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WO2024238956A1 - Compositions d'agents de liaison de promédicament et procédés d'utilisation - Google Patents

Compositions d'agents de liaison de promédicament et procédés d'utilisation Download PDF

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WO2024238956A1
WO2024238956A1 PCT/US2024/030018 US2024030018W WO2024238956A1 WO 2024238956 A1 WO2024238956 A1 WO 2024238956A1 US 2024030018 W US2024030018 W US 2024030018W WO 2024238956 A1 WO2024238956 A1 WO 2024238956A1
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cancer
compound
seq
phage
cell
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Jonathan H. BOYCE
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Ohio State Innovation Foundation
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/547—Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B20/00—Methods specially adapted for identifying library members
    • C40B20/04—Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present disclosure relates protease-cleavable linkers, compositions, and methods of use thereof for treating and/or preventing diseases, including, but not limited to cancer and bacterial infections. Also disclosed herein are methods of making and screening for protease- cleavable linkers.
  • BACKGROUND Patients with aggressive types of cancers such as, for example, pancreatic cancer, metastatic triple negative breast cancer, and metastatic triple negative breast cancer tend to have limited options to ensure long-term survival. The identification, diagnosis, and treatment for these and other types of cancers is critical. When surgery is not beneficial, such as in advanced pancreatic cancer, the only option is often traditional chemotherapy, and the side effects reduce patient quality of life.
  • the present invention also provides methods of purifying compositions comprising the protease- cleavable linker.
  • the present invention also provides compounds and/or compositions comprising a protease-cleavable linker.
  • a method of screening for a protease-cleavable linker comprising: a) a negative selection process comprising: i) incubating a substrate display library comprising one or more peptide sequences in a first complex biological matrix, ii) generating a first cleaved phage-containing peptide sequence and an uncleaved phage- containing peptide sequence, and iii) separating the uncleaved phage-containing peptide sequence from the first cleaved phage-containing peptide sequence; b) a positive selection process comprising: i) contacting the uncleaved phage-containing peptide sequence with a second complex biological matrix, and ii) producing a second
  • the first complex biological matrix comprises healthy blood serum or plasma, healthy organ tissue homogenates, media comprising healthy organ tissue homogenates, healthy organoid homogenates derived from healthy cells, conditioned media comprising healthy organoids derived from healthy cells, healthy cell lines, conditioned media comprised of healthy cell lines, or combinations thereof.
  • the second complex biological matrix comprises cancer tissue homogenates, media comprising cancer tissue homogenates, cancer organoid homogenates, conditioned media comprising cancer organoid homogenates, malignant ascites, malignant cystic fluid, cancer cell lines, conditioned media comprising cancer cell lines, or combinations thereof.
  • the second complex biological matrix comprises periplasmic extracts isolated from Gram-negative bacteria, bacteria lysates from Gram-positive or Gram- negative bacteria, or combinations thereof.
  • the negative selection process comprises a control cell or a microenvironment comprising the control cell.
  • the positive selection process comprises a cell of interest or a microenvironment comprising the cell of interest.
  • the cell of interest comprises a Gram-negative bacterium or a Gram-positive bacterium.
  • the gram-negative bacterium comprises A. nosocomialis, P. aeruginosa, E. cloacae, E. coli, A. baumannii, E. aerogenes, K. pneumonia, S. typhi, H. influenzae, L. pneumophilia, V. cholerae, Y. enterocolitica, B. pertussis, B. melitensis, H. pylori, S. dysenteriae, C. jejuni, M. catarrhalis, B. cepacia, or S. enterica.
  • the gram-positive bacterium comprises S. aureus, S. epidermidis, S. pneumoniae, S. pyogenes, C. difficile, C. perfringens, or L. monocytogenes.
  • the cell of interest comprises a cancer cell.
  • the cancer cell comprises hepatocellular carcinoma, cholangiocarcinoma, combined hepatocholangiocarcinoma, ovarian carcinoma, uterine carcinoma, triple negative breast cancer, glioblastoma, or pancreatic carcinoma.
  • the cell of interest is either the same or different in the at least two iterations of steps a) through e).
  • the first and second nucleic acid sequences encode the one or more protease-cleavable linkers.
  • the first and second nucleic acid Docket No.103361-473WO1 sequence is identified using Next Generation Sequencing (NGS).
  • NGS Next Generation Sequencing
  • the method further comprises identifying cleavage kinetics in the presence of the first complex biological matrix and the second complex biological matrix.
  • a compound comprising a targeting moiety, a therapeutic agent, and a cleavable linker, wherein the cleavable linker comprises a peptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a chiral variant thereof.
  • the cleavable linker is directly or indirectly linked to the targeting moiety and the therapeutic agent.
  • the targeting moiety comprises a siderophore.
  • the targeting moiety comprises an HMM siderophore, an enterobactin, an aerobactin, a yersiniabactin, or derivatives thereof.
  • the HMM siderophore comprises Compound I .
  • the enterobactin comprises Compound II .
  • the aerobactin comprises Compound III .
  • the versiniabactin IV .
  • the therapeutic agent comprises an antibiotic.
  • the antibiotic targets a cytoplasmic protein, a periplasmic protein, or a bacterial membrane protein.
  • the antibiotic comprises an amine- or aniline- containing antibiotic.
  • the antibiotic comprises a macrolide antibiotic, oxazolidinone antibiotic, cyclic lipopeptide antibiotic, or fluoroquinolone antibiotic.
  • the antibiotic comprises solithromycin, daptomycin, or eperezolid.
  • the therapeutic agent comprises an anti-cancer chemotherapeutic agent.
  • the anti-cancer chemotherapeutic agent comprises an apoptosis agent, an anti-mitotic agent, a kinase inhibitor, an antimetabolite, an alkylating agent, paclitaxel, docetaxel, a bifunctional proteolysis-targeting chimera, or a combination thereof.
  • the anti-cancer chemotherapeutic agent comprises doxorubicin, mitomycin, or a combination thereof.
  • FIG.1 shows the biopanning campaign workflow to optimize linkers that cleave selectively in each PLC subtype for protease-activated diagnostic development. Negative selections incorporate desired stability. Positive selections incorporate desired sensitivity.
  • FIG. 2 shows the validation of FRET diagnostic for each PLC subtype following sequence scoring from the phage platform.
  • FIG.3 shows the siderophore-chemotherapeutic conjugates (SCCs) and mechanism of proteolytic release inside Gram-negative bacteria. (1) An intact SCC, with a serum-stable peptide linker reaches the site of infection and (2,3) is actively transported to the periplasm via outer membrane proteins.
  • SCCs siderophore-chemotherapeutic conjugates
  • FIG.4 shows the workflow for discovering serum-stable linkers that cleave in periplasmic extract.
  • Step 1. Substrate phage display with negative and positive selections.
  • Step 2. NGS, data analysis, and candidate scoring.
  • Step 3. FRET pair and ACC conjugate synthesis and comparison of cleavage kinetics.
  • FIG.5 shows the peptide candidates with improved cleavage rates in periplasmic extract relative to serum.25 out of 28 peptides synthesized showed improved cleavage rates in periplasmic extract relative to serum, which validated the selection criteria.
  • AB-5075, AB- WC487, and AB-Nav18 are A. baumannii periplasmic extracts.
  • FIGS.6A, 6B, and 6C show cleavage rates and comparisons of cleavage linkers.
  • FIG.6A shows the cleavage rate of a candidate linker in serum and periplasmic extract.
  • FIGS 6B and 6C show the comparison of linker cleavage in 50 % mouse serum and periplasmic extract over 8 hours.
  • FIG 6B evaluates a linker that was discovered from phage platform with negative selections.
  • FIG 6C evaluates a linker that was discovered from phage platform without negative selections.
  • FIG.7 shows that the DAVNGEC (SEQ ID NO: 53) peptide demonstrated a 21-fold improvement in periplasmic extract versus serum.
  • FIG.8 shows that the A. baumannii extracts show better cleavage than non-A.baumannii extracts.
  • FIG.9 shows the ACC conjugates synthesized to identify an optimal scarless linker for LEHELGN (SEQ ID NO: 7).
  • FIG.10 shows the comparison between FRET pair cleavage and ACC release for LEHELGN (SEQ ID NO: 7).
  • FIGS.11A, 11B, 11C, 11D, 11E, and 11F show the scarless release of ACC in serum and A. baumannii periplasmic extract.
  • FIG.12 shows the cleavage rates of alanine-substituted Ac-LEHELG-ACC (SEQ ID NO: 9) variants over 8 hours in mouse serum and in periplasmic extracts from six different A. baumannii strains.
  • ACC is a turn-on fluorescent probe that only fluoresces if it is cleaved without an amino acid scar. ACC excitation maximum: 355 nm.
  • FIG.13 shows the cleavage rates ( ⁇ M/hour) of each Ac-LEXELG (SEQ ID NO: 5)-ACC peptide (where X represents 20 natural amino acids) in different periplasmic extracts of A. baumannii bacteria compared to 50% mouse serum over the course of 8 hours.
  • FIG.14 shows the cell viability assay in HL60 cells and shows the cleavable LEHELG (SEQ ID NO: 1)-mitomycin C conjugate is non-toxic relative to mitomycin C. DETAILED DESCRIPTION
  • the following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s).
  • a formulation may include an excipient
  • “Comprising” is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others.
  • Consisting essentially of'' when used to define compositions and methods shall mean including the recited elements, but excluding other elements of any essential significance to the combination.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of'' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.
  • An “increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, or more, increase so long as the increase is statistically significant.
  • a "decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • reduce or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.
  • prevent or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur.
  • nucleotide is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5-carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA.
  • nucleic acid is a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material.
  • Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base.
  • a nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).
  • a chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.
  • the word “vector” refers to any vehicle that carries a polynucleotide into a cell for the expression of the polynucleotide in the cell.
  • the vector may be, for example, a phagemid, a plasmid, a virus, a phage particle, or a nanoparticle. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself.
  • the vector is a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host cell.
  • control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation.
  • a “phagemid” refers to an engineered DNA-based cloning vector comprising both bacteriophage and plasmid properties and/or structures, such as for example a phagemid can comprise an origin of replication derived from a plasmid and an origin of replication derived from a bacteriophage.
  • Phagemid vectors are engineered as hybrid of filamentous phages and plasmids to generate a product that can be grown as a plasmid, and also packaged as a single stranded DNA in viral particles.
  • amino acid includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan
  • amino acid residue also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, ⁇ - alanine, ⁇ -Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3- Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N- Methylglycine, sarcosine, 3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, Docket No.103361-473WO1 6-N-Methyllysine, 2,4-Diamin
  • the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.
  • peptides, polypeptides, proteins, and compositions comprising peptides, polypeptides, and proteins are defined as a polymer of amino acids, typically of length ⁇ 100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).
  • a peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).
  • the term “variant” means a polypeptide derived from a parent polypeptide by one or more (several) alteration(s), i.e., a substitution, insertion, and/or deletion, at one or more (several) positions.
  • a substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1-3 amino acids immediately adjacent an amino acid occupying a position.
  • ‘immediately adjacent’ may be to the N-side (‘upstream’) or C-side (‘downstream’) of the amino acid occupying a position (‘the named amino acid’). Therefore, for an amino acid named/numbered ‘X,’ the insertion may be at position ‘X+1’ (‘downstream’) or at position ‘X ⁇ 1’ (‘upstream’).
  • the variant polypeptide can also be a chiral variant of the parent polypeptide, wherein the chiral variant is a mirror image of the parent and cannot be superimposed on the parent.
  • the chiral variant and parent polypeptides are “enantiomers” of one another; they are often distinguished as either “right-handed or (D)” or “left-handed or (L)”.
  • a “variant” of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L.
  • a variant polypeptide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polypeptide.
  • a variant polypeptide may have substantially the same Docket No.103361-473WO1 functional activity as a reference polypeptide.
  • screening refers to a method especially used in drug discovery in which data processing/control software, liquid handling devices, and sensitive detectors can allow for quick conductions of chemical, genetic, or pharmacological tests. This process allows one to quickly recognize active compounds, antibodies, genes and the like that modulate a particular biomolecular pathway. The results of these processes provide starting points for drug design or therapeutic treatments.
  • Compounds containing protease-cleavable linkers The present disclosure also provides compounds and/or compositions comprising a protease-cleavable linker. The present disclosure also provides methods of purifying compositions comprising the protease-cleavable linker.
  • a compound comprising a targeting moiety, a therapeutic agent, and a cleavable linker, wherein the cleavable linker comprises a peptide sequence selected from L-LEHELG (SEQ ID NO: 1), L-LEHALG (SEQ ID NO: 2), L-LEAELG (SEQ ID NO: 3), L-LEAALG (SEQ ID NO: 4), D-LEHELG (SEQ ID NO: 1), D-LEHALG (SEQ ID NO: 2), D-LEAELG (SEQ ID NO: 3), D-LEAALG (SEQ ID NO: 4), L-LEXELG (SEQ ID NO: 5), D-LEXELG (SEQ ID NO: 5), L-LEAELX (SEQ ID NO: 6), or D-LEAELX (SEQ ID NO: 6), wherein X is any chiral or achiral amino acid.
  • L-LEHELG SEQ ID NO: 1
  • L-LEHALG SEQ ID NO: 2
  • the cleavable linker is directly linked to the targeting moiety and the therapeutic agent. In some embodiments, the cleavable linker is indirectly linked to the targeting moiety.
  • the targeting moiety comprises a siderophore. In some embodiments, the targeting moiety comprises an HHM siderophore, enterobactin, or yersiniabactin. In some embodiments, the HMM siderophore comprises Compound I . Compound I In some embodiments, the enterobactin comprises Compound II Docket No.103361-473WO1 . Compound II In some embodiments, the aerobactin comprises Compound III .
  • the yersiniabactin comprises Compound IV .
  • the therapeutic agent comprises an antibiotic.
  • the antibiotic targets a cytosolic molecule, a periplasmic molecule, or a bacterial membrane molecule.
  • the antibiotic comprises a basic amine-containing antibiotic.
  • the antibiotic comprises an amine- or aniline-containing antibiotic.
  • the antibiotic includes but is not limited to solithromycin, penicillins (including, but not limited to amoxicillin, clavulanate and amoxicillin, ampicillin, Docket No.103361-473WO1 dicloxacillin, oxacillin, and penicillin V potassium), tetracyclins (including, but not limited to demeclocycline, doxycycline, eravacycline, minocycline, omadacycline, sarecycline, and tetracycline), cephalosporins (cefaclor, cefadroxil, cefdinir, cephalexin, cefprozil, cefepime, cefiderocol, cefotaxime, cefotetan, ceftaroline, ceftazidime, ceftriaxone, and cefuroxime), quinolones (also referred to as fluoroquinolones include, but are not limited to ciprofio
  • the therapeutic agent comprises an anti-cancer chemotherapeutic agent.
  • the anti-cancer chemotherapeutic agent comprises an apoptosis agent, an anti-mitotic agent, a kinase inhibitor, an antimetabolite, an alkylating agent, paclitaxel, docetaxel, a bifunctional proteolysis-targeting chimera, or a combination thereof.
  • the anti-cancer chemotherapeutic agent includes, but is not limited to doxorubicin, mitomycin, anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g.
  • goserelin and leuprolide anti-androgens (e.g. flutamide and bicalutamide), photodynamic therapies (e.g. verteporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g.
  • busulfan and treosulfan busulfan and treosulfan
  • triazenes e.g. dacarbazine, temozolomide
  • platinum containing compounds e.g. cisplatin, carboplatin, oxaliplatin
  • vinca alkaloids e.g. vincristine, vinblastine, vindesine, and vinorelbine
  • taxoids e.g.
  • paclitaxel or a paclitaxel equivalent such as nanoparticle albumin- bound paclitaxel (ABRAXANE), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel, e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g.
  • ABRAXANE nanoparticle albumin- bound paclitaxel
  • etoposide etoposide phosphate, teniposide, topotecan, 9- aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mytomycin C
  • anti-metabolites Docket No.103361-473WO1 DHFR inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonucleotide reductase inhibitors (e.g.
  • uracil analogs e.g.5- fluorouracil (5-FU), floxuridine, doxifluridine, raltitrexed, tegafur-uracil, capecitabine
  • cytosine analogs e.g. cytarabine (ara C), cytosine arabinoside, and fludarabine
  • purine analogs e.g. mercaptopurine and Thioguanine
  • Vitamin D3 analogs e.g. EB 1089, CB 1093, and KH 1060
  • isoprenylation inhibitors e.g.
  • lovastatin dopaminergic neurotoxins (e.g.1-methyl-4- phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), actinomycin (e.g. actinomycin D, dactinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g.
  • thapsigargin Ca 2+ ATPase inhibitors
  • imatinib thalidomide, lenalidomide
  • tyrosine kinase inhibitors e.g., axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTINTM, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP- 701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®,
  • axitinib
  • a method of screening for a protease-cleavable linker comprising: a) a negative selection process comprising: i) incubating a substrate display library comprising one or more peptide sequences in a first complex biological matrix, ii) generating a first cleaved phage-containing peptide sequence and an uncleaved phage- containing peptide sequence, and iii) separating the uncleaved phage-containing peptide sequence from the first cleaved phage-containing peptide sequence; b) a positive selection process comprising: i) contacting the uncleaved phage-containing peptide sequence with a second complex biological matrix, and ii) producing a second
  • a method of screening for two or more protease- cleavable linkers comprising: a) a negative selection process comprising: i) incubating a substrate display library comprising one or more peptide sequences in a first complex biological matrix, ii) generating a first cleaved phage-containing peptide sequence and an uncleaved phage-containing peptide sequence, and iii) separating the uncleaved phage- containing peptide sequence from the first cleaved phage-containing peptide sequence; b) a positive selection process comprising: i) contacting the uncleaved phage-containing peptide sequence with a second complex biological matrix, and ii) producing a second cleaved phage- containing peptide sequence; c) identifying a first nucleic acid sequence of the first cleaved phage-containing peptide sequence and identifying a second nucleic acid sequence
  • steps a) through d) are performed.
  • 2, 3, 4, 5, 6, 7, 8, or 9 iterations of steps a) through d) are performed.
  • the method screens 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, Docket No.103361-473WO1 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94
  • phagemid refers to a type of cloning vector engineered to be a hybrid of a bacteriophage and a plasmid to produce a vector that can grow as a plasmid and also be packaged as single stranded DNA in viral particles. It should be noted that the phagemid of any preceding aspect can comprise one or more components from a bacteriophage, one or more components from a plasmid, or any combination thereof, deemed necessary to achieved the desired function. In some embodiments, the phagemid of any preceding aspect comprises an origin of plasmid replication, an origin of bacteriophage replication, or a combination thereof.
  • the first complex biological matrix comprises healthy blood serum or plasma, healthy organ tissue homogenates, media comprising healthy organ tissue homogenates, healthy organoid homogenates derived from healthy cells, conditioned media comprising healthy organoids derived from healthy cells, healthy cell lines, conditioned media comprised of healthy cell lines, or combinations thereof.
  • the second complex biological matrix comprises cancer tissue homogenates, media comprising cancer tissue homogenates, cancer organoid homogenates, conditioned media comprising cancer organoid homogenates, malignant ascites, malignant cystic fluid, cancer cell lines, conditioned media comprising cancer cell lines, or combinations thereof.
  • the second complex biological matrix comprises periplasmic extracts isolated from Gram-negative bacteria, bacteria lysates from Gram-positive or Gram- negative bacteria, or combinations thereof.
  • the method comprises a negative selection process and a positive selection process.
  • the negative selection process comprises a control cell or a microenvironment comprising the control cell.
  • the positive selection process comprises a cell of interest or a microenvironment comprising the cell of interest.
  • the cell of interest comprises a gram-negative bacterium.
  • the gram-negative bacterium comprises A. nosocomialis, P. aeruginosa, E. cloacae, E. coli, A. baumannii, E.
  • the cell of interest comprises a gram-positive bacterium including, but not limited to S. aureus, S. epidermidis, S. pneumoniae, S. pyogenes, C. difficile, C. perfringens, or L. monocytogenes.
  • the cell of interest comprises a cancer cell.
  • the cancer cell is derived from a cancer including, but not limited to hepatocellular carcinoma (HCC), biliary cancer (such as, for example cholangiocarcinoma (CC)), combined hepatocholangiocarcinoma (cHCC-CC), ovarian carcinoma, uterine carcinoma, triple negative breast cancer, pancreatic carcinoma, glioblastoma, acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast),
  • HCC hepatocellular carcinoma
  • CC cholangio
  • Wilms' tumor, renal cell carcinoma), lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.
  • MMD myeloproliferative disorder
  • PV polycythemia Vera
  • ET essential thrombocytosis
  • AAMM agnogenic myeloid metaplasia
  • myelofibrosis MF
  • chronic idiopathic myelofibrosis chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)
  • neuroblastoma e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis
  • neuroendocrine cancer e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor
  • osteosarcoma ovarian cancer
  • cystadenocarcinoma ovarian embryonal carcinoma, ovarian adenocarcinoma
  • papillary adenocarcinoma pancreatic cancer
  • pancreatic cancer e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors
  • penile cancer e.g., Paget's disease of the pen
  • the cell of interest is the same in the at least two iterations of steps a) through e). In some embodiments, the cell of interest is different in the at least two iterations of steps a) through e).
  • the first and second nucleic acid sequences encode the one or more protease-cleavable linkers. In some embodiments, the first and second nucleic acid sequence is identified using Next Generation Sequencing (NGS). In some embodiments, the Docket No.103361-473WO1 method further comprises identifying cleavage kinetics in the presence of the first complex biological matrix and the second complex biological matrix.
  • Methods of preparing and/or purifying therapeutic compositions comprising a targeting moiety, a therapeutic agent, and a cleavable linker, the method comprising washing the therapeutic composition with about 1% acetic acid/acetonitrile, about 10mM ammonium acetate, or a combination thereof, binding the therapeutic composition to a reverse-phase column comprising about 0.1% acetic acid, wherein the column is a component on a liquid chromatography apparatus, and eluting the therapeutic composition from the column using a solution comprising 5% to 65% acetonitrile in water.
  • the method further comprises repeating the eluting step of any preceding aspect.
  • the solution comprises 5% to 47% acetonitrile in water.
  • the method further comprises purifying the therapeutic composition using high performance liquid chromatography.
  • the method of purifying a therapeutic composition of any preceding aspect comprising a cleavable linker comprising L-LEHELG (SEQ ID NO: 1), L- LEHALG (SEQ ID NO: 2), L-LEAELG (SEQ ID NO: 3), L-LEAALG (SEQ ID NO: 4), D- LEHELG (SEQ ID NO: 1), D-LEHALG (SEQ ID NO: 2), D-LEAELG (SEQ ID NO: 3), D- LEAALG (SEQ ID NO: 4), L-LEXELG (SEQ ID NO: 5), D-LEXELG (SEQ ID NO: 5), L- LEAELX (SEQ ID NO: 6), or D-LEAELX (SEQ ID NO: 6), wherein X is any chiral or achiral amino acid.
  • the therapeutic composition comprises a siderophore, L-LEXELG (SEQ ID NO: 5), and mitomycin C.
  • the therapeutic composition comprises a siderophore, D-LEXELG (SEQ ID NO: 5), and mitomycin C.
  • the X of SEQ ID NO: 5 is alanine, methionine, glycine, cysteine, aspartic acid, phenylalanine, glutamic acid, histidine, isoleucine, lysine, leucine, asparagine, glutamic acid, arginine, serine, threonine, valine, tryptophan, proline, or tyrosine.
  • LEXE is critical composition for serum stability.
  • LEXELG (SEQ ID NO: 5) comprises optimized properties for cleavage rate and serum stability.
  • variants of LEXELG (SEQ ID NO: 5) comprise LEMELG (SEQ ID NO: 57), LEAELG (SEQ ID NO: 3), LEGELG (SEQ ID NO: 58), LECELG (SEQ ID NO: 59), LEDELG (SEQ ID NO: 60), LEFELG (SEQ ID NO: 61), LEEELG (SEQ ID NO: 62), LEHELG (SEQ ID NO: 63), LEIELG (SEQ ID NO: 64), LEKELG (SEQ ID Docket No.103361-473WO1 NO: 65), LELELG (SEQ ID NO: 66), LENELG (SEQ ID NO: 67), LEQELG (SEQ ID NO: 68), LERELG (SEQ ID NO: 69), LESELG
  • pancreatic cancer is one of the deadliest and most difficult cancers to treat. It is projected that by 2030, pancreatic cancer will be the second leading cause of cancer-related deaths. Early diagnosis is challenging due to the cancer's progress being slow and asymptomatic. Surgery, radiation therapy, and chemotherapy are treatments that may extend survival or relieve symptoms, however, fewer than 20% of patients are candidates for surgical intervention due to metastasis. Immunotherapy (i.e. antibody drug conjugates (ADC)) remains an option for only a small number of cases and is normally ineffective due to the size of the ADC and the conjugate's inability to effectively penetrate the cancer stroma microenvironment.
  • ADC antibody drug conjugates
  • the tumor microenvironment of pancreatic cancers is heterogeneous with multiple proteases contributing to cancer progression and metastasis.
  • the present disclosure provides a method of developing selective cleavable linkers and their incorporation into targeted chemotherapeutics for pancreatic cancers that leverages the differences in protease populations between the tumor microenvironment and non-tumorous or healthy microenvironments. Because traditional chemotherapy is still dependent on toxic compounds that lack selectivity, their side effects reduce patient quality of life and lead to life- Docket No.103361-473WO1 threatening complications.
  • One method of mitigating chemotherapeutic toxicity is through the attachment of a peptide linker, which prevents cell permeability and/or target binding.
  • the peptide is cleaved by a protease that is upregulated within or proximal to tumor cells, releasing the active drug in the location of interest.
  • a protease that is upregulated within or proximal to tumor cells, releasing the active drug in the location of interest.
  • linkers e.g. Valine-Citrulline (Val-Cit)
  • ADCs FDA-approved antibody-drug conjugates
  • cleavable peptide prodrugs (without an antibody or targeting moiety) have not been FDA approved because existing protease-cleavable linkers lack the necessary selectivity to ensure site-specific delivery. Therefore, improvements in linker selectivity are drastically needed.
  • linkers are traditionally designed to target one or two proteases that are upregulated in cancer cells, and it is challenging to target a single protease because the human genome codes for more than 600 proteases, many with overlapping specificities.
  • a single protease may not be present in enough concentration for prodrug activation.
  • protease-activated peptide linker To simplify the challenge of optimizing a protease-activated peptide linker, the differences in overall protease activity between cancerous and healthy microenvironments is leveraged by screening complex biological mixtures (e.g. healthy and cancerous patient- derived tissue homogenates in addition to conditioned media from organoid cultures) with substrate phage display (SPD).
  • SPD substrate phage display
  • the platform benefits from a highly diverse 7-amino acid peptide phage library (containing up to 20 ⁇ 7 variants) and from the ability to remove peptides from the library that cleave in healthy microenvironments (negative selections) and identify remaining sequences that are cleaved selectively in tumor microenvironments (positive selections).
  • each biopanning campaign consists of multiple rounds of selection (positive and negative selections, along with biological replicates), and cancerous samples deriving from different patient populations may be used to increase the probability of site-specific payload release in a broad range of patients.
  • Homogenized pancreatic cancer tissues, malignant cystic fluids, homogenized necrosectomy samples, or conditioned media from cancerous organoid cultures can be used for positive selections.
  • Serum, homogenized healthy tissues e.g.
  • conditioned media from healthy organoid cultures can be used for negative selections to optimize linker stability in healthy microenvironments.
  • the phagemids from the Docket No.103361-473WO1 selections are barcoded and sequenced by Next Generation Sequencing (NGS), and the sequences are scored based on positive/negative enrichment as described in Example 5.
  • NGS Next Generation Sequencing
  • FRET fluorescence resonance energy transfer
  • ACC conjugates containing candidate linkers enable the evaluation of cleavage kinetics and identification of candidates for site-specific payload release in the various samples used in the biopanning selections (e.g. serum, tissue homogenates, and organoid conditioned media).
  • Candidate linkers can be conjugated to chemotherapeutics, and controls can be synthesized to interrogate on- target activity and prodrug selectivity (e.g. non-cleavable linker variants).
  • SPD was originally developed as a method to profile the substrate preferences of a single protease. Prior to the disclosure reported herein, SPD had never been successfully utilized for the development of cleavable prodrug linkers. Previous efforts by Whitney et al. to use SPD resulted in linkers that had serum instability and cleaved in healthy tissue extracts, which deprioritized prodrug applications. Recent efforts by Boyce et al. to utilize SPD for prodrug linkers led to candidates that were unstable in mouse and human serum, preventing application in an animal model.
  • SPD proteolytic profiling technologies that screen libraries of protease substrates (e.g. multiplex substrate profiling by mass spectrometry (MSP-MS), positional scanning of synthetic combinatorial libraries, and indexed arrays of fluorogenic substrates).
  • MSP-MS mass spectrometry
  • SPD enables the evaluation of maximal substrate diversity, with greater than 10 9 variants.
  • all existing proteolytic profiling technologies, including SPD were developed for single protease evaluations or comparisons of individual proteases and were not designed to interrogate the substrate profiles of complex biological mixtures with numerous proteases.
  • a phage-based platform that enables identification of linker candidates that cleave selectively in one set of microenvironments while remaining stable in others, allowing for the discovery of highly selective prodrug linkers.
  • the platform described is non-obvious because the proteases and their cleavage preferences are unknown with multiple cleavage sites for every enriched candidate identified, which complicates the discovery of consensus sequences. Indeed, for most applications of SPD, knowledge of the protease or identification of common cleavage motifs among multiple substrates is a critical component of the data analysis process. In contrast, this approach depends on the ability to identify sequences that are cleaved in one mixture over others independent of the proteases or their cleavage preferences.
  • An SPD platform was developed by incorporating negative selections (to remove linkers from a diverse linker library that cleave in healthy tissue homogenates and serum) and positive selections (to identify remaining Docket No.103361-473WO1 sequences that cleave in cancerous tissue homogenates, malignant pancreatic cystic fluid, and conditioned media from organoid cultures).
  • NGS analysis of both positive and negative selections are critical for identification of highly selective candidates and minimizing false positives.
  • Targeting multiple proteases may minimize resistance to prodrug therapeutics by increasing the evolutionary cost required to prevent conjugate activation through protease elimination. This platform is leveraged to achieve optimal stability and cleavage selectivity of discovered linkers for prodrug and diagnostic development.
  • the platforms and methods of the present disclosure can further comprise the following steps or features: 1) Separate biopanning campaigns incorporating positive selections conducted with primary cancer cells and/or metastatic cancer cells at various stages of progression; 2) Peptide prodrugs have potential to benefit patients that do not respond well to immunotherapy. 3) For effective linkers discovered in this platform, attachment of antibodies as targeting groups (e.g. those used in marketed antibody-drug conjugates) can be incorporated. Current linkers on the market undergo off-target release resulting in neutropenia; the linkers discovered by this platform have the potential to overcome this limitation.
  • euthanasia is completed by cervical dislocation.
  • the euthanized BL/6 mouse is placed on its back, and an incision is made with small surgical scissors.
  • Relevant organs e.g. kidney and liver
  • the organs are then processed on ice using the back of a 10-mL syringe plunger.
  • the resulting fluid is filtered through a cell strainer and diluted in phosphate-buffered saline (PBS). Docket No.103361-473WO1 Centrifugation facilitates removal of residual cell debris, followed by filtration through a 0.2 um filter.
  • the samples are then aliquoted into eight Eppendorf tubes for use in biopanning selections.
  • mice utilizes healthy BL/6 mice to sample diverse organs for use in negative selections of the substrate phage platform to improve the probability of identifying linkers that are stable in healthy mouse microenvironments.
  • Human tissue homogenates are procured through the Total Cancer Care (TCC) Program at Ohio State University. Those animals exhibiting clinical signs of disease or are severely lethargic, unresponsive to stimuli, have excessive weight loss (greater than 40% compared to other mice in this healthy cohort) or are unable to right themselves, are removed immediately and humanely euthanized.
  • TCC Total Cancer Care
  • PLC primary liver cancer
  • the diagnosis of unresectable PLC subtypes e.g.
  • HCC hepatocellular carcinoma
  • CC cholangiocarcinoma
  • cHCC- CC combined hepatocellular-cholangiocarcinoma
  • HCC patients benefit from targeted therapy, immunotherapy, and antiviral agents, while CC patients respond to traditional chemotherapy in addition to targeted therapy, and immunotherapy.
  • cHCC-CC patients have the poorest prognosis and mainly rely on major hepatectomy for optimal management; other treatments include radiation, yttrium-90 radioembolization, chemotherapy, combined radiation and chemotherapy, combined surgery and chemotherapy, and triple therapy (surgery, radiation, and chemotherapy).
  • the present disclosure provides a method of developing selective cleavable linkers for incorporation into targeted chemotherapeutics for PLC that leverages the differences in protease populations between the tumor microenvironment and non-tumorous or healthy microenvironments.
  • a peptide linker which prevents cell Docket No.103361-473WO1 permeability and/or target binding.
  • the peptide is cleaved by a protease that is upregulated within or proximal to tumor cells, releasing the active drug in the location of interest.
  • the most selective linkers e.g. Valine-Citrulline (Val-Cit)] in FDA- approved antibody-drug conjugates (ADCs) for immunotherapy undergo premature drug release causing neutropenia as a major side effect.
  • the differences in overall protease activity between cancerous and healthy microenvironments is leveraged by screening complex biological mixtures (e.g. conditioned media from organoids, patient-tissue homogenates, etc.) with SPD.
  • the platform benefits from a highly diverse 7- amino acid peptide phage library (containing up to 20 ⁇ 7 variants) and from the ability to remove peptides from the library that cleave in healthy microenvironments (negative selections) and identify remaining sequences that are cleaved selectively in tumor microenvironments (positive selections).
  • linkers may be developed that are either subtype-selective (e.g.
  • the present disclosure provides a method of developing a diagnostic for rapidly identifying cHCC-CC, CC, and HCC subtypes from a biopsied sample. Using this method, three distinct protease-activated turn-on fluorescent probes could be developed that specifically cleave in a homogenate of each PLC subtype.
  • the method comprises at least three separate biopanning campaigns (one for each PLC subtype), and each campaign consists of multiple rounds of selection and biological replicates.
  • PLC tissue homogenates deriving from different patient populations may improve the probability that protease-activated linkers discovered from this platform are effective for a broad range of patients.
  • the phagemids from the selections can be barcoded and sequenced by NGS, and the sequences can be scored based on positive/negative enrichment as described in Example 5.
  • Synthesis of FRET pairs and Docket No.103361-473WO1 evaluation of cleavage kinetics in HCC, CC, and cHCC-CC homogenates would inform which linkers provide optimal protease-activated diagnostics for each PLC subtype.
  • the present disclosure provides a method of developing protease-activated chemotherapeutic prodrugs that target all three subtypes of PLC: HCC, CC, and cHCC-CC.
  • the phage platform disclosed herein can be conducted with conditioned media from organoids or patient tissue homogenates that derive from each subtype. Serum, conditioned media from organoids, and/or tissue homogenates derived from healthy tissue (e.g. liver and kidney) can be used for negative selections to optimize linker stability in healthy microenvironments. Following biopanning selections, NGS, and sequence scoring, the sequences can be synthesized as FRET pairs and optimized for scarless prodrug release using turn-on fluorescent probes as described in Example 5.
  • Candidate linkers can be conjugated to chemotherapeutics, and controls can be synthesized to interrogate on-target activity and prodrug selectivity.
  • the potency of chemotherapeutic prodrugs and controls can be investigated in cell-viability assays utilizing healthy (kidney, liver) and cancerous organoid models (HCC, CC, and cHCC-CC).
  • the present disclosure combines SPD with tissue homogenates and organoids to address limitations that have prevented the ability to diagnose PLC subtypes.
  • the protease- activated imaging agents developed may improve the ability to diagnose and study cHCC-CC cases and facilitate further advances in interventional surgical imaging and monitoring therapeutic response (34).
  • HCC patients 80% of PLC cases tend to experience higher toxicity from traditional chemotherapy treatments [e.g. doxorubicin- and gemcitabine- based therapies, FOLFOX (5-fluorouracil, leucovorin, oxaliplatin), and PIAF (cisplatin/interferon alpha-2b/doxorubicin/5-fluorouracil)], with minimal benefit to overall survival (33-36).
  • chemotherapy remains the first-line standard-of-care for most patients with CC (15% of PLC cases).
  • Chemotherapy use for cHCC-CC patients (5% of PLC cases) is understudied because it is rare and challenging to diagnose due to similarities with HCC and/or CC.
  • Chemotherapeutic peptide prodrugs developed by the platform described herein, may enable patients with unresectable PLC to tolerate higher doses by increasing the therapeutic index and improve long-term survival. This strategy has the potential to benefit patients with any PLC subtype. Additionally, the peptide prodrugs developed by the platform described herein may especially benefit those patients that do not respond well to immunotherapy. Protease dysregulation in cancers often contributes to invasion and tumor spread. The protease mixtures in each tumor microenvironment have complex but specific proteolytic signatures that differ from those in healthy microenvironments. SPD is used to screen these Docket No.103361-473WO1 microenvironments and identify their preferred proteolytic substrates.
  • the phage libraries incorporate up to 20 ⁇ 7 unique peptide variants, which have the benefit of providing a large number of enriched candidates (e.g. sequences with desired cleavage properties), enhancing the probability of discovering shared substrates across all PLC subtypes that may lead to broad- spectrum PLC-targeting prodrugs.
  • the large number of enriched candidates also aid the discovery of unique substrates that cleave selectively in one PLC subtype over others, which may result in protease-activated imaging agents that are effective for PLC subtype diagnoses.
  • the platform described is non-obvious because the proteases and their cleavage preferences are unknown with multiple cleavage sites for every enriched candidate identified, which complicates the discovery of consensus sequences.
  • an SPD platform was developed by incorporating negative selections (to remove linkers from a diverse linker library that cleave in healthy tissue homogenates, organoid conditioned media, and serum) and positive selections (to identify remaining sequences that cleave in PLC tissue homogenates and tumoroid conditioned media).
  • negative selections to remove linkers from a diverse linker library that cleave in healthy tissue homogenates, organoid conditioned media, and serum
  • positive selections to identify remaining sequences that cleave in PLC tissue homogenates and tumoroid conditioned media.
  • NGS analysis of both positive and negative selections are critical for identification of highly selective candidates and minimizing false positives.
  • Targeting multiple proteases may minimize resistance to prodrug therapeutics by increasing the evolutionary cost required to prevent conjugate activation through protease elimination.
  • This platform is leveraged to achieve optimal stability and cleavage selectivity of discovered linkers for prodrug and diagnostic development.
  • the present disclosure provides a method of developing a diagnostic that can rapidly identify cHCC-CC, CC, and HCC subtypes from a biopsied sample. Application of the method describe herein would result in three unique protease-activated turn-on fluorescent probes that specifically cleave in a homogenate of each PLC subtype.
  • Linker selectivity can be optimized with the phage platform described in Example 5, using positive and negative selections in combination with NGS sequencing. The selections incorporate PLC-subtype homogenates.
  • the homogenates can be prepared on ice from 2 mg of tissue per 100 uL of phosphate-buffered saline (PBS) containing 1 mM ZnCl 2 .
  • PBS phosphate-buffered saline
  • separate biopanning campaigns can be conducted, each consisting of multiple rounds of selection, biological replicates, and PLC samples deriving from different patient populations.
  • the Docket No.103361-473WO1 biopanning campaigns begin with a randomized amino acid phage library and are summarized as follows.
  • Biopanning Campaign 1 (FIG 1): To identify sequences that release fluorescent probes in cHCC-CC homogenates with stability in HCC and CC homogenates, at least three rounds of SPD can be conducted with negative selections incorporating HCC and CC homogenates and positive selections incorporating cHCC-CC homogenates.
  • the negative selections remove sequences from the randomized amino acid phage library that cleave in HCC and CC homogenates, and the remaining sequences are subjected to positive selections for the identification of sequences that cleave selectively in cHCC-CC homogenates.
  • Biopanning Campaign 2 To identify sequences that rapidly release fluorescent probes in CC homogenates with stability in cHCC-CC and HCC homogenates, at least three rounds of SPD can be conducted with negative selections incorporating cHCC-CC and HCC homogenates and positive selections incorporating CC homogenates.
  • Biopanning Campaign 3 To identify sequences that rapidly release fluorescent probes in HCC homogenates with stability in cHCC-CC and CC homogenates, at least three rounds of SPD can be conducted with negative selections incorporating cHCC-CC and CC homogenates and positive selections incorporating HCC homogenates.
  • Each of the three biopanning campaigns described above can be optionally repeated with the following variables: 1) a separately prepared randomized amino acid phage library (see Methods Example 5 for phage library preparation), and 2) utilizing tissue homogenates from diverse patient populations (e.g. Asian, African, African American, Caucasian American, and Pacific Islander descent) [ideally, all three biopanning campaigns described above are repeated for each population and with a combination of homogenates from multiple populations].
  • the phagemids from the selections (positive and negative) can be sequenced by NGS, and the sequences scored based on positive/negative enrichment as described herein. Python scripts enable scoring of the sequences, and a selection of sequences with excellent scores can be synthesized as FRET pairs.
  • the first Python script converts DNA sequences to amino acid sequences for each sample.
  • the second script compares positive and negative selection read counts for each sequence from a single round.
  • the third script allows for comparison of the positive and negative selection counts across all rounds from each biopanning campaign.
  • Information from the third Python script is analyzed to create a focused candidate list based on positive/negative enrichment rankings and relative read counts in positive and negative selections across all rounds from each campaign.
  • the fourth script allows for scoring of each candidate as specified in Example 5. Docket No.103361-473WO1
  • the cleavage kinetics of each FRET substrate can be assessed in HCC, CC, and cHCC-CC homogenates by monitoring for fluorescence over the course of 8 hours (FIG 2).
  • the peptides that display the highest sensitivity and selectivity for a specific PLC subtype can be selected as protease-activated diagnostic candidates. After candidate linkers have been selected for each subtype, their sensitivity can be tested using homogenates of biopsied samples deriving from primary liver cancer patients to aid in the identification of PLC subtype. The conclusion from the assay results can be compared to the sample’s histopathological classification. A method to improve protease-activated agents to aid in the identification of cHCC-CC subtypes of PLC is contemplated. The diagnostic is contemplated to be used in parallel with histopathological methods to provide further confidence of subtype, which is often a challenging distinction that is critical for proper treatment.
  • the platform described herein is also contemplated to generate protease-activated chemotherapeutic prodrugs that target all three subtypes of PLC: HCC, CC, and cHCC-CC.
  • the homogenates from multiple PLC subtypes can be combined for positive selections in each round.
  • FRET pairs that display the highest selectivity for HCC, CC, and cHCC-CC proteolysis relative to healthy homogenate samples can be synthesized as ACC conjugates (with variable linker lengths) to optimize for scarless drug release as described in Example 5.
  • the scarless linkers can then be conjugated to chemotherapeutics using known chemical methods. Controls can be synthesized to interrogate on-target activity and probe of mechanism of action.
  • cleavable (L-amino acid linker) and non-cleavable (D-amino acid linker) variants can be synthesized for comparison to determine if cytotoxicity is due to proteolytic cleavage.
  • the platforms and methods of the present disclosure can further comprise the following steps or features: 1) Separate biopanning campaigns incorporating positive selections conducted with primary cancer cells and/or metastatic cancer cells at various stages of progression; 2) Peptide prodrugs have potential to benefit patients that do not respond well to immunotherapy. 3) For effective linkers discovered in this platform, attachment of antibodies as targeting groups (e.g. those used in marketed antibody-drug conjugates) can be incorporated.
  • Example 3 Platform Applications to ovarian cancer.
  • the present disclosure provides a method of developing peptide prodrug linkers that cleave selectively in ovarian cancer ascites and relevant ovarian cancer cell lines or organoid models.
  • linkers would display rapid cleavage in ovarian cancer ascites and malignant cystic fluids and stability in the presence of serum and diverse healthy tissue homogenates, including kidney and liver.
  • the method would incorporate positive and negative selections of SPD, followed by NGS sequencing, and sequence validation as described in Examples 2 and 5.
  • protease-activated prodrugs that target ovarian-cancer.
  • linker stability and selectivity remain a challenge.
  • FDA approved protease-activated cancer treatments e.g. ADCs
  • these medications are known to cause neutropenia as a common side effect due to premature drug release.
  • This disclosure addresses these challenges through a platform that discovers highly selective protease-cleavable linkers.
  • positive selections may include ovarian cancer ascites, malignant cystic fluid, conditioned media from ovarian cancer cell lines or organoids (e.g.
  • Negative selections may include, but are not limited to, serum and healthy tissue homogenates.
  • Candidate linkers can be identified and validated as described in Example 5. It should be understood that the platforms and methods of the present disclosure can further comprise the following steps or features: 1) Separate biopanning campaigns incorporating positive selections conducted with primary cancer cells and/or metastatic cancer cells at various stages of progression; 2) Peptide prodrugs have potential to benefit patients that do not respond well to immunotherapy. 3) For effective linkers discovered in this platform, attachment of antibodies as targeting groups (e.g. those used in marketed antibody-drug conjugates) can be incorporated.
  • targeting groups e.g. those used in marketed antibody-drug conjugates
  • TNBC triple-negative breast cancer
  • ER estrogen receptor
  • PR progesterone Docket No.103361-473WO1 receptor
  • HER2 human epidermal growth factor receptor 2
  • chemotherapeutics account for 90% treatment failure in mTNBC patients.
  • Alternative therapies such as antibody-drug conjugates (ADCs) are limited for mTNBC with only one being FDA-approved (Trodelvy, 2021).
  • ADCs antibody-drug conjugates
  • the present disclosure provides a method of developing improved targeted therapies for mTNBC through discovery of highly selective linkers for protease-activated chemotherapeutics.
  • Chemotherapeutic peptide prodrugs may enable the mTNBC patient to tolerate higher doses of chemotherapeutics by increasing the therapeutic index and increasing long-term survival.
  • prodrugs that result from this platform may especially benefit those patients that do not respond well to immunotherapy.
  • the present disclosure leverages the differences in overall protease activity between cancerous and healthy microenvironments by screening complex biological mixtures with SPD.
  • the platform benefits from a highly diverse peptide phage library and from the ability to remove peptides from the library that cleave in healthy microenvironments (negative selections) and identify remaining sequences that are cleaved selectively in tumor microenvironments (positive selections).
  • a linker LHELGN(SEQ ID NO: 7) was discovered to be 66-fold more stable in mouse serum than A. baumannii extract (see Example 5).
  • the linker was then used to convert chemotherapeutics into potent antibiotic prodrugs with minimal toxicity in human cell lines.
  • prodrug linkers that cleave in mTNBC ascites and patient-derived TNBC tissue homogenates (containing metastasized cancer tissue in the lungs, liver, and bone marrow and/or primary breast cancer tissue) but remain stable in the presence of serum and diverse healthy tissue homogenates (e.g. heart, kidney, liver, lung, bone marrow, and breast), negative and positive selections can be conducted utilizing the platform described in Example 5.
  • tissue homogenates conditioned media from 2D and 3D cultures can be incorporated into positive selections. Following NGS, the sequences can be scored and validated as described in Example 5.
  • the platforms and methods of the present disclosure can Docket No.103361-473WO1 further comprise the following steps or features: 1) Separate biopanning campaigns incorporating positive selections conducted with primary cancer cells and/or metastatic cancer cells at various stages of progression; 2) Peptide prodrugs have potential to benefit patients that do not respond well to immunotherapy. 3) For effective linkers discovered in this platform, attachment of antibodies as targeting groups (e.g. those used in marketed antibody-drug conjugates, such as Trodelvy) can be incorporated. Current linkers on the market undergo off-target release resulting in neutropenia; the linkers discovered by this platform have the potential to overcome this limitation.
  • targeting groups e.g. those used in marketed antibody-drug conjugates, such as Trodelvy
  • SACs Siderophore-antibiotic conjugates
  • Bacteria produce siderophores to sequester Fe(III), an essential nutrient.
  • SACs evolved in bacteria as defenses against other nearby microbes.
  • OM proteins enable SACs to actively transport to the periplasm (and in some species proceed to the cytoplasm), after which the conjugate may bind to its periplasmic targets.
  • protease activity In contrast to traditional proteolytic profiling methods that identify substrate preferences for individual proteases, a method is described that leverages differences in protease activity between complex biological mixtures, each representing microenvironments for host or pathogen. For example, serum and tissue homogenates would represent microenvironments of the host, and periplasmic extracts would represent the periplasm of G – bacteria. Analogous to a single protease, each biological mixture Docket No.103361-473WO1 has a unique proteolytic signature because of the combined activity from associated proteases. This strategy enables identification of a linker that cleaves selectively in one set of microenvironments while remaining stable in others. SPD was originally developed as a method to profile the substrate preferences of a single protease.
  • SPD had never been successfully utilized for the development of cleavable prodrug linkers.
  • Previous efforts by Whitney et al. to use SPD resulted in linkers that had serum instability and cleaved in healthy tissue extracts, which deprioritized prodrug applications.
  • Recent efforts by Boyce et al. to utilize SPD for prodrug linkers led to candidates that were unstable in mouse and human serum, preventing application in an animal model.
  • proteolytic profiling technologies that screen libraries of protease substrates (e.g. multiplex substrate profiling by mass spectrometry (MSP-MS), positional scanning of synthetic combinatorial libraries, and indexed arrays of fluorogenic substrates).
  • SPD Compared to other protease-profiling methods, SPD enables the evaluation of maximal substrate diversity, with greater than 10 9 variants.
  • all existing proteolytic profiling technologies, including SPD were developed for single protease evaluations or comparisons of individual proteases and were not designed to interrogate the substrate profiles of complex biological mixtures with numerous proteases.
  • a phage-based platform is described that enables identification of linker candidates that cleave selectively in one set of microenvironments while remaining stable in others, allowing for the discovery of highly selective prodrug linkers.
  • the platform described is non-obvious because the proteases and their cleavage preferences are unknown with multiple cleavage sites for every enriched candidate identified, which complicates the discovery of consensus sequences.
  • protease or identification of common cleavage motifs among multiple substrates is a critical component of the data analysis process.
  • this approach depends on the ability to identify sequences that are cleaved in one mixture over others independent of the proteases or their cleavage preferences.
  • Proteases in the periplasm a compartment that contains >20 known proteases for protein quality control, are targeted to ensure localization of SAC and target protease, as all SACs pass through the periplasm, including those that eventually proceed to cytoplasm. Targeting multiple proteases minimizes antimicrobial resistance by increasing the evolutionary cost required to prevent conjugate activation.
  • the platform described enabled the discovery of a linker that rapidly cleaved in a broad range of MDR pathogens, while maintaining stability in mouse Docket No.103361-473WO1 serum.
  • the linker enabled controlled and effective release of antibiotic warheads in nine G – strains (Table 1).
  • periplasmic extracts were isolated for conducting biopanning selections. This increases the probability of releasing the drug inside the periplasm, and after release from the peptide, the drug (e.g. chemotherapeutic or antibiotic) is free to diffuse through the inner membrane and bind to its target(s) in the cytoplasm.
  • FIG 3 outlines the process by which a siderophore-chemotherapeutic conjugate is used to facilitate drug delivery into G – bacteria.
  • negative selections with mouse serum were incorporated in the substrate phage platform. Negative selections remove sequences from the phage library that cleave in mouse serum, and the remaining sequences would presumably be serum stable. Positive selections with periplasmic extract would follow and enable the identification of sequences that are serum stable and cleave in a desired bacterial periplasmic environment.
  • step 1 Each round of selection included (FIG 4, step 1): 1) biotinylation of the AviTag, 2) immobilization of the phage library on streptavidin-coated 96-well plates 3) incubation with serum at 37 o C (negative selection), 4) incubation with periplasmic extract at 37 o C (positive selection), 5) amplification of phage- containing cleaved sequences from positive selections (evaluation of titers at this stage), and 6) phage purification and continuation with the next round of selection. Washes were implemented after steps 2 and 3 to remove sequences that stick to surfaces.
  • the phagemid DNA was isolated from phage- infected TG-1 cells (infected with the cleaved phage sequences from one selection) using a ThermoScientific GeneJET Plamid Miniprep Kit.
  • the DNA insert containing the randomized portion of the library was amplified by PCR, purified by DNA gel extraction, and barcoded with PCR using a Kapa HiFi PCR Kit.
  • the samples were submitted for NGS (SE_50). Sequences had an overall 92% high-quality read count, and each sample contained 5-10 million sequence reads.
  • the original library (with maximal diversity) was also sequenced for comparison to calculate sequence enrichment.
  • NGS of both the negative and positive selections enabled scoring of the sequences based on their relative read counts in periplasmic extract and mouse serum throughout all rounds of selection using customized Python scripts (FIG 4, step 2).
  • the sequences were prioritized as described in the Methods (NGS and Data Analysis).
  • the data pointed to more than 700 candidate sequences, and 28 sequences were selected for synthesis as FRET pairs. All FRET pairs were synthesized on solid phase as described in the Methods.
  • Each FRET pair contained a dinitrophenyl (DNP) quencher at the C-terminus and a 7- methoxycoumarin-4- acetic acid (MCA) fluorophore at the N-terminus; fluorescence occurs when the linker is cleaved at any amino acid position (FIG 4, step 3).25 out of the 28 FRET pairs synthesized showed improved cleavage rates in A. baumannii periplasmic extract relative to serum (up to 66-fold, FIG 5).
  • DNP dinitrophenyl
  • MCA methoxycoumarin-4- acetic acid
  • FIG 5 represents a peptide that is plotted as a ratio of its respective cleavage rates in periplasmic extract and serum [rate of cleavage in extract ( ⁇ M/h)/rate of cleavage in serum ( ⁇ M/h)].
  • the cleavage rates for one candidate are shown in FIG 6A for eight Gram-negative periplasmic extracts and serum (also see FIGS 7 and 8).
  • cleavage rates were much higher for A. baumannii and A. nosocomialis relative to non-Acinetobacter species (e.g. E. coli and E. cloacae), which speaks to the quality of the platform.
  • the P1 position is the amino acid directly adjacent to the cleavage site, which is conjugated to the ACC turn-on fluorophore.
  • the cleavage rates of ala-substituted variants were determined in mouse serum and in the periplasmic extracts from six strains of A. baumannii (FIG 12). Importantly, the parent peptide showed the highest stability in mouse serum compared to all ala-substituted variants, which speaks to the platform’s ability to discover peptide linkers that are stable in complex biological mixtures (e.g. serum).
  • the HMM siderophore (see FIG 3) was chosen due to its ability to actively transport antibiotics into diverse bacteria.
  • Solithromycin was chosen as a warhead to determine the quality of the cleavable linker in bacteria and to provide insight into the ability of the conjugate to actively transport through membranes.
  • Solithromycin is a broad- spectrum antibiotic (G + and G – ), and conjugates with this warhead require cleavage for activity, except in cases where the conjugate is actively transported to the cytoplasm, where the intact conjugate engages the target ribosome.
  • SCCs were synthesized through conjugation of an HMM siderophore to candidate linkers on solid phase (see Methods).
  • Mitomycin C was chosen as a chemotherapeutic for conjugation to the C-terminus due to its potency in G + and G – pathogens.
  • MICs were evaluated in G – and G + pathogens.
  • Controls e.g. conjugate without siderophore, conjugate without warhead, conjugate with non-cleavable D-linker
  • HMM siderophore-LEHELG SEQ ID NO: 1
  • mitomycin C EC501 ⁇ M, FIG 14
  • G – pathogens MIC 3-5 ⁇ M, P. aeruginosa, K. pneumoniae, E. aerogenes, and E. coli
  • G + pathogens MIC ⁇ 0.3 ⁇ M, S. aureus.
  • Materials All materials not described below were purchased from commercial suppliers and were of the highest grade available.
  • Mouse serum was purchased in 1.0 or 5.0 mL quantities from MP Biochemicals.
  • the enzyme BirA was expressed according to a known protocol.
  • the HMM siderophore was synthesized by a known protocol. Pathogens and Cell Lines Cell lines and bacteria strains were purchased from ATCC or acquired from BEI Resources. Reagents and Solvents DCM, DMF, THF, diethyl ether, and acetonitrile to be used in anhydrous reaction mixtures were dried by passage through activated alumina columns immediately prior to use. Hexanes used were ⁇ 85% n-hexane. Other commercial solvents and reagents were used as received, unless otherwise noted.
  • Fmoc-protected amino acids were purchased from GL Biochem and Chem-Impex International.2-(6-Chloro-1H- benzotriazole-1-yl)-1,1,3,3- Docket No.103361-473WO1 tetramethylaminium hexafluorophosphate (HCTU), trifluoroacetic acid (TFA) were purchased from Chem-Impex International and Millipore Sigma, respectively.4-Methylpiperidine was purchased from TCI.2,4-Dinitrophenyl (DNP) NovaTag resin was purchased from Millipore Sigma. Fmoc-7-amino-4-carbamoylmethylcoumarin (ACC) Rink amide resin was purchased from Kimia Corporation.
  • TLC thin-layer chromatography
  • Mitomycin conjugates were monitored at 220 nm, 254 nm, and 360 nm for HPLC purifications and UPLC-MS analysis. Mass spectra were obtained using a Waters Acquity UPLC system. Compound names, molecular formulas, and calculated exact masses for compounds were generated using the software built into CambridgeSoft- PerkinElmer's ChemDraw. Methods Periplasmic Extract Isolation: Bacteria were grown from glycerol stocks on LB agar at 37 °C for 18 hours. Added a single colony to 1000 mL of LB base broth. The flask was incubated with shaking until an OD 600 of 0.5 was reached ( ⁇ 5-6 hours).
  • the cultures were centrifuged at 12000 rpm for 10 minutes at 4 °C.
  • the supernatant was poured into bleach, so as not to disturb the pelleted cells.
  • Filter-sterilized 20 % sucrose in 30 mM Tris (50 mL, pH 8) was added to the cell pellets.
  • the pellets were gently suspended with a 50-mL serological pipet to achieve a homogenous Docket No.103361-473WO1 suspension, and the contents were mixed on a shaker for 10 minutes on ice in a cold room at 4 °C.
  • the suspension was then centrifuged at 4150 rpm for 20 minutes at 4 °C.
  • the supernatant was decanted into bleach so as not to disturb the pellets.
  • the filtrate was then concentrated in an Amicon Ultra-15 Centrifugal Filter Ultracel-10kDa (15- mL) by centrifuging at 4150 rpm for up to 15 minutes at 4 °C. The process was repeated until the periplasmic extract reached a volume of ⁇ 2-4 mL and a total protein concentration 1-5 mg/mL as indicated by a Nanodrop. For samples containing a higher total protein concentration than 5 mg/mL, the sample was diluted with Tris (50 mM, pH 8.0) to achieve a concentration ⁇ 5 mg/mL.
  • Tris 50 mM, pH 8.0
  • coli CJ236 was purchased from Lucigen. Pierce streptavidin-coated high-capacity plates were purchased from Docket No.103361-473WO1 ThermoFisher Scientific TM . gBlocks and primers were purchased from IDT.
  • the AviTag- displaying M13 phage libraries were constructed in the phagemid vector pCES1. After digesting the plasmid with ApaL1 and Not1-HF at 37 °C for 3 hours. The backbone was isolated by DNA gel and extracted.
  • Double-stranded DNA was generated according to Sidhu et al. (85, 86) and electroporated the CCC-dsDNA into E. coli TG1 using BioRad Gene Pulser Xcell Total System (1 cm cuvette, 1.8 kV field strength as a preset protocol, time constant: 4.1 seconds). The cells were rescued by adding 1 mL recovery media prewarmed to 37 °C. The media was transferred to a 15-mL culture tube and an additional 1 mL of recovery media was used to rinse the cuvette.
  • the 2-mL culture was incubated for 1 hour at 37 °C with shaking at 200 rpm, and the culture was then plated on three 15-cm LB (AMP) agar plates (0.4 mL culture per plate). The plates were incubated for 15 hours at 37 °C. Added 4-5 mL 2xYT (AMP) media to lift the colonies from the plate.
  • the library phagemid was isolated from the culture using a GeneJET Plasmid Miniprep Kit. The plasmid was incubated with Xh01 to remove the template from the library.
  • XhoI digestion Plasmid (20ug) was digested with 120U of XhoI enzyme in 200 ⁇ L solution for 2 hours.
  • the digestion was then subjected to a PCR clean-up using a Qiagen Kit.
  • the XhoI-cut phagemid was electroporated into TG-1 cells as described above and plated onto LB(AMP) agar plates. Following incubation at 37 °C for 15 hours, the colonies were lifted with 4-5 mL 2xYT (AMP) and transferred to 2xYT (AMP) [25- mL] to provide a culture with an OD of 0.1. The culture was incubated at 37 °C until an OD of 0.5 was achieved.
  • Helper phage was then added to the culture in a 20:1 phage-to-cells ratio, and Docket No.103361-473WO1 the culture was incubated at 37 °C without shaking for 25 minutes. The culture was then incubated at 37 °C with shaking for 25 minutes. The culture was centrifuged, the supernatant was removed, and the pellet was suspended in prewarmed 2xYT containing Kanamycin (25 ⁇ g/ ⁇ L) and Ampicillin (100 ⁇ g/ ⁇ L). The culture was incubated at 30 °C for 15 hours and centrifuged.
  • the supernatant was collected and the phage precipitated with 1/5 volumes of PEG/NaCl solution (20% PEG 8000 w/v, 2.5 M NaCl) on ice for 15 minutes.
  • a library size of 2.0x10 9 cfu/mL was generated.
  • the full protein sequence of the library M- GLNDIFEAQKIEWHE-GGSGG-XXXXXX-GGS-AAAHHHHHH-GAA EQKLISEEDLNGAA-gene 3 protein (g3p), where X is any amino acid.
  • Biotinylation of Phage Library For phage biotinylation, purified phages (4 x 10 12 cfu) were resuspended in 10 mM Tris (0.5 mL, pH 8.0) and concentrated in a 30 kDa MWCO tube; this process was repeated three times to ensure buffer exchange. Then was added water (40 ⁇ L), 2x biotinylation buffer (80 ⁇ L, composition of biotinylation buffer: 0.1 M Tris, 10 mM MgCl 2 , 2 mM Biotin, pH 8.0) to wash the 30 kDa MWCO tube and transfer the remaining solution to a 1.5-mL Eppendorf tube.
  • 2x biotinylation buffer 80 ⁇ L, composition of biotinylation buffer: 0.1 M Tris, 10 mM MgCl 2 , 2 mM Biotin, pH 8.0
  • Periplasmic extracts were diluted to 1-1.5 mg/mL for round 1 and concentrations were further diluted to 0.5-0.6 mg/mL for subsequent rounds. Serum was either used directly without dilution or as a 50% solution in Tris (50 mM, pH 8.0). Each periplasmic extract and serum was only allowed to undergo one freeze-thaw cycle to prevent degradation of protease activity. Phage samples contained 10 12 -10 14 phage as determined by titer analysis.
  • Step 1 Biopanning Procedure: High-binding capacity streptavidin 96-well plates (PierceTM, Thermo Fisher cat no: 15500) are blocked with 2 % BSA for 30 minutes and washed with phosphate-buffered saline (PBS) containing 0.1 % Tween 20 (PBST).
  • PBS phosphate-buffered saline
  • TG-1 culture [15 mL, OD 600 0.5; TG-1 culture was propagated in 2xYT media (without antibiotics) from a single colony grown on M9 minimal salts agar plates purchased from Teknova].
  • the two cultures were incubated at 37 °C without shaking for 25 minutes and subsequently with shaking for 25 minutes.
  • the cultures were centrifuged, supernatant removed, and the pellet was resuspended in 2xYT media (1 mL) and plated on two 15-cm LB/AMP agar plates and allowed to incubate at 37 °C for 18 hours.
  • Ampicillin resistant 2xYT media (20 mL) was added to each plate and the colonies were gently mixed on the agar surface and transferred to a 50-mL Falcon tube. An aliquot of the cultures was removed and miniprepped utilizing the GeneJET Plasmid Miniprep Kit to isolate the phagemid DNA. The cultures (100-200 uL, OD 600 10) were then added to ampicillin resistant 2xYT media (20 mL) until an OD 600 of 0.1 is achieved. The cultures were incubated with shaking at 37 °C until OD6000.5 is achieved ( ⁇ 45 minutes to 1.5 hours). Helper phage (10 uL, 2x10 13 cfu/mL) was added to each culture.
  • the cultures were then incubated at 37 °C without shaking for 25 minutes and with shaking for 25 minutes.
  • the cultures were centrifuged for 10 minutes at 4150 rpm, the supernatant was removed, and the pellets resuspended in prewarmed 2xYT (25 mL, 25 ⁇ g/mL kanamycin and 100 ⁇ g/mL ampicillin).
  • the cultures were incubated for 12 hours at 30 °C, centrifuged at 4150 rpm for 30 minutes or 15,000 rpm for 10 minutes, the supernatant was isolated, and the phage were precipitated by adding 5 mL of PEG/NaCl solution (20% PEG 8000 w/v, 2.5 M NaCl).
  • the supernatant/PEG salt solution mixture was allowed to sit on ice for 15-30 minutes before centrifuging to obtain a white pellet of M13 phage containing the amplified sequences of interest.
  • the phage were resuspended in PBS (1-2 mL, pH 7.4) and buffer exchanged into 10 mM Tris (pH 8) in a 5 mL Amicon filter tube (30 kDa).
  • Step 2 PCR Amplification and Barcoding: For each sample, the isolated phagemid DNA (2 ⁇ L, ⁇ 5 ng/uL) prepared as indicated above using a GeneJET Plasmid Miniprep Kit) was subjected to PCR amplification to amplify the insert containing the randomized portion of the library.
  • the following reverse and forward primers (2 ⁇ L, 10 ⁇ M) were added to the phagemid respectively: GAGTTCAGACGTGTGCTCTTCCACACTCTTTCCCATGGTGATGATGATGTGCGG (SEQ ID NO: 82) and AATGATACGGCGACCACCGAGATCTACACGCAGAAATTGAATGGCATGA (SEQ ID NO: 83).
  • Kapa HiFi fidelity buffer (5X, 10 ⁇ L), dNTPs (1 ⁇ L, 10 mM), H 2 O (32 ⁇ L), and KAPA HiFi polymerase (1 ⁇ L) were then added to the reaction mixture in a 0.2-mL PCR tube.
  • PCR amplification proceeded under the following conditions in sequence: 1) 95 °C for 3 minutes, 2) 98 °C for 20 seconds, 3) 65 °C for 15 seconds, 4) 72 °C for 15 seconds, 5) 72 °C for 1 minutes, 6) repeat steps 2-4 for 20 cycles, 7) 4 °C.
  • the product from the PCR reaction was purified by DNA gel and the relevant band was excised and the DNA was extracted using a GeneJET Gel Extraction Kit. The isolated phagemid DNA was barcoded.
  • the barcoding PCR involved the following components: plasmid (2 ⁇ L, 5 ng/ ⁇ L), Kapa HiFi fidelity buffer (5x, 10 ⁇ L), forward barcoding primer AATGATACGGCGACCACCGAGATCTACACGCAGAAAATTGAATGGCATGA (SEQ ID NO: 84) (5 ⁇ L, 0.1 M), dNTPs (1 ⁇ L, 10 mM), H 2 O (25 ⁇ L), True-Seq-barcode-primer CAAGCAGAAGACGGCATACGAGATXXXXXXXGTGACTGGAGTTCAGACGTGTGCT CTTCC (SEQ ID NO: 85) (5 ⁇ L, 0.1 ⁇ M, note: XXXXXX (SEQ ID NO: 86) represents the reverse compliment of the unique barcode used; a total of 47 unique barcodes were utilized in Docket No.103361-473WO1 these experiments), and KAPA HiFi Polymerase (1 ⁇ L).
  • the reaction mixture was subjected to the following PCR protocol: 1) 94 °C for 1 minute, 2) 94 °C for 30 seconds, 3) 60 °C for 30 seconds, 4) 72 °C for 1 minute, 5) repeat steps 2-4, 6) 94 °C for 10 minutes (at this stage, added 1 ⁇ L of a 10 ⁇ M hot start primer mix containing the following primers at this stage: AATGATACGGCGACCACC (SEQ ID NO: 87) and CAAGCAGAAGACGGCATAC (SEQ ID NO: 88); primers were obtained commercially from IDT and used as 100 ⁇ M stock solutions), 7) 94 °C for 30 seconds, 8) 55 °C for 30 seconds, 9) 72 °C for 1 minute, 10) repeat steps 7-9 twelve times, 11) 72 °C for 5 minutes.
  • the PCR product was diluted with water to a total volume of 100 ⁇ L and purified with the GeneJET PCR Purification Kit (note: isopropanol was used to improve the DNA yield).
  • a DNA gel was run on all samples to confirm the purity of each product. Any samples that contained more than one band were purified by DNA gel extraction as described above. From these experiments, a total of 150 samples were barcoded, which included cleavage data from each positive (periplasmic extract) and negative (mouse serum) selection conducted for each of up to nine rounds of selection, along with biological replicates. The concentration of each sample was adjusted to 3 ng/ ⁇ L (23.7 nM) as determined using the Quant-iT Qubit dsDNA HS Assay Kit and a Qubit 4 fluorometer.
  • Each phagemid sample were combined into four LowBind 1.5-mL Eppendorf tubes in 10 ⁇ L quantities ( ⁇ 38 samples per tube). A DNA gel was run on each combination to reveal a single band.
  • the custom Read1 primer for NGS sequencing is GACCACCGAGATCTACACGCAGAAAATTGAATGGCATGA (SEQ ID NO: 89) (100 uM, 10 uL), which was added to a separate LowBind 1.5-mL Eppendorf tube.
  • the library construct size for each sample was 192 base pairs. The samples were analyzed by HiSeq_SE50.
  • the workflow for data analysis was conducted in three steps: 1) The positive/negative read count was compared for each sequence in each round of selection, which enables the identification of sequences that were found more frequently in positive selections than in negative selections for each individual round; 2) The pos/negative read counts for sequences were compared across multiple rounds of selection to identify sequences that consistently cleaved more frequently in positive selections over negative selections; 3) Select sequences are scored and ranked based on their frequency of reads in positive relative to negative selections across the entire data set. The extent of sequence enrichment in subsequent rounds of selection relative to the initial na ⁇ ve library was also considered when prioritizing sequences for synthesis and evaluation of cleavage kinetics. A total of thirty sequences were prioritized for evaluation.
  • step 1) any sequence that has a positive read count greater than or equal to 100 and a positive/negative read count greater than or equal to 3 would be counted in category 1 for any round of selection
  • step 2) any sequence that has a positive/negative read count less than 3 but greater than or equal to 1.5 would be counted in category 2
  • step 3) any sequence that has a positive/negative read count between 1 and 1.5 would be counted in category 3
  • step 4) any sequence that has a negative/positive read count between 1 and 1.5 would be counted in category 4
  • step 5) any sequence that has a negative/positive read count greater than 1.5 could be counted in category 5
  • step 6) any sequence that has a positive read count less than 5 and a negative round count less than 5 would be considered insignificant and not included in the analysis.
  • Sequences that were found primarily in categories 1 and 2, with minimal occurrences in category 5 across all rounds of selection were prioritized.
  • one sequence (SADLNNQ, SEQ ID NO: 40) that was highly enriched in rounds 6, 7, 8, and 9 was also prioritized, which was independent of the scoring platform described.
  • a total of 28 sequences were selected for synthesis and evaluation.
  • a total of 25 sequences showed improved cleavage rates in periplasmic extract relative to serum. Three sequences were false positives.
  • Fluorescence Assays Cleavage of MCA-Linker-DNP (FRET Pairs) and Ac-Linker-ACC conjugates in Periplasmic Extract and Serum: Docket No.103361-473WO1 Periplasmic extracts and mouse serum were not diluted prior to use in fluorescence assays and were allowed to undergo only one freeze-thaw cycle prior to use. The total protein for different periplasmic extracts used in these experiments are listed above. Solutions for substrate (40 ⁇ M, 2X) were prepared in Tris (pH 8.0) from 2 mg/mL stock solutions in DMSO.
  • Each 40 ⁇ M solution (25 ⁇ L) was then transferred to a 96-well plate (opaque, 200 ⁇ L/well round bottom) in triplicate. Flash frozen periplasmic extract and commercial mouse serum were slowly thawed in a cold room at 4°C over 15 min. Periplasmic extract (25 ⁇ L, Total Protein: 1-5 mg/mL as listed above) or mouse serum (25 ⁇ L, Total Protein: > 50 mg/mL) was transferred in triplicate to wells containing substrate in a cold room at 4 °C.
  • the plate was sealed with a transparent polyolefin silicone film (Thermofisher TM Nuc TM Sealing Tape, 12-565-513) and placed in a plate reader at 37 °C with shaking at 225 rpm for 8 hours.
  • MCA excitation maximum 325.
  • MCA emission maximum 392 nm.
  • ACC excitation maximum 355 nm.
  • ACC emission maximum is 460 nm.
  • MIC Minimum Inhibitory Concentration
  • Broth and Agar Preparation Mueller-Hinton II (MH-II, cation-adjusted) broth was prepared from solid MH-II (22 g) and MilliQ H2O (1 L), which was autoclaved in a presterilized 1-L glass bottle.
  • the autoclaved MH-II media contained dipyridyl (DP, 200 ⁇ M) for MIC assays involving siderophore conjugates.
  • MH-II agar plates were prepared from autoclaved MH-II (22 g), Bacto agar (17 g), and MilliQ water (1 L).
  • Bacteria Culture Preparation Bacteria were streaked on MH-II agar plates from glycerol stocks. The streaked plates were placed in an incubator at 37 °C without shaking for 18- 20 hours. A sterilized pipet tip was gently touched on the surface of a single colony and added to autoclaved MH-II broth (5 mL) in a sterilized 14-mL culture tube. The cultures were incubated for 18 hours at 37 °C with shaking at 215 rpm.
  • the cultures were diluted 200-fold into autoclaved MH-II broth (4 mL) and incubated at 37 °C with shaking at 215 rpm for 3-5 hours, or until an OD 600 of 0.5 was achieved.
  • An aliquot of the culture (1 mL) was transferred to a sterilized culture tube and diluted with filter-sterilized PBS ( ⁇ 7 mL) until an OD 600 of 0.13- 0.15 was achieved.
  • An aliquot (11.5 ⁇ L-10 ⁇ L) of the cultures was then transferred into MH-II media (15-mL) to achieve a concentration of 1x10 5 cells/mL.
  • the diluted cultures (90 ⁇ L/well) were then added to substrate (10 ⁇ L at the following concentrations: 640 ⁇ g/mL, 320 ⁇ g/mL, 160 ⁇ g/mL, 80 ⁇ g/mL, 40 ⁇ g/mL, 20 ⁇ g/mL, 10 ⁇ g/mL, and 0 ⁇ g/mL in sterilized 96-well plates to achieve a final substrate concentration of 64 ⁇ g/mL, 32 ⁇ g/mL, 16 ⁇ g/mL, 8 ⁇ g/mL, 4 ⁇ g/mL, Docket No.103361-473WO1 2 ⁇ g/mL, 1 ⁇ g/mL, and 0 ⁇ g/mL.
  • HL-60 cells were cultured at 37 °C in a 5 % CO2 atmosphere. The cells were grown in Iscove’s Modified Dulbecco’s Medium, supplemented with 20 % fetal bovine serum (FBS) and 0.1 % penicillin-streptomycin. Media was replaced every other day until cells reached the desired confluency.
  • FBS fetal bovine serum
  • SPPS Solid-phase peptide synthesis
  • Each amino acid (5.0 equiv, 0.5 M in DMF) was attached via two sequential coupling steps with HCTU (5.0 equiv, 0.5 M in DMF) and DIEA (10 equiv, 0.5 M in DMF). At least three washes were conducted after Fmoc deprotection and coupling steps. Resin cleavage from 2-chlorotrityl resin was accomplished with AcOH/TFE/DCM (1:1:3) to provide peptide without removal of acid-sensitive protecting groups.
  • a Biotage peptide synthesizer was used to couple amino acids P1-P7 (15 equiv, 500 mM) to DNP NovaTag resin (63 mg, 0.025 mmol) with HCTU (15 equiv, 500 mM) and DIEA (30 equiv, 500 mM) using microwave-assisted synthesis (55 °C, 5 minutes). Deprotections with 20% 4-methylpiperidine were conducted at 75 oC (3 minutes). Swelling of the starting P1-conjugated Dnp NovaTagTM resin (Novabiochem, 0.30-0.60 mmol/g loading) was achieved in DMF (70 oC, 20 minutes).
  • Reaction cycle includes Fmoc deprotection, washing, coupling, and post-coupling washing steps. After the final Fmoc deprotection of the last amino acid, the resin is washed with DMF (3 x 5 mL), MeOH (3 x 5 mL), and DCM (3 x 5 mL). The resin is then dried under reduced pressure.
  • Step 2 Synthesis of MCA-Linker-DNP-resin: Attachment of MCA fluorophore to N-terminus. The starting resin was swelled in DCM (1 x 1.5 mL) prior to use.
  • Chem., 2002, 67, 910) are prepared from ACC-rink amide AM resin (Fmoc-ACC resin) by the following general procedure: To a 50-mL glass solid- phase peptide synthesis vessel containing Fmoc-ACC resin (300 mg, 0.045 mmol) was added DMF (15 mL), and the resin was swelled by mixing for 15 min. Fmoc deprotection was achieved by mixing 4-methylpiperidine (10 mL, 40% in DMF) with the ACC resin for 3 min, followed by mixing 4-methylpiperidine (10 mL, 20% in DMF) for 10 min. The resin was then washed with DMF (4x6 mL), with mixing for 3 minutes each.
  • the Fmoc-amino acid-ACC resin (0.045 mmol) prepared from Step 1 was then transferred to a 10-mL Biotage reaction vessel, and the remainder of the amino acids were coupled on a Biotage peptide synthesizer. Deprotections were accomplished with 20% 4-methylpiperidine at 75 oC (3 minutes). Amino acid couplings were accomplished with Fmoc-amino acid-OH (15 equiv, 500 mM), HCTU (15 equiv, 500 mM), and DIEA (30 equiv, 500 mM) using microwave-assisted synthesis (55 °C, 5 minutes). Swelling was achieved in DMF (70 oC, 20 minutes).
  • Reaction cycle includes Fmoc deprotection, washing, coupling, and post-coupling washing steps. After the final Fmoc deprotection of the last amino acid, the resin is washed with DMF (3 x 5 mL), MeOH (3 x 5 mL), and DCM (3 x 5 mL). The resin is then dried under reduced pressure.
  • S tep 3 Preparation of Ac-Linker-ACC resin : Fmoc deprotection and acetylation.
  • the Fmoc-Linker-ACC resin from Step 2 was Fmoc deprotected by mixing 40% 4- methylpiperidine (10 mL) for 3 min, followed by mixing 20% 4-methylpiperidine for 10 minutes.
  • Step 1 Preparation of Fmoc-glycine-2-chlorotrityl resin: To a 500-mL flame-dried, round-bottom flask was added dried Fmoc-glycine (446 mg, 1.5 mmol, predried on high vacuum for 12 hours).
  • the flask was purged with argon, and the amino acid was then suspended in anhydrous DCM (20 mL). The flask was sonicated for 5 minutes to assist with solubilizing the amino acid.
  • Dry 2-chlorotrityl resin (Chem-Impex, 2.00 g, 3.0 mmol, 1.0-2.0 mmol/g, predried on high vacuum for 12 hours) was added to the amino acid suspension in one portion, and the sides of the flask were rinsed with additional anhydrous DCM (10 mL).
  • DIEA 0.6 mL, 3.45 mmol
  • the resin was then filtered through a 50-mL solid phase peptide synthesis vessel and washed with DCM (3 x 20 mL). Resin capping was achieved by adding a 2.5% solution of MeOH (0.4 mL) and DIEA (0.4 mL) in DCM (15.2 mL), and the solid phase peptide synthesis vessel was rotated for 30 minutes on a nutator. The resin was then washed with DCM (3 x 20 mL) and dried on high vacuum to provide 2.37 g (1.79 mmol, 0.755 mmol/g, 60% yield) of Fmoc-glycine-2-chlorotrityl resin.
  • Step 2 Preparation of Fmoc-LEHELG-2-chlorotrityl resin: To a fritted SPPS reaction vessel containing Fmoc-Gly-2-chlorotrityl resin (1.78 g, 0.537 mmol/g loading) was added 20% 4-methylpiperidine in DMF (50 mL), followed by nitrogen mixing for 20 minutes. After removing the deprotection solution, the resin was washed with DMF (4 x 100 mL) with mixing for 5 minutes each. This process was repeated.
  • the final resin was washed with DMF (3 x 100 mL), MeOH (2 x 100 mL), and DCM (3 x 100 mL), and dried under reduced pressure to provide Fmoc-LEHELG-2-chlorotrityl resin (1.79 g, 0.686 mmol, 0.383 mmol/g loading, 72 %).
  • Step 3 Preparation of LEHELG-2-chlorotrityl resin: To the Fmoc-LEHELG-2- chlorotrityl resin (407.0 mg, 0.16 mmol 0.383 mmol/g) isolated from Step 2 was added 20% 4- methylpiperidine, followed by mixing for 10 minutes (2 x 6 mL) on a nutator. After each Fmoc- deprotection, the resin was washed with DMF (4 x 6 mL, mixed for 3 minutes/wash) and DCM (3 x 6 mL, allowed to sit for 3 minutes/wash without mixing).
  • Step 4 Preparation of Protected-HMM Siderophore-LEHELG-OH: To a fritted 10- mL Biotage peptide synthesis vessel containing LEHELG-2-chlorotrityl resin (149.0 mg, 0.057 mmol 0.383 mmol/g) from Step 3 was added a solution of protected HMM siderophore (183 ⁇ L, 500 mM in DMF, 1.6 equiv) (Boyce et al.
  • reaction mixture was filtered into a 20- mL scintillation vial and the resin was rinsed with DCM (3x3mL). The combined filtrate was concentrated under reduced pressure. The crude residue was sonicated and azeotropped with benzene (3 x 5 mL) to provide protected-HMM siderophore-LEHELG-OH (73.6 mg, 72 %) without the need for further purification.
  • Step 5 Preparation of Protected-HMM Siderophore-LEHELG-Solithromycin bis- TFA salt: To a 5-mL flame-dried, round-bottom flask was added the protected-HMM siderophore-LEHELG-OH (26.9 mg, 15.1 ⁇ mol) from Step 4, solithromycin (25.0 mg, 29.6 ⁇ mol, 1.96 equiv), HOBt (20% water, 5.79 mg, 30.2 ⁇ mol, 2.0 equiv), and EDC (4.35 mg, 22.7 ⁇ mol, 1.5 equiv).
  • the reaction mixture was concentrated under reduced pressure and azeotroped with benzene (3 x 3 mL) with sonication between azeotropes.
  • the solid residue was washed with Et 2 O (3 x 5 mL) and filtered through a cotton plug.
  • the crude solid was dissolved in DMSO:H2O (1:1, 4 mL) and purified by HPLC (5 - 80 % MeCN in H2O with 0.1 % TFA) to provide HMM siderophore-LEHELG-solithromycin tri-TFA salt (3C, 9.7 mg, 60 %) as a white powder after lyophilization.
  • the flask was purged with argon, followed by the addition of anhydrous MeCN (1.10 mL, 0.02 M) and triethylamine (6.7 uL, 47.7 ⁇ mol, 2.2 equiv) forming a suspension.
  • the reaction mixture was cooled to 0 °C and allowed to stir for 10 minutes at this temperature.
  • p- Nitrophenyl chloroformate (9.6 mg, 47.7 ⁇ mol, 2.2 equiv) was then added. After stirring for 5 min, 4-dimethylaminopyridine (0.50 mg, 4.3 ⁇ mol, 0.2 equiv) was added and the reaction mixture was allowed to stir for 5 minutes at 0 °C.
  • HMM siderophore-LEHELG-OPNP (18.7 mg, 86 %) as a white solid.
  • Exact mass calculated for C56H71N11O20: 1217.49, ESI LCMS: [M+H] 1218.4 Docket No.103361-473WO1 Step 2.
  • the reaction flask was purged with argon and anhydrous DMF (0.48 mL) was added. The reaction mixture was stirred for 30 minutes at room temperature. Triethylamine (7.9 ⁇ L, 0.6 M in DMF, 1.0 equiv) was then added over the course of 3 minutes. The reaction mixture was allowed to stir for 45 minutes at room temperature. Another equivalent of triethylamine (7.9 ⁇ L, 0.6 M in DMF, 1.0 equiv) was then added, and the reaction was allowed to stir for an additional 30 minutes.
  • ACC conjugates prepared by the General procedure for the synthesis of ACC Conjugates: Docket No.103361-473WO1 Docket No.103361-473WO1 Table 4. HMM Siderophore-Linker-Siderophore and HMM Siderophore-Linker-Mitomycin C conjugates: Docket No.103361-473WO1
  • X refers to any chiral amino acid including, but not limited to alanine, methionine, glycine, cysteine, aspartic acid, phenylalanine, glutamic acid, histidine, isoleucine, lysine, leucine, asparagine, glutamic acid, arginine, serine, threonine, valine, tryptophan, proline, and tyrosine.
  • X refers to any nucleotide including, but not limited to adenosine, cytosine, guanine, and thymine.

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Abstract

L'invention concerne des agents de liaison clivables par des protéases et des compositions de ceux-ci destinées à être utilisées pour le traitement et/ou la prévention de maladies, comprenant, sans y être limitées, le cancer. L'invention concerne également des procédés de fabrication et de criblage d'agents de liaison clivables par des protéases.
PCT/US2024/030018 2023-05-18 2024-05-17 Compositions d'agents de liaison de promédicament et procédés d'utilisation Pending WO2024238956A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022179572A1 (fr) * 2021-02-25 2022-09-01 Shanghai Allygen Biologics Co., Ltd. Conjugués de ciblage avec des agents thérapeutiques et oligonucléotides et leurs utilisations
US20220324975A1 (en) * 2019-06-05 2022-10-13 Chugai Seiyaku Kabushiki Kaisha Antibody cleavage site binding molecule
US20220387610A1 (en) * 2019-09-18 2022-12-08 Brandeis University Peptide-conjugated prodrugs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220324975A1 (en) * 2019-06-05 2022-10-13 Chugai Seiyaku Kabushiki Kaisha Antibody cleavage site binding molecule
US20220387610A1 (en) * 2019-09-18 2022-12-08 Brandeis University Peptide-conjugated prodrugs
WO2022179572A1 (fr) * 2021-02-25 2022-09-01 Shanghai Allygen Biologics Co., Ltd. Conjugués de ciblage avec des agents thérapeutiques et oligonucléotides et leurs utilisations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BORIS RATNIKOV, PIOTR CIEPLAK & JEFFREY W. SMITH: "High Throughput Substrate Phage Display for Protease Profiling", PROTEASES AND CANCER, vol. 2009, no. 539, 1 January 2009 (2009-01-01), pages 93 - 114, XP009559056, ISBN: 978-1-60327-002-1 *
BOYCE JONATHAN H., DANG BOBO, ARY BEATRICE, EDMONDSON QUINN, CRAIK CHARLES S., DEGRADO WILLIAM F., SEIPLE IAN B.: "Platform to Discover Protease-Activated Antibiotics and Application to Siderophore–Antibiotic Conjugates", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, vol. 142, no. 51, 23 December 2020 (2020-12-23), pages 21310 - 21321, XP055920230, ISSN: 0002-7863, DOI: 10.1021/jacs.0c06987 *

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