condition.

[0059] Unless otherwise indicated, the term “physiological conditions” refers to conditions typically found in a live mammalian body, and in particular an aqueous environment with a pH of from about 7 to about 7.5 (e.g., that found in plasma, serum).

[0060] The terms “subject” and “subjects” refer to an animal, such as a non-primate mammal (e.g., cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., monkey, a chimpanzee, human). Preferred subjects are human (e.g., adult humans).

[0061] As used herein, the term “such as” is accorded the same meaning as “include”, “includes”, and “including”.

[0062] A “therapeutically effective amount” of a composition (e.g., a compound) is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or condition, or to delay or minimize one or more symptoms associated with the disease or condition. A “therapeutically effective amount” of a composition means an amount, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of the disease or condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces, or avoids symptoms or causes of a disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0063] The terms “treat,” “treating” and “treatment” contemplate an action that occurs while a patient is suffering from a specified disease or disorder, which reduces the severity of the disease or disorder or retards or slows the progression of the disease or disorder. As used herein, the term includes management of a disease or disorder, which means maintaining the current state, severity, or degree of a disease or condition (e.g., preventing it from growing worse).

[0064] As used herein, the term “UC Publications” means the following publications: International Patent Application Publication WO2024 / 211779 to Yu et al:, U.S. patent no. 10,130,711 to Oneto et al , U.S. patent no. 10,342,882 to Oneto et al:, and U.S. patent publication no. 2022 / 0387633 to Uam and Zhang.

[0065] A wavy line ” that intersects a bond in a chemical structure indicates the point of attachment of the bond that the wavy bond intersects in the chemical structure to the remainder of a molecule.

[0066] Unless otherwise indicated, an adjective before a string of nouns should be construed to apply to each. For example, the phrase “optionally substituted pyridyl, pyrazyl, or furanyl” means the same as “optionally substituted pyridyl, optionally substituted pyrazyl, or optionally substituted furanyl”. The meanings of some abbreviations used herein are provided below:

[0067] Abbreviations

[0068] 4.2. COMPONENTS AND STEPS

[0069] This invention is directed, in part, to a two-component, two-step (TCTS) process for delivering a payload (e.g., a dye or therapeutic agent) to a target, such as a cell or an extracellular microenvironment.

[0070] The extracellular microenvironment includes the extracellular matrix, extracellular vesicles, and proteins found therein (e.g., cytokines and matrix proteins, such as collagen and fibronectin).

[0071] In particular embodiments of the invention, a first composition, or “component”, is contacted with a target (in vitro or by administering the compound to a subject). A second composition, or “component”, is then contacted with the target (in vitro or by administering the compound to a patient) sometime thereafter.

[0072] The first component comprises one or more transformable peptide monomers (TPMs), which are so named because they contain a peptidic moiety. Each TPM is a compound that has a hydrophobic end and a hydrophilic end such that in an aqueous environment (e.g. , plasma), the molecules form a micelle, or nanoparticle. Between the hydrophobic and hydrophilic ends of each TPM is a P-sheet-forming peptide, optionally flanked by linkers. When nanoparticles of the first composition come into contact with the target, they break apart to form fibrils due to the interactions between the target and the TPMs’ P-sheet-forming peptides.

[0073] When the first component is made of just one kind of TPM, its hydrophilic portion comprises a reactive moiety, which is capable of undergoing a bioorthogonal (e.g., a bioorthogonal Diels-Alder) reaction under physiological conditions. However, this invention also contemplates first components comprising a plurality of micelle-forming TPMs, wherein some contain a reactive group at their hydrophilic ends and others contain a targeting agent that binds at or in a target.

[0074] As shown in FIGS. 1A and IB, after the first component has been delivered to the target, a second component is contacted with it (e.g., by administration to a subject). The compound(s) forming the second component contain(s) a reactive group that can react with the reactive group(s) in the TPM(s) of the first component. The reactive group is connected to a payload (e.g., a drug or therapeutic agent) by a cleavable linker. When the second component contacts the first, the reactive groups undergo bioorthogonal coupling to form an intermediate compound to which the payload is attached by a cleavable linker. That linker is then chemically or enzymatically cleaved to release the payload at or in the target.

[0075] Each of the first and second components contains at least one compound having a reactive moiety. Each component may also contain dye or therapeutic moieties, which are discussed below.

[0076] 4.2.1. Reactive Moieties

[0077] The first and second components of the invention each comprise compounds having a reactive moiety (referred to herein as X or Y), which can undergo a biorthogonal reaction with the other reactive moiety when the components come into contact at or within a target.

[0078] Bioorthogonal chemistry is a term that was introduced over 20 years ago to refer to chemical reactions that can proceed under physiological conditions without interfering with biological processes and biomolecules. A variety of different types of bioorthogonal reactions are known, including Staudinger reactions, carbonyl ligation, metal-catalyzed reactions, and strain-promoted azide-alkyne cycloaddition (SPAAC) reactions. See, e.g., Mitry, M.M.A., et al., “In Vivo Applications of Bioorthogonal Reactions: Chemistry and Targeting Mechanisms” Chem. Eur. J., (2023) 29 e202203942. A particular type of bioorthogonal reaction is a Diels-Alder reaction. As used herein, the terms “Diels-Alder reaction” and “Diels-Alder cycloaddition reaction” encompass inverse electron-demand Diels-Alder (iEDDA) reactions between an electron-poor diene and an electron-rich dienophile.

[0079] Reactive moieties suitable for use in this invention are known in the art. See, e.g., Venrooij, K.R., et al., “Mutually Orthogonal Bioorthogonal Reactions: Selective Chemistries for Labeling Multiple Biomolecules Simultaneously” Topics in Current Chemistry (2024) 382:24; Smeenk, M.L.W.J., et al., “Recent developments in bioorthogonal chemistry and the orthogonality within” Current Opinion in Chemical Biology, (2021) 60:79-88. Particular methods of the invention use a tetrazine or a triazine as the diene. A preferred diene is methyltetrazine. Particular methods of the invention use a strained cyclooctene, such as trans- cyclooctene, as the dienophile.

[0080] 4.2.2. Dyes

[0081] Dyes useful in the present invention include those commonly used for staining and biomedical imaging. See, e.g., Johnson, I., Histochemical Journal, 20: 123-140 (1998), and The Molecular Probes® Handbook, 11th Edition, ed. Johnson and Spence, Life Technologies, Carlsbad, Calif., 2010. Particular dyes useful in various embodiments of this invention are described in the UC Publications.

[0082] Dyes useful in this invention include fluorescent dyes, metal chelators and chelated metals (e.g., chelated radioisotopes), and imaging agents (e.g., MRI contrast agents).

[0083] Examples of fluorescent dyes include benzylidene imidazolinone dyes, bis-pyrenes, cyanine dyes, indigo dyes, and triarylmethane dyes. Benzylidene imidazolinone dyes include 3,5-difluoro-4- hydroxybenzylidene imidazolinone (DFHBI). Bis-pyrene dyes include l,4-bis(octyloxy)-2,5- bis(ethynylpyrene)benzene, l,10-bis-(l-pyrene)decane, and bis-pyrene appended bis-triazolylated 1,4- dihydropyridine. Cyanine dyes can be streptocyanines (also known as open chain cyanines), hemicyanines, and closed chain cyanines. Examples include indocyanine green (ICG), Cy3, Cy5, Cy 5.5, and C7.

[0084] Particular dyes useful in embodiments of this invention include Alexa Fluor dyes (e.g., Alexa 680), Cy5, Cy5.5, l,r-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD), and 1,1'- dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR).

[0085] Metal (e.g., radiolabel) chelators that can be include bis-pyridines, porphyrins, 1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), and l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA).

[0086] Porphyrins useful in this invention may be substituted or unsubstituted. Examples include protoporphyrin IX, octaethylporphyrin, tetraphenyl porphyrin, pyropheophorbide-a, pheophorbide, pheophorbide-a, chlorin e6, purpurin, and purpurinimide. A particular porphyrin is pheophorbide-a.

[0087] Examples of imaging agents include quantum dots, paramagnetic agents, and radionuclides. Examples of paramagnetic agents include chelated Fe(III) and Gd(III) particles. Gd(III) chelators include gadobenic acid, gadobutrol, gadodiamide, gadopentetic acid, gadoteric acid, gadoteridol, and gadoversetamide. Examples of radionucleotides include3H,nC,13N,18F,19F,6oCo,64Cu,67Cu,68Ga,83Rb,90Sr,90Yt, "Tc, "mTc,i nIn,1231,1241,1251,1291,137Cs,177Lu,186Re,188Re,211At, Rn, Ra, Th, U, Pu, and241Am. 4.2.3. Therapeutic Agents

[0088] Therapeutic agents that may be used in embodiments of this invention include antibiotics and anti-fungals, anti-inflammatory drugs, chemotherapeutic agents, and immunomodulatory agents known in the art. Particular agents are described in the UC Publications.

[0089] Examples of antibiotics and anti-fungals include amphotericin, daptomycin, echinocandins, fluconazole (FLC), nystatin, posaconazole, and voriconazole.

[0090] Examples of immunomodulatory agents include immunosuppressants such as cyclosporin A, resiquimod, gardiquimod, and imiquimod.

[0091] Particular kinds of chemotherapeutic agents include alkylating agents, antifolates, thymidylate synthase inhibitors, microtubule inhibitors, purine analogs, pyrimidine analogs, radiopharmaceuticals, ribonucleotide reductase inhibitors, DNA-methyltransferase inhibitors, topoisomerase I inhibitors, and topoisomerase II inhibitors.

[0092] Some examples of chemotherapeutic agent include auristatins (e.g., MMAE, MMAF), calicheamicin, carmustine, cyclic AMP, deoxycholic acid or deoxycholate, doxorubicin, doxycycline, etoposide, fluconazole, gardiquimod, imiqumod, irinotecan, ixabepilone, maytansinoids (e.g., DM1, DM4), patupilone, platinum drugs, podophyllotoxin, rapamycin, resiquimod, stimulator of interferon gene (STING) agonists, taxanes, and pharmaceutically acceptable salts, metabolites (e.g., SN-38), and prodrugs thereof. Examples of taxanes include Baccatin III, cabazitaxel, 10-deacetylbaccatin, docetaxel, hongdoushan A, hongdoushan B, hongdoushan C, paclitaxel, and Vinca alkaloids (e.g., vinblastine, vincristine).

[0093] Other chemotherapeutic agents include chelated radioisotopes, or radiometal chelates, such as chelated32P,60Co,90Yt,1311,177Lu,188Re,223Ra, and225Ac.

[0094] 4.2.4. First Component

[0095] The first component used in methods of this invention comprises one or more transformable peptide monomers, which may be characterized as “reactive TPMs” or “targeting TPMs”. (In some instances, the first component is referred to as a “TPM-based component” or “TPM-based composition”.) In preferred embodiments of this invention, the first component comprises at least one reactive TPM and optionally one or more targeting TPMs. Both types can be prepared using synthetic techniques known in the art (See, e.g., the UC Publications) and described herein.

[0096] Reactive TPMs

[0097] Reactive TPMs of the invention contain a reactive moiety that can undergo bioorthogonal coupling. Particular reactive TPMs are compounds of the formula:

[0098] AEL^ BEL^-X or

[0099] X-L^-BUL^-A1 wherein: A1is a hydrophobic moiety; B1is a peptide that forms a p-sheet; each of L1Aand L1Bis independently absent or is a linker; and X is a reactive moiety capable of undergoing a bioorthogonal reaction.

[0100] The hydrophobic moiety A1drives the TPM to form micelles, or nanoparticles, in an aqueous environment at physiological pH (e.g., in plasma). The size and physiological characteristics of the nanoparticles may be adjusted by varying the size and nature of the linkers, L1Aand L1B.

[0101] This invention contemplates the use of bioorthogonal reactions and moieties known in the art. In particular embodiments of the invention, X is capable of undergoing a Diels-Alder coupling reaction.

[0102] Targeting TPMs

[0103] In particular embodiments of this invention, the first component further comprises a targeting TPM. Targeting TPMs contain a ligand that binds to a receptor expressed on or in the target. Particular targeting TPMs are compounds of the formula:

[0104] A2-L2A-B2-L2B-C or

[0105] C-L2A-B2-L2B-A2wherein: A2is a hydrophobic moiety; B2is a peptide that forms a p-sheet; each of L2Aand L2Bis independently absent or is a linker; and C is a hydrophilic targeting agent that binds to a receptor, which receptor is expressed within or on the surface of the target.

[0106] In some embodiments of the invention, A1is the same as A2. In some, B1and B2are the same. In some, L1Ais the same as L2A. In some, L1Bis the same as L2B.

[0107] Some embodiments of the invention comprise a plurality of different targeting TMPs (e.g., TPMs with different C groups).

[0108] Nanoparticles that contain targeting TPMs recognize the target to which targeting agent C binds. When contacted with the target, the TPMs lose their nanoparticle structure and forms fibrils at the targeting site due to interactions (e.g, hydrogen-bonding) between their p-sheet forming peptides (B1, B2) and the target, as shown in FIGs. 1A and IB.

[0109] Hydrophobic Moieties

[0110] Hydrophobic moieties useful in the present invention include suitable hydrophobic moieties known in the art. Hydrophobicity and hydrophilicity are commonly measured by the log P values of the compounds using the octane-water reference system. Values lower than 0 indicate hydrophilicity whereas values higher than 0 indicate hydrophobicity. Hydrophobic moieties useful in the present invention includes moieties with log P values of at least 1. In some embodiments, hydrophobic moieties useful in the present invention have a log P value of at least 1 .5. In some embodiments, hydrophobic moieties useful in the present invention have a log P value of 1.5-15. Examples of hydrophobic moieties include cholesterol, vitamin D and derivatives thereof, vitamin E and derivatives thereof, some dyes, and some therapeutic agents (e.g., small molecule drugs, radiometal chelators). In some embodiments of the invention, A1and / or A2is cholesterol, vitamin D or a derivative thereof, or vitamin E or a derivative thereof. In some embodiments, the hydrophobic moiety is a dye or therapeutic agent. In some embodiments, the hydrophobic moiety is a chemotherapeutic agent, deoxycholate, fluorescent dye, immunomodulatory agent, porphyrin, toll-like receptor agonist, or stimulator of interferon gene (STING) agonist. Examples of dyes include those described herein, provided that they are hydrophobic (a characteristic readily determined from the art or by simple experiment). Examples of therapeutic agents include those described herein, provided that they are hydrophobic.

[0111] Beta-Sheet Peptides

[0112] The P-sheet-forming peptides (B1, B2) contained within the TPMs of this invention form a -sheet under physiological conditions. P-sheet peptide sequences useful in the present invention include those known in the art. See, e.g., Chafekar et al., “Branched KLVFF tetramers strongly potentiate inhibition of beta-amyloid aggregation” Chemhiochem. 2007 Oct. 15; 8(15): 1857-64. In some embodiments, the peptide comprises a peptide sequence from a p-sheet peptide domain of an p-amyloid peptide, green fluorescent protein, an interleukin, or an immunoglobulin. In some embodiments, the peptide comprises a peptide sequence from a p-sheet peptide domain of a p-amyloid peptide. In some embodiments, the P-amyloid peptide is P-amyloid.

[0113] Any suitable P-sheet forming peptide may be used in this invention. In some embodiments, the P-sheet forming peptide is a P-sheet forming peptide sequence 5-50 amino acids in length. In some embodiments, the p-sheet forming peptide is a p-sheet forming peptide sequence 5-40 amino acids in length. In some embodiments, the p-sheet forming peptide is a p-sheet forming peptide sequence 5-30 amino acids in length. In some embodiments, the p-sheet forming peptide is a p-sheet forming peptide sequence 5-25 amino acids in length. In some embodiments, the p-sheet forming peptide is a p-sheet forming peptide sequence 5-20 amino acids in length. In some embodiments, the P-sheet forming peptide is a p-sheet forming peptide sequence 5-15 amino acids in length. In some embodiments, the p-sheet forming peptide is a p-sheet forming peptide sequence about 5-10 amino acids in length.

[0114] In some embodiments of the invention, B1and / or B2comprises an amino acid sequence having at least 40% (e.g., at least 60%, 70%, 80%, 90%) sequence identity with at least one of:

[0115] KLVVF [SEQ. ID No. 1];

[0116] FVVLK [SEQ. ID No. 2], which is the reverse of KLVVF;

[0117] FFVLK [SEQ. ID No. 3] and its reverse;

[0118] KLVFF [SEQ. ID No. 4] and its reverse;

[0119] KLVFF derivatives such as KLVFF AE [SEQ. ID No. 5] and its reverse;

[0120] VQIVYK (Tau amyloid core) [SEQ. ID No. 6] and its reverse;

[0121] DFNKF (calcitonin fragment; forms cross-P) [SEQ. ID No. 7] and its reverse; or

[0122] (XZXZ)p (amphipathic repeats forming fibrillar P-sheets, X = hydrophobic, Z = hydrophilic, where p is 1-10). In some embodiments, B1and / or B2is the hydrogel-forming [3-sheet peptide RADA 16 (AC-(RADA)4-C0NH2) or its reverse.

[0123] In some embodiments, B1and / or B2comprises an amino acid sequence containing one or more modified (e.g., artificial) amino acid residues, which amino acid sequence is a functional equivalent of any of the sequences in SEQ. ID Nos. 1-7 insofar as it forms a p-sheet at the target. Examples of modified amino acid residues include acylated, fluorinated, and methylated derivatives of lysine (K), leucine (L), valine (V), and phenylalanine (F).

[0124] Hydrophilic Targeting Agents

[0125] The hydrophilic portion C of a targeting TPM is a targeting agent, or targeting ligand, that binds to a receptor within or on the surface of a target. Examples of targets include cancerous cells (e.g., solid tumors) and other cells in the tumor microenvironment, such as fibroblasts, immune cells, and endothelial cells. Targets also include extracellular vessels, which can, for example, promote angiogenesis, cancer cell adhesion, migration, and invasion and facilitate communication between cells in the tumor microenvironment. See, e.g., Kalluri, R., McAndrews, K.M., “The role of extracellular vesicles in cancer” Ce / / (2023) 186: 1610-1623.

[0126] Whatever the target, it should contain — or have attached to it — one or more receptors to which a targeting agent binds. In particular embodiments of the invention, the receptor recognized by the targeting agent is overexpressed on or in the target, which overexpression may be associated with a disease or condition. Targeting ligands can be used to target transmembrane receptors such as integrins and epidermal growth factor receptors to delivery compounds, drugs, or components of interest to the cell or extracellular environment. Hydrophilic targeting ligands include peptides.

[0127] Examples of receptors to which the targeting agent may bind include a3pi integrin, avP3 integrin, a4pi integrin, c-MET, epidermal growth factor receptor (EGFR), HER2, and toll-like receptors.

[0128] Examples of a3pi integrin targeting agents include LLY13 [SEQ. ID No. 12] and LXY30 [SEQ. ID No. 13], Examples of a4pi integrin targeting agents include LLP2A [SEQ. ID No. 11]. Examples of avP3 integrin targeting agents include PLZ4 [SEQ. ID No. 14] and the cyclic peptide LXW64. These agents are known in the art. See, e.g., UC Publications. Other integrin targeting agents are peptides or peptidomimetics having at least 80% (e.g., 85%, 90%, 95%) sequence identity with any of SEQ. ID Nos. 11-13 and prodrugs thereof.

[0129] Examples of EGFR targeting agents — also referred to as “EGFR ligands” — include P22 [SEQ. ID No. 8], GE11 [SEQ. ID No. 9], and peptides or peptidomimetics having at least 80% (e.g., 85%, 90%, 95%) sequence identity therewith.

[0130] Examples of HERZ targeting agents — also referred to as “HERZ ligands” — include amphiregulin, anti-HERZ monoclonal antibodies (e.g., trastuzumab and pertuzumab), betacellulin, EGF, epigen, epiregulin, HB-EGF, neuregulin 1, neuregulin 2, neuregulin 3, neuregulin 4, and TGF -alpha. Particular HERZ ligands include HERZPep [SEQ. ID No. 10] and peptides or peptidomimetics having at least 80% (e.g., 85%, 90%, 95%) sequence identity therewith. Examples of compounds that bind to toll-like receptors include CpG oligonucleotides (CpG ODN), which are toll-like receptor agonists comprising cytosine-guanosine dinucleotide motifs. The two nucleotides can be linked by a phosphodiester linker or a modified phosphorothioate linker.

[0131] In particular embodiments of the invention, the targeting agent is an DUPA, an EGFR ligand, folate, a HER2 ligand, an LLP2A prodrug, LLP2A, LXY30, LXW64, a luteinizing hormone releasing hormone (LHRH) peptide, PLZ4 [SEQ. ID No. 14], or a toll-like receptor agonist.

[0132] Linkers

[0133] In the first component, one or both of the moieties L1and L2may be absent. However, linkers are used in many embodiments of the invention to, for example, facilitate recognition of the targeting agent at its receptor, adjust the size and bioavailability of nanoparticles composed of the first component, and facilitate the coupling of its reactive moiety.

[0134] Linkers L1and L2are biocompatible and resistant to cleavage in plasma and include those described in the UC Publications. Examples of suitable linkers include (2-[2- [2- (amino)ethoxy] ethoxy] acetic acid) (AEEA, AeeA), [(8-amino-3,6-dioxa-octyl)succinamic acid (EBES), lysine, cleavage-resistant peptides (i.e., peptides resistant to cleavage in plasma), polyethylene glycol (PEG), and combinations thereof.

[0135] In some embodiments, the linker is a combination of one or more AEEA and optionally one or more lysine (K) residues, such as (AEEA)n(e.g., AEEA-AEEA), and (AEEA)nK(AEEA)m(e.g., AEEA-K(AEEA), AEEA-AEEA-K(AEEA)), wherein n is 1-10 and m is 1-10.

[0136] In some embodiments, the linker is EBES or one of the following structures:

[0137] Other moieties that may be used to provide L1and L2include peptides and peptide-based linkers that are resistant to enzymatic- and environmentally-induced cleavage, and which are preferably not recognized by targets in the body. Examples of cleavage-resistant peptides include (GS)n, GSG, GG, and (EK)nwhere n is 1-10, and combinations thereof.

[0138] Exemplary First Components

[0139] Particular first components of the invention contain at least one reactive TPM and at least one targeting TPM. Examples of some reactive and targeting TPMs are provided below and described in detail elsewhere herein: Exemplary TPMs

[0140] Some embodiments comprise a plurality of different reactive TPMs and / or a plurality of different targeting TPMs. Preferred embodiments comprise a reactive TPM and a plurality of different targeting TPMs. In some embodiments, the first component comprises reactive and targeting TPMs in a (reactive TPM : targeting TPM) molar ratio of about 1: 10, 1:5, 2:5, 2:3, 1: 1, 3:2, 5:2, 5: 1, or 10: 1.

[0141] Nanoparticles

[0142] When not interacting with the target, the first component of the TCTS will form a micelle (also referred to herein as a “nanoparticle”) in an aqueous environment. In this way, the P-sheet forming peptides do form fibrils until they reach the target.

[0143] In some embodiments, the present invention provides a nanoparticle having an interior and an exterior, the nanoparticle comprising a plurality of compounds of the present invention, wherein each compound self-assembles in an aqueous solvent to form the nanoparticle such that a hydrophobic pocket is formed in the interior of the nanoparticle, and a hydrophilic group self-assembles on the exterior of the nanoparticle. In some embodiments, the nanoparticle further comprises a hydrophobic drug or an imaging agent sequestered in the hydrophobic pocket of the nanoparticle.

[0144] The diameter of the nanoparticle of the present invention can be of any size that allows for its delivery to the target. In some embodiments, the nanoparticle has a diameter of 5 to 100 nm. In some embodiments, the nanoparticle has a diameter of 10 to 100 nm. In some embodiments, the nanoparticle has a diameter of 15 to 80 nm. In some embodiments, the nanoparticle has a diameter of 25 to 60 nm. In some embodiments, the nanoparticle has a diameter of about 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, or about 70 nm. In some embodiments, the nanoparticle has a diameter of about 20 nm or about 30 nm. In some embodiments, the nanoparticle has a diameter of about 20 run. In some embodiments, the nanoparticle has a diameter of about 30 nm.

[0145] 4.2.5. Second Component

[0146] The second component, or compound, used in methods of this invention has the formula:

[0147] D-L3-X wherein: D is payload (e.g., a dye or therapeutic agent); L3is a linker; and X is or comprises a reactive moiety. In some embodiments, L3is a cleavable linker, which can be cleaved at or in the target (e.g., by an esterase or by environmental conditions at the target). In others, it is a non-cleavable linker (e.g. , as described above for L1A and 1Band L2A and 2B). Unlike the first component, the second component need not — although it still may — form nanoparticles in an aqueous environment (e.g., when administered intravenously to a subject). Unlike the linkers used in the first component, linker L3may be cleavable so that the dye or therapeutic agent D can be released at or in the target. In some embodiments (e.g., when the payload is a dye or radiometal used for imaging), L3is not cleavable. In others (e.g., when the payload is a therapeutic agent), L3is cleavable, and is cleaved in or at the target. Compounds of the second component can be prepared by methods known in the art.

[0148] Payloads

[0149] The payload D may be a dye or therapeutic agent. In particular embodiments, D is hydrophilic, although that need not be the case. The second component may, but need not, form micelles in an aqueous environment. Examples of dyes include those described herein. Examples of therapeutic agents include those described herein.

[0150] Cleavable Linkers

[0151] In some embodiments of the invention, L3, the linker that connects D to X, is not cleavable. In others, however, it is.

[0152] Cleavable linkers are designed to cleave at the target, thereby releasing X. Examples of cleavable linkers are well known in the art. See, e.g., Sheyi R., et al., “An Assurance for Controlled Delivery of Antibody-Drug Conjugate” Pharmaceutics (2022) 14(2):396; and Wharton T. and Spring DR., “Advances in the Release of Amide -Containing Molecules” Chemistry (2025) 31(16).

[0153] Linkers may be chemically or enzymatically cleavable. Chemically cleavable linkers are sensitive to environmental factors, such as pH and reduction. Enzymatically cleavable linkers include peptide- based linkers that are susceptible to proteolysis, p-glucuronide-based linkers (e.g, p-glucuronidase sensitive linkers), p-galactosidase sensitive linkers, phosphate linkers (e.g., phosphatase sensitive linkers), and carbamates and esters, which are sensitive to esterase cleavage.

[0154] Particular cleavable linkers useful in embodiments of this invention include linkers sensitive to reductive environments (e.g., disulfide bonds), acidic pH (e.g., hydrazones, maleamic acid linkers) and linkers sensitive to esterase cleavage (e.g., esters).

[0155] Examples of some cleavable linkers include: reduction sensitive linkers containing a disulfide, pyridyl disulfide, or dithiobenzyl moiety; pH sensitive linkers containing an acetone hydrazone, benzaldehyde hydrazone, N-(2-hydroxyethyl)maleamic acid, N-(2-aminoethyl)maleamic acid, cis- aconityl, 1,2-diol-based acetal, p-methoxybenzaldehyde acetal, imine / Schiff base, or oxazolidine moiety; esterase sensitive linkers containing a simple ester (e.g., succinate, glutarate ester), p-aminobenzyl ester (PAB ester), acetoxymethyl ester (AM ester), phenyl ester, or benzyl ester moiety; protease sensitive linkers containing a Valine-Citrulline (Val-Cit), Gly-Phe-Leu-Gly, Pro-Leu-Gly-Leu-Ala-Gly, or Ala-Ala-Asn moiety; and other enzyme sensitive linkers containing a p-aminobenzyl glucuronide, galactosyl carbamate, or phosphate ester moiety. Exemplary Second Components

[0156] Some particular second components suitable for use in embodiments of this invention include those listed below.

[0157] 4.2.6. Component Delivery and Effect

[0158] In the TCTS methods of this invention, the first component is administered as a composition of transformable peptide monomers. The composition can be prepared by mixing at least one reactive and one targeting TPM in a vehicle suitable for administration to a subject. The vehicle and route of administration used should ensure that micelles, or nanoparticles, of the first component are formed or maintained in the plasma of the subject. A preferred route of administration is intravenous and suitable vehicles (e.g., aqueous saline solutions), are well known in the art.

[0159] After the first component has had sufficient time to contact the target and form fibrils, the second component is administered. The time T between the first and second administrations will vary depending on a number of factors, including the nature of the components themselves (e.g., the targeting ligand used), the size and bioavailability of the nanoparticles, and the target itself. The time T is preferably of sufficient duration to allow un-targeted nanoparticles — nanoparticles that did not reach the target to form fibrils — to flush from the subject’s body prior to administration of the second component. Specific values for T may be determined by observing fibril formation in vivo (e.g. , in a test animal) or in a subject’s cells (e.g., tumor cells) tested ex vivo using methods described herein and elsewhere. See, e.g., UC Publications. Values of T may also be determined by urinalysis for TPMs themselves or their components.

[0160] In particular embodiments of this invention, the time T between administration of the first and second components to a subject is at least 1 hour (e.g., at least 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 1 week, or 2 weeks). 4.3. METHODS OF USE

[0161] This invention encompasses methods of delivering a payload (e.g. , a dye or therapeutic agent) to a target, such as a cell or an extracellular microenvironment (e.g., extracellular matrix, extracellular vesicles, exosomes, and cytokines surrounding a cell). In particular methods of the invention, the target is ex vivo or within a test subject (e.g. , a mouse, rat, dog, monkey). In others, the target is within the body of a patient (e.g., a human patient).

[0162] One embodiment of the invention is a method of treating a disease or disorder associated with the overexpression of a target, which comprises administering a first component to a patient in need thereof, following by the administration of a second component sometime thereafter. Examples of such diseases and conditions include cancer and chronic inflammation (e.g., some forms of diabetes, cardiovascular diseases, infectious diseases, and neurodegenerative diseases).

[0163] One embodiment is a method of treating cancer, which comprises administering a first component to a patient in need thereof, following by the administration of a second component sometime thereafter. Examples of cancers include solid tumors, such as bladder cancer, brain tumors (e.g., glioblastoma, meningioma), breast cancer, cervical cancer, colorectal cancer, endometrial (uterine) cancer, esophageal cancer, gastric cancers, head and neck cancers, kidney (renal cell) cancer, liver cancer, lung cancer, melanomas, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, testicular cancer, and thyroid cancer. Particular cancers are lung cancer (e.g., non-small cell lung cancer) and ovarian cancer.

[0164] This invention also encompasses a method of imaging a target, which comprise delivering an imaging to the target. Particular methods comprises administering a first component to a subject having the target followed by the administration of a second component sometime thereafter, wherein the imaging agent is provided in the first component (e.g., as A1or A2) or in the second (e.g., as D).

[0165] 4.4. PHARMACEUTICAL FORMULATIONS

[0166] Compounds and compositions disclosed herein may be systemically administered in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable, edible carrier. They may be in the form of a single unit oral dosage form (e.g., enclosed in hard or soft shell gelatin capsules or compressed into tablets), although preferred formulations are suitable for intravenous administration.

[0167] Compositions of this invention are preferably administered intravenously or intraperitoneally by infusion or injection. Solutions of an active compound or its salts may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0168] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid, and stable under manufacture and storage conditions. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0169] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate carrier with various of the other ingredients enumerated above, as required, followed by fdter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-fdtered solutions.

[0170] 4.5. EXAMPLES

[0171] The following examples include descriptions of particular embodiments of the invention. These embodiments focus on the use of the TCTS drug delivery method with an EGFR / a3pi dual targeting fibrillar transformable nanoplatform and bioorthogonal click chemistry for on-demand delivery of therapeutic payloads such as cabazitaxel to tumor sites, sparing healthy tissues. This approach is believed to be particularly useful in the treatment of non-small cell lung cancer and ovarian cancer.

[0172] The nanoparticles are self-assembled from transformable peptide monomers (TPMs). At the N-terminus of the TPMs, trans-cyclooctene (TCO), P22 (EGFR targeting), and LXY30 (a3pi targeting) are linked respectively. Pheophorbide a (Pa), a highly hydrophobic porphyrin based photosensitizer, is conjugated at the C-terminus. Upon self-assembly, the N-terminal hydrophilic moieties are displayed on the surface of the nanoparticle and the hydrophobic moieties reside inside the core. After intravenous (i.v.) administration, the nanoparticles are preferentially taken up by tumor tissues via enhanced permeability and retention (EPR) effects and undergo receptor mediated (EGFR and a3piintegrin) transformation inside the tumor microenvironment (TME) upon interacting with the tumor cell membrane.

[0173] After the administration of TNPs, when the nanofibers are cleared from healthy tissues (around 1 or 2 days), the methyltetrazine drug conjugate (drug = cabazitaxel) is administered intravenously and then undergo in situ click reaction with the TCO displayed on the nanofibrillar network at the tumor sites. After the in situ click reaction, the unreacted drug conjugates are rapidly cleared from the body. As a result, a much higher level of cabazitaxel is delivered to the tumor sites. Cabazitaxel is released continuously at the tumor sites by esterase at the TME, maximizing cytotoxic effects against the tumor cells. This novel dual function nanoplatform EGFR dimerization suppression and targeted cabazitaxel delivery is expected to be highly efficacious against NSCLC. This approach is meticulously designed to maximize therapeutic effectiveness while mitigating the potential for toxicity in normal tissues.

[0174] 4.5.1. Synthesis of TPM(P22)

[0175] This compound has the structure P22-GSG-KLVFFK(Pa). The synthetic route of TPM(P22) (sequence: YHWYGYTPENVIGSGKLVFFK(Pa) is shown in FIG. 2. The linear peptide sequence Boc- YHWYGYTPENVIGSGKLVFFK(Dde) was assembled on of Rink Amide AM resin (loading capacity: 0.68 mmol / g) using the microwave peptide synthesizer. Selective deprotection of the l-(4,4-Dimethyl- 2,6-dioxocyclohex-l-ylidene)ethyl (Dde) on C-terminal lysine residue was performed using 2% hydrazine monohydrate in DMF for 5 min, followed by a second treatment for 10 min. Deprotected resin was then coupled to Pa via the s-amino group of lysine. The resin was treated with 1.5 equiv Pa, 4 equiv Oxyma, and 4 equiv DIC in DCM / DMF (2: 1, v / v) for 12 h under light-protected conditions. The synthesized compound is characterized by ESI-MS (FIG. 3).

[0176] 4.5.2. Synthesis of TPM(LXY30)

[0177] This compound has the structure LXY30-(EK)4-KLVFF-K(Pa). Synthesis of TPM(LXY30). The synthetic route of TPM(LXY30) (sequence: cdG-Phe(3,5-diF)-G-Hyp-NcR-AeeA-EKEKEKEK-AeeA- KLVFFK(Pa)) is shown in FIG. 4. The linear sequence Fmoc-c(Mmt)dG-Phe(3,5-diF)-G-Hyp- Nc(Mmt)R-AeeA-EKEKEKEK-AeeA-KLVFFK(Dde) was assembled on Rink Amide CEM resin (loading capacity: 0.18 mmol / g) using the microwave peptide synthesizer. Fmoc was retained on the peptide following synthesis. After drying the resin, 4 -Methoxytrityl (Mmt) groups were removed using 2% TFA in DCM (15 mb, 2 min, 5 times). Completeness of deprotection was confirmed by a positive result in the Ellman test. Disulfide cyclization was carried out using 2 equiv N-chlorosuccinimide (NCS) in DMF (10 pmol / mL) for 15 minutes. Successful cyclization was verified by a negative result in the Ellman test. N-terminal fluorenylmethyloxycarbonyl (Fmoc) was removed using 20% 4 -methylpiperidine in DMF for 5 min, followed by a second treatment for 15 min. tert-Butoxycarbonyl (Boc) protection was achieved using 10 equiv Boc anhydride and 20 equiv DIEA in DCM for 8 h. Dde deprotection of lysine was performed with hydroxylamine 2.4 M hydrochloride and 1.8 M imidazole in NMP / DMF (2: 1, v / v) for 8 h. Coupling to Pa was done with 1.5 equiv Pa, 4 equiv Oxyma, and 4 equiv DIC in DCM / DMF (2: 1, v / v) for 12 h. The synthesized compound is characterized by ESI-MS (FIG. 5).

[0178] 4.5.3. Synthesis of TPM(TCO) I and TMP(TCO) II

[0179] TPM(TCO) I is a first version of TPM(TCO). The synthetic route of TPM(TCO) I (sequence: sequence: TCO-AeeA-EKEKEKEK-AeeA-KLVFFK(Pa)) is shown in FIG. 6. The linear peptide sequence AeeA-EKEKEKEK-AeeA-KLVFFK(Dde) was assembled on Rink Amide CEM resin (loading capacity: 0.18 mmol / g) with the microwave peptide synthesizer. After deprotection, the resin was treated with 2 equiv trans-cyclooctene-N-hydroxysuccinimide ester (TCO-NHS) and 4 equiv DIEA in DMF for 12 h to couple TCO to the N-terminus. Dde deprotection of lysine was performed using 2.4 M hydroxylamine hydrochloride and 1.8 M imidazole in NMP / DMF (2: 1, v / v) for 8 h. Pa was conjugated using 1.5 equiv Pa, 4 equiv Oxyma, and 4 equiv DIC in DCM / DMF (2: 1, v / v) for 12 h. The synthesized compound is characterized by ESI-MS (FIG. 7).

[0180] TPM(TCO) II is the second version of TPM(TCO). The sequence is sequence: sequence: TCO-G- Nal2-TXA-EKEKEKEK-AeeA-KLVFFK(Pa). This compound is synthesized using analogously to TPM(TCO) I with similar method. The synthesized compound is characterized by ESI-MS (FIG. 8).

[0181] 4.5.4. Synthesis of CBZ-SS-PEG4-mTz

[0182] The synthetic route of CBZ-SS-PEG-mTz is shown in FIG. 9B. 1.86g of 2,2’ -dithiodiacetic acid (DTDA) was suspended in 45 mb of dichloromethane (DCM and 1 mb of DMF, then added 2.106g of DCC. The resulting mixture was stirred at room temperature under argon atmosphere for 6 days. After fdtration, the liquid was added to a solution of 255mg of mTz-PEG4-NH2 HC1 salt and 489 uL of DIEA in 2 mb of DMF and 23 mb of DCM. The resulting solution was stirred overnight and DCM was removed by rotovap. The residue liquid was diluted with 50% acetonitrile (ACN) and H2O, then submitted for HPLC purification. The collected solution was lyophilized to give mTz-SS-COOH as a red powder. To a solution of 107 mg of mTz-SS-COOH, 117 mg of EDC.HC1, 30 mg of DMAP, 106 uL of DIEA in 3 mb DMF, 203 mg CBZ was added. The solution was stirred at room temperature for 2 days. The solution was added to ice water and the solid was collected by filtration, then purified by HPLC to give designed product CBZ-SS-PEG4-mTz. The identity of the compound was confirmed by ESI-MS (FIG. 10).

[0183] 4.5.5. Synthesis of PTX-PEG-mTz

[0184] The synthetic approach is shown in FIG. 11. 435 mg of PTX was added to a solution of 244mg of mTz-PEG4 acid, 174 uL of DIC, 14 mg of DMAP in 7 mb of DCM. The resulting mixture was stirred at room temperature for 2 days. The solution was submitted for silica gel column purification (DCM: MeOH=30: 1), the product contains small portion of impurity, A second run of silica gel column purification (ethyl acetate) was performed to give pure product PTX-PEG-mTz. The identity was confirmed by ESI-MS. (FIG. 12).

[0185] 4.5.6. Synthesis of OXA-PEG4-mTz

[0186] The synthetic scheme is shown in FIG 13. 500mg of Oxaliplatin (OXA) was added to 100 mb of acetic acid, then 4.2 mb of 30% H2O2 was added. The resulting solution was stirred at room temperature for 70 min, then poured into 1 L of cold tert-butyl methyl ether (TBME). The white precipitate was collected by centrifuge and filtration, washed with TBME and dried to give OXA(OAcOH). 250mg OXA(OAcOH) was added to a solution of 230mg mTz-PEG4-COOH, 122mg of EDC.HC1, 85 mg DMAP in 4 mb DMF. The resulting mixture was stirred at room temperature for 1 day. The solution was diluted with 50% acetonitrile (ACN) and H2O, then submitted for HPLC purification. The collected solution was lyophilized to give 0XA-PEG4-mTz as a powder. The identity was confirmed by ESI-MS (FIG. 14).

[0187] 4.5.7. Synthesis of CBZ-PEG4-mTz, DMl-PEG4-mTz, Resiquimod-PEG4-mTz, and MMAE-PEG4-mTz

[0188] Synthetic methods for the preparation of the captioned compounds are shown in FIGS. 15A-15C, respectively. A carboxyl group is first introduced to the drug through an ester bond, which is then used to couple to the amino group of methyl -tetrazine (mTz)-PEG-amine in the presence of HCTU / DIEA. mTzAeeA-DOTA is synthesized using standard coupling chemistry and product identity is confirmed by ESI-MS.

[0189] 4.5.8. Synthesis of DOTAGA-d-(AeeA)4-mTz

[0190] The synthetic approach is shown in FIG. 16. First, intermediate DOTAGA-d-(AeeA)4-k was synthesized by CEM microwave peptide synthesizer on solid phase, followed by TFA cleavage and HPLC purification. 1 lOmg of mTz-NHS was added to a solution of 432 mg of DOTAGA-d-(AeeA)4-k and 373 uL of DIEA in 4 mL of DMF. The resulting mixture was stirred at room temperature for 4 h. The liquid was added to cold TBME to precipitate, followed by HPLC purification to give DOTAGA-d- (AeeA)4-mTz. The identity was confirmed by ESI-MS (FIG. 17).

[0191] 4.5.9. Synthesis of DOTAGA-PEG8-m Tz

[0192] The synthetic approach is shown in FIG. 18. 340 mg of B0C-PEG8-NH2 was added to a solution of 516mg of DOTA-GA(tBu)4, 156mg of Oxyma and 228 uL of DIC. The resulting mixture was stirred at room temperature overnight. The liquid was added to cold TBME to precipitate. The solid was treated with a mixture of 95% TFA, 2.5% TiS and 2.5% H2O overnight. After TFA was removed, cold TBME was added to give precipitate which was reacted with mTz-NHS in presence of 8 eq. of DIEA to give DOTAGA-PEG8-mTz. The identity was confirmed by ESI-MS (FIG. 19).

[0193] 4.5.10. Preparation of Hybrid TNPs

[0194] In this example, two versions of transformable peptide monomers (TPMs) were prepared. TNPs I were composed of three transformable peptide monomers: TPM(P22), TPM(LXY30), and TPM(TCO) I. The optimized molar ratio of these three TPMs was TPM(P22) / TPM(LXY30) / TPM(TCO) I = 28 / 42 / 30. TCNPs II were composed of two TPMs: TPM(LXY30) and TPM(TCO) II, with an optimized molar ratio of 50 / 50.

[0195] To prepare TNPs, a dimethyl sulfoxide (DMSO) solution containing compositional peptides at a total concentration of 20-100 mM was premixed. An aliquot of the premixed DMSO solution was transferred to the bottom of an Eppendorf tube or centrifuge tube. A 100-fold volume of 5% dextrose in water (D5W) was added to the DMSO solution, followed immediately by pipette flushing and vortexing for 30 seconds. The formulation was then allowed to stand for 1-3 hours at room temperature to facilitate peptide self-assembly and spontaneous homogenization. The resulting nanoparticle formulation was further diluted with D5W to the desired concentration for subsequent studies.

[0196] 4.5.11. Particle Characterization

[0197] Size Measurement of Nanoparticles

[0198] An aliquot (200 pL) of the nanoparticle suspension was transferred into a cuvette, and the hydrodynamic diameter of the nanoparticles was measured at room temperature using dynamic light scattering (DLS) with a Zetasizer ZS90 (Malvern Instruments Ltd., Malvern, Worcestershire, UK). The size values were analyzed based on the number mean of hydrodynamic diameters. Each sample was measured three times to calculate the size deviation.

[0199] Characterization of Nanoparticle Morphology

[0200] The morphology of the nanoparticles was examined using a transmission electron microscope (TEM) (FEI Talos 120C, Thermo Fisher Scientific, Hillsboro, Oregon). A 400-mesh carbon-coated copper TEM grid was used for sample preparation. An aliquot (40 pL) of the diluted nanoparticle suspension (20 pM total peptide) was applied to the grid, allowing it to adsorb for 30 minutes. Excess liquid was then blotted with filter paper to form a thin film, which was dried at room temperature for 5-10 minutes. The sample film was subsequently stained with 40 pL of 2% (w / v) uranyl acetate for 15 minutes, followed by blotting with filter paper. After air-drying at room temperature, the grid was transferred to the TEM for imaging.

[0201] The particle size and morphology of TNPs I and TNPs II are shown in FIGS. 20A and 20B, respectively.

[0202] Stability of TNPs in the presence of human plasma and protease

[0203] As can be seen in FIG. 21, TNPs II were incubated with 80% human plasma from a healthy donor at 37°C for 3 days. The peptide concentrations were determined by integration of the TPM(LXY30) peak (area) from reversed-phase high-performance liquid chromatography (RP-HPLC) chromatograms at UV 413 nm . The half-life of LXY 30 in the plasma was calculated using the formula 11 / 2= 1 / kobs • ln(2) . Assays were performed in triplicate.

[0204] 4.5.12. Confocal laser scanning microscopy (CLSM) and scanning electron microscope (SEM) validation of TNPs structural transformation on cell surfaces

[0205] EGFR+a3pi+cancer cells (human ovarian cancer SKOV-3 cells and human lung cancer A549 cells) were treated with TNPs (50 pM) for 24 h, and their interactions were examined using CLSM. CLSM imaging revealed a stepwise morphological transformation of TNPs I upon binding to SKOV-3 and A549 cells, initially forming short rods and subsequently elongating into nanofibers (FIG. 22). A similar transformation was observed from TNPs II on A549 cells and Mouse Lewis Lung Carcinoma cells (50 pM) (FIG. 23A). The SEM image of LLC cells treated with TNPs II (FIG. 23B) clearly show the formation of filament structures on the cells’ surface.

[0206] 4.5.13. Determination of Toxicity and Immunogenicity

[0207] Acute toxicity of TNPs II was investigated. Three male and three female FVB mice were intravenously injected with TNPs at three doses (10, 20, and 40 mg / kg). After 24h of injection, blood was collected and submitted for Complete Blood Count (CBC) and Comprehensive metabolic panel (CMP) studies. FIG. 24 provides data obtained from these experiments. In particular, Table 1 provides the complete blood count (CBC) of TNPs and Table 2 provides a comprehensive metabolic panel (CMP) of TNPs.

[0208] Conclusion: TNPs with all three tested doses did not show acute toxicity after one dose. All mice were alive and did not lose weight. All the indexes from CBC and CMP test were in the normal range of FVB, or close to the indexes of control group (non-treated FVB mice).

[0209] To study the sub-chronic toxicity of the PTX-PEG4-mTz and determine the maximum tolerated dose (MTD), three male and three female FVB mice were intravenously injected with PTX-PEG4-mTz at incremental dose (20, 40, and 60 mg / kg) once a week for 4 weeks. The weight and survival of the mice were monitored during the four weeks of treatment. After the last injection, the mice were euthanized, and their complete blood and plasma samples were collected to run the Complete Blood Count (CBC) and Comprehensive metabolic panel (CMP) tests (FIG. 25). The effect of the compound on the body weights of the mice is shown in FIG. 26.

[0210] These results indicate that the MTD of PTX-PEG4-mTz is >60 mg / kg. The mice treated with the dose 20, 40, and 60 mg / kg had 100% survival and did not lose significant weight. All the indexes from CBC test were in the normal range of FVB, or close to the index of control group (non-treated FVB mice). For CMP test, the glucose level of mice treated by 60 mg / kg PTX-PEG4-mTz was higher than the normal range.

[0211] 4.6. TABLE OF SEQUENCES

[0212] Sequences of certain peptides and peptidomimetics described herein are provided in the table below: All publications (e.g., patents and patent applications) cited above are incorporated herein by reference in their entireties.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a reactive transformable peptide monomer (TPM) of formula:A^L^-B^L^-X orX-L^-B^L^-A1wherein:A1is a hydrophobic moiety;B1is a peptide that forms a p-sheet; each of L1Aand L1Bis independently absent or is a linker; andX is a reactive moiety capable of undergoing a bioorthogonal reaction; which composition forms a micelle in water.

2. The composition of claim 1, which further comprises a targeting TPM of formula:A2-L2A-B2-L2B-C orC-L2A-B2-L2B-A2wherein:A2is a hydrophobic moiety;B2is a peptide that forms a p-sheet; each of L2Aand L2Bis independently absent or is a linker; andC is a hydrophilic targeting agent that binds to a receptor, which receptor is expressed within or on the surface of the target.

3. The composition of claim 1, wherein A1is a dye or a therapeutic agent.

4. The composition of claim 2, wherein A2is a dye or a therapeutic agent.

5. The composition of claim 3 or 4, wherein the dye is a chelated metal, fluorescent dye, imaging agent, or metal chelator.

6. The composition of claim 5, wherein the dye is a porphyrin (e.g., protoporphyrin IX, octaethylporphyrin, tetraphenyl porphyrin, pyropheophorbide-a, pheophorbide, pheophorbide-a, chlorin e6, purpurin, purpurinimide).

7. The composition of claim 4, wherein the therapeutic agent is an antibiotic or anti-fungal, chemotherapeutic agent, immunomodulatory agent, or anti-inflammatory drug.

8. The composition of claim 7, wherein the chemotherapeutic agent is an alkylating agent, antifolate, thymidylate synthase inhibitor, microtubule inhibitor, purine analog, pyrimidine analog,radiopharmaceutical, ribonucleotide reductase inhibitor, DNA-methyltransferase inhibitor, topoisomerase I inhibitor, or topoisomerase II inhibitor.

9. The method of claim 7, wherein the chemotherapeutic agent is auristatin (e.g., MMAE, MMAF), calicheamicin, carmustine, cyclic AMP, deoxycholic acid or deoxycholate, doxorubicin, doxycycline, etoposide, fluconazole, gardiquimod, imiqumod, irinotecan, ixabepilone, maytansinoid (e.g., DM1, DM4), patupilone, platinum drugs, podophyllotoxin, rapamycin, resiquimod, stimulator of interferon gene (STING) agonist, taxane, or a pharmaceutically acceptable salt, metabolite, or prodrug thereof.

10. The composition of claim 2, wherein A2is the same as A1.

11. The composition of claim 1, wherein B1is a peptide that forms a p-sheet, which peptide is a P-sheet peptide domain of an p-amyloid peptide, green fluorescent protein, an interleukin, or an immunoglobulin, or a derivative thereof.

12. The composition of claim 2, wherein B2is a peptide that forms a P-sheet, which is a P- sheet peptide domain of an p-amyloid peptide, green fluorescent protein, an interleukin, or an immunoglobulin .

13. The composition of claim 11 or 12, wherein the peptide comprises an amino acid sequence having at least 60% (e.g., at least 80%) sequence identity with any of SEQ. ID Nos. 1-4.

14. The composition of claim 13, wherein the peptide is of SEQ. ID Nos. 1 or 2.

15. The composition of any of claims 2-14, wherein B1and B2are the same.

16. The composition of any of the previous claims, wherein L1Ais absent.

17. The composition of any of the previous claims, wherein L1Bis absent.

18. The composition of any of the previous claims, wherein at least one of L1Aand L1Bis (2-[2-[2- (amino)ethoxy] ethoxy] acetic acid) (AEEA, AeeA), [(8-amino-3,6-dioxa-octyl)succinamic acid (EBES), lysine, a cleavage-resistant peptide (i.e., peptides resistant to cleavage in plasma), polyethylene glycol (PEG), or a combination thereof.

19. The composition of claim 18, wherein the cleavage-resistant peptide is (GS)n, GSG, GG, and (EK)nwhere n is 1-10, and combinations thereof.

20. The composition of any of claims 2-19, wherein L2Ais absent.

21. The composition of any of claims 2-20, wherein L2Bis absent.

22. The composition of any of claims 2-21, wherein at least one of L2Aand L2Bis (2-[2-[2-(amino)ethoxy] ethoxy] acetic acid) (AEEA, AeeA), [(8-amino-3,6-dioxa-octyl)succinamic acid (EBES),lysine, a cleavage-resistant peptide (i.e., peptides resistant to cleavage in plasma), polyethylene glycol (PEG), or a combination thereof.

23. The composition of claim 22, wherein the cleavage-resistant peptide is (GS)n, GSG, GG, and (EK)nwhere n is 1-10, and combinations thereof.

24. The composition of any of claims 2-23, wherein C binds to a3pi integrin, avP3 integrin, a4pi integrin, c-MET, epidermal growth factor receptor (EGFR), HER2, or a toll-like receptor.

25. The composition of claim 24, wherein C is an a3pi integrin targeting agent (e.g., LLY13 [SEQ. ID No. 12], LXY30 [SEQ. ID No. 13]), an a4pi integrin targeting agent (e.g., LLP2A [SEQ. ID No. 11]), an avP3 integrin targeting agent (e.g., the cyclic peptide LXW64), and peptides or peptidomimetics having at least 80% (e.g., 85%, 90%, 95%) sequence identity therewith, and prodrugs thereof.

26. The composition of claim 24, wherein C is an EGFR targeting agent, such as P22 [SEQ. ID No. 5], GE11 [SEQ. ID No. 6], peptides having at least 80% (e.g., 85%, 90%, 95%) sequence identity therewith, and prodrugs thereof.

27. The composition of claim 24, wherein C is a HER2 targeting agent, such as HER2Pep [SEQ. ID No. 7], peptides having at least 80% (e.g, 85%, 90%, 95%) sequence identity therewith, and prodrugs thereof.

28. The composition of claim 24, wherein C is DUPA, folate, a luteinizing hormone releasing hormone (LHRH) peptide, or a toll-like receptor agonist.

29. The composition of any of the previous claims, wherein the bioorthogonal reaction is a Diels-Alder reaction.

30. The composition of claim 29, wherein X is a dienophile.

31. The composition of claim 30, wherein X is an acylazetine, a cyclopropane, a norbomene, or a trans -cyclooctene (e.g., trans -cyclooctene).

32. The composition of any of claims 29, wherein X is a diene.

33. The composition of claim 32, wherein X is a tetrazine (e.g. , methyltetrazine) or a triazine.

34. The composition of any of the previous claims, wherein the micelle has a diameter of from about 5 nm and 100 nm (e.g., from about 15 to 80 or 25 to 60 nm).

35. The composition of any of the previous claims, wherein the reactive TPM is: TCO-Aee A-EKEKEKEK-Aee A-KLVFFK(Pa) ; or TCO-G-Nal2-TXA-EKEKEKEK-AeeA-KLVFFK(Pa).

36. The composition of any claims 2-35, wherein the targeting TPM is:P22-GSG-KLVFFK(Pa); orLXY30-(EK)4-KLVFF-K(Pa).

37. The composition of any claims 2-36, which composition comprises at least one reactive TPM and at least one targeting TPM in a molar ratio of about 1 : 10 to about 10: 1 (reactive TPM(s) : targeting TPM(s)).

38. A pharmaceutical composition comprising a composition of any of claims 1-37 and a pharmaceutically acceptable excipient.

39. The pharmaceutical composition of claim 38, which is provided as a solid that can be reconstituted in a vehicle suitable for intravenous or intraperitoneal administration to a subject.

40. The pharmaceutical composition of claim 39, which comprises nanoparticles suspended in a vehicle suitable for intravenous or intraperitoneal administration to a subject.

41. A kit comprising a pharmaceutical composition of any of claims 38-40.

42. The kit of claim 41, which further comprises a second pharmaceutical composition, which second composition is a compound of formula D-L3-Y, wherein:D is a dye or a therapeutic agent (e.g., a radiometal chelate);L3is a linker (e.g., a cleavable linker); andY is a reactive moiety capable of undergoing a bioorthogonal reaction.

43. A method of delivering a chemical moiety D to a target, comprising: contacting the target with a first component, which first component is a composition of any of claims 2-37; and contacting the target with a second component, which second component is a compound of formula D-L3- Y ; wherein: the target is a cell or an extracellular microenvironment;D is a dye or a therapeutic agent;L3is a linker (e.g., a cleavable linker);Y is a reactive moiety capable of undergoing a bioorthogonal reaction with X; and the second component is contacted with the target at a time T after the first component is contacted with the target, wherein T is at least 1 hour (e.g., at least 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 1 week, or 2 weeks).

44. The method of claim 43, wherein D is a dye.

45. The method of claim 44, wherein the dye is a chelated metal, fluorescent dye, imaging agent, or metal chelator.

46. The method of claim 44, wherein the dye is a porphyrin (e.g. , protoporphyrin IX, octaethylporphyrin, tetraphenyl porphyrin, pyropheophorbide-a, pheophorbide, pheophorbide-a, chlorin e6, purpurin, purpurinimide).

47. The method of claim 44, wherein D is a therapeutic agent.

48. The method of claim 47, wherein the therapeutic agent is an antibiotic or anti-fungal, chemotherapeutic agent, immunomodulatory agent, or anti-inflammatory drug.

49. The method of claim 48, wherein the chemotherapeutic agent is an alkylating agent, antifolate, thymidylate synthase inhibitor, microtubule inhibitor, purine analog, pyrimidine analog, radiopharmaceutical, ribonucleotide reductase inhibitor, DNA-methyltransferase inhibitor, topoisomerase I inhibitor, or topoisomerase II inhibitor.

50. The method of claim 48, wherein the chemotherapeutic agent is auristatin (e.g., MMAE, MMAF), calicheamicin, carmustine, cyclic AMP, deoxycholic acid or deoxycholate, doxorubicin, doxycycline, etoposide, fluconazole, gardiquimod, imiqumod, irinotecan, ixabepilone, maytansinoid (e.g., DM1, DM4), patupilone, platinum drugs, podophyllotoxin, rapamycin, resiquimod, STING agonist, taxane, or a pharmaceutically acceptable salt, metabolite, or prodrug thereof.

51. The method of claim 47, wherein D is a radiometal chelate.

52. The method of any of claims 43-51, wherein L3is a chemically cleavable linker.

53. The method of claim 52, wherein L3is a reduction-sensitive linker (e.g., comprising a disulfide, pyridyl disulfide, or dithiobenzyl moiety) or a pH-sensitive linker (e.g., comprising an acetone hydrazone, benzaldehyde hydrazone, N-(2-hydroxyethyl)maleamic acid, N-(2-aminoethyl)maleamic acid, cis-aconityl, 1,2-diol-based acetal, p-methoxybenzaldehyde acetal, imine / Schiff base, or oxazolidine moiety).

54. The method of any of claims 43-51, wherein L3is an enzymatically cleavable linker.

55. The method of claim 54, wherein L3is an esterase-sensitive linker (e.g., comprising a succinate, glutarate, p-aminobenzyl ester, acetoxymethyl ester, phenyl ester, or benzyl ester moiety), a protease -sensitive linker (e.g., comprising a Valine-Citrulline (Val-Cit), Gly-Phe-Leu-Gly, Pro-Leu- Gly-Leu-Ala-Gly, or Ala-Ala-Asn moiety), or a p-aminobenzyl glucuronide, galactosyl carbamate, or phosphate ester moiety.

56. The method of any of claims 43-55, wherein the bioorthogonal reaction is a Diels-Alder reaction.

57. The method of claim 56, wherein Y is a dienophile.

58. The method of claim 57, wherein Y is an acylazetine, a cyclopropane, a norbomene, or a / raw.s'-cyclooctcnc (e.g., / ram-cyclooctcnc).

59. The method of any of claim 56, wherein Y is a diene.

60. The method of claim 59, wherein Y is a tetrazine (e.g., methyltetrazine) or a triazine.

61. The method of any of claims 43-58, wherein second component is:CBZ-SS-PEG4-mTz;CB-PEG-mTz;OXA-PEG4-mTz;CTX-PEG-mTz;DMl-PEG-mTz;Resiquimod-PEG-mTz;DOTAGA-d-(AeeA)4-mTz; orDOTAGA-PEG8-mTz.

62. A method of treating a disease or condition in a patient, comprising: administering to the patient a therapeutically effective amount of a first component; and administering to the patient a therapeutically effective amount of a second component; such that the second component is administered at a time T after the first component is administered, wherein T is at least 1 hour (e.g., at least 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 1 week, or 2 weeks); wherein: the first component comprises: a reactive transformable peptide monomer (TPM) of formula A^L^-B^L^-X or X-L^-BkL^-A1; and a targeting TMP of formula A2-L2A-B2-L2B-C or C-L2A-B2-L2B-A2; the second component comprises a compound of formula D-L3-Y ;A1is a hydrophobic moiety;A2is a hydrophobic moiety;B1is a peptide that forms a p-sheet;B2is a peptide that forms a p-sheet; each of L1Aand L1Bis independently absent or is a linker; each of L2Aand L2Bis independently absent or is a linker; andL3is a linker (e.g., a cleavable linker);D is a therapeutic agent;X is a reactive moiety capable of undergoing a bioorthogonal reaction with Y ; andY is a reactive moiety capable of undergoing a bioorthogonal reaction with X.

63. The method of claim 62, wherein the disease or condition is cancer.

64. The method of claim 63, wherein the cancer is bladder cancer, brain tumor (e.g., glioblastoma, meningioma), breast cancer, cervical cancer, colorectal cancer, endometrial (uterine) cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney (renal cell) cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, testicular cancer, or thyroid cancer.

65. The method of claim 64, wherein the cancer is non-small cell lung cancer or ovarian cancer.

66. A method of imaging a target in a subject, comprising: administering to the subject an amount of a first component; and administering to the subject an amount of a second component; such that the second component is administered at a time T after the first component is administered, wherein T is at least 1 hour (e.g., at least 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 1 week, or 2 weeks); wherein: the first component comprises: a reactive transformable peptide monomer (TPM) of formula A^L^-B^L^-X or X-L^-B^L^-A1; and a targeting TMP of formula A2-L2A-B2-L2B-C or C-L2A-B2-L2B-A2; the second component comprises a compound of formula D-L3-Y ;A1is a hydrophobic moiety;A2is a hydrophobic moiety;B1is a peptide that forms a p-sheet;B2is a peptide that forms a p-sheet; each of L1Aand L1Bis independently absent or is a linker; each of L2Aand L2Bis independently absent or is a linker; andL3is a linker (e.g., a cleavable linker);D is a dye;X is a reactive moiety capable of undergoing a bioorthogonal reaction with Y ; andY is a reactive moiety capable of undergoing a bioorthogonal reaction with X.

67. The method of claim 66, wherein L3is not cleavable.

68. The method of claim 67, wherein is L3is AEEA, EBES, lysine, a cleavage-resistant peptide, PEG, or a combination thereof.

69. The method of any of claims 66-68, wherein the dye is an imaging agent.

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