WO2015130846A2 - Compositions and methods for the site-specific modification of polypeptides - Google Patents

Compositions and methods for the site-specific modification of polypeptides Download PDF

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WO2015130846A2
WO2015130846A2 PCT/US2015/017601 US2015017601W WO2015130846A2 WO 2015130846 A2 WO2015130846 A2 WO 2015130846A2 US 2015017601 W US2015017601 W US 2015017601W WO 2015130846 A2 WO2015130846 A2 WO 2015130846A2
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seq
amino acid
polypeptide
agent
srta
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PCT/US2015/017601
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French (fr)
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WO2015130846A3 (en
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Ashutosh Chilkoti
Joseph J. BELLUCCI
Jayanta Bhattacharyya
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Duke University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment

Definitions

  • compositions and methods for the site-specific modification of polypeptides with agents relate to compositions and methods for the site-specific modification of polypeptides with agents.
  • Small molecule drugs that are too toxic to be administered on their own can be directed to diseased tissues by covalent conjugation to a targeting protein.
  • ADCs antibody-drug conjugates
  • Antibodies contain eight cysteines involved in interchain disulfide bonds and a large, variable number of lysines that can serve as attachment sites for small molecules. While the average number of conjugated drugs can be controlled, the product is a heterogeneous mixture of antibodies containing between zero and eight drugs that are stochastically attached to available conjugation sites.
  • Both the number of conjugated drugs and their locations in the protein may negatively impact the efficacy of ADC treatment. For instance, modification of lysines in the antibody's variable regions can reduce affinity for its target, thereby increasing the amount of circulating ADC and the likelihood of off-target toxicity. Similarly, modification of lysines or cysteines in the Fc domain can affect the bioavailability of the drug by altering ADC clearance.
  • the method may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZG (SEQ ID NO: 72) wherein X and Z are independently any amino acid, and wherein the ⁇ -amino group of the lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
  • SertA sortase A
  • the method may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid and wherein the carboxy- terminus of the amino acid Z of LPXZ (SEQ ID NO: 75) is modified to a methyl ester, and wherein the ⁇ -amino group of the lysine of the first polypeptide and the Z amino acid of LPXZ (SEQ ID NO: 75) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
  • SertA sortase A
  • the method may include administering the agent-first polypeptide conjugate formed by methods of conjugating an agent to a first polypeptide.
  • the method of conjugating an agent to a first polypeptide may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZG (SEQ ID NO: 72) wherein X and Z are independently any amino acid, and wherein the ⁇ -amino group of the lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
  • SertA sortase A
  • the method of conjugating an agent to a first polypeptide may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid and wherein the carboxy- terminus of the amino acid Z of LPXZ (SEQ ID NO: 75) is modified to a methyl ester, and wherein the ⁇ -amino group of the lysine of the first polypeptide and the Z amino acid of LPXZ (SEQ ID NO: 75) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
  • SertA sortase A
  • agent-first polypeptide conjugates may be formed by methods of conjugating an agent to a first polypeptide.
  • the method of conjugating an agent to a first polypeptide may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZG (SEQ ID NO: 72) wherein X and Z are independently any amino acid, and wherein the ⁇ -amino group of the lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
  • SertA sortase A
  • the method of conjugating an agent to a first polypeptide may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid and wherein the carboxy- terminus of the amino acid Z of LPXZ (SEQ ID NO: 75) is modified to a methyl ester, and wherein the ⁇ -amino group of the lysine of the first polypeptide and the Z amino acid of LPXZ (SEQ ID NO: 75) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
  • SertA sortase A
  • FIG. 1 is a schematic of pilin assembly by sortases in Gram-positive bacteria,
  • SpaA Secreted pilin monomer protein
  • the pilin polymer extends after the intermediate undergoes nucleophilic attack by the ⁇ -amino group of a lysine in the next monomer, forming an isopeptide bond
  • a housekeeping sortase terminates polymerization by anchoring the assembled pilin chain to a pentaglycine branch in the peptidoglycan through a native peptide bond.
  • FIG. 2 is a schematic of sortase A from S. aureus catalyzing isopeptide bond formation at the pilin domain lysine, (a) Schematic of the polymerization reaction model for isopeptide bond formation, (b) SDS-PAGE of the reaction product, (c) Maximum monomer consumption in the polymerization reaction was achieved between 29°C and 34°C. (d) MALDI- TOF mass spectrometry of a tryptic digest of reaction products.
  • FIG. 3 is a schematic of sortase catalyzing site-specific protein-small molecule conjugation, (a) Overview of the reaction used to conjugate biotin to the pilin domain in the Fn3- PLNs-ELP (SEQ ID NO: 13) fusion protein, (b) SDS-PAGE of biotinylation reactions with Fn3- ELP (SEQ ID NO: 14; 36 kDa) and Fn3-PLN 3 -ELP (SEQ ID NO: 13; 43 kDa).
  • FIG. 4 is a gel and images of the sortase-catalyzed biotinylation reaction generating bioactive protein-small molecule conjugates, (a) Western blot of the CEX load, non-bound (NB), and elution (Elu) fractions for biotinylated Fn3-PLN 3 -ELP. (b) Fluorescence microscopy to show the uptake of biotinylated Fn3-PLN 3 -ELP, indicating nuclei, glycoproteins (for cell morphology), and intracellular biotin. (c) Fluorescence microscopy to show the reduced uptake of biotinylated Fn3-PLN 3 -ELP with a 10-fold molar excess of unlabeled Fn3-ELP.
  • FIG. 5 is schematic overview of the transpeptidation reaction catalyzed by SrtA in S. aureus
  • SrtA breaks the peptide bond between the threonine and glycine residues and forms an enzyme-substrate intermediate through a thioester bond between the threonine residue and the catalytic cysteine
  • the acyl-enzyme intermediate is stable but is resolved after nucleophilic attack by the oamino group of a pentaglycine branch of the cell wall
  • the catalytic thiol of SrtA is regenerated and the substrate protein is anchored to the peptidoglycan through a native peptide bond.
  • FIG. 6 are graphs of the thermal responsiveness of the SrtA-ELP fusion protein used in this study, (a) Absorbance at 350 nm versus temperature for various SrtA-ELP concentrations, (b) Transition temperature versus SrtA-ELP concentration.
  • FIG. 7 is a graph analyzing the reaction product components for polymerization of ELP-GLP1 (SEQ ID NO: 70).
  • FIG. 8 is a schematic diagram and a gel to demonstrate the effect of pH on the efficiency of the ELP-GLP1 (SEQ ID NO: 70) polymerization reaction, (a) Schematic of the reaction, (b) SDS-PAGE of products after reacting SrtA-ELP (SEQ ID NO: 1 1 ) with ELP-GLP1 (SEQ ID NO: 70) at various pHs.
  • FIG. 9 is the structure of biotin-LPET (SEQ ID NO: 29).
  • FIG. 10 is a schematic diagram and spectra for the SrtA-ELP-catalyzed reaction of biotin-LPETGRAGG (SEQ ID NO: 10) peptide and Fn3-PLN 3 -ELP (SEQ ID NO: 13).
  • FIG. 11 is a schematic diagram and spectra for the conjugation of dabcyl to ELP- GLP1 (SEQ ID NO: 70).
  • FIG. 12 is a schematic diagram and spectra for the analysis of peptides of ELP- GLP1 (SEQ ID NO: 70) conjugated with dabcyl-LPETG-edans (LPETG is SEQ ID NO: 15) without including triglycine in the trypsinization reaction, (a) Schematic of the reaction, (b) The MALDI-TOF spectrum for the unpurified reaction product.
  • FIG. 13 is a graph and a gel for the purification of biotinylated Fn3-PLN 3 -ELP (SEQ ID NO: 13).
  • FIG. 14 are graphs for the quantitation of the biotin: protein molar ratio for the Fn3- PLN 3 -ELP (SEQ ID NO: 13) biotinylation reaction using fluorescent (a) and colorimetric (b) HABA displacement assays.
  • FIG. 15 are additional images for analysis of the uptake of biotinylated Fn3-PLN 3 - ELP (SEQ ID NO: 13) by HUVECs.
  • FIG. 16 are graphs from flow cytometry to confirm ⁇ ⁇ ⁇ 3 expression on HUVECs.
  • HUVECs were stained with either biotinylated mouse lgG1 ⁇ (filled histogram), unreacted Fn3-PLN 3 -ELP (left peak, open histogram), or biotinylated Fn3-PLN 3 -ELP (right peak, open histogram) followed by secondary detection with streptavidin-FITC.
  • FIG. 17 is an exemplary schematic diagram of a reaction catalyzed by SrtA to form the agent-first polypeptide conjugate.
  • FIG. 18 shows that FITC can be conjugated to substrate proteins by sortase- mediated isopeptide ligation
  • FIG. 19 is a schematic diagram of the isopeptide ligation reaction catalyzed by sortase A with peptide substrates, (a) Scheme showing the sortase-mediated isopeptide ligation of a peptide containing the pilin domain with a peptide containing the sortase recognition sequence LPETG (SEQ ID NO: 15) and an amino-terminal biotin. (b) MALDI-TOF mass spectra with and without SrtA-ELP (SEQ ID NO: 1 1 ).
  • FIG. 20 shows that the His-tagged SrtA (H6-SrtA; SEQ ID NO: 49) achieved site- specific conjugation of biotin to the pilin domain lysine, (a) SDS-PAGE of reactions of H6-SrtA (SEQ ID NO: 49) and Fn3-PLN3-ELP (SEQ ID NO: 13) with or without biotin-LPETGRAGG (SEQ ID NO: 10) peptide, (b) Western blot of the gel in panel (a) using streptavidin-Cy5 to detect biotinylated protein, (c) The MALDI-TOF mass spectrum of tryptic peptides of the unpurified 20°C biotinylation reaction.
  • H6-SrtA SEQ ID NO: 49
  • Fn3-PLN3-ELP SEQ ID NO: 13
  • biotin-LPETGRAGG SEQ ID NO: 10
  • FIG. 21 is a fully annotated version of the MS1 spectrum for LC-MS/MS corresponding to FIG. 24a and FIG. 13.
  • MS mass spectrometry
  • EICs Extracted ion chromatograms
  • FIG. 24 shows that sortase-mediated isopeptide ligation can be used to modify monoclonal antibodies (mAbs).
  • mAbs monoclonal antibodies
  • FIG. 25 are graphs for the quantitation of the biotin-to-protein molar ratio for anti- Her2 with one pilin domain inserted at the carboxy-terminus of each heavy chain.
  • FIG. 26 are additional images for the SK-OV-3 immunofluorescence of FIG. 24.
  • FIG. 27 shows reaction of SrtA with various truncated isopeptide attachment sequences
  • SrtA-ELP SEQ I D NO: 1 1
  • biotin- LPETGRAGG SEQ I D NO: 10
  • Fn3-I PA-ELP fusion proteins SEQ I D NO: 10
  • MALDI-TOF spectra of the reaction products for fusion proteins containing IPA sequences MALDI-TOF spectra of the reaction products for fusion proteins containing IPA sequences.
  • FIG. 28 is a Western Blot of reactions with SrtA, biotin-LPETGRAGG (SEQ I D NO: 10), and a panel of polypeptides with mutant pilin domains.
  • bioactive conjugates are a result of site-specific protein modifications that make use of lysine chemistry for attachment of one or more agents to proteins.
  • the method centers on a previously uncharacterized activity of the transpeptidase sortase A (SrtA), which catalyzes the covalent attachment of two moieties through a site-specific isopeptide bond.
  • SertA transpeptidase sortase A
  • a first polypeptide is contacted with sortase A (SrtA) and at least one agent conjugated to a second polypeptide.
  • the first polypeptide includes at least one lysine, such as, for example, in an amino acid sequence consisting of ZX 1 X 2 X 3 VX 4 VYPKH (SEQ I D NO: 1 ), where Xi, X 2 , X 3 , and X 4 are independently any amino acid and Z is any hydrophobic amino acid.
  • the second polypeptide includes an amino acid sequence consisting of, for example, LPXZG (SEQ I D NO: 72), wherein X and Z are independently any amino acid.
  • the resulting agent-first polypeptide conjugate includes an isopeptide bond between the ⁇ -amino group of the lysine, such as the lysine of ZX 1 X 2 X 3 VX 4 VYPKH (SEQ I D NO: 1 ), and the Z amino acid of LPXZG (SEQ I D NO: 72).
  • the method results in site-specific protein modifications that contribute to the rapidly expanding repertoire of therapeutic protein-drug and protein-polymer conjugates.
  • the SrtA catalyzed reaction was applied in vitro to generate recombinant protein polymers with unique, branched structures as well as receptor-targeting proteins loaded with multiple small molecules.
  • the reaction displays a level of control over the site of conjugation; the modification occurred exclusively at a lysine ⁇ -amino group within an engineered site in the protein substrate. Accordingly, the method disclosed herein provides a unique chemistry for the generation of lysine-modified protein conjugates and allows the precise control of the number of conjugated molecules as well their location.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • the term "about” refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • Polymer as used herein is intended to encompass a homopolymer, heteropolymer, block polymer, co-polymer, ter-polymer, etc., and blends, combinations and mixtures thereof.
  • polymers include, but are not limited to, functionalized polymers, such as a polymer comprising 5-vinyltetrazole monomer units and having a molecular weight distribution less than 2.0.
  • the polymer may be or contain one or more of a star block copolymer, a linear polymer, a branched polymer, a hyperbranched polymer, a dendritic polymer, a comb polymer, a graft polymer, a brush polymer, a bottle-brush copolymer and a crosslinked structure, such as a block copolymer comprising a block of 5-vinyltetrazole monomer units.
  • Polymers include, without limitation, polyesters, poly(meth)acrylamides, poly(meth)acrylates, polyethers, polystyrenes, polynorbornenes and monomers that have unsaturated bonds. For example, amphiphilic comb polymers are described in U.S.
  • amphiphilic comb-type polymers may be present in the form of copolymers, containing a backbone formed of a hydrophobic, water- insoluble polymer and side chains formed of short, hydrophilic non-cell binding polymers.
  • polyalkylenes such as polyethylene and polypropylene; polychloroprene; polyvinyl ethers; such as polyvinyl acetate); polyvinyl halides such as polyvinyl chloride); polysiloxanes; polystyrenes; polyurethanes; polyacrylates; such as poly(methyl (meth)acrylate), poly(ethyl (meth)acrylate), poly(n-butyl(meth)acrylate), poly(isobutyl (meth)acrylate), poly(tert-butyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl
  • polymers may include useful derivatives, including polymers having substitutions, additions of chemical groups, for example, alkyl groups, alkylene groups, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art.
  • the polymers may include zwitterionic polymers such as, for example, polyphosphorycholine, polycarboxybetaine, and polysulfobetaine.
  • the polymers may have side chains of betaine, carboxybetaine, sulfobetaine, oligoethylene glycol (OEG), sarcosine or polyethyleneglycol (PEG).
  • poly(oligoethyleneglycol methacrylate) poly(OEGMA)
  • Poly(OEGMA) may be hydrophilic, water-soluble, non-fouling, non-toxic and non-immunogenic due to the OEG side chains.
  • Polynucleotide as used herein can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequence.
  • the polynucleotide can be nucleic acid, natural or synthetic, DNA, genomic DNA, cDNA, RNA, or a hybrid, where the polynucleotide can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine.
  • Polynucleotides can be obtained by chemical synthesis methods or by recombinant methods.
  • a "peptide” or “polypeptide” is a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as antibodies.
  • Subject as used herein can mean a mammal that wants to or is in need of being treated with the herein described agent-first polypeptide conjugate.
  • the mammal can be a human, dog, cat, horse, cow, pig, mouse, rat, or non-human primate such as, for example, chimpanzee, gorilla, orangutan, and gibbon.
  • Treatment when referring to protection of an animal from a disease, means preventing, suppressing, repressing, ameliorating, or completely eliminating the disease.
  • Preventing the disease involves administering a composition of the present invention to an animal prior to onset of the disease.
  • Suppressing the disease involves administering a composition of the present invention to an animal after induction of the disease but before its clinical appearance.
  • Repressing or ameliorating the disease involves administering a composition of the present invention to an animal after clinical appearance of the disease.
  • substantially identical can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% over a region of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100 amino acids.
  • Variant used herein with respect to a polynucleotide means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a polynucleotide that is substantially identical to a referenced polynucleotide or the complement thereof; or (iv) a polynucleotide that hybridizes under stringent conditions to the referenced polynucleotide, complement thereof, or a sequences substantially identical thereto.
  • a “variant” can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity.
  • biological activity include the ability to be bound by a specific antibody or to promote an immune response.
  • Variant can mean a substantially identical sequence.
  • Variant can mean a functional fragment thereof.
  • Variant can also mean multiple copies of a polypeptide. The multiple copies can be in tandem or separated by a linker.
  • Variant can also mean a polypeptide with an amino acid sequence that is substantially identical to a referenced polypeptide with an amino acid sequence that retains at least one biological activity.
  • a conservative substitution of an amino acid i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids. See Kyte et al., J. Mol. Biol. 1982, 157, 105-132. The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ⁇ 2 are substituted.
  • hydrophibicity of amino acids can also be used to reveal substitutions that would result in polypeptides retaining biological function.
  • a consideration of the hydrophilicity of amino acids in the context of a polypeptide permits calculation of the greatest local average hydrophilicity of that polypeptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity, as discussed in U.S. Patent No. 4,554,101 , which is fully incorporated herein by reference.
  • Substitution of amino acids having similar hydrophilicity values can result in polypeptides retaining biological activity, for example immunogenicity, as is understood in the art.
  • Substitutions can be performed with amino acids having hydrophilicity values within ⁇ 2 of each other.
  • hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
  • a variant can be a polynucleotide sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof.
  • the polynucleotide sequence can be 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof.
  • a variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof.
  • the amino acid sequence can be 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.
  • SrtA recognizes the primary sequence LPXTG (SEQ ID NO: 3, where X is any amino acid) in a protein and cleaves the peptide bond between threonine and glycine, forming a stable intermediate that joins the catalytic thiol in SrtA to the carboxyl group of threonine in a thioester bond.
  • This intermediate undergoes nucleophilic attack by the oamino group of an oligoglycine branch in the peptidoglycan, generating a native peptide bond that anchors the substrate protein to the cell wall (FIG. 5).
  • SrtA recognizes an amino acid sequence consisting of LPXZG (SEQ ID NO: 72, where X and Z are independently any amino acid) and cleaves the peptide bond between the Z amino acid and the glycine of LPXZG (SEQ ID NO: 72) and forms a thioester bond between the catalytic thiol in SrtA and the carboxyl group of the Z amino acid.
  • SrtA forms an isopeptide bond between the ⁇ -amino group of any solvent-accessible, nucleophilic lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72).
  • SrtA forms an isopeptide bond between the ⁇ -amino group of any solvent-accessible, nucleophilic lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72).
  • An exemplary schematic diagram of the process is shown in FIG. 17, wherein the Z amino acid is threonine and ZX 1 X2X 3 VX 4 VYPKH (SEQ ID NO: 1 ) comprises the lysine of the first polypeptide.
  • the SrtA may be any SrtA, such as S. aureus SrtA.
  • SrtA may be from a Gram positive bacterium, such as, for example, bacteria in a genus selected from Staphylococcus, Streptococcus, Enterococcus, Bacillus, Corynebacterium, Nocardia, Clostridium, Actinobacteria, and Listeria.
  • SrtA is from S. aureaus.
  • the SrtA may be wild-type SrtA or a variant thereof.
  • the SrtA may include a hydrophobic, membrane-binding domain at the amino terminal end.
  • SrtA can comprise the full length wild-type S. aureus SrtA, a variant thereof, a fragment thereof, or a combination thereof.
  • SrtA may be immobilized on, for example, beads or or resin or other solid support.
  • SrtA is a recombinant polypeptide corresponding the wild-type S. aureus having a deletion of the amino-terminal 59 amino acids to remove a hydrophobic, membrane-binding domain.
  • SrtA is a recombinant polypeptide or fusion protein comprising a purification tag, such as, for example, an elastin-like polypeptide (ELP).
  • ELP is a peptide polymer comprising repeats of the pentapeptide VPGXG (SEQ ID NO: 51 ) where X is any amino acid except proline.
  • SrtA may be membrane- bound.
  • SrtA may be insoluble.
  • SrtA may be soluble.
  • SrtA may comprise an amino acid sequence consisting of SEQ ID NO: 6, which is encoded by a polynucleotide sequence of SEQ ID NO: 7.
  • SEQ ID NO: 6 refers to the full-length wild-type S. aureus polypeptide sequence.
  • SrtA can comprise a polypeptide having an amino acid sequence that is 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over the entire length of the amino acid sequence of SEQ ID NO: 6.
  • SrtA may comprise an amino acid sequence consisting of SEQ ID NO: 4, which is encoded by a polynucleotide sequence of SEQ ID NO: 5.
  • SEQ ID NO: 4 refers to the wild-type S. aureus polypeptide sequence with a deletion of the amino-terminal 59 amino acids. This removes a hydrophobic, membrane-binding domain and makes the resulting protein soluble.
  • SrtA can comprise a polypeptide having an amino acid sequence that is 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over the entire length of the amino acid sequence of SEQ ID NO: 4.
  • Certain Gram-positive bacteria use SrtA, and homologs thereof, to assemble pili- fibrous polymers of structural proteins that extend from the bacterial surface and are implicated in adhesion and biofilm formation.
  • the initial step for pilin formation is the same as that used to anchor proteins to the cell wall and generates a thioester intermediate upon cleavage of the LPXTG (SEQ ID NO: 3) motif.
  • Extension of a pilin polymer proceeds when the thioester undergoes nucleophilic attack by the ⁇ -amino group of a lysine residue in another sortase-linked pilin monomer (FIG. 1 ). This process repeats and is stochastically terminated by a "housekeeping" sortase that anchors the pilin polymer to the cell wall through a native peptide bond at the a-amino group of oligoglycine.
  • the pilin polymer is therefore composed of a series of branched monomers linked by isopeptide bonds, with each monomer retaining an unmodified a-amino group.
  • the nucleophilic lysine used in polymer extension is contained in a "pilin domain" with the sequence WX 1 X 2 X 3 VX 4 VYPKI-I (SEQ ID NO: 2), wherein X ⁇ ,, X 2 , X 3 , and X 4 are non-conserved amino acid residues.
  • the SrtA may recognize the primary sequence LPXTG (SEQ ID NO: 3, where X is any amino acid) in a protein and cleave the peptide bond between threonine and glycine, forming a stable intermediate.
  • this intermediate undergoes nucleophilic attack by the a-amino group of an oligoglycine branch in the peptidoglycan, generating a native peptide bond that anchors the substrate protein to the cell wall (FIG. 5).
  • certain amino acid substitutions are also tolerated in the LPXTG (SEQ ID NO: 3) sequence at the first, fourth, and fifth positions that affect the rate but not the overall outcome of the reaction.
  • SrtA recognizes the primary sequence LPXZG (SEQ ID NO: 72, wherein X and Z are independently any amino acid) in a protein and cleave the peptide bond between the Z amino acid and glycine, forming a stable intermediate.
  • the linkage between X and Z amino acids of LPXZG (SEQ ID NO: 72) does not have to be a peptide bond.
  • the linkage between X and Z amino acids of LPXZG (SEQ ID NO: 72) is a desipeptide bond.
  • SrtA recognizes the primary sequence LPXZ in a protein, wherein the carboxy- terminus of the amino acid Z of LPXZ is modified to another functional group, for example, a methyl ester rather than a carboxylic acid.
  • SrtA recognizes the primary sequence LPGAG (SEQ ID NO: 73) in a protein and cleaves the peptide bond between the alanine and glycine, forming a stable intermediate.
  • SrtA recognizes the primary sequence LPETG (SEQ ID NO: 15) in a protein and cleaves the peptide bond between the threonine and glycine, forming a stable intermediate.
  • Solution conditions for SrtA activity are flexible and may vary.
  • the temperature for SrtA activity may be about 20°C to about 45°C, about 20°C to about 42°C, about 22°C to about 40°C, about 25°C to about 35°C, about 30°C to about 34°C, or about 32°C to about 33°C.
  • conditions for SrtA activity may include a temperature of about 20°C to about 42°C.
  • conditions for SrtA activity may include a temperature of about 25°C.
  • conditions for SrtA activity may include a temperature of about 33°C.
  • the pH for SrtA activity may be about pH 4 to about pH 1 1 , pH 4.5 to about pH 10, pH 5 to about pH 9.5, pH 6 to about pH 9, or pH 7 to about pH 8.5. In some embodiments, conditions for SrtA activity may include a pH 5 to about pH 9.5. In some embodiments, conditions for SrtA activity may include a pH of about 8.5.
  • the molar ratio of substrate:enzyme for SrtA activity may be about 1 :2 to about 5:1 , about 1 :1 to about 4:1 , or about 2:1 to about 3:1. In some embodiments, conditions for SrtA activity may include molar ratio of substrate:enzyme of about 1 :2 to about 5:1 .
  • conditions for SrtA activity may include a buffer (comprising 50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI 2 , pH 8.5) and incubation for 18- 24 hours.
  • a buffer comprising 50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI 2 , pH 8.5.
  • the location of the pilin domain within a protein may impact its solvent accessibility and may hence affect both kinetics and yield.
  • the precise sequence of the pilin domain sequence may also impact both reaction yield and kinetics.
  • the pilin domain may tolerate truncations, which may make it easier to engineer this sequence into a variety of solvent accessible loops within various proteins.
  • agent-first polypeptide conjugate comprised of a first polypeptide conjugated to an agent via a portion or fragment of a second polypeptide.
  • agent is selectively conjugated to a specific lysine of the first polypeptide via an amino acid of the second polypeptide in a reaction catalyzed by SrtA.
  • SrtA The formation of agent-first polypeptide conjugate is catalyzed by SrtA.
  • the first polypeptide comprises at least one lysine.
  • the at least one lysine is solvent-accessible.
  • the at least one lysine is nucleophilic.
  • the at least one lysine may have a side chain amino group that is nucleophilic.
  • the at least one lysine of the first polypeptide comprises a nucleophilic nitrogen atom.
  • the at least one lysine of the first polypeptide comprises a side chain comprising an uncharged primary amino group. In some embodiments, the at least one lysine of the first polypeptide has a pKa of less than 10.53. In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of KH. In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of PKH.
  • the first polypeptide comprises at least one amino acid sequence consisting of ZX 1 X 2 X 3 VX 4 VYPKH (SEQ ID NO: 1 ), wherein X ⁇ ,, X 2 , X 3 , and X 4 are independently any amino acid residue and Z is any hydrophobic amino acid residue.
  • Hydrophobic amino acid residues include, for example, A, V, I, L, M, F, Y, and W.
  • Z is W.
  • Z is F.
  • the first polypeptide comprises at least one amino acid sequence consisting of WX 1 X 2 X 3 VX 4 VYPKH (SEQ ID NO: 2), wherein X 1 ; X 2 , X 3 , and X 4 are independently any amino acid residue.
  • the first polypeptide comprises at least one amino acid sequence consisting of WLQDVHVYPKH (SEQ ID NO: 45).
  • the first polypeptide comprises at least one amino acid sequence consisting of WLQDVHVYPK (SEQ ID NO: 71 ).
  • the first polypeptide comprises at least one amino acid sequence consisting of VHVYPKH (SEQ ID NO: 46).
  • the first polypeptide comprises at least one amino acid sequence consisting of VYPKH (SEQ ID NO: 47). In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of YPKH (SEQ ID NO: 48). The first polypeptide facilitates administration of the agent.
  • the first polypeptide may further comprise an additional polypeptide such as, for example, an antibody, enzyme, therapeutic protein, fibronectin III (Fn3) domain, Fn3 domain from the human tenascin protein (TN domain), Designed Ankyrin Repeat Domain (DARPIN), affibody, scFv, and dsFv, or any combination thereof.
  • the first polypeptide further comprises an antibody.
  • the first polypeptide further comprises a TN domain, DARPINS, affibodies, and/or scFvs.
  • the first polypeptide further comprises a Fn3 domain.
  • the Fn3 domain may be a recombinant polypeptide comprising an amino acid sequence of SEQ ID NO: 8, encoded by a polynucleotide sequence of SEQ ID NO: 9.
  • the first polypeptide comprises two or more lysines.
  • the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to a different lysine.
  • the first polypeptide comprises two or more lysines, each with a pKa less than 10.53.
  • the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to a different lysine, each with a pKa less than 10.53.
  • the first polypeptide comprises two or more amino acid sequences consisting of ZX 1 X2X 3 VX 4 VYPKH (SEQ ID NO: 1 ).
  • the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of ZX X 2 z VX 4 VYPKH (SEQ ID NO: 1 ).
  • the agent is conjugated to the lysine of SEQ ID NO: 1.
  • the first polypeptide comprises two or more amino acid sequences consisting of WX 1 X 2 X 3 VX 4 VYPKH (SEQ ID NO: 2).
  • the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of WX 1 X 2 X 3 VX 4 VYPKH (SEQ ID NO: 2).
  • the first polypeptide comprises two or more amino acid sequences consisting of WLQDVHVYPK (SEQ ID NO: 71 ).
  • the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of WLQDVHVYPK (SEQ ID NO: 71 ).
  • the first polypeptide comprises two or more amino acid sequences consisting of WLQDVHVYPKH (SEQ ID NO: 45).
  • the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of WLQDVHVYPKH (SEQ ID NO: 45).
  • the first polypeptide comprises two or more amino acid sequences consisting of VHVYPKH (SEQ ID NO: 46).
  • the agent- first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of VHVYPKH (SEQ ID NO: 46).
  • the first polypeptide comprises two or more amino acid sequences consisting of VYPKH (SEQ ID NO: 47).
  • the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of VYPKH (SEQ ID NO: 47).
  • the first polypeptide comprises two or more amino acid sequences consisting of YPKH (SEQ ID NO: 48). In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of YPKH (SEQ ID NO: 48). In some embodiments, the first polypeptide comprises two or more amino acid sequences consisting of PKH. In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of PKH. In some embodiments, the first polypeptide comprises two or more amino acid sequences consisting of KH. In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of KH. b. Agent
  • the agent is any composition that elicits a desired effect in the subject upon administration.
  • the agent may be selected from the group consisting of polynucleotide, polypeptide, chemotherapeutic agent, vaccine, hormone, cytokine, anti-viral, steroid, opiate, anti-inflammatory, anti-convulsant, polymerization initiator, and polymer.
  • the agent comprises a protein.
  • the agent comprises an antibody.
  • the agent comprises a peptide drug.
  • the agent comprises a small molecule.
  • the small molecule comprises a detectable label.
  • the small molecule comprises a fluorophore.
  • the agent comprises a small molecule chemotherapeutic.
  • the agent comprises a polymerization initiator or a polymer or a combination thereof.
  • a polymer may be in situ polymerized from the polymerization initiator.
  • a polymerization initiator is a molecule that assists in beginning polymerization by interacting with a polypeptide (e.g., the second polypeptide detailed below) and a monomer to form a polymer.
  • Polymerization may include, for example, atom transfer radical polymerization (ATRP), reversible addition- fragmentation chain transfer (RAFT) polymerization, nitroxide mediated radical polymerization (NMP), ring-opening metathesis polymerization (ROMP), and combinations thereof.
  • ATRP atom transfer radical polymerization
  • RAFT reversible addition- fragmentation chain transfer
  • NMP nitroxide mediated radical polymerization
  • ROMP ring-opening metathesis polymerization
  • polymerization initiators include those compatible with ATRP such as, without limitation, N-(2- aminoethyl)-2-bromo-2-methylpropanamide, N-(2-aminoethyl)-2-chloro-2-methylpropanamide, 2-bromo-N-(2-(2-hydrazinylacetamido) ethyl)-2-methylpropanamide, 2-chloro-N-(2-(2- hydrazinylacetamido) ethyl)-2-methylpropanamide.
  • RAFT chain transfer agent
  • R can be cysteine, hydrazine, hydroxylamine
  • Z can be phenyl, alkyl, phthalimidomethyl
  • traditional radical polymerization initiators including those such as AIBN which are cleaved to initiate the polymerization.
  • polymerization initiators also include those compatible with ROMP such as, without limitation, A- B, where A can be cysteine, hydrazine, hydroxylamine, and B can be olefins.
  • the agent is a polymerization initiator comprising an ATRP initiator, followed by in situ ATRP polymerization from the initiator. In some embodiments, the agent is a polymerization initiator comprising a RAFT agent, followed by in situ RAFT polymerization from the initiator.
  • ATRP catalyst system may include, but are not limited to, copper halides and ligands, where ligands can be derivatives of 2, 2'-bipyridine, other ⁇ -accepting, chelating nitrogen-based ligands such as 2-iminopyridines and some aliphatic polyamines.
  • RAFT catalyst system may include water soluble radical generating compounds, such as 4, 4'-azobis(4-cyanopentanoic acid), 2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2'-Azobis[2-(2-imidazolin-2- yl)propane]-disulfate dehydrate, 2,2'-Azobis(2-ethylpropionamidine)-dihydrochloride.
  • water soluble radical generating compounds such as 4, 4'-azobis(4-cyanopentanoic acid), 2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2'-Azobis[2-(2-imidazolin-2- yl)propane]-disulfate dehydrate, 2,2'-Azobis(2-ethylpropionamidine)-dihydrochloride.
  • ROMP catalyst systems may include, but are not limited to, soluble Grubbs catalysts, such as tetraethylene glycol substituted ruthenium benzylidene, ruthenium alkylidene with triaryl phosphate ligands, or ruthenium alkylidene with ligands with quaternary ammonium.
  • Other conditions used for polymerization may include, for example, that the polymerization be carried out under low oxygen, for example, under a noble or non-reactive gas such as argon, and/or for a time of at least about 5 min, at least about 15 min, at least about 60 min, and optionally no more than about 12 hours, no more than about 24 hours, no more than about 48 hours.
  • Polymerization may be carried out, for example, at a temperature of at least about 5°C, at least about 10°C, at least about 15°C, at least about 20°C, at least about 30°C, at least about 40°C, at least about 50°C, at least about 60°C, at least about 70°C, at least about 80°C, at least about 90°C or at least about 100°C.
  • the agent comprises a polymerization initiator
  • monomers include, but are not limited to, lactic acid, epichlorohydrin, acrylate, methacylate, acrylamide, methacrylamide, norbornene, and oxanorbornene.
  • Examples of monomer structures that may be used in ROMP, NMP, ATRP and RAFT, and other components and techniques that may be used are described in U.S. Patent Publication No. 201 10294189, the entire disclosure of which is herein incorporated by reference in its entirety.
  • the second polypeptide links the agent to the first polypeptide and may be described as a linker.
  • the terms “portion” and “fragment” are used interchangeably herein.
  • the second polypeptide comprises an amino acid sequence consisting of LPXZG (SEQ ID NO: 72), wherein X and Z are independently any amino acid residue. L is at the N-terminal end and G is at the C-terminal end of LPXZG (SEQ ID NO: 72).
  • the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75), wherein X and Z are independently any amino acid residue and wherein the carboxy-terminus of Z is modified to another functional group, for example, a methyl ester.
  • the linkage between X and Z of SEQ ID NO: 72 or 75 may be, for example, a peptide bond or a desipeptide bond.
  • the second polypeptide comprises an amino acid sequence consisting of LPXTG (SEQ ID NO: 3), wherein X is any amino acid residue.
  • X is E.
  • second polypeptide comprises an amino acid sequence consisting of LPETG (SEQ ID NO: 15).
  • the second polypeptide comprises an amino acid sequence consisting of LPGAG (SEQ ID NO: 73).
  • the second polypeptide may include additional amino acids C-terminal to LPXZG (SEQ ID NO: 72).
  • the second polypeptide may include at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids C-terminal to LPXZG (SEQ ID NO: 72).
  • the second polypeptide may include a variety of chemical modifications to the carboxyl group of the C-terminal end of LPXZG (SEQ ID NO: 72) such as, for example, conversion to a methyl ester or amide.
  • the second polypeptide comprises an amino acid sequence consisting of LPETGRAGG (SEQ ID NO: 10).
  • the side chain of the last amino acid at the carboxy end of the second polypeptide does not include a carboxyl group.
  • amino acid sequence carboxy-terminal to LPXZG can be any amino acid sequence or other chemical moiety, as long as there is not a carboxyl group at the carboxy-terminus of the LPXZG (SEQ ID NO: 72) peptide.
  • Other amino acids can also be present amino-terminal to LPXZG (SEQ ID NO: 72).
  • the agent is at the amino terminal end of the second polypeptide, or a fragment thereof.
  • the agent is amino terminal to LPXZG (SEQ ID NO: 72) of the second polypeptide, or a fragment thereof.
  • the agent can be conjugated to the LPXZG (SEQ ID NO: 72) through an amino acid or other chemical linker.
  • the agent does not have to be conjugated to the amino terminus of the LPXZG (SEQ ID NO: 72) peptide if additional amino acids are located amino-terminal to the LPXZG (SEQ ID NO: 72) sequence.
  • the agent can be conjugated to a lysine, a cysteine, or an unnatural amino acid in a peptide containing the LPXZG (SEQ ID NO: 72) sequence, as long as the agent is conjugated amino-terminal to the LPXZG (SEQ ID NO: 72) enzyme recognition site.
  • the agent is conjugated to the second polypeptide, or a fragment thereof, amino- terminal to the LPXZG (SEQ ID NO: 72) sequence.
  • the present invention is directed to a method of conjugating an agent to a first polypeptide.
  • the method may comprise contacting the first polypeptide with SrtA and at least one agent conjugated to a second polypeptide, to form an agent-first polypeptide conjugate.
  • the present invention is directed to a method of drug delivery.
  • the method may comprise administering the agent-first polypeptide complex.
  • the agent-first polypeptide complex may be administered to a subject.
  • agent-first polypeptide conjugate as detailed herein may have improved properties for delivery of the agent or the first polypeptide.
  • an agent-first polypeptide conjugate produced as described herein may show improvement in one or more of solubility, stability, pharmacokinetics, immunogenicity and biodistribution or bioaccumulation at the cell, tissue, disease site, or organ level.
  • the agent-first polypeptide conjugate provides a longer plasma half-life than the first polypeptide by itself. 5.
  • agent-first polypeptide conjugate as detailed above can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration.
  • the agent-first polypeptide conjugate can be administered prophylactically or therapeutically.
  • the agent-first polypeptide conjugates can be administered in an amount sufficient to induce a response.
  • the agent-first polypeptide conjugates are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect.
  • An amount adequate to accomplish this is defined as "therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the agent-first polypeptide conjugate regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.
  • the agent-first polypeptide conjugate can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 1997, 15, 617-648); Feigner et al. (U.S. Patent No. 5,580,859, issued Dec. 3, 1996); Feigner (U.S. Patent No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Patent No. 5,679,647, issued Oct. 21 , 1997), the contents of all of which are incorporated herein by reference in their entirety.
  • the polypeptide or agent of the agent-first polypeptide conjugate can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun.
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound, depends, for example, on the route of administration.
  • the agent-first polypeptide conjugates can be delivered via a variety of routes.
  • Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery.
  • Other routes include oral administration, intranasal, intravaginal, transdermal, intravenous, and epidermal routes.
  • the agent-first polypeptide conjugate can be a liquid preparation such as a suspension, syrup, or elixir.
  • the agent-first polypeptide conjugate can be incorporated into liposomes, microspheres, or other polymer matrices (such as by a method described in Feigner et al., U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed. 1993), the contents of which are incorporated herein by reference in their entirety).
  • Liposomes can consist of phospholipids or other lipids, and can be nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • the vaccine can be administered via electroporation, such as by a method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference.
  • the electroporation can be by a method and/or apparatus described in U.S. Patent Nos. 6,302,874; 5,676,646; 6,241 ,701 ; 6,233,482; 6,216,034; 6,208,893; 6, 192,270; 6,181 ,964; 6, 150, 148; 6, 120,493; 6,096,020; 6,068,650; and 5,702,359, the contents of which are incorporated herein by reference in their entirety.
  • the electroporation can be carried out via a minimally invasive device.
  • ELP-GLP1 (SEQ ID NO: 70), Fn3-ELP (SEQ ID NO: 14), and Fn3-PLN 3 -ELP (SEQ ID NO: 13) fusion proteins were genetically assembled in pET24b vectors using synthetic DNA fragments from Integrated DNA Technologies (Coralville, IA) for the pilin domain and sortase recognition sites. Fusion proteins were expressed in BL21 (DE3) E. coli (EdgeBio, Gaithersburg, MD) and purified as described previously (Bellucci et al. Angew Chem. Int. Ed. Engl. 2013, 52, 3703-3708).
  • SrtA-ELP (SEQ ID NO: 1 1 ; 100 ⁇ ) was incubated with ELP-GLP1 (SEQ ID NO: 70; 500 ⁇ initial concentration) for 18 hours at 33°C in reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI 2 , pH 8.5). The temperature was varied for FIG. 2b and the pH was varied for FIG. 8b. Reaction products were visualized by SDS-PAGE using Stain-free any kD Mini- PROTEAN TGX polyacrylamide gels from Bio-Rad (Hercules, CA).
  • the unpurified reaction product was incubated with MS grade trypsin (Thermo Scientific Pierce, Rockford, IL) for 4-6 hours at 20°C at a trypsin:substrate ratio of approximately 1 :20 (m/m). Trypsinized samples were diluted 1 :40 into oCyano-4-hydroxycinnamic acid (CHCA) matrix, spotted, and analyzed by MALDI-TOF using an Applied Biosystems Voyager-DE Pro system with a nitrogen laser.
  • MS grade trypsin Thermo Scientific Pierce, Rockford, IL
  • SrtA-ELP (SEQ ID NO: 1 1 ) at 100 ⁇ was incubated with biotin-LPETGRAGG (SEQ ID NO: 10) peptide purchased from GenScript (Picastaway, NJ) at 800 ⁇ in reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI 2 , pH 8.5) for 2 hours at 20°C to load the enzyme with the biotinylated peptide substrate.
  • reaction buffer 50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI 2 , pH 8.5
  • dabcyl-LPETG-edans peptide (LPETG is SEQ ID NO: 15) from AnaSpec (Fremont, CA) was used at 500 ⁇ for FIG. 11 and FIG. 12.
  • Fn3-ELP SEQ ID NO: 14
  • Fn3-PLN 3 -ELP SEQ ID NO: 13
  • Fn3-ELP (SEQ ID NO: 14) or Fn3-PLN 3 -ELP (SEQ ID NO: 13) was reacted at 50 ⁇ with SrtA-ELP (SEQ ID NO: 1 1 ) at 100 ⁇ and biotin-LPETGRAGG (SEQ ID NO: 10) peptide or FITC-LPETGRAGG (SEQ ID NO: 10) peptide at 500 ⁇ in reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI 2 ).
  • Pilin domain peptide was purchased from Genscript (Piscataway, NJ) and reacted at 10 ⁇ with SrtA-ELP (SEQ ID NO: 1 1 ) at 100 ⁇ and biotin-LPETGRAGG (SEQ ID NO: 10) peptide at 500 ⁇ in reaction buffer.
  • Purified antibodies were reacted at 0.5 mg/mL with SrtA-ELP (SEQ ID NO: 1 1 ) at 10 ⁇ ( ⁇ 2-fold excess over the mAb) and biotin-LPETGRAGG (SEQ ID NO: 10) peptide at 500 ⁇ ( ⁇ 100-fold excess over available pilin domains) in reaction buffer. Reactions conducted overnight at 32°C. Reactions were quenched by adding triglycine peptide to 10 mM.
  • reaction product was run on SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). The blot was blocked with 5% nonfat milk and probed with 1 mg/mL streptavidin-Cy5 conjugate (Life Technologies, Grand Island, NY) at a 1 :20,000 dilution in Tris-buffered saline with 1 % Tween-20 and 0.5% nonfat milk. Fluorescent bands were detected with a Typhoon variable mode laser scanner (GE Healthcare Life Sciences, Pittsburgh, PA).
  • the molar ratio of biotin:protein was determined using biotinylated Fn3-PLN 3 -ELP (SEQ ID NO: 13) purified by cation exchange chromatography.
  • the biotinylated Fn3-PLN3-ELP purified by cation exchange chromatography was digested with MS-grade trypsin overnight at 20°C at a trypsin:substrate ratio of approximately 1 :20 (m/m).
  • biotin :protein ratio was assessed by comparison of the integrated areas of summed extracted ion chromatograms for ions containing either biotinylated or non-biotinylated pilin domain lysines.
  • Fn3-PLN 3 -ELP (SEQ ID NO: 13) reactions the biotin:protein ratio was assessed using a fluorescence biotin quantitation kit (Thermo Scientific) that measured the increase in 520 nm fluorescence of avidin-conjugated DyLight fluorophore when bound HABA was displaced by protein-linked biotin.
  • the assay was performed in the microplate format per the manufacturer's instructions and the moles of biotin in each sample tested were within the linear range of a standard curve developed using biocytin.
  • the results of the fluorescent assay were confirmed using a colorimetric quantitation kit (Thermo) that measured the decrease in HABA 500 nm absorbance when displaced from avidin by protein-bound biotin.
  • the assay was performed in the cuvette format per the manufacturer's instructions. Unreacted Fn3-PLN 3 -ELP (SEQ ID NO: 13) was also tested in both assays.
  • Biotinylated horseradish peroxidase (HRP) that was provided with the colorimetric assay provided a positive control.
  • Protein concentrations used in the assay were obtained using the bicinchoninic acid (BCA) assay. Measurements of biotin and protein concentrations were performed in triplicate for each sample and standard.
  • HUVEC human umbilical cord vein endothelial cells
  • HBSS Hank's buffered saline solution
  • SK-OV-3 cells were maintained in McCoy's 5a medium with 10% fetal bovine serum, 5 units/mL penicillin, and 5 ⁇ g/mL streptomycin (Gibco, by Thermo Fisher Scientific, Waltham, MA). Cells were attached to glass coverslips overnight.
  • Cells were washed in PBS, fixed with 4% paraformaldehyde in PBS at room temperature for 15 minutes, and stained with a 1 :200 dilution of 0.5 mg/mL anti-Her2 biotinylated by reaction with SrtA or a biotinylated murine lgG1 isotype control antibody, followed by a 1 :200 dilution of 1 mg/mL streptavidin-FITC conjugate. Cells were analyzed with a Nikon TE2000-U inverted fluorescent microscope with a 60x 1.25NA oil immersion objective.
  • SrtA The ability of SrtA to carry out isopeptide ligation was confirmed by reacting a LPETGRAGG (SEQ ID NO: 10) peptide containing an amino-terminal biotin with a pilin domain peptide (VGGSWLQDVHVYPKHGGSGR; SEQ ID NO: 50).
  • SrtA was produced as a fusion protein with an elastin-like polypeptide (ELP), which is a peptide polymer composed of repeats of the pentapeptide VPGXG (SEQ ID NO: 51 ) where X is any amino acid except proline.
  • ELPs and their fusions phase separate in aqueous solution when heated above a characteristic transition temperature (FIG.
  • ELPs are inert, i.e., they impart no new bioactivity beyond phase transition behavior to their fused peptide or protein partner, and provide efficient purification tags that allow easy recovery of ELP fusions without column chromatography by exploiting their phase transition behavior.
  • FIG. 6 Shown in FIG. 6 are graphs of the thermal responsiveness of the SrtA-ELP fusion protein used in this study. As shown in FIG.
  • absorbance at 350 nm was measured for 100, 75, 50, and 25 ⁇ SrtA-ELP concentrations as the temperature was ramped from 15°C-35°C at 1 °C/min in sortase reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI 2 , pH 8.5).
  • the transition temperature was defined as the inflection point of the absorbance as a function of temperature.
  • plotting the transition temperature versus SrtA-ELP concentration allowed prediction of the SrtA-ELP transition temperature at a particular concentration by interpolation or extrapolation.
  • Turbidity profiles for protein-ELP fusions were characterized using a Cary 300 Bio UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA) and concentrations were determined by the Beer-Lambert Law using calculated extinction coefficients and the absorbance at 280 nm measured using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE).
  • Glucagon-like peptide-1 (GLP1 , SEQ ID NO: 69) was chosen to test the SrtA- catalyzed isopeptide ligation reaction, as it is a pharmaceutically relevant peptide with two internal lysines that could serve as off-target sites for isopeptide bond formation.
  • GLP1 (SEQ ID NO: 69) was fused to both the pilin domain and the sortase LPETG (SEQ ID NO: 15) recognition sequence, generating a polypeptide that was expressed as a fusion protein with an ELP.
  • each ELP-GLP1 (SEQ ID NO: 70) contained both the pilin domain and LPETG (SEQ ID NO: 15) enzyme recognition sequence, it was polymerized by incubation with SrtA-ELP (SEQ ID NO: 1 1 ) according to the reaction shown in FIG. 2a, wherein predicted tryptic peptides of the polymerization product are boxed.
  • SDS-PAGE of the reaction product (FIG. 2b) showed a ladder of polymers with different numbers of subunits, showing polymerization of the 41 kDa ELP-GLP1 (SEQ ID NO: 70) monomer at all temperatures tested except reactions stored at - 20°C.
  • the unpurified reaction product was treated with proteomics-grade trypsin, and the resulting peptides were analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry.
  • Trypsin normally cleaves a peptide bond on the carboxy-terminal side of lysine or arginine residues, but cannot cleave at lysines with modified ⁇ -amino groups.
  • the MALDI-TOF spectrometry of a tryptic digest of 33°C reaction product showed the presence of an ion at m/z 2809.4 from the branched junction of two monomers (labeled 5 in FIG. 2d and Table 1 ).
  • FIG. 2d shows the isopeptide linkage at the pilin domain lysine (ion 5) along with other ions that correspond to unmodified peptides from the ELP-GLP1 (SEQ ID NO: 70) and SrtA-ELP (SEQ ID NO: 1 1 ) fusion proteins. Shown in Table 1 are the tryptic peptide sequences for peaks identified in the MALDI-TOF spectrum in FIG. 2d.
  • GAGLPET SEQ ID NO: 21 + SK 625.7 + 233.2 Not N/A
  • GAGLPET SEQ ID NO: 21 + 625.7 + 3272.6 Not N/A
  • VK (SEQ ID NO: 25) 3898.3
  • GAGLPET SEQ ID NO: 21 + 625.7 + 1802.1 Not N/A
  • GAGLPET SEQ ID NO: 21 + 625.7 + 2029.3 Not N/A
  • the SrtA reaction mechanism was used to generate protein-small molecule conjugates where the site of conjugation could be controlled.
  • Two genetic-level fusion proteins were generated: a fibronectin type III (Fn3) domain fused to an ELP with 3 intervening copies of the pilin domain (Fn3-PLN 3 -ELP; SEQ ID NO: 13), and a Fn3-ELP (SEQ ID NO: 14) lacking the pilin domain as a negative control.
  • Multiple copies of the pilin domain were incorporated to confirm that the isopeptide ligation can be carried out at an internal site, and because a payload of several small molecules per protein is typically necessary to attain a therapeutically relevant dose in protein-drug conjugates.
  • the Fn3 domain is an attractive targeting molecule for drug delivery because it mimics antibodies but does not contain their complex quaternary structure, glycosylation, or intramolecular disulfide bonds, allowing easy production in Escherichia coli.
  • the Fn3 scaffold is similar to the immunoglobulin fold in antibodies and contains loop regions that can be mutated and affinity matured against a target, similar to the complementarity determining regions in antibodies.
  • the Fn3 variant used had a RGDWXE (SEQ ID NO: 44) element in its FG loop that targets the ⁇ ⁇ ⁇ 3 integrin, which is upregulated in the endothelial cells of angiogenic tumor vasculature.
  • the model small molecules biotin and dabcyl were selected to explore the versatility of the conjugation reaction.
  • a synthetic LPETGRAGG (SEQ ID NO: 10) peptide with an amino- terminal biotin and SrtA-ELP (SEQ ID NO: 1 1 ) were incubated with either the Fn3-ELP (SEQ ID NO: 14) or Fn3-PLN 3 -ELP (SEQ ID NO: 13) fusion (overview of the reaction is shown in FIG. 3a, along with the expected tryptic peptides of the reaction product being boxed).
  • SrtA-ELP SEQ ID NO: 1 1
  • biotin-LPETGRAGG SEQ ID NO: 10
  • Fn3-PLN 3 -ELP SEQ ID NO: 13
  • SDS-PAGE SDS-PAGE and the corresponding Western blot are shown in FIG. 3b and FIG. 3c, respectively.
  • Lanes 3 and 4 show control reactions containing Fn3-ELP (SEQ ID NO: 14) and Fn3-PLN 3 -ELP (SEQ ID NO: 13), respectively, where the biotin-LPETGRAGG (SEQ ID NO: 10) peptide was excluded. As expected, no bands are visualized in the Western blot for the reactions in lanes 3 and 4.
  • Table 2 Included in Table 2 are the predicted molecular weight, the experimentally measured molecular weight (m/z value - H + mass), and the difference between these two values.
  • the complete sequences of SrtA-ELP (SEQ ID NO: 10) and Fn3-PLN 3 -ELP (SEQ ID NO: 13) are provided for reference.
  • the structure of biotin-LPET (SEQ ID NO: 29) is shown in FIG. 9. Excellent agreement between the predicted and measured peptide molecular weights was observed for all ions in the MALDI-TOF spectra.
  • biotinylation was specific to the pilin domain lysine, as peptides corresponding to biotinylation at the oamino group of Fn3-PLN 3 -ELP (SEQ ID NO: 13) or through the ⁇ -amino groups of any of the 3 lysines in Fn3 were not found in the MALDI-TOF spectrum.
  • biotin- LPET (SEQ ID NO: 29) 3870.1
  • VYPK (SEQ ID NO: 32)
  • Biotin-LPET (SEQ ID NO: 29) + 666.8+804. Not N/A
  • Biotin-LPET SEQ ID NO: 29 + 666.8+672. Not N/A
  • a commercially available dabcyl-LPETG-edans (LPETG is SEQ ID NO: 15) reagent was successfully conjugated to the ELP-GLP1 (SEQ ID NO: 70) construct, and the tryptic digest of the reaction product was analyzed by MALDI-TOF mass spectrometry (FIG. 11 and Table 3). Shown in FIG. 11 a is the structure of dabcyl-LPETG-edans peptide (SEQ ID NO: 15). Dabcyl absorbs at the 337 nm wavelength of the N 2 laser used in MALDI-TOF, and reproducibly lost a 132 Da segment due to fragmentation (the proposed fragment is highlighted).
  • the ELP-GLP1 (SEQ ID NO: 70) is the same protein used in the polymerization model, and therefore, both polymerization and dabcyl conjugation can occur through the pilin domain as shown in the schematic of FIG. 11 b. This conjugation was also site-specific for the ⁇ -amino group of the pilin domain lysine, demonstrating that the reaction can be used with different small molecule and protein substrates.
  • the MALDI-TOF spectrum is shown in FIG. 11 c. The spectrum shows the presence of both conjugated dabcyl (ion 1 ) and the branched polymer junction (ion 5) at the pilin domain lysine.
  • ion 1-132 For dabcyl attachment, another ion is present (ion 1-132) that corresponds to attachment at the pilin domain lysine but with the loss of 132 Da. This same loss is also observed for ions corresponding to the reaction side product, dabcyl- LPETGGG (SEQ ID NO: 33), as indicated on the spectrum. Both conjugation and polymerization reactions are specific for isopeptide-linked attachment at the pilin domain lysine ⁇ -amino group, as all other ions in the spectra map to unmodified segments of SrtA-ELP (SEQ ID NO: 1 1 ) or ELP-GLP1 (SEQ ID NO: 70).
  • Dabcyl- Dabcyl-LPETGGG (SEQ ID NO: 33) + 691 .8 + 925.2 -0.2
  • Dabcyl- Dabcyl-LPETGGG (SEQ ID NO: 33) + 925.0 - 793.3 -0.3
  • Dabcyl-LPET (SEQ ID NO: 29) + MSK 691 .8 + Not N/A
  • Dabcyl-LPET (SEQ ID NO: 29) + SK 691 .8 + Not N/A
  • FIG. 12a is a schematic of the reaction without including triglycine in the trypsinization reaction. Omission of triglycine during trypsinization resulted in more complicated MALDI-TOF spectra with peaks corresponding to peptides linked to LPET-bearing substrate molecules (LPET is SEQ ID NO: 29) through an oamino group (FIG. 12 and Table 4). As shown in FIG.
  • the MALDI-TOF spectrum for the unpurified reaction product shows the presence of many ions corresponding to tryptic peptides with amino-terminal glycine residues that had reacted with the SrtA acyl-enzyme intermediate to form native peptide bonds with dabcyl-LPET (ions 1 , 3, 5, and 7; LPET is SEQ ID NO: 29). These ions were only observed for tryptic peptides that had an amino-terminal glycine residue and were not present when excess triglycine is added to the trypsinization reaction (compare to FIG. 11 ).
  • Peak Peptide Peptide sequence Predicted Found Difference label source mass mass (Da)
  • H6-SrtA (SEQ ID NO: 49) appeared as a biotinylated band at -25 kDa in reactions containing biotin-LPETGRAGG (SEQ ID NO: 10) because the thioester intermediate of H6-SrtA (SEQ ID NO: 49) and biotin-LPET (SEQ ID NO: 15) is stable in SDS-PAGE.
  • a disulfide-linked dimer of the enzyme also appeared as a biotinylated protein in the blot due to the amino-terminal glycine of the enzyme, which can be biotinylated by the native peptide transpeptidation reaction at its o amino group. As shown in FIG.
  • the MALDI-TOF mass spectrum of tryptic peptides of the unpurified 20°C biotinylation reaction contained ions that corresponded to the same biotinylated pilin domain peptides (ions 1 and 2) as in FIG. 22a, as well as a peptide corresponding to the amino-terminus of the H6-SrtA (SEQ ID NO: 49) enzyme biotinylated through a native peptide bond at the terminal glycine residue. This ruled out the potential influence of the ELP tag on the enzyme's activity and suggested that protein modification by isopeptide ligation is a general in vitro function of SrtA.
  • FITC Fluorescein isothiocyanate
  • FITC-Ahx-LPETGRAGG (SEQ ID NO: 68) peptide was reacted with Fn3-PLN3-ELP (SEQ ID NO: 13) and SrtA-ELP (SEQ ID NO: 1 1 ) overnight at 32°C in reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI 2 ). As shown in FIG. 18b, the reaction product was analyzed by SDS-PAGE and total protein was visualized (upper panel).
  • the MALDI-TOF spectrum showed the presence of two ions with m/z 3040.7 and 4148.7, which correspond to the predicted molecular weight of pilin domain tryptic peptides linked to FITC-Ahx-LPET (SEQ ID NO: 52) through an isopeptide bond at the pilin domain lysine. As with the biotin conjugation reactions, these peptides contain a missed tryptic digestion at the pilin domain lysine because trypsin is unable to cleave at these lysines after isopeptide bond formation.
  • the chemical structure of the FITC- Ahx-LPET (SEQ ID NO: 52) moiety attached to the pilin domain lysine is shown in FIG. 18d. This demonstrated that the reaction is not restricted to biotin and can be used with bulkier small molecule substrates. Table 5. Corresponding peak table for the MALDI-TOF spectra of FIG. 18.
  • FIG. 13a shows the absorbance at 280 nm, pH, and conductivity traces for the cation exchange chromatography.
  • the biotinylation reaction product was loaded on a HiTrap SP FF 5 ml. column at 5 mL/min, washed in 20 mM Tris, pH 6.0, and eluted in a gradient to 100% 1 M NaCI, 20 mM Tris, pH 6.0 over 50 minutes. Non-bound (NB) and elution (Elu) fractions were collected. As shown in FIG.
  • This assay measures the increase in intensity at 520 nm of an avidin-conjugated DyLight fluorophore upon displacement of the quencher 4'-hydroxyazobenzene-2-carboxylic acid (HABA) by protein-linked biotin.
  • HABA 4'-hydroxyazobenzene-2-carboxylic acid
  • Table 6 A peak table with the ions shown in the MS1 spectrum of FIG. 21.
  • FIG. 23b is an outline of the observed fragmentation pattern and the nomenclature used to classify daughter ions, corresponding to the daughter ion chemical structures in FIG. 23c. The spectrum showed four ions with multiple charge states whose masses confirmed the expected chemical structures for biotin-LPET (SEQ ID NO: 29) linked to the pilin domain lysine through an isopeptide bond (ions 1-4).
  • P and yi P correspond to daughter ions from fragmentation events within the isopeptide-linked LPET moiety. These fragments corresponded to y- and b-type ions produced by breaking peptide bonds along the pilin domain backbone (ions 1 , 3, and 4) as well as within the LPET (SEQ ID NO: 29) linker region (ions 1 and 2).
  • HABA 4'-hydroxyazobenzene-2-carboxylic acid
  • HUVECs human umbilical vein endothelial cells
  • Fn3-PLN 3 -ELP 100 nM biotinylated Fn3-PLN 3 -ELP (SEQ ID NO: 13) for 30 minutes, then washed and immediately fixed, permeabilized, and stained with streptavidin-FITC to detect intracellular biotin.
  • Cells showed significant uptake of Fn3-PLN 3 -ELP (SEQ ID NO: 13) by fluorescence microscopy, as evidenced by punctate fluorescence in the FITC channel (FIG. 4b).
  • FIG. 4b is a merged image showing nuclei (blue), glycoproteins (for cell morphology, red), and intracellular biotin (green). Punctate intracellular fluorescence in the green channel suggested that biotinylated Fn3-PLN 3 -ELP was internalized. Uptake of the biotinylated reagent appeared to be receptor-mediated, as it was dramatically reduced by saturating ⁇ ⁇ ⁇ 3 dimers with a 10-fold molar excess of unlabeled ligand during incubation of the cells with biotinylated Fn3-PLN 3 -ELP (SEQ ID NO: 13) (FIG. 4c).
  • FIG. 4c is a merged image showing nuclei (blue), glycoproteins (for cell morphology, red), and intracellular biotin (green). Punctate intracellular fluorescence in the green channel suggested that biotinylated Fn3-PLN 3 -ELP was internalized. Uptake of the biotiny
  • FIG. 4c shows HUVECs incubated with biotinylated Fn3-PLN 3 -ELP (SEQ ID NO: 13) and a 10-fold molar excess of unlabeled Fn3-ELP (SEQ ID NO: 14) had dramatically reduced uptake of the biotinylated ligand compared to FIG. 4b.
  • 16b shows flow cytometry for HUVECs stained with either biotinylated mouse lgG1 ⁇ (filled histogram), unreacted Fn3-PLN 3 -ELP (SEQ ID NO: 13; left peak, open histogram), or biotinylated Fn3-PLN 3 -ELP (SEQ ID NO: 13; right peak, open histogram) followed by secondary detection with a 1 :200 dilution of 0.5 mg/mL streptavidin- FITC. Cells were analyzed immediately using a BD LSRII flow cytometer (BD Biosciences, San Jose, CA).
  • biotinylated Fn3-PLN 3 -ELP could also be used as a detection reagent in flow cytometry but provided lower signal intensity compared to the monoclonal antibody, which was expected due to its lower affinity ( ⁇ 10 "8 M) and monovalency.
  • SrtA isopeptide transpeptidation reaction can be used to produce bioactive protein-small molecule conjugates, as the reaction conditions and conjugation did not destabilize the Fn3 structure and allowed it to maintain affinity for its target antigen.
  • SrtA-ELP SEQ ID NO: 1 1
  • biotin-LPETGRAGG SEQ ID NO: 10
  • SDS-PAGE and an anti-biotin Western blot indicated that the pilin domain was required for biotinylation of antibodies.
  • Non-reduced anti-Her2 containing the pilin domain on its heavy chain was biotinylated in the reaction, and this modification mapped exclusively to the heavy chain when the antibody's interchain disulfide bonds were reduced prior to electrophoresis.
  • FIG. 24c shows immunofluorescence of the Her2-overexpressing cell line SK-OV-3 using biotinylated anti-Her2 containing a pilin domain on its heavy chain followed by secondary staining with streptavidin-FITC conjugate
  • FIG. 23d shows immunofluorescence of SK-OV-3 using a biotinylated isotype control antibody in the primary staining step (with nuclear stain and FITC; scale bars are 15 ⁇ ).
  • the biotinylated antibody also retained antigen targeting, as it showed efficient labeling of Her2 on SK-OV-3 human ovarian adenocarcinoma cells (FIG. 24c) compared to a biotinylated isotype control antibody (FIG.
  • FIG. 26 Additional images from both groups are shown in FIG. 26.
  • FIGS. 26a-c cells were stained with a 1 :200 dilution of anti-Her2 (with a pilin domain on each heavy chain) biotinylated by reaction with sortase.
  • FIGS. 26d-f cells were stained with a 1 :200 dilution of biotinylated murine lgG1 isotype control antibody. Biotin in both groups was detected by secondary staining with streptavidin-FITC conjugate (green), nuclei were stained with Hoechst 33342 (blue), and scale bars were 15 ⁇ .
  • Antibody was biotinylated by overnight reaction at 32°C with SrtA-ELP (SEQ ID NO: 1 1 ) and biotin-LPETGRAGG (SEQ ID NO: 10) peptide in 2- and 100-fold molar excess, respectively. Purified antibody was dialyzed extensively (30 diavolumes) with PBS to remove unreacted biotin. Protein-bound biotin was quantified using a fluorescence biotin detection kit with a known concentration of biocytin as a standard, according to the manufacturer's instructions. Protein concentration was determined by BCA assay using a bovine serum albumin standard per the manufacturer's instructions.
  • Biotin and protein content were evaluated for undiluted antibody as well as 4 dilutions of the conjugate (1 :4, 1 :6, 1 :8, and 1 :10). Plotting the concentration of biotin versus the concentration of protein for each sample gave a slope of 1.8 with a standard error of 0.1 , which indicates that 90% of the pilin domains in the antibody sample were biotinylated in the isopeptide ligation reaction. That is, using the HABA displacement assay, it was determined that there were 1.8 ⁇ 0.1 biotins conjugated per antibody out of 2 possible pilin domain attachment sites (FIG. 25, wherein all samples and standards were measured in triplicate and error bars indicate standard deviations). This equated to a conversion of 90%, which is consistent with reported yields for other enzymatic reactions at the carboxy- termini of antibody heavy chains.
  • the length and sequence of the first polypeptide was examined for function in the sortase-mediated isopeptide ligation reaction using truncated isopeptide attachment (IPA) sequences.
  • IPA truncated isopeptide attachment
  • SrtA-ELP SEQ ID NO: 1 1
  • biotin-LPETGRAGG SEQ ID NO: 10
  • SDS-PAGE of the reaction products and Western blot with streptavidin-Cy5 indicated that fusion proteins containing any of the truncated IPA sequences were biotinylated by reaction with SrtA, while no biotinylation was observed when a fusion protein without any IPA sequence was reacted.
  • the reaction products were trypsinized and analyzed with MALDI-TOF. As shown in FIG.
  • biotin was installed at the IPA sequence lysine in each of the different versions of the IPA sequence, with all other ions corresponded to unmodified peptides from the Fn3-IPA-ELP and SrtA-ELP (SEQ ID NO: 1 1 ) fusion proteins. Accordingly, SrtA was able to catalyze the isopeptide ligation reaction when the first polypeptide comprised YPKH (SEQ ID NO: 48), VYPKH (SEQ ID NO: 47), VHVYPKH (SEQ ID NO: 46), or WLQDVHVYPKH (SEQ ID NO: 45).
  • a panel of deletion mutants was generated and tested for activity in an isopeptide ligation reaction with biotin-LPETGRAGG (SEQ ID NO: 10) and SrtA.
  • the basis of the panel was Fn3 conjugated to a pilin domain and ELP (Fn3-PLN-ELP; SEQ ID NO: 76), with linker sequences N-terminal and C-terminal to the pilin domain.
  • the N-terminal and C-terminal linkers were glycine- and serine-rich flexible spacers with the sequences GGTSGSGSGGGSGG (SEQ ID NO: 87) and GGSGR (SEQ ID NO: 88), respectively.
  • Deletion mutants were named according to the section of the pilin domain that was removed and are shown in Table 7; for example, ⁇ 4 (SEQ ID NO: 78) lacked the N-terminal 4 amino acids of the native pilin domain WLQDVHVYPKH (SEQ ID NO: 45) to be VHVYPKH (SEQ ID NO: 46).
  • a modified version of Fn3 was also generated, in which the FG loop was mutated from PRGDWNEGS (SEQ ID NO: 89) to PKHGSPASS (SEQ ID NO: 90) to include a PKH sequence for isopeptide ligation.
  • each peptide was reacted with biotin-LPETGRAGG (SEQ ID NO: 10) and SrtA as detailed in Example 1 , i.e., 100 ⁇ SrtA-ELP was incubated with 800 ⁇ biotin-LPETGRAGG (SEQ ID NO: 10) in reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI 2 , pH 8.5) for 2 hours at 20°C before Fn3-PLN-ELP (SEQ ID NO: 76) or variant was added to a final concentration of 50 ⁇ and then incubated for 18 hours at 33°C. Samples of each reaction were analyzed for biotin via Western Blot. Results are shown in FIG.
  • a method of conjugating an agent to a first polypeptide comprising: contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZG (SEQ ID NO: 72) wherein X and Z are independently any amino acid, and wherein the ⁇ -amino group of the lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
  • SertA sortase A
  • a method of conjugating an agent to a first polypeptide comprising: contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid and wherein the carboxy- terminus of the amino acid Z of LPXZ (SEQ ID NO: 75) is modified to a methyl ester, and wherein the ⁇ - amino group of the lysine of the first polypeptide and the Z amino acid of LPXZ (SEQ ID NO: 75) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
  • SertA sortase A
  • Clause 4 The method of any one of the above clauses, wherein the at least one lysine of the first polypeptide comprises a nucleophilic nitrogen atom.
  • Clause 6 The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of KH.
  • Clause 7 The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of PKH.
  • Clause 8 The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of YPKH (SEQ ID NO: 48).
  • Clause 1 1 . The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of WLQDVHVYPK (SEQ ID NO: 71 ).
  • Clause 14 The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of WLQDVHVYPKH (SEQ ID NO: 45).
  • Clause 16 The method of clause 15, wherein the SrtA is from Staphylococcus aureus.
  • Clause 18 The method of any one of the above clauses, wherein the SrtA is a recombinant polypeptide comprising an amino acid sequence of SEQ ID NO: 4.
  • Clause 19 The method of any one of the above clauses, wherein the SrtA is a recombinant polypeptide comprising an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 5.
  • Clause 20 The method of any one of the above clauses, wherein the agent is selected from the group consisting of polynucleotide, polypeptide, chemotherapeutic agent, vaccine, hormone, cytokine, anti-viral, steroid, opiate, anti-inflammatory, anti-convulsant, polymerization initiator, and polymer.
  • the agent is selected from the group consisting of polynucleotide, polypeptide, chemotherapeutic agent, vaccine, hormone, cytokine, anti-viral, steroid, opiate, anti-inflammatory, anti-convulsant, polymerization initiator, and polymer.
  • Clause 21 The method of any one of the above clauses, wherein the first polypeptide comprises one lysine with a pKa of less than 10.53.
  • Clause 22 The method of any one of the above clauses, wherein the first polypeptide comprises two or more lysines, each with a pKa of less than 10.53.
  • Clause 23 The method of clause 21 , wherein the conjugate formed comprises two or more agents, each agent conjugated to an independent lysine.
  • Clause 24 The method of any one of the above clauses, wherein the first polypeptide comprises one amino acid sequence consisting of ⁇ 2 ⁇ 3 VX 4 VYPKH (SEQ ID NO: 1 ), wherein X-i , X 2 , X 3 , and X4 are independently any amino acid and Z is any hydrophobic residue.
  • Clause 25 The method of any one of the above clauses, wherein the first polypeptide comprises two or more amino acid sequences consisting of ZX 1 X 2 X 3 VX 4 VYPKH (SEQ ID NO: 1 ), wherein X-i , X 2 , X 3 , and X4 are independently any amino acid and Z is any hydrophobic residue.
  • Clause 26 The method of clause 25, wherein the conjugate formed comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of ZX 1 X 2 X 3 VX 4 VYPKH (SEQ ID NO: 1 ), wherein X-,, X 2 , X 3 , and X 4 are independently any amino acid and Z is any hydrophobic residue.
  • Clause 27 The method of any one of the above clauses, wherein the first polypeptide further comprises an additional polypeptide selected from the group consisting of antibody, enzyme, therapeutic protein, fibronectin III (Fn3) domain, TN domain, DARPIN, affibody, scFv, and dsFv, or any combination thereof.
  • additional polypeptide selected from the group consisting of antibody, enzyme, therapeutic protein, fibronectin III (Fn3) domain, TN domain, DARPIN, affibody, scFv, and dsFv, or any combination thereof.
  • Clause 28 The method of any one of the above clauses, wherein the first polypeptide further comprises a fibronectin III (Fn3) domain.
  • Fn3 fibronectin III
  • Clause 30 The method of clause 28, wherein the Fn3 domain is a recombinant polypeptide comprising an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 9.
  • Clause 32 The method of any one of clauses 1 and 3-31 , wherein the agent is conjugated to the second polypeptide amino-terminal to the LPXZG (SEQ ID NO: 72) sequence, wherein X and Z are independently any amino acid.
  • Clause 33 The method of any one of clauses 1 and 3-32, wherein the second polypeptide comprises an amino acid sequence consisting of LPXTG (SEQ ID NO: 3), where X is any amino acid.
  • Clause 34 The method of any one of clauses 1 and 3-33, wherein the second polypeptide comprises an amino acid sequence consisting of LPETG (SEQ ID NO: 15).
  • Clause 35 The method of any one of clauses 1 and 3-32, wherein the second polypeptide comprises an amino acid sequence consisting of LPGAG (SEQ ID NO: 73).
  • Clause 36 The method of any one of clauses 1 and 3-34, wherein the second polypeptide comprises an amino acid sequence consisting of LPETGRAGG (SEQ ID NO: 10).
  • Clause 39 The method of any one of clauses 1 and 3-38, wherein the SrtA recognizes LPXZG (SEQ ID NO: 72) of the second polypeptide.
  • Clause 40 The method of any one of clauses 1 and 3-39, wherein the SrtA cleaves the bond between the Z amino acid and the glycine of LPXZG (SEQ ID NO: 72) and forms a thioester bond between the catalytic thiol in SrtA and the carboxyl group of the Z amino acid.
  • Clause 41 The method of clause 40, wherein the thioester bond between the catalytic thiol in SrtA and the carboxyl group of the Z amino acid forms an intermediate.
  • Clause 42 The method of clause 41 , wherein the intermediate undergoes nucleophilic attack by the ⁇ -amino group of the lysine.
  • Clause 43 The method of any one of clauses 40-42, wherein LPXZG (SEQ ID NO: 72) is LPXTG (SEQ ID NO: 3) and the Z amino acid is threonine.
  • Clause 44 The method of any one of the above clauses, wherein nucleophilic attack by the ⁇ -amino group of the lysine forms an isopeptide bond between the first polypeptide and the second polypeptide to form the agent-first polypeptide conjugate.
  • Clause 45 The method of any one of the above clauses, wherein the agent-first polypeptide conjugate comprises a fragment of the second polypeptide.
  • Clause 46 The method of clause 45, wherein the fragment of the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid.
  • Clause 47 The method of clause 46, wherein the fragment of the second polypeptide comprises an amino acid sequence consisting of LPXT (SEQ ID NO: 74) wherein X is any amino acid.
  • Clause 48 The method of any one of clauses 45-47, wherein the agent-first polypeptide conjugate does not comprise the G of LPXZG (SEQ ID NO: 72; wherein X and Z are independently any amino acid) of the second polypeptide.
  • Clause 49 The method of any one of clauses 1 and 3-48, wherein the formation of the agent-first polypeptide conjugate forms a third polypeptide comprising the G of LPXZG (SEQ ID NO: 72; wherein X and Z are independently any amino acid) and the C-terminal fragment thereto of the second polypeptide.
  • Clause 50 A method of drug delivery, the method comprising administering the agent-first polypeptide conjugate formed by the method of any one of clauses 1 -49.
  • Clause 51 An agent-first polypeptide conjugate formed by the method of any one of clauses 1 -49.
  • LPETGRAGG may be conjugated to biotin or FITC, for example
  • Fn3-PLN3-ELP sequence, amino acid may be conjugated to biotin or FITC for example MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGWLQDVHVYPKHG GSGRGSGGWLQDVHVYPKHGGSGRGSGGWLQDVHVYPKHGGSGRGVGV-(VPGVG) 60
  • LPETG may be conjugated to biotin and/or dabcyl and/or edans, for example.
  • LPET may be conjugated to biotin or dabcyl or FITC, for exampl SEQ ID NO: 30
  • LPETGGG may be conjugated to biotin or dabcyl, for example.
  • Pilin domain of ⁇ 4 deletion mutant first polypeptide, amino acid, artificial VHVYPKH SEQ ID NO: 47
  • Pilin domain of ⁇ 6 deletion mutant first polypeptide, amino acid, artificial
  • VPGXG (wherein X is any amino acid except proline)
  • Ahx-LPET wherein Ahx is the hydrophobic spacer aminohexanoic acid; may be conjugated to FITC, for example
  • HGGSGRGSGGWLQDVHVYPuHGGSGRGSGGWLQDVHVYPKHGGSGR, wherein u lysine isopeptide-linked to biotin-LPETG
  • Ahx-LPETGRAGG wherein Ahx is the hydrophobic spacer aminohexanoic acid; may be conjugated to FITC, for example.
  • LPXZG (wherein X and Z are independently any amino acid)

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Abstract

Provided herein are methods of conjugating an agent to a first polypeptide, wherein the first polypeptide comprises at least one lysine. The lysine may have a pKa of less than 10.53. The method may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZG (SEQ ID NO: 72) wherein X and Z are independently any amino acid, and wherein the ε-amino group of the lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.

Description

COMPOSITIONS AND METHODS FOR THE SITE-SPECIFIC
MODIFICATION OF POLYPEPTIDES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application No. 61/944,489, filed February 25, 2014, U.S. Provisional Patent Application No. 61/985, 167, filed April 28, 2014, U.S. Provisional Patent Application No. 62/058,523, filed October 1 , 2014, U.S. Provisional Patent Application No. 62/062,054, filed October 9, 2014, and U.S. Provisional Patent Application No. 62/064,921 , filed October 16, 2014, each of which is incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant R01 GM061232 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD
[0003] This disclosure relates to compositions and methods for the site-specific modification of polypeptides with agents.
INTRODUCTION
[0004] Small molecule drugs that are too toxic to be administered on their own can be directed to diseased tissues by covalent conjugation to a targeting protein. For example, linking antibodies to extremely cytotoxic small molecule drugs is a promising strategy for the targeted treatment of a variety of cancers. These antibody-drug conjugates (ADCs) may be produced using chemical crosslinking agents that modify the side chains of reactive lysine or cysteine residues in the antibody. Antibodies contain eight cysteines involved in interchain disulfide bonds and a large, variable number of lysines that can serve as attachment sites for small molecules. While the average number of conjugated drugs can be controlled, the product is a heterogeneous mixture of antibodies containing between zero and eight drugs that are stochastically attached to available conjugation sites. Both the number of conjugated drugs and their locations in the protein may negatively impact the efficacy of ADC treatment. For instance, modification of lysines in the antibody's variable regions can reduce affinity for its target, thereby increasing the amount of circulating ADC and the likelihood of off-target toxicity. Similarly, modification of lysines or cysteines in the Fc domain can affect the bioavailability of the drug by altering ADC clearance.
[0005] As such, many of the chemical reactions available for drug attachment are nonspecific and cannot prevent modification at sites on the protein that lower its targeting ability or increase its rate of elimination from the body. Improved conjugation methods that tightly control the site of protein modification by maximizing efficacy and minimizing side effects are therefore highly desirable.
SUMMARY
[0006] Provided herein are methods of conjugating an agent to a first polypeptide. The method may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZG (SEQ ID NO: 72) wherein X and Z are independently any amino acid, and wherein the ε-amino group of the lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
[0007] Further provided are methods of conjugating an agent to a first polypeptide. The method may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid and wherein the carboxy- terminus of the amino acid Z of LPXZ (SEQ ID NO: 75) is modified to a methyl ester, and wherein the ε-amino group of the lysine of the first polypeptide and the Z amino acid of LPXZ (SEQ ID NO: 75) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
[0008] Further provided are methods of drug delivery. The method may include administering the agent-first polypeptide conjugate formed by methods of conjugating an agent to a first polypeptide. The method of conjugating an agent to a first polypeptide may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZG (SEQ ID NO: 72) wherein X and Z are independently any amino acid, and wherein the ε-amino group of the lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate. The method of conjugating an agent to a first polypeptide may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid and wherein the carboxy- terminus of the amino acid Z of LPXZ (SEQ ID NO: 75) is modified to a methyl ester, and wherein the ε-amino group of the lysine of the first polypeptide and the Z amino acid of LPXZ (SEQ ID NO: 75) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
[0009] Further provided are agent-first polypeptide conjugates. The agent-first polypeptide conjugates may be formed by methods of conjugating an agent to a first polypeptide. The method of conjugating an agent to a first polypeptide may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZG (SEQ ID NO: 72) wherein X and Z are independently any amino acid, and wherein the ε-amino group of the lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate. The method of conjugating an agent to a first polypeptide may include contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid and wherein the carboxy- terminus of the amino acid Z of LPXZ (SEQ ID NO: 75) is modified to a methyl ester, and wherein the ε-amino group of the lysine of the first polypeptide and the Z amino acid of LPXZ (SEQ ID NO: 75) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a schematic of pilin assembly by sortases in Gram-positive bacteria, (a) Secreted pilin monomer protein (SpaA) is cleaved by a pilin-specific sortase, forming a thioester-linked intermediate, (b) The pilin polymer extends after the intermediate undergoes nucleophilic attack by the ε-amino group of a lysine in the next monomer, forming an isopeptide bond, (c) After a stochastic number of extension steps, a housekeeping sortase terminates polymerization by anchoring the assembled pilin chain to a pentaglycine branch in the peptidoglycan through a native peptide bond.
[0011] FIG. 2 is a schematic of sortase A from S. aureus catalyzing isopeptide bond formation at the pilin domain lysine, (a) Schematic of the polymerization reaction model for isopeptide bond formation, (b) SDS-PAGE of the reaction product, (c) Maximum monomer consumption in the polymerization reaction was achieved between 29°C and 34°C. (d) MALDI- TOF mass spectrometry of a tryptic digest of reaction products.
[0012] FIG. 3 is a schematic of sortase catalyzing site-specific protein-small molecule conjugation, (a) Overview of the reaction used to conjugate biotin to the pilin domain in the Fn3- PLNs-ELP (SEQ ID NO: 13) fusion protein, (b) SDS-PAGE of biotinylation reactions with Fn3- ELP (SEQ ID NO: 14; 36 kDa) and Fn3-PLN3-ELP (SEQ ID NO: 13; 43 kDa). (c) Western blot with streptavidin-Cy5 of biotinylation reactions with Fn3-ELP (SEQ ID NO: 14; 36 kDa) and Fn3- PLNs-ELP (SEQ ID NO: 13; 43 kDa). (d) MALDI-TOF spectrum of the unpurified Fn3-PLN3-ELP (SEQ ID NO: 13) biotinylation reaction product, corresponding to lane 2 of the Western blot in panel (c), after trypsinization. (e) MALDI-TOF spectrum of tryptic peptides from Fn3-PLN3-ELP (SEQ ID NO: 13) reacted without biotin-modified LPETG (SEQ ID NO: 15) peptide, corresponding to lane 4 of the Western blot in panel (c).
[0013] FIG. 4 is a gel and images of the sortase-catalyzed biotinylation reaction generating bioactive protein-small molecule conjugates, (a) Western blot of the CEX load, non-bound (NB), and elution (Elu) fractions for biotinylated Fn3-PLN3-ELP. (b) Fluorescence microscopy to show the uptake of biotinylated Fn3-PLN3-ELP, indicating nuclei, glycoproteins (for cell morphology), and intracellular biotin. (c) Fluorescence microscopy to show the reduced uptake of biotinylated Fn3-PLN3-ELP with a 10-fold molar excess of unlabeled Fn3-ELP.
[0014] FIG. 5 is schematic overview of the transpeptidation reaction catalyzed by SrtA in S. aureus, (a) As proteins bearing the LPXTG (SEQ ID NO: 3) sortase recognition site are secreted through the cell membrane, SrtA breaks the peptide bond between the threonine and glycine residues and forms an enzyme-substrate intermediate through a thioester bond between the threonine residue and the catalytic cysteine, (b) The acyl-enzyme intermediate is stable but is resolved after nucleophilic attack by the oamino group of a pentaglycine branch of the cell wall, (c) After the transpeptidation reaction is complete, the catalytic thiol of SrtA is regenerated and the substrate protein is anchored to the peptidoglycan through a native peptide bond.
[0015] FIG. 6 are graphs of the thermal responsiveness of the SrtA-ELP fusion protein used in this study, (a) Absorbance at 350 nm versus temperature for various SrtA-ELP concentrations, (b) Transition temperature versus SrtA-ELP concentration.
[0016] FIG. 7 is a graph analyzing the reaction product components for polymerization of ELP-GLP1 (SEQ ID NO: 70).
[0017] FIG. 8 is a schematic diagram and a gel to demonstrate the effect of pH on the efficiency of the ELP-GLP1 (SEQ ID NO: 70) polymerization reaction, (a) Schematic of the reaction, (b) SDS-PAGE of products after reacting SrtA-ELP (SEQ ID NO: 1 1 ) with ELP-GLP1 (SEQ ID NO: 70) at various pHs.
[0018] FIG. 9 is the structure of biotin-LPET (SEQ ID NO: 29).
[0019] FIG. 10 is a schematic diagram and spectra for the SrtA-ELP-catalyzed reaction of biotin-LPETGRAGG (SEQ ID NO: 10) peptide and Fn3-PLN3-ELP (SEQ ID NO: 13). (a) Schematic of the SrtA-ELP-catalyzed reaction of biotin-LPETGRAGG (SEQ ID NO: 10) peptide and Fn3-PLN3-ELP (SEQ ID NO: 13), showing all peptides that would be expected for different sites of biotin conjugation, (b)-(c) Control reactions for the MALDI-TOF spectrum shown in FIG. 3d. (b) MALDI-TOF spectrum for biotinylation reaction containing Fn3-ELP (SEQ ID NO: 14; no pilin domains) and biotin-LPETGRAGG (SEQ ID NO: 10) peptide, (c) MALDI-TOF spectrum for reaction of Fn3-ELP (SEQ ID NO: 14; no pilin domains) without biotin-LPETGRAGG (SEQ ID NO: 10) peptide.
[0020] FIG. 11 is a schematic diagram and spectra for the conjugation of dabcyl to ELP- GLP1 (SEQ ID NO: 70). (a) Structure of dabcyl-LPETG-edans peptide (SEQ ID NO: 15). (b) Schematic for the conjugation of dabcyl to ELP-GLP1 (SEQ ID NO: 70). (c) MALDI-TOF spectra for the unpurified reaction product. [0021] FIG. 12 is a schematic diagram and spectra for the analysis of peptides of ELP- GLP1 (SEQ ID NO: 70) conjugated with dabcyl-LPETG-edans (LPETG is SEQ ID NO: 15) without including triglycine in the trypsinization reaction, (a) Schematic of the reaction, (b) The MALDI-TOF spectrum for the unpurified reaction product.
[0022] FIG. 13 is a graph and a gel for the purification of biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13). (a) Absorbance at 280 nm, pH, and conductivity traces for the cation exchange chromatography (CEX). (b) SDS-PAGE of the CEX load, non-bound, and elution fractions of biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13).
[0023] FIG. 14 are graphs for the quantitation of the biotin: protein molar ratio for the Fn3- PLN3-ELP (SEQ ID NO: 13) biotinylation reaction using fluorescent (a) and colorimetric (b) HABA displacement assays.
[0024] FIG. 15 are additional images for analysis of the uptake of biotinylated Fn3-PLN3- ELP (SEQ ID NO: 13) by HUVECs. (a-c) HUVECs treated with 100 nM biotinylated Fn3-PLN3- ELP (SEQ ID NO: 13). (d-f) HUVECs treated with 100 nM biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13) and a 10-fold molar excess of unlabeled Fn3-ELP (SEQ ID NO: 14).
[0025] FIG. 16 are graphs from flow cytometry to confirm αγβ3 expression on HUVECs. (a) Cells were stained with biotinylated isotype control antibody (black, filled histogram) or a biotinylated anti-human CD51/CD61 (blue, open histogram) monoclonal antibody (eBioscience catalog number 13-0519), washed, and stained with streptavidin-FITC. (b) HUVECs were stained with either biotinylated mouse lgG1 κ (filled histogram), unreacted Fn3-PLN3-ELP (left peak, open histogram), or biotinylated Fn3-PLN3-ELP (right peak, open histogram) followed by secondary detection with streptavidin-FITC.
[0026] FIG. 17 is an exemplary schematic diagram of a reaction catalyzed by SrtA to form the agent-first polypeptide conjugate.
[0027] FIG. 18 shows that FITC can be conjugated to substrate proteins by sortase- mediated isopeptide ligation, (a) Schematic of the reaction of FITC-Ahx-LPETGRAGG (SEQ ID NO: 68) peptide with Fn3-PLN3-ELP (SEQ ID NO: 13) and SrtA-ELP (SEQ ID NO: 1 1 ). (b) SDS-PAGE of the reaction product (upper panel). 488 nm laser excitation and a 520 BP 40 nm emission filter for FITC (lower panel), (c) MALDI-TOF spectrum, (d) The chemical structure of the FITC-Ahx-LPET (SEQ ID NO: 52) moiety attached to the pilin domain lysine. [0028] FIG. 19 is a schematic diagram of the isopeptide ligation reaction catalyzed by sortase A with peptide substrates, (a) Scheme showing the sortase-mediated isopeptide ligation of a peptide containing the pilin domain with a peptide containing the sortase recognition sequence LPETG (SEQ ID NO: 15) and an amino-terminal biotin. (b) MALDI-TOF mass spectra with and without SrtA-ELP (SEQ ID NO: 1 1 ).
[0029] FIG. 20 shows that the His-tagged SrtA (H6-SrtA; SEQ ID NO: 49) achieved site- specific conjugation of biotin to the pilin domain lysine, (a) SDS-PAGE of reactions of H6-SrtA (SEQ ID NO: 49) and Fn3-PLN3-ELP (SEQ ID NO: 13) with or without biotin-LPETGRAGG (SEQ ID NO: 10) peptide, (b) Western blot of the gel in panel (a) using streptavidin-Cy5 to detect biotinylated protein, (c) The MALDI-TOF mass spectrum of tryptic peptides of the unpurified 20°C biotinylation reaction.
[0030] FIG. 21 is a fully annotated version of the MS1 spectrum for LC-MS/MS corresponding to FIG. 24a and FIG. 13.
[0031] FIG. 22 shows tandem mass spectrometry (MS) of tryptic peptides of biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13), which confirmed biotinylation at the pilin domain lysine through an isopeptide bond, (a) MS2 spectrum of daughter ions produced by isolating the +4 charge state (m/z = 691 ) of a biotinylated pilin domain peptide, (b) An outline of the observed fragmentation pattern and the nomenclature used to classify daughter ions, corresponding to the daughter ion chemical structures in panel (c). (d) Extracted ion chromatograms (EICs) for individual ions were summed for peptides containing biotinylated (light lines) or non-biotinylated (dark lines) pilin domain lysines, (e) Quantitation of the biotin:protein molar ratio using a colorimetric assay measuring HABA displacement from avidin.
[0032] FIG. 23 is (a) MS2 spectrum of daughter ions produced by isolating the +3 charge state (m/z = 920.4) of a biotinylated pilin domain peptide, (b) An outline of the observed fragmentation pattern and the nomenclature used to classify daughter ions, corresponding to the daughter ion chemical structures in panel (c). Peptide bond cleavage resulted in a charged amino-terminal fragment (b ions) or a charged carboxy-terminal fragment (y ions).
[0033] FIG. 24 shows that sortase-mediated isopeptide ligation can be used to modify monoclonal antibodies (mAbs). (a) Schematic of the reaction between an anti-Her2 mAb, genetically modified to contain the pilin domain at the heavy chain carboxy-terminus, and biotin- LPETGRAGG (SEQ ID NO: 10) peptide and SrtA-ELP (SEQ ID NO: 1 1 ). (b) SDS-PAGE and an anti-biotin Western blot of reaction products, (c) Immunofluorescence of the Her2- overexpressing cell line SK-OV-3 using biotinylated anti-Her2 containing a pilin domain on its heavy chain followed by secondary staining with streptavidin-FITC conjugate, (d) Immunofluorescence of SK-OV-3 using a biotinylated isotype control antibody in the primary staining step.
[0034] FIG. 25 are graphs for the quantitation of the biotin-to-protein molar ratio for anti- Her2 with one pilin domain inserted at the carboxy-terminus of each heavy chain.
[0035] FIG. 26 are additional images for the SK-OV-3 immunofluorescence of FIG. 24.
[0036] FIG. 27 shows reaction of SrtA with various truncated isopeptide attachment sequences, (a) A schematic diagram of the reaction of SrtA-ELP (SEQ I D NO: 1 1 ) and biotin- LPETGRAGG (SEQ I D NO: 10) and Fn3-I PA-ELP fusion proteins, (b) SDS-PAGE and Western blot with streptavidin-Cy5 of the reaction products for fusion proteins containing isopeptide attachment (I PA) sequences, (c) MALDI-TOF spectra of the reaction products for fusion proteins containing IPA sequences.
[0037] FIG. 28 is a Western Blot of reactions with SrtA, biotin-LPETGRAGG (SEQ I D NO: 10), and a panel of polypeptides with mutant pilin domains.
DETAILED DESCRIPTION
[0038] Provided herein is a method for making bioactive conjugates. These bioactive conjugates are a result of site-specific protein modifications that make use of lysine chemistry for attachment of one or more agents to proteins. The method centers on a previously uncharacterized activity of the transpeptidase sortase A (SrtA), which catalyzes the covalent attachment of two moieties through a site-specific isopeptide bond. A first polypeptide is contacted with sortase A (SrtA) and at least one agent conjugated to a second polypeptide. The first polypeptide includes at least one lysine, such as, for example, in an amino acid sequence consisting of ZX1X2X3VX4VYPKH (SEQ I D NO: 1 ), where Xi, X2, X3, and X4 are independently any amino acid and Z is any hydrophobic amino acid. The second polypeptide includes an amino acid sequence consisting of, for example, LPXZG (SEQ I D NO: 72), wherein X and Z are independently any amino acid. The resulting agent-first polypeptide conjugate includes an isopeptide bond between the ε-amino group of the lysine, such as the lysine of ZX1X2X3VX4VYPKH (SEQ I D NO: 1 ), and the Z amino acid of LPXZG (SEQ I D NO: 72). The method results in site-specific protein modifications that contribute to the rapidly expanding repertoire of therapeutic protein-drug and protein-polymer conjugates.
[0039] The SrtA catalyzed reaction was applied in vitro to generate recombinant protein polymers with unique, branched structures as well as receptor-targeting proteins loaded with multiple small molecules. The reaction displays a level of control over the site of conjugation; the modification occurred exclusively at a lysine ε-amino group within an engineered site in the protein substrate. Accordingly, the method disclosed herein provides a unique chemistry for the generation of lysine-modified protein conjugates and allows the precise control of the number of conjugated molecules as well their location.
1. Definitions
[0040] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of" and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.
[0041] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. [0043] The term "about" as used herein as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain aspects, the term "about" refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
[0044] "Polymer" as used herein is intended to encompass a homopolymer, heteropolymer, block polymer, co-polymer, ter-polymer, etc., and blends, combinations and mixtures thereof. Examples of polymers include, but are not limited to, functionalized polymers, such as a polymer comprising 5-vinyltetrazole monomer units and having a molecular weight distribution less than 2.0. The polymer may be or contain one or more of a star block copolymer, a linear polymer, a branched polymer, a hyperbranched polymer, a dendritic polymer, a comb polymer, a graft polymer, a brush polymer, a bottle-brush copolymer and a crosslinked structure, such as a block copolymer comprising a block of 5-vinyltetrazole monomer units. Polymers include, without limitation, polyesters, poly(meth)acrylamides, poly(meth)acrylates, polyethers, polystyrenes, polynorbornenes and monomers that have unsaturated bonds. For example, amphiphilic comb polymers are described in U.S. Patent Application Publication No. 2007/00871 14 and in U.S. Patent No. 6,207,749 to Mayes et al., the disclosure of each of which is herein incorporated by reference in its entirety. The amphiphilic comb-type polymers may be present in the form of copolymers, containing a backbone formed of a hydrophobic, water- insoluble polymer and side chains formed of short, hydrophilic non-cell binding polymers. Examples of other polymers include, but are not limited to, polyalkylenes such as polyethylene and polypropylene; polychloroprene; polyvinyl ethers; such as polyvinyl acetate); polyvinyl halides such as polyvinyl chloride); polysiloxanes; polystyrenes; polyurethanes; polyacrylates; such as poly(methyl (meth)acrylate), poly(ethyl (meth)acrylate), poly(n-butyl(meth)acrylate), poly(isobutyl (meth)acrylate), poly(tert-butyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate); polyacrylamides such as poly(acrylamide), poly(methacrylamide), poly(ethyl acrylamide), poly(ethyl methacrylamide), poly(N-isopropyl acrylamide), poly(n, iso, and tert-butyl acrylamide); and copolymers and mixtures thereof. These polymers may include useful derivatives, including polymers having substitutions, additions of chemical groups, for example, alkyl groups, alkylene groups, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art. The polymers may include zwitterionic polymers such as, for example, polyphosphorycholine, polycarboxybetaine, and polysulfobetaine. The polymers may have side chains of betaine, carboxybetaine, sulfobetaine, oligoethylene glycol (OEG), sarcosine or polyethyleneglycol (PEG). For example, poly(oligoethyleneglycol methacrylate) (poly(OEGMA)) may be used. Poly(OEGMA) may be hydrophilic, water-soluble, non-fouling, non-toxic and non-immunogenic due to the OEG side chains.
[0045] "Polynucleotide" as used herein can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequence. The polynucleotide can be nucleic acid, natural or synthetic, DNA, genomic DNA, cDNA, RNA, or a hybrid, where the polynucleotide can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis methods or by recombinant methods.
[0046] A "peptide" or "polypeptide" is a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as antibodies.
[0047] "Subject" as used herein can mean a mammal that wants to or is in need of being treated with the herein described agent-first polypeptide conjugate. The mammal can be a human, dog, cat, horse, cow, pig, mouse, rat, or non-human primate such as, for example, chimpanzee, gorilla, orangutan, and gibbon.
[0048] "Treatment" or "treating," when referring to protection of an animal from a disease, means preventing, suppressing, repressing, ameliorating, or completely eliminating the disease. Preventing the disease involves administering a composition of the present invention to an animal prior to onset of the disease. Suppressing the disease involves administering a composition of the present invention to an animal after induction of the disease but before its clinical appearance. Repressing or ameliorating the disease involves administering a composition of the present invention to an animal after clinical appearance of the disease.
[0049] "Substantially identical" can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% over a region of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100 amino acids.
[0050] "Variant" used herein with respect to a polynucleotide means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a polynucleotide that is substantially identical to a referenced polynucleotide or the complement thereof; or (iv) a polynucleotide that hybridizes under stringent conditions to the referenced polynucleotide, complement thereof, or a sequences substantially identical thereto.
[0051] A "variant" can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or to promote an immune response. Variant can mean a substantially identical sequence. Variant can mean a functional fragment thereof. Variant can also mean multiple copies of a polypeptide. The multiple copies can be in tandem or separated by a linker. Variant can also mean a polypeptide with an amino acid sequence that is substantially identical to a referenced polypeptide with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids. See Kyte et al., J. Mol. Biol. 1982, 157, 105-132. The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophibicity of amino acids can also be used to reveal substitutions that would result in polypeptides retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a polypeptide permits calculation of the greatest local average hydrophilicity of that polypeptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity, as discussed in U.S. Patent No. 4,554,101 , which is fully incorporated herein by reference. Substitution of amino acids having similar hydrophilicity values can result in polypeptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
[0052] A variant can be a polynucleotide sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The polynucleotide sequence can be 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence can be 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.
2. SrtA
[0053] In its canonical function in Staphylococcus aureus, SrtA recognizes the primary sequence LPXTG (SEQ ID NO: 3, where X is any amino acid) in a protein and cleaves the peptide bond between threonine and glycine, forming a stable intermediate that joins the catalytic thiol in SrtA to the carboxyl group of threonine in a thioester bond. This intermediate undergoes nucleophilic attack by the oamino group of an oligoglycine branch in the peptidoglycan, generating a native peptide bond that anchors the substrate protein to the cell wall (FIG. 5).
[0054] According to the compositions and methods detailed herein, SrtA recognizes an amino acid sequence consisting of LPXZG (SEQ ID NO: 72, where X and Z are independently any amino acid) and cleaves the peptide bond between the Z amino acid and the glycine of LPXZG (SEQ ID NO: 72) and forms a thioester bond between the catalytic thiol in SrtA and the carboxyl group of the Z amino acid. The thioester bond between the catalytic thiol in SrtA and the carboxyl group of the Z amino acid forms an intermediate, and the intermediate undergoes nucleophilic attack by the ε-amino group of the lysine of first polypeptide to form an isopeptide bond between the ε-amino group of the lysine and the Z amino acid of LPXZG (SEQ ID NO: 72). In some embodiments, SrtA forms an isopeptide bond between the ε-amino group of any solvent-accessible, nucleophilic lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72). An exemplary schematic diagram of the process is shown in FIG. 17, wherein the Z amino acid is threonine and ZX1X2X3VX4VYPKH (SEQ ID NO: 1 ) comprises the lysine of the first polypeptide.
[0055] The SrtA may be any SrtA, such as S. aureus SrtA. SrtA may be from a Gram positive bacterium, such as, for example, bacteria in a genus selected from Staphylococcus, Streptococcus, Enterococcus, Bacillus, Corynebacterium, Nocardia, Clostridium, Actinobacteria, and Listeria. In some embodiments, SrtA is from S. aureaus. The SrtA may be wild-type SrtA or a variant thereof.
[0056] The SrtA may include a hydrophobic, membrane-binding domain at the amino terminal end. SrtA can comprise the full length wild-type S. aureus SrtA, a variant thereof, a fragment thereof, or a combination thereof. SrtA may be immobilized on, for example, beads or or resin or other solid support. In some embodiments, SrtA is a recombinant polypeptide corresponding the wild-type S. aureus having a deletion of the amino-terminal 59 amino acids to remove a hydrophobic, membrane-binding domain. In some embodiments, SrtA is a recombinant polypeptide or fusion protein comprising a purification tag, such as, for example, an elastin-like polypeptide (ELP). ELP is a peptide polymer comprising repeats of the pentapeptide VPGXG (SEQ ID NO: 51 ) where X is any amino acid except proline. SrtA may be membrane- bound. SrtA may be insoluble. SrtA may be soluble.
[0057] SrtA may comprise an amino acid sequence consisting of SEQ ID NO: 6, which is encoded by a polynucleotide sequence of SEQ ID NO: 7. SEQ ID NO: 6 refers to the full-length wild-type S. aureus polypeptide sequence. SrtA can comprise a polypeptide having an amino acid sequence that is 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over the entire length of the amino acid sequence of SEQ ID NO: 6.
[0058] SrtA may comprise an amino acid sequence consisting of SEQ ID NO: 4, which is encoded by a polynucleotide sequence of SEQ ID NO: 5. SEQ ID NO: 4 refers to the wild-type S. aureus polypeptide sequence with a deletion of the amino-terminal 59 amino acids. This removes a hydrophobic, membrane-binding domain and makes the resulting protein soluble. SrtA can comprise a polypeptide having an amino acid sequence that is 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over the entire length of the amino acid sequence of SEQ ID NO: 4. [0059] Certain Gram-positive bacteria use SrtA, and homologs thereof, to assemble pili- fibrous polymers of structural proteins that extend from the bacterial surface and are implicated in adhesion and biofilm formation. The initial step for pilin formation is the same as that used to anchor proteins to the cell wall and generates a thioester intermediate upon cleavage of the LPXTG (SEQ ID NO: 3) motif. Extension of a pilin polymer proceeds when the thioester undergoes nucleophilic attack by the ε-amino group of a lysine residue in another sortase-linked pilin monomer (FIG. 1 ). This process repeats and is stochastically terminated by a "housekeeping" sortase that anchors the pilin polymer to the cell wall through a native peptide bond at the a-amino group of oligoglycine. The pilin polymer is therefore composed of a series of branched monomers linked by isopeptide bonds, with each monomer retaining an unmodified a-amino group. In Corynebacterium diphtheriae and Actinomyces naeslrmdii, the nucleophilic lysine used in polymer extension is contained in a "pilin domain" with the sequence WX1X2X3VX4VYPKI-I (SEQ ID NO: 2), wherein X·,, X2, X3, and X4 are non-conserved amino acid residues.
[0060] The SrtA may recognize the primary sequence LPXTG (SEQ ID NO: 3, where X is any amino acid) in a protein and cleave the peptide bond between threonine and glycine, forming a stable intermediate. In S. aureus, this intermediate undergoes nucleophilic attack by the a-amino group of an oligoglycine branch in the peptidoglycan, generating a native peptide bond that anchors the substrate protein to the cell wall (FIG. 5). In addition to "X", certain amino acid substitutions are also tolerated in the LPXTG (SEQ ID NO: 3) sequence at the first, fourth, and fifth positions that affect the rate but not the overall outcome of the reaction. These mutations can be made according to Kruger et al. (Biochemistry 2004, 43, 1541-1551 ), Bellucci et al. {Angew Chem. Int. Ed. Engl. 2013, 52, 3703-3708), Williamson, et al. {Angew. Chemie Int. Ed. 2012, 51, 9377-9380), and Antos, et al. {J. Am. Chem. Soc. 2009, 131, 10800-10801 ). In some embodiments, SrtA recognizes the primary sequence LPXZG (SEQ ID NO: 72, wherein X and Z are independently any amino acid) in a protein and cleave the peptide bond between the Z amino acid and glycine, forming a stable intermediate. The linkage between X and Z amino acids of LPXZG (SEQ ID NO: 72) does not have to be a peptide bond. In some embodiments, the linkage between X and Z amino acids of LPXZG (SEQ ID NO: 72) is a desipeptide bond. In some embodiments, SrtA recognizes the primary sequence LPXZ in a protein, wherein the carboxy- terminus of the amino acid Z of LPXZ is modified to another functional group, for example, a methyl ester rather than a carboxylic acid. In some embodiments, SrtA recognizes the primary sequence LPGAG (SEQ ID NO: 73) in a protein and cleaves the peptide bond between the alanine and glycine, forming a stable intermediate. In some embodiments, SrtA recognizes the primary sequence LPETG (SEQ ID NO: 15) in a protein and cleaves the peptide bond between the threonine and glycine, forming a stable intermediate.
[0061] Solution conditions for SrtA activity are flexible and may vary. The temperature for SrtA activity may be about 20°C to about 45°C, about 20°C to about 42°C, about 22°C to about 40°C, about 25°C to about 35°C, about 30°C to about 34°C, or about 32°C to about 33°C. In some embodiments, conditions for SrtA activity may include a temperature of about 20°C to about 42°C. In some embodiments, conditions for SrtA activity may include a temperature of about 25°C. In some embodiments, conditions for SrtA activity may include a temperature of about 33°C. The pH for SrtA activity may be about pH 4 to about pH 1 1 , pH 4.5 to about pH 10, pH 5 to about pH 9.5, pH 6 to about pH 9, or pH 7 to about pH 8.5. In some embodiments, conditions for SrtA activity may include a pH 5 to about pH 9.5. In some embodiments, conditions for SrtA activity may include a pH of about 8.5. The molar ratio of substrate:enzyme for SrtA activity may be about 1 :2 to about 5:1 , about 1 :1 to about 4:1 , or about 2:1 to about 3:1. In some embodiments, conditions for SrtA activity may include molar ratio of substrate:enzyme of about 1 :2 to about 5:1 . In some embodiments, conditions for SrtA activity may include a buffer (comprising 50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI2, pH 8.5) and incubation for 18- 24 hours. However, none of the investigated ranges for these variables or the concentrations in the reaction buffer are known to impose strict limits on the reaction, and significant modifications to these conditions may be tolerated.
[0062] Several factors may affect the SrtA reaction yield and kinetics. The location of the pilin domain within a protein may impact its solvent accessibility and may hence affect both kinetics and yield. The precise sequence of the pilin domain sequence may also impact both reaction yield and kinetics. The pilin domain may tolerate truncations, which may make it easier to engineer this sequence into a variety of solvent accessible loops within various proteins.
3. Agent-First Polypeptide Conjugate
[0063] Further provided herein is an agent-first polypeptide conjugate comprised of a first polypeptide conjugated to an agent via a portion or fragment of a second polypeptide. The terms "complex" and "construct" and "conjugate" are used interchangeably herein. The agent is selectively conjugated to a specific lysine of the first polypeptide via an amino acid of the second polypeptide in a reaction catalyzed by SrtA. The formation of agent-first polypeptide conjugate is catalyzed by SrtA. a. First Polypeptide
[0064] The first polypeptide comprises at least one lysine. In some embodiments, the at least one lysine is solvent-accessible. In some embodiments, the at least one lysine is nucleophilic. The at least one lysine may have a side chain amino group that is nucleophilic. The nucleophilicity may be due to the particular 3-dimensional local protein structure. For example, a lysine in a hydrophobic or positively-charged patch of the protein structure would be more nucleophilic (pKa < 10.53) than a typical lysine (pKa = 10.53). In some embodiments, the at least one lysine of the first polypeptide comprises a nucleophilic nitrogen atom. In some embodiments, the at least one lysine of the first polypeptide comprises a side chain comprising an uncharged primary amino group. In some embodiments, the at least one lysine of the first polypeptide has a pKa of less than 10.53. In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of KH. In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of PKH. In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of ZX1X2X3VX4VYPKH (SEQ ID NO: 1 ), wherein X·,, X2, X3, and X4 are independently any amino acid residue and Z is any hydrophobic amino acid residue. Hydrophobic amino acid residues include, for example, A, V, I, L, M, F, Y, and W. In some embodiments, Z is W. In some embodiments, Z is F. In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of WX1X2X3VX4VYPKH (SEQ ID NO: 2), wherein X1 ; X2, X3, and X4 are independently any amino acid residue. In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of WLQDVHVYPKH (SEQ ID NO: 45). In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of WLQDVHVYPK (SEQ ID NO: 71 ). In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of VHVYPKH (SEQ ID NO: 46). In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of VYPKH (SEQ ID NO: 47). In some embodiments, the first polypeptide comprises at least one amino acid sequence consisting of YPKH (SEQ ID NO: 48). The first polypeptide facilitates administration of the agent.
[0065] The first polypeptide may further comprise an additional polypeptide such as, for example, an antibody, enzyme, therapeutic protein, fibronectin III (Fn3) domain, Fn3 domain from the human tenascin protein (TN domain), Designed Ankyrin Repeat Domain (DARPIN), affibody, scFv, and dsFv, or any combination thereof. In some embodiments, the first polypeptide further comprises an antibody. In some embodiments, the first polypeptide further comprises a TN domain, DARPINS, affibodies, and/or scFvs. In some embodiments, the first polypeptide further comprises a Fn3 domain. The Fn3 domain may be a recombinant polypeptide comprising an amino acid sequence of SEQ ID NO: 8, encoded by a polynucleotide sequence of SEQ ID NO: 9.
[0066] In some embodiments, the first polypeptide comprises two or more lysines. In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to a different lysine. In some embodiments, the first polypeptide comprises two or more lysines, each with a pKa less than 10.53. In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to a different lysine, each with a pKa less than 10.53. In some embodiments, the first polypeptide comprises two or more amino acid sequences consisting of ZX1X2X3VX4VYPKH (SEQ ID NO: 1 ). In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of ZX X2 z VX4VYPKH (SEQ ID NO: 1 ). For example, as shown in FIG. 17 the agent is conjugated to the lysine of SEQ ID NO: 1. In some embodiments, the first polypeptide comprises two or more amino acid sequences consisting of WX1X2X3VX4VYPKH (SEQ ID NO: 2). In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of WX1X2X3VX4VYPKH (SEQ ID NO: 2). In some embodiments, the first polypeptide comprises two or more amino acid sequences consisting of WLQDVHVYPK (SEQ ID NO: 71 ). In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of WLQDVHVYPK (SEQ ID NO: 71 ). In some embodiments, the first polypeptide comprises two or more amino acid sequences consisting of WLQDVHVYPKH (SEQ ID NO: 45). In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of WLQDVHVYPKH (SEQ ID NO: 45). In some embodiments, the first polypeptide comprises two or more amino acid sequences consisting of VHVYPKH (SEQ ID NO: 46). In such embodiments, the agent- first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of VHVYPKH (SEQ ID NO: 46). In some embodiments, the first polypeptide comprises two or more amino acid sequences consisting of VYPKH (SEQ ID NO: 47). In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of VYPKH (SEQ ID NO: 47). In some embodiments, the first polypeptide comprises two or more amino acid sequences consisting of YPKH (SEQ ID NO: 48). In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of YPKH (SEQ ID NO: 48). In some embodiments, the first polypeptide comprises two or more amino acid sequences consisting of PKH. In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of PKH. In some embodiments, the first polypeptide comprises two or more amino acid sequences consisting of KH. In such embodiments, the agent-first polypeptide conjugate comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of KH. b. Agent
[0067] The agent is any composition that elicits a desired effect in the subject upon administration. The agent may be selected from the group consisting of polynucleotide, polypeptide, chemotherapeutic agent, vaccine, hormone, cytokine, anti-viral, steroid, opiate, anti-inflammatory, anti-convulsant, polymerization initiator, and polymer. In some embodiments, the agent comprises a protein. In some embodiments, the agent comprises an antibody. In some embodiments, the agent comprises a peptide drug. In some embodiments, the agent comprises a small molecule. In some embodiments, the small molecule comprises a detectable label. In some embodiments, the small molecule comprises a fluorophore. In some embodiments, the agent comprises a small molecule chemotherapeutic.
[0068] In some embodiments, the agent comprises a polymerization initiator or a polymer or a combination thereof. In embodiments wherein the agent comprises a polymerization initiator, a polymer may be in situ polymerized from the polymerization initiator. A polymerization initiator is a molecule that assists in beginning polymerization by interacting with a polypeptide (e.g., the second polypeptide detailed below) and a monomer to form a polymer. Polymerization may include, for example, atom transfer radical polymerization (ATRP), reversible addition- fragmentation chain transfer (RAFT) polymerization, nitroxide mediated radical polymerization (NMP), ring-opening metathesis polymerization (ROMP), and combinations thereof. Examples of polymerization initiators include those compatible with ATRP such as, without limitation, N-(2- aminoethyl)-2-bromo-2-methylpropanamide, N-(2-aminoethyl)-2-chloro-2-methylpropanamide, 2-bromo-N-(2-(2-hydrazinylacetamido) ethyl)-2-methylpropanamide, 2-chloro-N-(2-(2- hydrazinylacetamido) ethyl)-2-methylpropanamide. Examples of polymerization initiators and systems also include those compatible with RAFT such as, without limitation, a chain transfer agent (CTA), ZC(=S)SR, where R can be cysteine, hydrazine, hydroxylamine, and Z can be phenyl, alkyl, phthalimidomethyl, coupled with traditional radical polymerization initiators including those such as AIBN which are cleaved to initiate the polymerization. Examples of polymerization initiators also include those compatible with ROMP such as, without limitation, A- B, where A can be cysteine, hydrazine, hydroxylamine, and B can be olefins. In some embodiments, the agent is a polymerization initiator comprising an ATRP initiator, followed by in situ ATRP polymerization from the initiator. In some embodiments, the agent is a polymerization initiator comprising a RAFT agent, followed by in situ RAFT polymerization from the initiator.
[0069] Polymerization may be facilitated by the inclusion of a catalyst solution. For example, ATRP catalyst system may include, but are not limited to, copper halides and ligands, where ligands can be derivatives of 2, 2'-bipyridine, other ττ-accepting, chelating nitrogen-based ligands such as 2-iminopyridines and some aliphatic polyamines. RAFT catalyst system may include water soluble radical generating compounds, such as 4, 4'-azobis(4-cyanopentanoic acid), 2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2'-Azobis[2-(2-imidazolin-2- yl)propane]-disulfate dehydrate, 2,2'-Azobis(2-ethylpropionamidine)-dihydrochloride. ROMP catalyst systems may include, but are not limited to, soluble Grubbs catalysts, such as tetraethylene glycol substituted ruthenium benzylidene, ruthenium alkylidene with triaryl phosphate ligands, or ruthenium alkylidene with ligands with quaternary ammonium. Other conditions used for polymerization may include, for example, that the polymerization be carried out under low oxygen, for example, under a noble or non-reactive gas such as argon, and/or for a time of at least about 5 min, at least about 15 min, at least about 60 min, and optionally no more than about 12 hours, no more than about 24 hours, no more than about 48 hours. Polymerization may be carried out, for example, at a temperature of at least about 5°C, at least about 10°C, at least about 15°C, at least about 20°C, at least about 30°C, at least about 40°C, at least about 50°C, at least about 60°C, at least about 70°C, at least about 80°C, at least about 90°C or at least about 100°C.
[0070] In embodiments wherein the agent comprises a polymerization initiator, a variety of monomers may be suitable for the polymerization. Exemplary monomers include, but are not limited to, lactic acid, epichlorohydrin, acrylate, methacylate, acrylamide, methacrylamide, norbornene, and oxanorbornene. Examples of monomer structures that may be used in ROMP, NMP, ATRP and RAFT, and other components and techniques that may be used are described in U.S. Patent Publication No. 201 10294189, the entire disclosure of which is herein incorporated by reference in its entirety. c. Second Polypeptide
[0071] A portion or fragment of the second polypeptide links the agent to the first polypeptide and may be described as a linker. The terms "portion" and "fragment" are used interchangeably herein. In some embodiments, the second polypeptide comprises an amino acid sequence consisting of LPXZG (SEQ ID NO: 72), wherein X and Z are independently any amino acid residue. L is at the N-terminal end and G is at the C-terminal end of LPXZG (SEQ ID NO: 72). In some embodiments, the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75), wherein X and Z are independently any amino acid residue and wherein the carboxy-terminus of Z is modified to another functional group, for example, a methyl ester. The linkage between X and Z of SEQ ID NO: 72 or 75 may be, for example, a peptide bond or a desipeptide bond. In some embodiments, the second polypeptide comprises an amino acid sequence consisting of LPXTG (SEQ ID NO: 3), wherein X is any amino acid residue. In some embodiments, X is E. In some embodiments, second polypeptide comprises an amino acid sequence consisting of LPETG (SEQ ID NO: 15). In some embodiments, the second polypeptide comprises an amino acid sequence consisting of LPGAG (SEQ ID NO: 73). The second polypeptide may include additional amino acids C-terminal to LPXZG (SEQ ID NO: 72). For example, the second polypeptide may include at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids C-terminal to LPXZG (SEQ ID NO: 72). The second polypeptide may include a variety of chemical modifications to the carboxyl group of the C-terminal end of LPXZG (SEQ ID NO: 72) such as, for example, conversion to a methyl ester or amide. In some embodiments, the second polypeptide comprises an amino acid sequence consisting of LPETGRAGG (SEQ ID NO: 10). In some embodiments, the side chain of the last amino acid at the carboxy end of the second polypeptide does not include a carboxyl group. The amino acid sequence carboxy-terminal to LPXZG (SEQ ID NO: 72) can be any amino acid sequence or other chemical moiety, as long as there is not a carboxyl group at the carboxy-terminus of the LPXZG (SEQ ID NO: 72) peptide. Other amino acids can also be present amino-terminal to LPXZG (SEQ ID NO: 72).
[0072] In some embodiments, the agent is at the amino terminal end of the second polypeptide, or a fragment thereof. In some embodiments, the agent is amino terminal to LPXZG (SEQ ID NO: 72) of the second polypeptide, or a fragment thereof. The agent can be conjugated to the LPXZG (SEQ ID NO: 72) through an amino acid or other chemical linker. The agent does not have to be conjugated to the amino terminus of the LPXZG (SEQ ID NO: 72) peptide if additional amino acids are located amino-terminal to the LPXZG (SEQ ID NO: 72) sequence. For example, the agent can be conjugated to a lysine, a cysteine, or an unnatural amino acid in a peptide containing the LPXZG (SEQ ID NO: 72) sequence, as long as the agent is conjugated amino-terminal to the LPXZG (SEQ ID NO: 72) enzyme recognition site. In some embodiments, the agent is conjugated to the second polypeptide, or a fragment thereof, amino- terminal to the LPXZG (SEQ ID NO: 72) sequence.
4. Methods a. Methods of Conjugating an Agent to a First Polypeptide
[0073] The present invention is directed to a method of conjugating an agent to a first polypeptide. The method may comprise contacting the first polypeptide with SrtA and at least one agent conjugated to a second polypeptide, to form an agent-first polypeptide conjugate. b. Methods of Drug Delivery
[0074] The present invention is directed to a method of drug delivery. The method may comprise administering the agent-first polypeptide complex. The agent-first polypeptide complex may be administered to a subject.
[0075] The agent-first polypeptide conjugate as detailed herein may have improved properties for delivery of the agent or the first polypeptide. For example, an agent-first polypeptide conjugate produced as described herein may show improvement in one or more of solubility, stability, pharmacokinetics, immunogenicity and biodistribution or bioaccumulation at the cell, tissue, disease site, or organ level. In some embodiments, the agent-first polypeptide conjugate provides a longer plasma half-life than the first polypeptide by itself. 5. Administration
[0076] The agent-first polypeptide conjugate as detailed above can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration.
[0077] The agent-first polypeptide conjugate can be administered prophylactically or therapeutically. In prophylactic administration, the agent-first polypeptide conjugates can be administered in an amount sufficient to induce a response. In therapeutic applications, the agent-first polypeptide conjugates are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, e.g., the particular composition of the agent-first polypeptide conjugate regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.
[0078] The agent-first polypeptide conjugate can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 1997, 15, 617-648); Feigner et al. (U.S. Patent No. 5,580,859, issued Dec. 3, 1996); Feigner (U.S. Patent No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Patent No. 5,679,647, issued Oct. 21 , 1997), the contents of all of which are incorporated herein by reference in their entirety. The polypeptide or agent of the agent-first polypeptide conjugate can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration.
[0079] The agent-first polypeptide conjugates can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, intravaginal, transdermal, intravenous, and epidermal routes.
[0080] The agent-first polypeptide conjugate can be a liquid preparation such as a suspension, syrup, or elixir. The agent-first polypeptide conjugate can be incorporated into liposomes, microspheres, or other polymer matrices (such as by a method described in Feigner et al., U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed. 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can consist of phospholipids or other lipids, and can be nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
[0081] The vaccine can be administered via electroporation, such as by a method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. The electroporation can be by a method and/or apparatus described in U.S. Patent Nos. 6,302,874; 5,676,646; 6,241 ,701 ; 6,233,482; 6,216,034; 6,208,893; 6, 192,270; 6,181 ,964; 6, 150, 148; 6, 120,493; 6,096,020; 6,068,650; and 5,702,359, the contents of which are incorporated herein by reference in their entirety. The electroporation can be carried out via a minimally invasive device.
EXAMPLES Example 1 Materials and Methods [0082] Production of fusion proteins
[0083] The amino-terminal 59 amino acids were removed from the S. aureus SrtA gene by polymerase chain reaction. The resulting fragment was linked genetically to a segment coding for ELP-(VPGVG)24o in a pET24b (Merck KGaA, Darmstadt, Germany) E. coli expression vector. The genes for GLP1 (SEQ ID NO: 69) and Fn3 (SEQ ID NO: 8) were available from previous studies. ELP-GLP1 (SEQ ID NO: 70), Fn3-ELP (SEQ ID NO: 14), and Fn3-PLN3-ELP (SEQ ID NO: 13) fusion proteins were genetically assembled in pET24b vectors using synthetic DNA fragments from Integrated DNA Technologies (Coralville, IA) for the pilin domain and sortase recognition sites. Fusion proteins were expressed in BL21 (DE3) E. coli (EdgeBio, Gaithersburg, MD) and purified as described previously (Bellucci et al. Angew Chem. Int. Ed. Engl. 2013, 52, 3703-3708).
[0084] Antibodies
[0085] Genes encoding the heavy and light chains of the murine lgG1 κ anti-human Her2 antibody (clone 4D5) were cloned from a hybridoma obtained through the Duke University Health System Cell Culture Facility (Durham, North Carolina). The 3' end of the heavy chain DNA was modified to contain one copy of the coding sequence for a pilin domain peptide, and the modified and unmodified antibodies were expressed by transient transfection of HEK293 cells using the Expi293 system (Life Technologies, Grand Island, NY). Secreted antibodies were purified from the culture supernatant by protein G chromatography (Thermo Scientific, Rockford, IL).
[0086] Polymerization reactions
[0087] SrtA-ELP (SEQ ID NO: 1 1 ; 100 μΜ) was incubated with ELP-GLP1 (SEQ ID NO: 70; 500 μΜ initial concentration) for 18 hours at 33°C in reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI2, pH 8.5). The temperature was varied for FIG. 2b and the pH was varied for FIG. 8b. Reaction products were visualized by SDS-PAGE using Stain-free any kD Mini- PROTEAN TGX polyacrylamide gels from Bio-Rad (Hercules, CA).
[0088] For analysis of the conjugation site, the unpurified reaction product was incubated with MS grade trypsin (Thermo Scientific Pierce, Rockford, IL) for 4-6 hours at 20°C at a trypsin:substrate ratio of approximately 1 :20 (m/m). Trypsinized samples were diluted 1 :40 into oCyano-4-hydroxycinnamic acid (CHCA) matrix, spotted, and analyzed by MALDI-TOF using an Applied Biosystems Voyager-DE Pro system with a nitrogen laser.
[0089] Protein-small molecule conjugation reactions
[0090] SrtA-ELP (SEQ ID NO: 1 1 ) at 100 μΜ was incubated with biotin-LPETGRAGG (SEQ ID NO: 10) peptide purchased from GenScript (Picastaway, NJ) at 800 μΜ in reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI2, pH 8.5) for 2 hours at 20°C to load the enzyme with the biotinylated peptide substrate. Alternatively, dabcyl-LPETG-edans peptide (LPETG is SEQ ID NO: 15) from AnaSpec (Fremont, CA) was used at 500 μΜ for FIG. 11 and FIG. 12. Either Fn3-ELP (SEQ ID NO: 14) or Fn3-PLN3-ELP (SEQ ID NO: 13) were added to a final concentration of 50 μΜ, the temperature was increased to 33°C, and the mixture was incubated for 18 hours at 33°C.
[0091] Alternatively, Fn3-ELP (SEQ ID NO: 14) or Fn3-PLN3-ELP (SEQ ID NO: 13) was reacted at 50 μΜ with SrtA-ELP (SEQ ID NO: 1 1 ) at 100 μΜ and biotin-LPETGRAGG (SEQ ID NO: 10) peptide or FITC-LPETGRAGG (SEQ ID NO: 10) peptide at 500 μΜ in reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI2). Pilin domain peptide was purchased from Genscript (Piscataway, NJ) and reacted at 10 μΜ with SrtA-ELP (SEQ ID NO: 1 1 ) at 100 μΜ and biotin-LPETGRAGG (SEQ ID NO: 10) peptide at 500 μΜ in reaction buffer. Purified antibodies were reacted at 0.5 mg/mL with SrtA-ELP (SEQ ID NO: 1 1 ) at 10 μΜ (~2-fold excess over the mAb) and biotin-LPETGRAGG (SEQ ID NO: 10) peptide at 500 μΜ (~100-fold excess over available pilin domains) in reaction buffer. Reactions conducted overnight at 32°C. Reactions were quenched by adding triglycine peptide to 10 mM.
[0092] To assess the protein components that were biotinylated, the reaction product was run on SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). The blot was blocked with 5% nonfat milk and probed with 1 mg/mL streptavidin-Cy5 conjugate (Life Technologies, Grand Island, NY) at a 1 :20,000 dilution in Tris-buffered saline with 1 % Tween-20 and 0.5% nonfat milk. Fluorescent bands were detected with a Typhoon variable mode laser scanner (GE Healthcare Life Sciences, Pittsburgh, PA).
[0093] To determine the biotin conjugation site, the unpurified reaction product was incubated with MS grade trypsin for 4-6 hours at 20°C at a trypsin:substrate ratio of approximately 1 :20 (m/m). Trypsinized samples were diluted 1 :40 into a-CHCA matrix, spotted, and analyzed by MALDI-TOF. The MS-Digest function of Protein Prospector v 5.10.14 (available at http://prospector.ucsf.edu/prospector/mshome.htm) was used to calculate expected tryptic peptide masses for recombinant protein sequences directly translated from DNA sequencing.
[0094] LC-MS/MS and Quantitation of biotin :protein molar ratios
[0095] The molar ratio of biotin:protein was determined using biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13) purified by cation exchange chromatography. The biotinylated Fn3-PLN3-ELP purified by cation exchange chromatography was digested with MS-grade trypsin overnight at 20°C at a trypsin:substrate ratio of approximately 1 :20 (m/m). Desalted peptides in 50 mM ammonium bicarbonate were separated by reverse-phase high pressure liquid chromatography using a Supelco Ascentis (5 cm x 1 mm, 3 μηη) C18 column over an elution gradient of 5-50% buffer B (5% H2O, 95% acetonitrile). MS data was collected using an Agilent 1 100 series quadrupole ion trap (Agilent Technologies, Santa Clara, CA). For fragmentation and MS2, parent ions were isolated in the trap and fragmented by collisions with helium. Parent and daughter ions were identified using the MS-Digest and MS-Product functions of ProteinProspector v 5.10.14. [0096] The biotin :protein ratio was assessed by comparison of the integrated areas of summed extracted ion chromatograms for ions containing either biotinylated or non-biotinylated pilin domain lysines. For Fn3-PLN3-ELP (SEQ ID NO: 13) reactions, the biotin:protein ratio was assessed using a fluorescence biotin quantitation kit (Thermo Scientific) that measured the increase in 520 nm fluorescence of avidin-conjugated DyLight fluorophore when bound HABA was displaced by protein-linked biotin. The assay was performed in the microplate format per the manufacturer's instructions and the moles of biotin in each sample tested were within the linear range of a standard curve developed using biocytin. The results of the fluorescent assay were confirmed using a colorimetric quantitation kit (Thermo) that measured the decrease in HABA 500 nm absorbance when displaced from avidin by protein-bound biotin. The assay was performed in the cuvette format per the manufacturer's instructions. Unreacted Fn3-PLN3-ELP (SEQ ID NO: 13) was also tested in both assays. Biotinylated horseradish peroxidase (HRP) that was provided with the colorimetric assay provided a positive control. Protein concentrations used in the assay were obtained using the bicinchoninic acid (BCA) assay. Measurements of biotin and protein concentrations were performed in triplicate for each sample and standard.
[0097] Immunofluorescence
[0098] Clonetics human umbilical cord vein endothelial cells (HUVEC, passage 3) were obtained from the Duke University Health System Cell Culture Facility (Durham, North Carolina) were grown in EBM-2 supplemented with EGM-2 BulletKit components (Lonza). HUVEC were attached to glass coverslips at 150,000 cells/mL overnight. Cells were washed in Hank's buffered saline solution (HBSS), then treated for 30 minutes at room temperature with 100 nM biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13) in HBSS. In a separate group, cells were treated with 100 nM biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13) and 1000 nM unlabeled Fn3-ELP (SEQ ID NO: 14) in HBSS for 30 minutes at room temperature. Both groups were washed, stained with 5 μg/mL wheat germ agglutinin-Alexa 594 conjugate and 1 μg/mL Hoechst 33342 for 10 minutes, then fixed with 4% paraformaldehyde in PBS at room temperature for 15 minutes. Fixed cells were permeabilized with 0.2% Tween-20 for 10 minutes and stained with a 1 :200 dilution of 0.5 mg/mL streptavidin-FITC conjugate prior to visualization with a Nikon TE2000-U inverted fluorescent microscope.
[0099] SK-OV-3 cells were maintained in McCoy's 5a medium with 10% fetal bovine serum, 5 units/mL penicillin, and 5 μg/mL streptomycin (Gibco, by Thermo Fisher Scientific, Waltham, MA). Cells were attached to glass coverslips overnight. Cells were washed in PBS, fixed with 4% paraformaldehyde in PBS at room temperature for 15 minutes, and stained with a 1 :200 dilution of 0.5 mg/mL anti-Her2 biotinylated by reaction with SrtA or a biotinylated murine lgG1 isotype control antibody, followed by a 1 :200 dilution of 1 mg/mL streptavidin-FITC conjugate. Cells were analyzed with a Nikon TE2000-U inverted fluorescent microscope with a 60x 1.25NA oil immersion objective.
Example 2
Sortase-Catalyzed Isopeptide Ligation Of Two Peptides
[00100] The ability of SrtA to carry out isopeptide ligation was confirmed by reacting a LPETGRAGG (SEQ ID NO: 10) peptide containing an amino-terminal biotin with a pilin domain peptide (VGGSWLQDVHVYPKHGGSGR; SEQ ID NO: 50). SrtA was produced as a fusion protein with an elastin-like polypeptide (ELP), which is a peptide polymer composed of repeats of the pentapeptide VPGXG (SEQ ID NO: 51 ) where X is any amino acid except proline. ELPs and their fusions phase separate in aqueous solution when heated above a characteristic transition temperature (FIG. 6) to form micron-size aggregates that can be isolated from host cell proteins by centrifugation. ELPs are inert, i.e., they impart no new bioactivity beyond phase transition behavior to their fused peptide or protein partner, and provide efficient purification tags that allow easy recovery of ELP fusions without column chromatography by exploiting their phase transition behavior. Shown in FIG. 6 are graphs of the thermal responsiveness of the SrtA-ELP fusion protein used in this study. As shown in FIG. 6a, absorbance at 350 nm was measured for 100, 75, 50, and 25 μΜ SrtA-ELP concentrations as the temperature was ramped from 15°C-35°C at 1 °C/min in sortase reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI2, pH 8.5). The transition temperature was defined as the inflection point of the absorbance as a function of temperature. As shown in FIG. 6b, plotting the transition temperature versus SrtA-ELP concentration allowed prediction of the SrtA-ELP transition temperature at a particular concentration by interpolation or extrapolation. Turbidity profiles for protein-ELP fusions were characterized using a Cary 300 Bio UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA) and concentrations were determined by the Beer-Lambert Law using calculated extinction coefficients and the absorbance at 280 nm measured using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE).
[00101] After incubating the two peptides biotin-LPETGRAGG (SEQ ID NO: 10) and VGGSWLQDVHVYPKHGGSGR (SEQ ID NO: 50) with the SrtA-ELP (SEQ ID NO: 1 1 ) overnight, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated the formation of a species that corresponded to the isopeptide-linked biotin-LPET (SEQ ID NO: 29) and the pilin domain peptide (SEQ ID NO: 50) (FIG. 19). The schematic of the reaction is shown in FIG. 19a. MALDI-TOF mass spectrometry (FIG. 19b) indicated the formation of a reaction product with a molecular weight corresponding to peptides linked through an isopeptide bond when SrtA-ELP (SEQ ID NO: 1 1 ) was included in the reaction (upper panel), but not when the enzyme was omitted from the reaction mixture (lower panel).
Example 3
Sortase-Catalyzed Isopeptide Ligation Demonstrated By A Protein Polymerization Model
[00102] Glucagon-like peptide-1 (GLP1 , SEQ ID NO: 69) was chosen to test the SrtA- catalyzed isopeptide ligation reaction, as it is a pharmaceutically relevant peptide with two internal lysines that could serve as off-target sites for isopeptide bond formation. GLP1 (SEQ ID NO: 69) was fused to both the pilin domain and the sortase LPETG (SEQ ID NO: 15) recognition sequence, generating a polypeptide that was expressed as a fusion protein with an ELP. Because each ELP-GLP1 (SEQ ID NO: 70) contained both the pilin domain and LPETG (SEQ ID NO: 15) enzyme recognition sequence, it was polymerized by incubation with SrtA-ELP (SEQ ID NO: 1 1 ) according to the reaction shown in FIG. 2a, wherein predicted tryptic peptides of the polymerization product are boxed. SDS-PAGE of the reaction product (FIG. 2b) showed a ladder of polymers with different numbers of subunits, showing polymerization of the 41 kDa ELP-GLP1 (SEQ ID NO: 70) monomer at all temperatures tested except reactions stored at - 20°C. Polymerization was temperature-dependent, and greater than 90% monomer consumption was achieved between 29°C and 34°C (FIG. 2c, wherein conversions are reported as the average of 3 independent reactions carried out at each temperature that were analyzed by SDS-PAGE and densitometry; error bars indicate standard deviations). This temperature range also showed the greatest fraction of high molecular weight (>250 kDa) polymers, with this population comprising 50% of the reaction product by densitometry (FIG. 7). For FIG. 7, SDS-PAGE bands in FIG. 2b were analyzed by densitometry and the background-corrected volumes were reported as a fraction of the overall converted product for n = 2, 3, 4, and greater than or equal to 5, as molecular weights above 250 kDa cannot be accurately estimated by this method. This analysis indicated that in reactions between 29°C and 32°C, approximately 50% of the converted monomer was converted to high molecular weight products (>250 kDa). Therefore 33°C was selected for all subsequent reactions. 100 μΜ SrtA-ELP (SEQ ID NO: 1 1 ) was reacted with ELP-GLP1 (SEQ ID NO: 70) at a starting concentration of 500 μΜ at 33°C for 18 hours at various pHs. Reaction efficiency appeared to have little dependence on pH, though slightly more monomer was consumed in reactions run at pH 8.0 and 8.5. pH had a minor effect on reaction efficiency between pH 5.5 and pH 10, with the highest qualitative yield at pH 8-8.5 (FIG. 8). Based on this data, pH 8.5 was selected for all subsequent reactions.
[00103] Assuming that nucleophilic attack by a primary amine resolves the thioester intermediate, it is possible that polymerization proceeds through the terminal a-amino group of the ELP-GLP1 (SEQ ID NO: 70) fusion rather than the ε-amino group of the pilin domain lysine. If the ε-amino group acts as the nucleophile, the junction of two monomers will be branched as shown in FIG. 2a, whereas a linear junction would arise from head-to-tail polymerization through the terminal a-amino group. To determine the location of the polymer junction, the unpurified reaction product was treated with proteomics-grade trypsin, and the resulting peptides were analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Trypsin normally cleaves a peptide bond on the carboxy-terminal side of lysine or arginine residues, but cannot cleave at lysines with modified ε-amino groups. The MALDI-TOF spectrometry of a tryptic digest of 33°C reaction product showed the presence of an ion at m/z 2809.4 from the branched junction of two monomers (labeled 5 in FIG. 2d and Table 1 ). This ion corresponded to a peptide with a single missed digestion at the pilin domain lysine and an additional mass of 625.7 Da, which indicated that amino acids from the carboxy-terminus of another monomer had been joined to the pilin domain lysine through an isopeptide bond. FIG. 2d shows the isopeptide linkage at the pilin domain lysine (ion 5) along with other ions that correspond to unmodified peptides from the ELP-GLP1 (SEQ ID NO: 70) and SrtA-ELP (SEQ ID NO: 1 1 ) fusion proteins. Shown in Table 1 are the tryptic peptide sequences for peaks identified in the MALDI-TOF spectrum in FIG. 2d. Included are the predicted molecular weight, the experimentally measured molecular weight (calculated by subtracting the mass of H+ from the m/z value), and the difference between these two values. Excellent agreement between the predicted and measured peptide molecular weights was observed for all ions in the MALDI-TOF spectrum. Notably, no signal was observed at the predicted m/z values for ions that would have arisen from modification of either of the two lysines in GLP1 (SEQ ID NO: 69) or at the amino- terminus of the protein, suggesting that SrtA-mediated conjugation was specific for the pilin domain lysine. Table 1. Tryptic peptide sequences for peaks identified in the MALDI-TOF spectrum of FIG. 2d for the reaction with SrtA-ELP (SEQ ID NO: 22) and ELP-PLN-GLP-LPETG (SEQ ID NO: 12). Peptides corresponding to polymerization through the a-amino group of ELP-GLP1 (SEQ ID NO: 70) or through the ε-amino group of the GLP1 (SEQ ID NO: 69) lysine were not found in the MALDI-TOF spectrum.
Figure imgf000032_0001
amino group) 990.1
GAGLPET (SEQ ID NO: 21 ) + SK 625.7 + 233.2 Not N/A
(attachment to N-terminus through o found
amino group, methionine loss) 858.9
GAGLPET (SEQ ID NO: 21 ) + 625.7 + 3272.6 Not N/A
GAHGEGTFTSDVSSYLEEQAAKEFIAWL found
VK (SEQ ID NO: 25) 3898.3
(attachment to lysine ε-amino group in
GLP- 1 )
GAGLPET (SEQ ID NO: 21 ) + 625.7 + 1802.1 Not N/A
EFIAWLVKGAGLPETGGG (SEQ ID NO: found
26) 2427.8
GAGLPET (SEQ ID NO: 21 ) + 625.7 + 2029.3 Not N/A
EFIAWLVKGAGLPETGRAGG (SEQ ID found
NO: 27) 2655.0
Example 4
Site-Specific Conjugation Of Small Molecules To The Fn3 Protein
[00104] The SrtA reaction mechanism was used to generate protein-small molecule conjugates where the site of conjugation could be controlled. Two genetic-level fusion proteins were generated: a fibronectin type III (Fn3) domain fused to an ELP with 3 intervening copies of the pilin domain (Fn3-PLN3-ELP; SEQ ID NO: 13), and a Fn3-ELP (SEQ ID NO: 14) lacking the pilin domain as a negative control. Multiple copies of the pilin domain were incorporated to confirm that the isopeptide ligation can be carried out at an internal site, and because a payload of several small molecules per protein is typically necessary to attain a therapeutically relevant dose in protein-drug conjugates. The Fn3 domain is an attractive targeting molecule for drug delivery because it mimics antibodies but does not contain their complex quaternary structure, glycosylation, or intramolecular disulfide bonds, allowing easy production in Escherichia coli. The Fn3 scaffold is similar to the immunoglobulin fold in antibodies and contains loop regions that can be mutated and affinity matured against a target, similar to the complementarity determining regions in antibodies. The Fn3 variant used had a RGDWXE (SEQ ID NO: 44) element in its FG loop that targets the αγβ3 integrin, which is upregulated in the endothelial cells of angiogenic tumor vasculature.
[00105] The model small molecules biotin and dabcyl were selected to explore the versatility of the conjugation reaction. A synthetic LPETGRAGG (SEQ ID NO: 10) peptide with an amino- terminal biotin and SrtA-ELP (SEQ ID NO: 1 1 ) were incubated with either the Fn3-ELP (SEQ ID NO: 14) or Fn3-PLN3-ELP (SEQ ID NO: 13) fusion (overview of the reaction is shown in FIG. 3a, along with the expected tryptic peptides of the reaction product being boxed). SrtA-ELP (SEQ ID NO: 1 1 ) and biotin-LPETGRAGG (SEQ ID NO: 10) were used at 2- and 10-fold molar excess to Fn3-PLN3-ELP (SEQ ID NO: 13), respectively. After overnight reaction at 33°C, the product was analyzed by Western blot using a streptavidin-Cy5 conjugate to detect biotinylated protein. SDS-PAGE and the corresponding Western blot are shown in FIG. 3b and FIG. 3c, respectively. Only the reaction of biotin-LPETGRAGG (SEQ ID NO: 10) peptide with Fn3-PLN3- ELP (SEQ ID NO: 13) resulted in biotinylated target protein, which suggested that the pilin domain was required for biotinylation. Fn3-ELP (SEQ ID NO: 14) was not biotinylated, as no band was visualized in lane 1 of the Western blot at the expected molecular weight of 36 kDa, despite the fact that Fn3-ELP (SEQ ID NO: 14) contains three lysine residues and a terminal primary amine that offer sites for off-target conjugation. Only SrtA-ELP (SEQ ID NO: 1 1 ) was visualized in this reaction (at approximately 120 kDa) because the thioester-linked SrtA-biotin intermediate is stable in SDS-PAGE. In contrast, biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13) appeared as a 43 kDa band in lane 2 of the Western blot, along with SrtA-ELP (SEQ ID NO: 1 1 ) at 120 kDa. Lanes 3 and 4 show control reactions containing Fn3-ELP (SEQ ID NO: 14) and Fn3-PLN3-ELP (SEQ ID NO: 13), respectively, where the biotin-LPETGRAGG (SEQ ID NO: 10) peptide was excluded. As expected, no bands are visualized in the Western blot for the reactions in lanes 3 and 4.
[00106] In order to determine the biotinylation site and the selectivity of the reaction for the pilin domain, the unpurified product was digested with proteomics-grade trypsin. As shown in FIG. 3d with MALDI-TOF mass spectrometry, biotin was installed specifically at the pilin domain lysine (ions 1 and 2), with all other ions corresponding to unmodified peptides from the Fn3-PLN3-ELP (SEQ ID NO: 13) and SrtA-ELP (SEQ ID NO: 1 1 ) fusion proteins. The spectrum of FIG. 3e shows the same peaks as in FIG. 3d except those corresponding to ions 1 and 2, confirming that these ions arise from biotinylation of the pilin domain lysine when both the biotinylated LPETG (SEQ ID NO: 15) peptide and the pilin domain are present in the reaction. MALDI-TOF mass spectrometry indicated the presence of peptides with a missed digestion at the pilin domain lysine and an additional mass of 666.8 Da (matching the mass of biotin- LPET) in the reaction of Fn3-PLN3-ELP (SEQ ID NO: 13) with biotin-LPETGRAGG (ions 1 and 2 in FIG. 3d; LPETGRAGG is SEQ ID NO: 10). These ions corresponded to m/z values of both 2763.5 and 3871 .2 because the tryptic peptide of the amino-terminal copy of the pilin domain contained some residues from the Fn3 sequence. These ions were not present in the mass spectrum when the reaction did not contain biotin-LPETGRAGG (SEQ ID NO: 10) peptide (FIG. 3e), and all other ions in the spectra mapped to expected, unmodified segments of the Fn3-PLN3-ELP (SEQ ID NO: 13) or SrtA-ELP (SEQ ID NO: 1 1 ) fusion proteins (Table 2). Included in Table 2 are the predicted molecular weight, the experimentally measured molecular weight (m/z value - H+ mass), and the difference between these two values. The complete sequences of SrtA-ELP (SEQ ID NO: 10) and Fn3-PLN3-ELP (SEQ ID NO: 13) are provided for reference. The structure of biotin-LPET (SEQ ID NO: 29) is shown in FIG. 9. Excellent agreement between the predicted and measured peptide molecular weights was observed for all ions in the MALDI-TOF spectra. Importantly, no ions corresponding to biotinylation at any of the 3 lysines in the Fn3 domain or at the Fn3 amino-terminus were observed in the MALDI-TOF spectra (Table 2). Moreover, no biotinylated peptides were observed for reactions with Fn3-ELP lacking the pilin domain (FIG. 10). As shown in FIG. 10b, the MALDI-TOF spectrum for biotinylation reaction containing Fn3-ELP (SEQ ID NO: 14; no pilin domains) and biotin-LPETGRAGG (SEQ ID NO: 10) peptide did not contain any ions corresponding to biotinylated peptides (this spectrum corresponds to lane 1 of the Western blot in FIG. 3c). As shown in FIG. 10c, the MALDI-TOF spectrum for reaction of Fn3-ELP (SEQ ID NO: 14; no pilin domains) without biotin-LPETGRAGG (SEQ ID NO: 10) peptide did not contain ions corresponding to biotinylated peptides (this spectrum corresponds to lane 3 of the Western blot in FIG. 3c). That is, biotinylation was specific to the pilin domain lysine, as peptides corresponding to biotinylation at the oamino group of Fn3-PLN3-ELP (SEQ ID NO: 13) or through the ε-amino groups of any of the 3 lysines in Fn3 were not found in the MALDI-TOF spectrum.
Table 2. Peaks identified in the MALDI-TOF spectrum of FIG. 3d and FIG. 10 resulting from tryptic digestion of the SrtA-ELP-catalyzed reaction of biotin-LPETG (SEQ ID NO: 15) with Fn3- PLN3-ELP (SEQ ID NO: 13).
SrtA-ELP amino acid sequence:
MGQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNJSJAGH TFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLIT CDDYNEKTGVWEKRKIFVATEVKALVTMGVG-(VPGVG)24o (SEQ ID NO: 1 1 )
Fn3-PLN3-ELP amino acid sequence:
MVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRJTYGETGGNSPVQEFTVPGSKSTATJSGL KPGVDYTJTVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGWLQDVHVYPKH GGSGRGSGGWLQDVHVYPKHGGSGRGSGGWLQDVHVYPKHGGSGRGVGV-(VPGVG)60 (SEQ ID NO: 13)
Fn3-ELP amino acid sequence:
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKP GVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGVG-(VPGVG)60 (SEQ ID NO: 14)
Peak Peptide sequence Predicted Found Difference Labei mass (Da) mass (Da)
[m/z-H+]
(Da)
1 TGGTSGGTSGSGSGGGSGGWLQDVH 3203.3+66 3870.2 -0.1
VYPKHGGSGR (SEQ ID NO: 28) + 6.8 =
biotin- LPET (SEQ ID NO: 29) 3870.1
2 GSGGWLQDVHVYPKHGGSGR (SEQ 2095.3+66 2762.5 -0.4
ID NO: 30) + biotin-LPET (SEQ ID NO: 6.8 =
29) (attachment to lysine ε-amino group) 2762.1
3 GSGGWLQDVHVYPK (SEQ ID NO: 31 ) 1543.7 1543.5 0.2
4 TGGTSGGTSGSGSGGGSGGWLQDVH 2651 .8 2651.6 0.2
VYPK (SEQ ID NO: 32)
Biotin- Biotin-LPETGGG (SEQ ID NO: 33) + 666.8+189. 900.6 -0.6 LPETGGG 2Na+ 2+44 =
900.0
SRT28-42 EPVYPGPATPEQLNR (SEQ ID NO: 34) 1668.9 1669.0 -0.1
SRT 5_42 VAGYIEIPDADIKEPVYPGPATPEQLNR 3054.4 3053.5 0.9
(SEQ ID NO: 23)
SRT121-149 QLTLITCDDYNEKTGVWEKRKIFVATEV 3430.0 3430.8 -0.8
K (SEQ ID NO: 35)
SRT43-77 GVSFAEENESLDDQNISIAGHTFIDRPN 3972.3 3972.3 0.0
YQFTNLK (SEQ ID NO: 24) Fn3eo-94 GDWNEGSKPISINYR (SEQ ID NO: 36) 1736.9 1736.2 0.7
Fn335-55 ITYGETGGNSPVQEFTVPGSK (SEQ ID 2169.4 2168.4 1.0
NO: 37)
Fn 380-94* Biotin-LPET (SEQ ID NO: 29) + 666.8+173 2403.7 0.0
GDWNEGSKPISINYR (SEQ ID NO: 36) 6.9 =
(through a-amino group) 2403.7
Fn356-79 STATISGLKPGVDYTITVYAVTPR (SEQ 251 1 .9 2512.1 -0.2
ID NO: 38)
Fn38-3i D L E WAAT PTS L L I S W D AP AVTV R 2525 > 9 2526.2 -0.3
(SEQ ID NO: 39)
Biotin-LPET (SEQ ID NO: 29) + 666.8+804. Not N/A
MVSDVPR (SEQ ID NO: 40) 0 = 1470.8 found
(attachment to amino-terminus through
a-amino group)
Biotin-LPET (SEQ ID NO: 29) + 666.8+672. Not N/A
VSDVPR (SEQ ID NO: 41 ) (attachment 8 = 1339.6 found
to amino-terminus through a-amino
group, methionine loss)
STATISGLKPGVDYTITVYAVTPR (SEQ 251 1 .9+66 Not N/A
ID NO: 38) + biotin-LPET (SEQ ID NO: 6.8 = found
29) (attachment to lysine ε-amino group 3178.7
in Fn3)
ITYGETGGNSPVQEFTVPGSKSTATIS 4662.2+66 Not N/A
GLKPGVDYTITVY AVTPR (SEQ ID NO: 6.8 = found
42) + biotin-LPET (SEQ ID NO: 29) 5329.0
(attachment to lysine c-amino group in
Fn3)
[00107] A commercially available dabcyl-LPETG-edans (LPETG is SEQ ID NO: 15) reagent was successfully conjugated to the ELP-GLP1 (SEQ ID NO: 70) construct, and the tryptic digest of the reaction product was analyzed by MALDI-TOF mass spectrometry (FIG. 11 and Table 3). Shown in FIG. 11 a is the structure of dabcyl-LPETG-edans peptide (SEQ ID NO: 15). Dabcyl absorbs at the 337 nm wavelength of the N2 laser used in MALDI-TOF, and reproducibly lost a 132 Da segment due to fragmentation (the proposed fragment is highlighted). The ELP-GLP1 (SEQ ID NO: 70) is the same protein used in the polymerization model, and therefore, both polymerization and dabcyl conjugation can occur through the pilin domain as shown in the schematic of FIG. 11 b. This conjugation was also site-specific for the ε-amino group of the pilin domain lysine, demonstrating that the reaction can be used with different small molecule and protein substrates. The MALDI-TOF spectrum is shown in FIG. 11 c. The spectrum shows the presence of both conjugated dabcyl (ion 1 ) and the branched polymer junction (ion 5) at the pilin domain lysine. For dabcyl attachment, another ion is present (ion 1-132) that corresponds to attachment at the pilin domain lysine but with the loss of 132 Da. This same loss is also observed for ions corresponding to the reaction side product, dabcyl- LPETGGG (SEQ ID NO: 33), as indicated on the spectrum. Both conjugation and polymerization reactions are specific for isopeptide-linked attachment at the pilin domain lysine ε-amino group, as all other ions in the spectra map to unmodified segments of SrtA-ELP (SEQ ID NO: 1 1 ) or ELP-GLP1 (SEQ ID NO: 70).
Table 3. Corresponding peak table for MALDI-TOF spectra for the tryptic peptides of FIG. 11c from the SrtA-ELP (SEQ ID NO: 1 1 ) reaction with ELP-PLN-GLP-LPETG (SEQ ID NO: 12) and Dabcyl-LPETG-Edans (SEQ ID NO: 15).
Figure imgf000038_0001
(SEQ ID NO: 20) + Dabcyl-LPET (SEQ 132 =
ID NO: 29) 132 Da (due to dabcyl 2742.2
fragmentation)
2 GSSGGWLQDVHVYPK (SEQ ID NO: 1630.8 1629.3 -.5
16)
3 GAHGEGTFTSDVSSYLEEQAAK (SEQ 2285.4 2284.5 0.9
ID NO: 18)
4 EFIAWLVK (SEQ ID NO: 19) 1007.2 1006.3 0.9
5 GSSGGWLQDVHVYPKHGGSGR 2808.1 2807.1 1.0
(SEQ ID NO: 20) + GAGLPETG (SEQ
ID NO: 43)
Dabcyl- Dabcyl-LPETGGG (SEQ ID NO: 33) + 691 .8 + 925.2 -0.2
LPETGGG 2Na+ 189 + 44
= 925.0
Dabcyl- Dabcyl-LPETGGG (SEQ ID NO: 33) + 925.0 - 793.3 -0.3
LPETGGG 2Na+ - 132 Da (due to dabcyl 132 =
- 132 fragmentation) 793.0
SRT 5_42 VAGYIEIPDADIKEPVYPGPATPEQLN 3054.4 3054.1 0.3
R (SEQ ID NO: 23)
SRT43-77 GVSFAEENESLDDQNISIAGHTFIDRP 3972.3 3971 .1 1.2
NYQFTNLK (SEQ ID NO: 24)
Dabcyl-LPET (SEQ ID NO: 29) + MSK 691 .8 + Not N/A
(attachment to amino terminus through 364.4 = found
a-amino group) 1056.2
Dabcyl-LPET (SEQ ID NO: 29) + SK 691 .8 + Not N/A
(attachment to N-terminus through o 233.2 = found
amino group, methionine loss) 925.0
[00108] Because the reaction product was directly digested with trypsin, the presence of the SrtA-ELP (SEQ ID NO: 1 1 ) fusion required the addition of excess triglycine peptide during trypsinization, as many of the peptides generated by trypsin contained amino-terminal glycine residues and were able to resolve the SrtA-thioester intermediate through the well-characterized SrtA native transpeptidation reaction. Because triglycine is a preferred nucleophile in this reaction, its presence mitigated the formation of these modified peptides.
[00109] FIG. 12a is a schematic of the reaction without including triglycine in the trypsinization reaction. Omission of triglycine during trypsinization resulted in more complicated MALDI-TOF spectra with peaks corresponding to peptides linked to LPET-bearing substrate molecules (LPET is SEQ ID NO: 29) through an oamino group (FIG. 12 and Table 4). As shown in FIG. 12b, the MALDI-TOF spectrum for the unpurified reaction product shows the presence of many ions corresponding to tryptic peptides with amino-terminal glycine residues that had reacted with the SrtA acyl-enzyme intermediate to form native peptide bonds with dabcyl-LPET (ions 1 , 3, 5, and 7; LPET is SEQ ID NO: 29). These ions were only observed for tryptic peptides that had an amino-terminal glycine residue and were not present when excess triglycine is added to the trypsinization reaction (compare to FIG. 11 ). The spectrum nonetheless confirmed isopeptide ligation of dabcyl-LPET (SEQ ID NO: 29) to the ε-amino group of the pilin domain lysine (ions 2 and 5), though one of these (ion 5) has two conjugated dabcyl-LPET (SEQ ID NO: 29) moieties, one through the oamino group of the peptide and the other through the ε-amino group of the pilin domain lysine.
Table 4. Corresponding peak table for the MALDI-TOF spectra of FIG. 12 for the SrtA-ELP (SEQ ID NO: 1 1 ) reaction with ELP-PLN-GLP-LPETG (SEQ ID NO: 12) and Dabcyl-LPETG- Edans (SEQ ID NO: 15).
SrtA-ELP amino acid sequence:
MGQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHT
FIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITC
DDYNEKTGVWEKRKIFVATEVKALVTMGVG-(VPGVG)24o (SEQ ID NO: 1 1 )
ELP-PLN-GLP-LPETG amino acid sequence:
MSKGPGVG-(VPGVG)so-
VPGSGLVPRGSSGGWLQDVHVYPKHGGSGRGAHGEGTFTSDVSSYLEEQAAKEFIAWLVK
GAGLPETGRAGG (SEQ ID NO: 12)
Peak Peptide Peptide sequence Predicted Found Difference label source mass mass (Da)
(Da) [m/z-
H+]
(Da) 1 PLN Dabcyl-LPET (SEQ ID NO: 29) + 1630.8 + 2323.0 -0.4
GSSGGWLQDVHVYPK (SEQ ID NO: 691 .8 =
16) (through a-amino group) 2322.6
2 PLN GSSGGWLQDVHVYPKHGGSGR 2182.4 + 2876.9 -2.7
(SEQ ID NO: 20) + Dabcyl-LPET 691 .8 =
(SEQ ID NO: 29) (through lysine ε- 2874.2
amino group)
3 GLP1 Dabcyl-LPET (SEQ ID NO: 29) + 2285.4 + 2977.8 -0.6
GAHGEGTFTSDVSSYLEEQAAK 691 .8 =
(SEQ ID NO: 18) (through a-amino 2977.2
group)
4 SRT-I5.42 VAGYIEIPDADIKEPVYPGPATPEQL 3054.4 3055.8 -1 .4
NR (SEQ ID NO: 23)
5 PLN GSSGGWLQDVHVYPKHGGSGR 2812.4 + 3566.2 -0.2
(SEQ ID NO: 20) + 2 Dabcyl-LPET 2*691 .8 =
(SEQ ID NO: 29) (through both lysine 3566.0
ε-amino group and a-amino group)
6 SRT43-77 GVSFAEENESLDDQNISIAGHTFIDR 3972.3 3973.8 -1 .5
PNYQFTNLK (SEQ ID NO: 24)
7 SRT43-77 Dabcyl-LPET (SEQ ID NO: 29) + 3972.3 + 4664.6 -0.5
GVSFAEENESLDDQNISIAGHTFIDR 691 .8 =
PNYQFTNLK (SEQ ID NO: 24) 4664.1
(through a-amino group)
[00110] The same SrtA linked to a hexahistidine tag (H6-SrtA; SEQ ID NO: 49), rather than an ELP, and purified by immobilized metal affinity chromatography also biotinylated the Fn3- PLN3-ELP (SEQ ID NO: 13) through isopeptide bonds at pilin domain lysines, as assessed by Western blotting and MALDI-TOF (FIG. 20). Briefly, SDS-PAGE (FIG. 20a) of reactions of H6- SrtA (SEQ ID NO: 49) and Fn3-PLN3-ELP (SEQ ID NO: 13) were run overnight at a range of temperatures from 21-42°C with or without biotin-LPETGRAGG (SEQ ID NO: 10) peptide. Western blot (FIG. 20b) of the SDS-PAGE using streptavidin-Cy5 was done to detect biotinylated protein. Only reactions containing both the pilin domain and the biotin- LPETGRAGG (SEQ ID NO: 10) peptide resulted in biotinylation of the target protein at 43 kDa. H6-SrtA (SEQ ID NO: 49) appeared as a biotinylated band at -25 kDa in reactions containing biotin-LPETGRAGG (SEQ ID NO: 10) because the thioester intermediate of H6-SrtA (SEQ ID NO: 49) and biotin-LPET (SEQ ID NO: 15) is stable in SDS-PAGE. A disulfide-linked dimer of the enzyme also appeared as a biotinylated protein in the blot due to the amino-terminal glycine of the enzyme, which can be biotinylated by the native peptide transpeptidation reaction at its o amino group. As shown in FIG. 20c, the MALDI-TOF mass spectrum of tryptic peptides of the unpurified 20°C biotinylation reaction contained ions that corresponded to the same biotinylated pilin domain peptides (ions 1 and 2) as in FIG. 22a, as well as a peptide corresponding to the amino-terminus of the H6-SrtA (SEQ ID NO: 49) enzyme biotinylated through a native peptide bond at the terminal glycine residue. This ruled out the potential influence of the ELP tag on the enzyme's activity and suggested that protein modification by isopeptide ligation is a general in vitro function of SrtA.
[00111] Fluorescein isothiocyanate (FITC) coupled to an LPETGRAGG peptide (SEQ ID NO: 10) was attached to the Fn3-PLN3-ELP (SEQ ID NO: 13) by isopeptide ligation (FIG. 18 and Table 5), and conjugation to the pilin domain lysine was confirmed by in-gel fluorescence detection and MALDI-TOF mass spectrometry. Briefly, FITC-Ahx-LPETGRAGG (SEQ ID NO: 68) peptide was reacted with Fn3-PLN3-ELP (SEQ ID NO: 13) and SrtA-ELP (SEQ ID NO: 1 1 ) overnight at 32°C in reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI2). As shown in FIG. 18b, the reaction product was analyzed by SDS-PAGE and total protein was visualized (upper panel). Scanning the gel with a Typhoon scanner with 488 nm laser excitation and a 520 BP 40 nm emission filter showed that Fn3-PLN3-ELP (SEQ ID NO: 13) was conjugated to FITC (lower panel). SrtA-ELP (SEQ ID NO: 1 1 ) was also visible in this image because the thioester reaction intermediate of SrtA and the FITC-LPET peptide (SEQ ID NO: 29) is stable in SDS- PAGE. As shown in FIG. 18c, the reaction product was trypsinized and the resulting peptides were analyzed by MALDI-TOF mass spectrometry. The MALDI-TOF spectrum showed the presence of two ions with m/z 3040.7 and 4148.7, which correspond to the predicted molecular weight of pilin domain tryptic peptides linked to FITC-Ahx-LPET (SEQ ID NO: 52) through an isopeptide bond at the pilin domain lysine. As with the biotin conjugation reactions, these peptides contain a missed tryptic digestion at the pilin domain lysine because trypsin is unable to cleave at these lysines after isopeptide bond formation. The chemical structure of the FITC- Ahx-LPET (SEQ ID NO: 52) moiety attached to the pilin domain lysine is shown in FIG. 18d. This demonstrated that the reaction is not restricted to biotin and can be used with bulkier small molecule substrates. Table 5. Corresponding peak table for the MALDI-TOF spectra of FIG. 18.
Figure imgf000043_0001
[00112] The biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13) was purified from the reaction mixture using cation exchange chromatography (FIG. 13). FIG. 13a shows the absorbance at 280 nm, pH, and conductivity traces for the cation exchange chromatography. The biotinylation reaction product was loaded on a HiTrap SP FF 5 ml. column at 5 mL/min, washed in 20 mM Tris, pH 6.0, and eluted in a gradient to 100% 1 M NaCI, 20 mM Tris, pH 6.0 over 50 minutes. Non-bound (NB) and elution (Elu) fractions were collected. As shown in FIG. 13b, SDS-PAGE of the CEX load, non-bound, and elution fractions indicated that Fn3-PLN3-ELP (SEQ ID NO: 13) was selectively recovered in the elution fraction. The biotinylation of the recovered fractions was confirmed by Western blot (FIG. 4a), in which the Western blot included the CEX load, non- bound (NB), and elution (Elu) fractions using streptavidin-Cy5, indicating successful recovery of biotinylated Fn3-PLN3-ELP in the elution. A fluorescent biotin quantitation kit (Thermo Scientific) was utilized to determine the molar ratio of biotin to protein. This assay measures the increase in intensity at 520 nm of an avidin-conjugated DyLight fluorophore upon displacement of the quencher 4'-hydroxyazobenzene-2-carboxylic acid (HABA) by protein-linked biotin. Using this technique, it was determined that the molar ratio of biotin to protein was approximately 1.9, whereas unreacted Fn3-PLN3-ELP (SEQ ID NO: 13) gave no signal above background. These results were confirmed using a colorimetric assay that measures the decrease in absorbance of HABA at 500 nm when displaced from avidin by protein-bound biotin (FIG. 14). Unreacted Fn3-PLN3-ELP (SEQ ID NO: 13) did not give significant signal above background. Biotinylated horseradish peroxidase (HRP) that was provided with the colorimetric assay provided a positive control.
[00113] The biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13) that was purified from the reaction mixture using cation exchange chromatography (FIG. 13b) was also trypsinized, and the resulting peptides were analyzed by liquid chromatographyelectrospray ionization tandem mass spectrometry (LC-MS/MS). This achieved two aims: (1 ) conclusive identification of the isopeptide bond in the linker between biotin and the pilin domain and (2) quantitation of the average number of biotin molecules per protein by comparing the relative amounts of peptides with modified and unmodified pilin domain lysines. The MS1 spectrum (FIG. 21 , Table 6) showed the presence of biotinylated pilin domain peptides and other, unmodified peptides from the Fn3-PLN3-ELP (SEQ ID NO: 13) sequence, with biotinylation only observed for peptides containing the pilin domain lysine, in agreement with the results obtained using MALDI-TOF mass spectrometry.
Table 6. A peak table with the ions shown in the MS1 spectrum of FIG. 21.
Figure imgf000044_0001
Figure imgf000045_0001
[00114] To provide direct evidence for the isopeptide conjugation of biotin to the pilin domain lysine, the biotinylated pilin domain peptide was fragmented by low-energy collisions with helium. Shown in FIG. 22a is the MS2 spectrum of daughter ions produced by isolating the +4 charge state (m/z = 691 ) of a biotinylated pilin domain peptide. The MS2 spectrum for the parent ion with z = +4 (FIG. 22a) contained daughter ions formed by breaking peptide bonds in the protein backbone (the fragmentation pattern is outlined in FIG. 22b). The spectrum shows four ions with multiple charge states. Several of these daughter ions corresponded to peptides with an isopeptide junction between biotin-LPET (SEQ ID NO: 29) and the pilin domain lysine (ions 1 -4, with the chemical structures shown in FIG. 22c), confirming the expected chemical structures for biotin-LPET (SEQ ID NO: 29) linked to the pilin domain lysine through an isopeptide bond (ions 1-4). These fragments correspond to y- and b- type ions produced by breaking peptide bonds along the pilin domain backbone (ions 1 , 3, and 4) as well as within the LPET (SEQ ID NO: 29) linker region (ions 1 and 2). Peptide bond cleavage resulted in a charged amino-terminal fragment (b ions) or a charged carboxy-terminal fragment (y ions). b|P and yiP corresponded to daughter ions from fragmentation events within the isopeptide-linked LPET moiety.
[00115] A similar fragmentation pattern was observed for the parent ion with z = +3 (FIG. 23). FIG. 23a is MS2 spectrum of daughter ions produced by isolating the +3 charge state (m/z = 920.4) of a biotinylated pilin domain peptide. FIG. 23b is an outline of the observed fragmentation pattern and the nomenclature used to classify daughter ions, corresponding to the daughter ion chemical structures in FIG. 23c. The spectrum showed four ions with multiple charge states whose masses confirmed the expected chemical structures for biotin-LPET (SEQ ID NO: 29) linked to the pilin domain lysine through an isopeptide bond (ions 1-4). b|P and yiP correspond to daughter ions from fragmentation events within the isopeptide-linked LPET moiety. These fragments corresponded to y- and b-type ions produced by breaking peptide bonds along the pilin domain backbone (ions 1 , 3, and 4) as well as within the LPET (SEQ ID NO: 29) linker region (ions 1 and 2). The presence of ions 1 -4 in the MS2 spectra for multiple charge states conclusively showed that the sortase-mediated reaction installed biotin-LPET (SEQ ID NO: 29) at the pilin domain lysine through an isopeptide bond, as the daughter ions produced by tandem-MS provided an unambiguous signature that specifically confirmed the identity of the biotinylated parent ion. Extracted ion chromatograms (EICs) from MS1 for peptides with biotinylated or unmodified pilin domain lysines tracked the intensity of these ions over time as they eluted from the chromatography column. This allowed the relative abundance of biotinylated versus unmodified lysines to be determined by comparing the integrated areas of their corresponding EICs. As shown in FIG. 22d, extracted ion chromatograms (EICs) for individual ions were summed for peptides containing biotinylated (light lines) or non-biotinylated (dark lines) pilin domain lysines. By summing the EIC intensities for all identified ions containing either biotinylated or unmodified pilin domain lysine residues, it was determined that 75% of the pilin domain lysines were biotinylated, which corresponded to an average of 2.2 biotinylated pilin domains per Fn3-PLN3-ELP protein (SEQ ID NO: 13) (FIG. 22d). To verify this result, a colorimetric 4'-hydroxyazobenzene-2-carboxylic acid (HABA) biotin quantitation assay was used to determine the molar ratio of biotin to protein, which yielded a molar ratio of biotin to protein of 1.7 ± 0.2, whereas unreacted Fn3-PLN3-ELP (SEQ ID NO: 13) gave no signal above background (FIG. 22e). The biotin:protein molar ratio was quantitated using a colorimetric assay measuring HABA displacement from avidin. A chemically-crosslinked biotin-horseradish peroxidase (HRP) conjugate was analyzed as a positive control, and results are shown in FIG. 22e.
[00116] To demonstrate that the biotinylated fusion protein retained the targeting affinity of the Fn3 domain, its uptake by human umbilical vein endothelial cells (HUVECs) was investigated. HUVECs were treated with 100 nM biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13) for 30 minutes, then washed and immediately fixed, permeabilized, and stained with streptavidin-FITC to detect intracellular biotin. Cells showed significant uptake of Fn3-PLN3-ELP (SEQ ID NO: 13) by fluorescence microscopy, as evidenced by punctate fluorescence in the FITC channel (FIG. 4b). FIG. 4b is a merged image showing nuclei (blue), glycoproteins (for cell morphology, red), and intracellular biotin (green). Punctate intracellular fluorescence in the green channel suggested that biotinylated Fn3-PLN3-ELP was internalized. Uptake of the biotinylated reagent appeared to be receptor-mediated, as it was dramatically reduced by saturating αγβ3 dimers with a 10-fold molar excess of unlabeled ligand during incubation of the cells with biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13) (FIG. 4c). FIG. 4c shows HUVECs incubated with biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13) and a 10-fold molar excess of unlabeled Fn3-ELP (SEQ ID NO: 14) had dramatically reduced uptake of the biotinylated ligand compared to FIG. 4b. This suggested that receptor-mediated endocytosis of biotinylated Fn3- PLN3-ELP (SEQ ID NO: 13) was blocked by saturation of the αγβ3 integrin dimer with unlabeled ligand. Additional images from both treatment groups are available in FIG. 15, wherein nuclei were stained with Hoechst 33342 (blue), glycoproteins with wheat germ agglutinin-Aiexa 594 conjugate (red), and biotinylated protein with streptavidin-FITC (green). It was confirmed that these cells expressed the αγβ3 integrin by performing flow cytometry with an antibody that recognizes an epitope spanning the integrin dimer (FIG. 16). For FIG. 16a, cells were harvested using Hanks-based enzyme-free dissociation buffer, and 2.5x105 cells were stained with a 1 :100 dilution of 0.5 mg/mL biotinylated isotype control antibody (black, filled histogram) or a 1 :100 dilution of 0.5 mg/mL biotinylated anti-human CD51/CD61 (blue, open histogram) monoclonal antibody (eBioscience catalog number 13-0519), washed, and stained with a 1 :200 dilution of 0.5 mg/mL streptavidin-FITC. FIG. 16b shows flow cytometry for HUVECs stained with either biotinylated mouse lgG1 κ (filled histogram), unreacted Fn3-PLN3-ELP (SEQ ID NO: 13; left peak, open histogram), or biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13; right peak, open histogram) followed by secondary detection with a 1 :200 dilution of 0.5 mg/mL streptavidin- FITC. Cells were analyzed immediately using a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). Interestingly, biotinylated Fn3-PLN3-ELP (SEQ ID NO: 13) could also be used as a detection reagent in flow cytometry but provided lower signal intensity compared to the monoclonal antibody, which was expected due to its lower affinity (~10"8 M) and monovalency. This data demonstrates that the SrtA isopeptide transpeptidation reaction can be used to produce bioactive protein-small molecule conjugates, as the reaction conditions and conjugation did not destabilize the Fn3 structure and allowed it to maintain affinity for its target antigen.
Example 5
Site-Specific Conjugation Of Small Molecules To An Antibody
[00117] Having characterized the isopeptide ligation reaction with the Fn3 domain, the generality of this reaction was demonstrated by modifying a more complex protein. To examine the feasibility of isopeptide ligation to generate antibody-drug conjugates, the 4D5 monoclonal antibody against the human epidermal growth factor receptor 2 (Her2) was cloned and genetically modified to contain a pilin domain at the carboxy-termini of its heavy chains. The recombinant antibody was incubated with SrtA-ELP (SEQ ID NO: 1 1 ) and biotin-LPETGRAGG (SEQ ID NO: 10) peptide overnight (schematic shown in FIG. 24a). SrtA-ELP (SEQ ID NO: 1 1 ) and biotin-LPETGRAGG (SEQ ID NO: 10) were used at 2- and 100-fold molar excess to the mAb, respectively. The product was compared to several control reactions run in parallel to assess the specificity of biotinylation of the pilin domain. SDS-PAGE and an anti-biotin Western blot indicated that the pilin domain was required for biotinylation of antibodies. Non-reduced anti-Her2 containing the pilin domain on its heavy chain was biotinylated in the reaction, and this modification mapped exclusively to the heavy chain when the antibody's interchain disulfide bonds were reduced prior to electrophoresis. No biotinylation was observed for reactions on a panel of antibodies including anti-Her2 without the pilin domain modification, as well as all murine IgG isotypes and both kappa and lambda murine light chain variants. That is, based on an anti-biotin Western blot (FIG. 24b), SrtA-ELP (SEQ ID NO: 1 1 ) did not biotinylate any sites on the anti-Her2 antibody when it did not contain the pilin domain. Additionally, no biotinylation of any of four murine IgG isotype controls (lgG1 , lgG2a, lgG2b, or lgG3), or either class of murine light chain (kappa or lambda) was detected. Only the anti-Her2 heavy chain modified to contain the pilin domain was biotinylated by SrtA-ELP (SEQ ID NO: 1 1 ), which, consistent with the data for the Fn3 domain, strongly suggested that isopeptide ligation is specific for this amino acid sequence. The reaction was specific for the pilin domain, as no biotinylation was seen at any of the 86 off-target lysines in the antibody. FIG. 24c shows immunofluorescence of the Her2-overexpressing cell line SK-OV-3 using biotinylated anti-Her2 containing a pilin domain on its heavy chain followed by secondary staining with streptavidin-FITC conjugate, and FIG. 23d shows immunofluorescence of SK-OV-3 using a biotinylated isotype control antibody in the primary staining step (with nuclear stain and FITC; scale bars are 15 μηη). The biotinylated antibody also retained antigen targeting, as it showed efficient labeling of Her2 on SK-OV-3 human ovarian adenocarcinoma cells (FIG. 24c) compared to a biotinylated isotype control antibody (FIG. 24d) in immunofluorescence microscopy. Additional images from both groups are shown in FIG. 26. For FIGS. 26a-c, cells were stained with a 1 :200 dilution of anti-Her2 (with a pilin domain on each heavy chain) biotinylated by reaction with sortase. For FIGS. 26d-f, cells were stained with a 1 :200 dilution of biotinylated murine lgG1 isotype control antibody. Biotin in both groups was detected by secondary staining with streptavidin-FITC conjugate (green), nuclei were stained with Hoechst 33342 (blue), and scale bars were 15 μηη.
[00118] Antibody was biotinylated by overnight reaction at 32°C with SrtA-ELP (SEQ ID NO: 1 1 ) and biotin-LPETGRAGG (SEQ ID NO: 10) peptide in 2- and 100-fold molar excess, respectively. Purified antibody was dialyzed extensively (30 diavolumes) with PBS to remove unreacted biotin. Protein-bound biotin was quantified using a fluorescence biotin detection kit with a known concentration of biocytin as a standard, according to the manufacturer's instructions. Protein concentration was determined by BCA assay using a bovine serum albumin standard per the manufacturer's instructions. Biotin and protein content were evaluated for undiluted antibody as well as 4 dilutions of the conjugate (1 :4, 1 :6, 1 :8, and 1 :10). Plotting the concentration of biotin versus the concentration of protein for each sample gave a slope of 1.8 with a standard error of 0.1 , which indicates that 90% of the pilin domains in the antibody sample were biotinylated in the isopeptide ligation reaction. That is, using the HABA displacement assay, it was determined that there were 1.8 ± 0.1 biotins conjugated per antibody out of 2 possible pilin domain attachment sites (FIG. 25, wherein all samples and standards were measured in triplicate and error bars indicate standard deviations). This equated to a conversion of 90%, which is consistent with reported yields for other enzymatic reactions at the carboxy- termini of antibody heavy chains.
Example 6
Analysis of Substrate Sequence Requirements
[00119] The length and sequence of the first polypeptide was examined for function in the sortase-mediated isopeptide ligation reaction using truncated isopeptide attachment (IPA) sequences. A schematic diagram of the reaction is shown in FIG. 27a. Fusion proteins of a fibronectin type 3 (Fn3) domain, i.e., a model targeting protein, and an elastin-like polypeptide were produced with a single intervening copy of each of the IPA sequences. SrtA-ELP (SEQ ID NO: 1 1 ) and biotin-LPETGRAGG (SEQ ID NO: 10) were incubated with each Fn3-IPA-ELP at 2- and 10-fold molar excess to Fn3-IPA-ELP, respectively. As shown in FIG. 27b, SDS-PAGE of the reaction products and Western blot with streptavidin-Cy5 indicated that fusion proteins containing any of the truncated IPA sequences were biotinylated by reaction with SrtA, while no biotinylation was observed when a fusion protein without any IPA sequence was reacted. The reaction products were trypsinized and analyzed with MALDI-TOF. As shown in FIG. 27c, biotin was installed at the IPA sequence lysine in each of the different versions of the IPA sequence, with all other ions corresponded to unmodified peptides from the Fn3-IPA-ELP and SrtA-ELP (SEQ ID NO: 1 1 ) fusion proteins. Accordingly, SrtA was able to catalyze the isopeptide ligation reaction when the first polypeptide comprised YPKH (SEQ ID NO: 48), VYPKH (SEQ ID NO: 47), VHVYPKH (SEQ ID NO: 46), or WLQDVHVYPKH (SEQ ID NO: 45).
Example 7
Further Analysis of Substrate Sequence Requirements with Deletion Mutants
[00120] A panel of deletion mutants was generated and tested for activity in an isopeptide ligation reaction with biotin-LPETGRAGG (SEQ ID NO: 10) and SrtA. The basis of the panel was Fn3 conjugated to a pilin domain and ELP (Fn3-PLN-ELP; SEQ ID NO: 76), with linker sequences N-terminal and C-terminal to the pilin domain. The N-terminal and C-terminal linkers were glycine- and serine-rich flexible spacers with the sequences GGTSGSGSGGGSGG (SEQ ID NO: 87) and GGSGR (SEQ ID NO: 88), respectively. Deletion mutants were named according to the section of the pilin domain that was removed and are shown in Table 7; for example, ΔΝ4 (SEQ ID NO: 78) lacked the N-terminal 4 amino acids of the native pilin domain WLQDVHVYPKH (SEQ ID NO: 45) to be VHVYPKH (SEQ ID NO: 46). A modified version of Fn3 was also generated, in which the FG loop was mutated from PRGDWNEGS (SEQ ID NO: 89) to PKHGSPASS (SEQ ID NO: 90) to include a PKH sequence for isopeptide ligation.
Table 7. Polypeptide sequences of deletion mutants.
Figure imgf000051_0001
VTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGS
GGKHGGSGRGVGV-(VPGVG)60
Δ01 SEQ ID NO: 83 MVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRJTYG
ETGGNSPVQEFTVPGSKSTATJSGLKPGVDYTJTVYA VTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGS GGWLQDVHVYPKGGSGRGVGV-(VPGVG)fin
ΔΝ-terminal SEQ ID NO: 84 MVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRJTYG linker ETGGNSPVQEFTVPGSKSTATJSGLKPGVDYTJTVYA
VTPRGDWNEGSKPISINYRTGGTSWLQDVHVYPKHG GSGRGVGV-(VPGVG)60
AC-terminal SEQ ID NO: 85 MVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRJTYG linker ETGGNSPVQEFTVPGSKSTATJSGLKPGVDYTJTVYA
VTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGS GGWLQDVHVYPKHSALVPRGVGV-iVPGVGW
PKH in Fn3 FG SEQ ID NO: 86 MVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRJTYG loop ETGGNSPVQEFTVPGSKSTATJSGLKPGVDYTJTVYA
VTPKHGSPASSKPISINYRTGGTSGGTSGSGSGGGSG GGSGFGGGVGV-(VPGVG)60
[00121] Each peptide was reacted with biotin-LPETGRAGG (SEQ ID NO: 10) and SrtA as detailed in Example 1 , i.e., 100 μΜ SrtA-ELP was incubated with 800 μΜ biotin-LPETGRAGG (SEQ ID NO: 10) in reaction buffer (50 mM Tris-HCI, 150 mM NaCI, 10 mM CaCI2, pH 8.5) for 2 hours at 20°C before Fn3-PLN-ELP (SEQ ID NO: 76) or variant was added to a final concentration of 50 μΜ and then incubated for 18 hours at 33°C. Samples of each reaction were analyzed for biotin via Western Blot. Results are shown in FIG. 28. All substrate proteins tested in the reactions showed biotinylation at the engineered isopeptide ligation site except for when the entire pilin domain was deleted. This confirmed that the attachment site was reduced to a solvent-accessible lysine with a local amino acid environment that enhances the nucleophilicity of the amino group of the lysine side chain.
[00122] The foregoing description of the specific aspects will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.
[00123] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.
[00124] All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.
[00125] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:
[00126] Clause 1. A method of conjugating an agent to a first polypeptide, the method comprising: contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZG (SEQ ID NO: 72) wherein X and Z are independently any amino acid, and wherein the ε-amino group of the lysine of the first polypeptide and the Z amino acid of LPXZG (SEQ ID NO: 72) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
[00127] Clause 2. A method of conjugating an agent to a first polypeptide, the method comprising: contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide, wherein the first polypeptide comprises at least one lysine, wherein the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid and wherein the carboxy- terminus of the amino acid Z of LPXZ (SEQ ID NO: 75) is modified to a methyl ester, and wherein the ε- amino group of the lysine of the first polypeptide and the Z amino acid of LPXZ (SEQ ID NO: 75) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
[00128] Clause 3. The method of clause 1 or 2, wherein the at least one lysine of the first polypeptide has a pKa of less than 10.53.
[00129] Clause 4. The method of any one of the above clauses, wherein the at least one lysine of the first polypeptide comprises a nucleophilic nitrogen atom.
[00130] Clause 5. The method of any one of the above clauses, wherein the at least one lysine of the first polypeptide comprises a side chain comprising an uncharged primary amino group.
[00131] Clause 6. The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of KH.
[00132] Clause 7. The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of PKH.
[00133] Clause 8. The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of YPKH (SEQ ID NO: 48).
[00134] Clause 9. The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of VYPKH (SEQ ID NO: 47).
[00135] Clause 10. The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of VHVYPKH (SEQ ID NO: 46).
[00136] Clause 1 1 . The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of WLQDVHVYPK (SEQ ID NO: 71 ).
[00137] Clause 12. The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of
Figure imgf000054_0001
(SEQ ID NO: 1 ), wherein X1 ; X2, X3, and X4 are independently any amino acid and Z is any hydrophobic residue. [00138] Clause 13. The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of WX1X2X3VX4VYPKI-I (SEQ ID NO: 2), wherein X1 ; X2, X3, and X4 are independently any amino acid.
[00139] Clause 14. The method of any one of the above clauses, wherein the first polypeptide comprises at least one amino acid sequence consisting of WLQDVHVYPKH (SEQ ID NO: 45).
[00140] Clause 15. The method of any one of the above clauses, wherein the SrtA is from a Gram-positive bacterium.
[00141] Clause 16. The method of clause 15, wherein the SrtA is from Staphylococcus aureus.
[00142] Clause 17. The method of any one of the above clauses, wherein the SrtA is soluble or is covalently linked to an insoluble support bead or resin.
[00143] Clause 18. The method of any one of the above clauses, wherein the SrtA is a recombinant polypeptide comprising an amino acid sequence of SEQ ID NO: 4.
[00144] Clause 19. The method of any one of the above clauses, wherein the SrtA is a recombinant polypeptide comprising an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 5.
[00145] Clause 20. The method of any one of the above clauses, wherein the agent is selected from the group consisting of polynucleotide, polypeptide, chemotherapeutic agent, vaccine, hormone, cytokine, anti-viral, steroid, opiate, anti-inflammatory, anti-convulsant, polymerization initiator, and polymer.
[00146] Clause 21 . The method of any one of the above clauses, wherein the first polypeptide comprises one lysine with a pKa of less than 10.53.
[00147] Clause 22. The method of any one of the above clauses, wherein the first polypeptide comprises two or more lysines, each with a pKa of less than 10.53.
[00148] Clause 23. The method of clause 21 , wherein the conjugate formed comprises two or more agents, each agent conjugated to an independent lysine. [00149] Clause 24. The method of any one of the above clauses, wherein the first polypeptide comprises one amino acid sequence consisting of ΖΧ^2Χ3 VX4VYPKH (SEQ ID NO: 1 ), wherein X-i , X2, X3, and X4 are independently any amino acid and Z is any hydrophobic residue.
[00150] Clause 25. The method of any one of the above clauses, wherein the first polypeptide comprises two or more amino acid sequences consisting of ZX1X2X3VX4VYPKH (SEQ ID NO: 1 ), wherein X-i , X2, X3, and X4 are independently any amino acid and Z is any hydrophobic residue.
[00151] Clause 26. The method of clause 25, wherein the conjugate formed comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of ZX1X2X3VX4VYPKH (SEQ ID NO: 1 ), wherein X-,, X2, X3, and X4 are independently any amino acid and Z is any hydrophobic residue.
[00152] Clause 27. The method of any one of the above clauses, wherein the first polypeptide further comprises an additional polypeptide selected from the group consisting of antibody, enzyme, therapeutic protein, fibronectin III (Fn3) domain, TN domain, DARPIN, affibody, scFv, and dsFv, or any combination thereof.
[00153] Clause 28. The method of any one of the above clauses, wherein the first polypeptide further comprises a fibronectin III (Fn3) domain.
[00154] Clause 29. The method of clause 28, wherein the Fn3 domain is a recombinant polypeptide comprising an amino acid sequence of SEQ ID NO: 8.
[00155] Clause 30. The method of clause 28, wherein the Fn3 domain is a recombinant polypeptide comprising an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 9.
[00156] Clause 31 . The method of any one of the above clauses, wherein the agent is conjugated to the amino terminal end of the second polypeptide.
[00157] Clause 32. The method of any one of clauses 1 and 3-31 , wherein the agent is conjugated to the second polypeptide amino-terminal to the LPXZG (SEQ ID NO: 72) sequence, wherein X and Z are independently any amino acid. [00158] Clause 33. The method of any one of clauses 1 and 3-32, wherein the second polypeptide comprises an amino acid sequence consisting of LPXTG (SEQ ID NO: 3), where X is any amino acid.
[00159] Clause 34. The method of any one of clauses 1 and 3-33, wherein the second polypeptide comprises an amino acid sequence consisting of LPETG (SEQ ID NO: 15).
[00160] Clause 35. The method of any one of clauses 1 and 3-32, wherein the second polypeptide comprises an amino acid sequence consisting of LPGAG (SEQ ID NO: 73).
[00161] Clause 36. The method of any one of clauses 1 and 3-34, wherein the second polypeptide comprises an amino acid sequence consisting of LPETGRAGG (SEQ ID NO: 10).
[00162] Clause 37. The method of any one of the above clauses, wherein the last amino acid at the carboxy end of the second polypeptide comprises a modified carboxyl group.
[00163] Clause 38. The method of clause 37, wherein the modified carboxyl group comprises a methyl ester.
[00164] Clause 39. The method of any one of clauses 1 and 3-38, wherein the SrtA recognizes LPXZG (SEQ ID NO: 72) of the second polypeptide.
[00165] Clause 40. The method of any one of clauses 1 and 3-39, wherein the SrtA cleaves the bond between the Z amino acid and the glycine of LPXZG (SEQ ID NO: 72) and forms a thioester bond between the catalytic thiol in SrtA and the carboxyl group of the Z amino acid.
[00166] Clause 41. The method of clause 40, wherein the thioester bond between the catalytic thiol in SrtA and the carboxyl group of the Z amino acid forms an intermediate.
[00167] Clause 42. The method of clause 41 , wherein the intermediate undergoes nucleophilic attack by the ε-amino group of the lysine.
[00168] Clause 43. The method of any one of clauses 40-42, wherein LPXZG (SEQ ID NO: 72) is LPXTG (SEQ ID NO: 3) and the Z amino acid is threonine.
[00169] Clause 44. The method of any one of the above clauses, wherein nucleophilic attack by the ε-amino group of the lysine forms an isopeptide bond between the first polypeptide and the second polypeptide to form the agent-first polypeptide conjugate. [00170] Clause 45. The method of any one of the above clauses, wherein the agent-first polypeptide conjugate comprises a fragment of the second polypeptide.
[00171] Clause 46. The method of clause 45, wherein the fragment of the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid.
[00172] Clause 47. The method of clause 46, wherein the fragment of the second polypeptide comprises an amino acid sequence consisting of LPXT (SEQ ID NO: 74) wherein X is any amino acid.
[00173] Clause 48. The method of any one of clauses 45-47, wherein the agent-first polypeptide conjugate does not comprise the G of LPXZG (SEQ ID NO: 72; wherein X and Z are independently any amino acid) of the second polypeptide.
[00174] Clause 49. The method of any one of clauses 1 and 3-48, wherein the formation of the agent-first polypeptide conjugate forms a third polypeptide comprising the G of LPXZG (SEQ ID NO: 72; wherein X and Z are independently any amino acid) and the C-terminal fragment thereto of the second polypeptide.
[00175] Clause 50. A method of drug delivery, the method comprising administering the agent-first polypeptide conjugate formed by the method of any one of clauses 1 -49.
[00176] Clause 51 . An agent-first polypeptide conjugate formed by the method of any one of clauses 1 -49.
SEQUENCES
SEQ ID NO: 1
Pilin domain, S. aureus, amino acid
ZX1X2X3VX4VYPKH (wherein X1 ; X2, X3, and X4 are independently any amino acid, and Z is any hydrophobic residue)
SEQ ID NO: 2
Pilin domain, S. aureus, amino acid
WX1X2X3VX4VYPKH (wherein X1 ; X2, X3, and X4 are independently any amino acid) SEQ ID NO: 3
SrtA recognition site, S. aureus, amino acid
LPXTG (where X is any amino acid)
SEQ ID NO: 4
Truncated S. aureus SrtA, amino acid
GQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFID RPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDY NEKTGVWEKRKIFVATEVKALVT
SEQ ID NO: 5
Truncated S. aureus SrtA, nucleotide
ggccaagctaaacctcaaattccgaaagataaatcgaaagtggcaggctatattgaaattccagatgctgatattaaagaaccagtgt atccaggaccagcaacacctgaacaattaaatagaggtgtaagctttgcagaagaaaatgaatcactagatgatcaaaatatttcaat tgcaggacacactttcattgaccgtccgaactatcaatttacaaatcttaaagcagccaaaaaaggtagtatggtgtactttaaagttggt aatgaaacacgtaagtataaaatgacaagtataagagatgttaagcctacagatgtaggagttctagatgaacaaaaaggtaaaga taaacaattaacattaattacttgtgatgattacaatgaaaagacaggcgtttgggaaaaacgtaaaatctttgtagctacagaagtcaa agcactagttact
SEQ ID NO: 6
Full length wild-type S. aureus SrtA, amino acid
MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDNKQQAKPQ IPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQF TNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVEVLDEQKGKDKQLTLITCDDYNEKTGV WEKRKIFVATEVK
SEQ ID NO: 7
Full length wild-type S. aureus SrtA, nucleotide
atgaaaaaat ggacaaatcg attaatgaca atcgctggtg tagtacttat cctagtggca gcatatttgt ttgctaaacc acatatcgat aattatcttc acgataaaga taaagatgaa aagattgaac aatatgataa aaatgtaaaa gaacaggcga gtaaagacaa taagcagcaa gctaaacctc aaattccgaa agataaatca aaagtggcag gctatattga aattccagat gctgatatta aagaaccagt atatccagga ccagcaacac ctgaacaatt aaatagaggt gtaagctttg cagaagaaaa tgaatcacta gatgatcaaa atatttcaat tgcaggacac actttcattg accgtccgaa ctatcaattt acaaatctta aagcagccaa aaaaggtagt atggtgtact ttaaagttgg taatgaaaca cgtaagtata aaatgacaag tataagagat gttaagccaa cagatgtaga agttctagat gaacaaaaag gtaaagataa acaattaaca ttaattactt gtgatgatta caatgaaaag acaggcgttt gggaaaaacg taaaatcttt gtagctacag aagtcaaata a
SEQ ID NO: 8
Fn3 domain, Homo sapiens, amino acid
VSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKP GVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGG
SEQ ID NO: 9
Fn3 domain, Homo sapiens, nucleotide
gttagcgatgttcctcgcgatctggaagtggtggcagcaacaccgacatctctgctgatctcttgggatgcgcctgcggtgaccgtgcgc tactatcgcatcacctatggcgaaaccggtggtaatagccctgtgcaggaatttaccgttccgggttctaaaagcacggcaacgatctc tggtctgaaaccgggtgtggattataccattacggtttatgcggtgaccccacgcggcgattggaatgaaggctctaaaccaatctcgat taactaccgcaccggcggtacatctggtggtacaagtggttctggatccggtggcggcagcggcggt
SEQ ID NO: 10
Experimental SrtA recognition site, S. aureus, amino acid
LPETGRAGG, may be conjugated to biotin or FITC, for example
SEQ ID NO: 1 1
SrtA-ELP sequence, amino acid
MGQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTF IDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDD YNEKTGVWEKRKIFVATEVKALVTMGVG-(VPGVG)24o
SEQ ID NO: 12
ELP-PLN-GLP-LPETG sequence, amino acid
MSKGPGVG-(VPGVG)80-
VPGSGLVPRGSSGGWLQDVHVYPKHGGSGRGAHGEGTFTSDVSSYLEEQAAKEFIAWLVKG AGLPETGRAGG
SEQ ID NO: 13
Fn3-PLN3-ELP sequence, amino acid, may be conjugated to biotin or FITC for example MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGWLQDVHVYPKHG GSGRGSGGWLQDVHVYPKHGGSGRGSGGWLQDVHVYPKHGGSGRGVGV-(VPGVG)60
SEQ ID NO: 14
Fn3-ELP sequence, amino acid
VSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKP GVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGVG-(VPGVG)60
SEQ ID NO: 15
SrtA recognition site, S. aureus, amino acid
LPETG, may be conjugated to biotin and/or dabcyl and/or edans, for example.
SEQ ID NO: 16
amino acid
GSSGGWLQDVHVYPK
SEQ ID NO: 17
amino acid
HGGSGR
SEQ ID NO: 18
amino acid
GAHGEGTFTSDVSSYLEEQAAK
SEQ ID NO: 19
amino acid
EFIAWLVK
SEQ ID NO: 20
amino acid
GSSGGWLQDVHVYPKHGGSGR
SEQ ID NO: 21 amino acid
GAG LPET
SEQ ID NO: 22
amino acid
GAGLPETGGG
SEQ ID NO: 23
SRT15_ 2 amino acid
VAGYIEIPDADIKEPVYPGPATPEQLNR
SEQ ID NO: 24
SRT43-77 amino acid
GVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLK
SEQ ID NO: 25
amino acid
GAHGEGTFTSDVSSYLEEQAAKEFIAWLVK
SEQ ID NO: 26
amino acid
EFIAWLVKGAGLPETGGG
SEQ ID NO: 27
amino acid
EFIAWLVKGAGLPETGRAGG
SEQ ID NO: 28
amino acid
TGGTSGGTSGSGSGGGSGGWLQDVHVYPKHGGSGR
SEQ ID NO: 29
amino acid
LPET, may be conjugated to biotin or dabcyl or FITC, for exampl SEQ ID NO: 30
amino acid
GSGGWLQDVHVYPKHGGSGR
SEQ ID NO: 31
amino acid
GSGGWLQDVHVYPK
SEQ ID NO: 32
amino acid
TGGTSGGTSGSGSGGGSGGWLQDVHVYPK
SEQ ID NO: 33
amino acid
LPETGGG, may be conjugated to biotin or dabcyl, for example.
SEQ ID NO: 34
SRT28-42 amino acid
EPVYPGPATPEQLNR
SEQ ID NO: 35
SRT -121-149 amino acid
QLTLITCDDYNEKTGVWEKRKIFVATEVK
SEQ ID NO: 36
Fn38o-94 amino acid
GDWNEGSKPISINYR
SEQ ID NO: 37
Fn335-55 amino acid
ITYGETGGNSPVQEFTVPGSK
SEQ ID NO: 38 Fn356-79 amino acid
STATI SG LKPGVDYTITVYAVTP R
SEQ ID NO: 39
Fn38-3i amino acid
DLEVVAATPTSLLISWDAPAVTVR
SEQ ID NO: 40
amino acid
MVSDVPR
SEQ ID NO: 41
amino acid
VSDVPR
SEQ ID NO: 42
amino acid
ITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTPR
SEQ ID NO: 43
amino acid
GAGLPETG
SEQ ID NO: 44
amino acid
RGDWXE
SEQ ID NO: 45
amino acid, sequence in first polypeptide, artificial
WLQDVHVYPKH
SEQ ID NO: 46
Pilin domain of ΔΝ4 deletion mutant first polypeptide, amino acid, artificial VHVYPKH SEQ ID NO: 47
Pilin domain of ΔΝ6 deletion mutant first polypeptide, amino acid, artificial
VYPKH
SEQ ID NO: 48
Pilin domain of ΔΝ7 deletion mutant first polypeptide, amino acid, artificial
YPKH
SEQ ID NO: 49
His-tagged SrtA (H6-SrtA), amino acid
MGSSHHHHHHSSGLVPRGSHMQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGV SFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTD VGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVK
SEQ ID NO: 50
Pilin domain peptide, amino acid
VGGSWLQDVHVYPKHGGSGR
SEQ ID NO: 51
ELP pentapeptide, amino acid
VPGXG (wherein X is any amino acid except proline)
SEQ ID NO: 52
amino acid
Ahx-LPET, wherein Ahx is the hydrophobic spacer aminohexanoic acid; may be conjugated to FITC, for example
SEQ ID NO: 53
amino acid
GSGGWLQDVHVYPuHGGSGR, wherein u = lysine isopeptide-linked to biotin-LPETG
SEQ ID NO: 54
amino acid ITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTPRGDWNEGSKPISINYR
SEQ ID NO: 55
amino acid
GDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGWLQDVHVYPK
SEQ ID NO: 56
amino acid
DLEVVAATPTSLLISWDAPAVTVRYYR
SEQ ID NO: 57
amino acid
TGGTSGGTSGSGSGGGSGGWLQDVHVYPuHGGSGR, wherein u = lysine isopeptide-linked to biotin-LPETG
SEQ ID NO: 58
amino acid
GDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGWLQDVH VYPuHGGSGR, wherein u = lysine isopeptide-linked to biotin-LPETG
SEQ ID NO: 59
amino acid
TGGTSGGTSGSGSGGGSGGWLQDVHVYPuHGGSGRGSGGWLQDVHVYPuHGGSGR, wherein u = lysine isopeptide-linked to biotin-LPETG
SEQ ID NO: 60
amino acid
TGGTSGGTSGSGSGGGSGGWLQDVHVYPuHGGSGRGSGGWLQDVHVYPuHGGSGRGSGG WLQDVHVYPuHGGSGR, wherein u = lysine isopeptide-linked to biotin-LPETG
SEQ ID NO: 61
amino acid
TGGTSGGTSGSGSGGGSGGWLQDVHVYPuHGGSGRGSGGWLQDVHVYPK, wherein u = lysine isopeptide-linked to biotin-LPETG SEQ ID NO: 62
amino acid
GDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGWLQDVHVYPuHGGSGR, wherein u = lysine isopeptide-linked to biotin-LPETG
SEQ ID NO: 63
amino acid
MVSDVPRDL E WAAT PTS L L I S W D AP AVTV RYY R
SEQ ID NO: 64
amino acid
TGGTSGGTSGSGSGGGSGGWLQDVHVYPuHGGSGRGSGGWLQDVHVYPKHGGSGR, wherein u = lysine isopeptide-linked to biotin-LPETG
SEQ ID NO: 65
amino acid
DLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQ EFTVPGSK
SEQ ID NO: 66
amino acid
HGGSGRGSGGWLQDVHVYPuHGGSGRGSGGWLQDVHVYPKHGGSGR, wherein u = lysine isopeptide-linked to biotin-LPETG
SEQ ID NO: 67
amino acid
ITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTPRTGGTSGGTSGSGSGGGSG GWLQDVHVYPK
SEQ ID NO: 68
amino acid
Ahx-LPETGRAGG, wherein Ahx is the hydrophobic spacer aminohexanoic acid; may be conjugated to FITC, for example. SEQ ID NO: 69
GLP1 , amino acid
GGSGRGAHGEGTFTSDVSSYLEEQAAKEFIAWLVK
SEQ ID NO: 70
ELP-GLP1 , amino acid
VPGXG GGSGRGAHGEGTFTSDVSSYLEEQAAKEFIAWLVK SEQ ID NO: 71
Pilin domain of AC1 deletion mutant, amino acid, artificial
WLQDVHVYPK
SEQ ID NO: 72
SrtA recognition site, S. aureus, amino acid
LPXZG (wherein X and Z are independently any amino acid)
SEQ ID NO: 73
SrtA recognition site, S. aureus, amino acid
LPGAG
SEQ ID NO: 74
SrtA recognition site remaining in second polypeptide after reaction, amino acid
LPXT (wherein X is any amino acid)
SEQ ID NO: 75
SrtA recognition site remaining in second polypeptide after reaction, amino acid
LPXZ (wherein X and Z are independently any amino acid)
SEQ ID NO: 76
Fn3-PLN-ELP for deletion mutant analysis, amino acid
MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGWLQDVHVYPKHG GSGRGVGV-(VPGVG)60 SEQ ID NO: 77
Fn3-ELP for deletion mutant analysis, amino acid
MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTS-GVGV-(VPGVG)60
SEQ ID NO: 78
ΔΝ4 for deletion mutant analysis, amino acid
MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGVHVYPKHGGSGR GVGV-(VPGVG)60
SEQ ID NO: 79
ΔΝ6 for deletion mutant analysis, amino acid
MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGVYPKHGGSGRGV GV-(VPGVG)60
SEQ ID NO: 80
ΔΝ7 for deletion mutant analysis, amino acid
MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGYPKHGGSGRGVG
V-(VPGVG)60
SEQ ID NO: 81
ΔΝ8 for deletion mutant analysis, amino acid
MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK
PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGPKHGGSGRGVGV-
(VPGVG)60
SEQ ID NO: 82
ΔΝ9 for deletion mutant analysis, amino acid MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK
PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGKHGGSGRGVGV-
(VPGVG)60
SEQ ID NO: 83
AC1 for deletion mutant analysis, amino acid
MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGWLQDVHVYPKGG SGRGVGV-(VPGVG)60
SEQ ID NO: 84
ΔΝ-terminal linker for deletion mutant analysis, amino acid
MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK
PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSWLQDVHVYPKHGGSGRGVGV-
(VPGVG)60
SEQ ID NO: 85
AC-terminal linker for deletion mutant analysis, amino acid
MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK
PGVDYTITVYAVTPRGDWNEGSKPISINYRTGGTSGGTSGSGSGGGSGGWLQDVHVYPKHSA
LVPRGVGV-(VPGVG)60
SEQ ID NO: 86
Fn3 with PKH mutation in Fn3 loop for deletion mutant analysis, amino acid
MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLK PGVDYTITVYAVTPKHGSPASSKPISINYRTGGTSGGTSGSGSGGGSGGGSGFGGGVGV- (VPGVG)60
SEQ ID NO: 87
N-terminal linker for deletion mutant analysis, amino acid
GGTSGSGSGGGSGG
SEQ ID NO: 88
C-terminal linker for deletion mutant analysis, amino acid GGSGR
SEQ ID NO: 89
FG loop of Fn3, amino acid
PRGDWNEGS
SEQ ID NO: 90
Mutant FG loop of Fn3, amino acid PKHGSPASS

Claims

CLAIMS We claim:
1. A method of conjugating an agent to a first polypeptide, the method comprising:
contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide,
wherein the first polypeptide comprises at least one lysine,
wherein the second polypeptide comprises an amino acid sequence consisting of
LPXZG (SEQ ID NO: 72) wherein X and Z are independently any amino acid, and
wherein the ε-amino group of the lysine of the first polypeptide and the Z amino acid of
LPXZG (SEQ ID NO: 72) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
2. A method of conjugating an agent to a first polypeptide, the method comprising:
contacting the first polypeptide with sortase A (SrtA) and at least one agent conjugated to a second polypeptide,
wherein the first polypeptide comprises at least one lysine,
wherein the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid and wherein the carboxy- terminus of the amino acid Z of LPXZ (SEQ ID NO: 75) is modified to a methyl ester, and
wherein the ε-amino group of the lysine of the first polypeptide and the Z amino acid of LPXZ (SEQ ID NO: 75) form an isopeptide bond to conjugate the agent to the first polypeptide, thereby forming an agent-first polypeptide conjugate.
3. The method of claim 1 or 2, wherein the at least one lysine of the first polypeptide has a pKa of less than 10.53.
4. The method of any one of the above claims, wherein the at least one lysine of the first polypeptide comprises a nucleophilic nitrogen atom.
5. The method of any one of the above claims, wherein the at least one lysine of the first polypeptide comprises a side chain comprising an uncharged primary amino group.
6. The method of any one of the above claims, wherein the first polypeptide comprises at least one amino acid sequence consisting of KH.
7. The method of any one of the above claims, wherein the first polypeptide comprises at least one amino acid sequence consisting of PKH.
8. The method of any one of the above claims, wherein the first polypeptide comprises at least one amino acid sequence consisting of YPKH (SEQ ID NO: 48).
9. The method of any one of the above claims, wherein the first polypeptide comprises at least one amino acid sequence consisting of VYPKH (SEQ ID NO: 47).
10. The method of any one of the above claims, wherein the first polypeptide comprises at least one amino acid sequence consisting of VHVYPKH (SEQ ID NO: 46).
1 1 . The method of any one of the above claims, wherein the first polypeptide comprises at least one amino acid sequence consisting of WLQDVHVYPK (SEQ ID NO: 71 ).
12. The method of any one of the above claims, wherein the first polypeptide comprises at least one amino acid sequence consisting of ZX X2 z VX4 VYPKH (SEQ ID NO: 1 ), wherein X1 ; X2, X3, and X4 are independently any amino acid and Z is any hydrophobic residue.
13. The method of any one of the above claims, wherein the first polypeptide comprises at least one amino acid sequence consisting of WX1X2X3VX4 VYPKH (SEQ ID NO: 2), wherein X-i , X2, X3, and X4 are independently any amino acid.
14. The method of any one of the above claims, wherein the first polypeptide comprises at least one amino acid sequence consisting of WLQDVH VYPKH (SEQ ID NO: 45).
15. The method of any one of the above claims, wherein the SrtA is from a Gram-positive bacterium.
16. The method of claim 15, wherein the SrtA is from Staphylococcus aureus.
17. The method of any one of the above claims, wherein the SrtA is soluble or is covalently linked to an insoluble support bead or resin.
18. The method of any one of the above claims, wherein the SrtA is a recombinant polypeptide comprising an amino acid sequence of SEQ ID NO: 4.
19. The method of any one of the above claims, wherein the SrtA is a recombinant polypeptide comprising an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 5.
20. The method of any one of the above claims, wherein the agent is selected from the group consisting of polynucleotide, polypeptide, chemotherapeutic agent, vaccine, hormone, cytokine, anti-viral, steroid, opiate, anti-inflammatory, anti-convulsant, polymerization initiator, and polymer.
21 . The method of any one of the above claims, wherein the first polypeptide comprises one lysine with a pKa of less than 10.53.
22. The method of any one of the above claims, wherein the first polypeptide comprises two or more lysines, each with a pKa of less than 10.53.
23. The method of claim 21 , wherein the conjugate formed comprises two or more agents, each agent conjugated to an independent lysine.
24. The method of any one of the above claims, wherein the first polypeptide comprises one amino acid sequence consisting of ZX1X2X3VX4VYPKH (SEQ ID NO: 1 ), wherein X1 ; X2, X3, and X4 are independently any amino acid and Z is any hydrophobic residue.
25. The method of any one of the above claims, wherein the first polypeptide comprises two or more amino acid sequences consisting of ZX1X2X3VX4VYPKH (SEQ ID NO: 1 ), wherein X1 ; X2, X3, and X4 are independently any amino acid and Z is any hydrophobic residue.
26. The method of claim 25, wherein the conjugate formed comprises two or more agents, each agent conjugated to an independent amino acid sequence consisting of ZX1X2X3VX4VYPKH (SEQ ID NO: 1 ), wherein X·,, X2, X3, and X4 are independently any amino acid and Z is any hydrophobic residue.
27. The method of any one of the above claims, wherein the first polypeptide further comprises an additional polypeptide selected from the group consisting of antibody, enzyme, therapeutic protein, fibronectin III (Fn3) domain, TN domain, DARPIN, affibody, scFv, and dsFv, or any combination thereof.
28. The method of any one of the above claims, wherein the first polypeptide further comprises a fibronectin III (Fn3) domain.
29. The method of claim 28, wherein the Fn3 domain is a recombinant polypeptide comprising an amino acid sequence of SEQ ID NO: 8.
30. The method of claim 28, wherein the Fn3 domain is a recombinant polypeptide comprising an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 9.
31 . The method of any one of the above claims, wherein the agent is conjugated to the amino terminal end of the second polypeptide.
32. The method of any one of claims 1 and 3-31 , wherein the agent is conjugated to the second polypeptide amino-terminal to the LPXZG (SEQ ID NO: 72) sequence, wherein X and Z are independently any amino acid.
33. The method of any one of claims 1 and 3-32, wherein the second polypeptide comprises an amino acid sequence consisting of LPXTG (SEQ ID NO: 3), where X is any amino acid.
34. The method of any one of claims 1 and 3-33, wherein the second polypeptide comprises an amino acid sequence consisting of LPETG (SEQ ID NO: 15).
35. The method of any one of claims 1 and 3-32, wherein the second polypeptide comprises an amino acid sequence consisting of LPGAG (SEQ ID NO: 73).
36. The method of any one of claims 1 and 3-34, wherein the second polypeptide comprises an amino acid sequence consisting of LPETGRAGG (SEQ ID NO: 10).
37. The method of any one of the above claims, wherein the last amino acid at the carboxy end of the second polypeptide comprises a modified carboxyl group.
38. The method of claim 37, wherein the modified carboxyl group comprises a methyl ester.
39. The method of any one of claims 1 and 3-38, wherein the SrtA recognizes LPXZG (SEQ ID NO: 72) of the second polypeptide.
40. The method of any one of claims 1 and 3-39, wherein the SrtA cleaves the bond between the Z amino acid and the glycine of LPXZG (SEQ ID NO: 72) and forms a thioester bond between the catalytic thiol in SrtA and the carboxyl group of the Z amino acid.
41 . The method of claim 40, wherein the thioester bond between the catalytic thiol in SrtA and the carboxyl group of the Z amino acid forms an intermediate.
42. The method of claim 41 , wherein the intermediate undergoes nucleophilic attack by the ε-amino group of the lysine.
43. The method of any one of claims 40-42, wherein LPXZG (SEQ ID NO: 72) is LPXTG (SEQ ID NO: 3) and the Z amino acid is threonine.
44. The method of any one of the above claims, wherein nucleophilic attack by the ε-amino group of the lysine forms an isopeptide bond between the first polypeptide and the second polypeptide to form the agent-first polypeptide conjugate.
45. The method of any one of the above claims, wherein the agent-first polypeptide conjugate comprises a fragment of the second polypeptide.
46. The method of claim 45, wherein the fragment of the second polypeptide comprises an amino acid sequence consisting of LPXZ (SEQ ID NO: 75) wherein X and Z are independently any amino acid.
47. The method of claim 46, wherein the fragment of the second polypeptide comprises an amino acid sequence consisting of LPXT (SEQ ID NO: 74) wherein X is any amino acid.
48. The method of any one of claims 45-47, wherein the agent-first polypeptide conjugate does not comprise the G of LPXZG (SEQ ID NO: 72; wherein X and Z are independently any amino acid) of the second polypeptide.
49. The method of any one of claims 1 and 3-48, wherein the formation of the agent-first polypeptide conjugate forms a third polypeptide comprising the G of LPXZG (SEQ ID NO: 72; wherein X and Z are independently any amino acid) and the C-terminal fragment thereto of the second polypeptide.
50. A method of drug delivery, the method comprising administering the agent-first polypeptide conjugate formed by the method of any one of claims 1 -49.
51 . An agent-first polypeptide conjugate formed by the method of any one of claims 1-49.
PCT/US2015/017601 2014-02-25 2015-02-25 Compositions and methods for the site-specific modification of polypeptides WO2015130846A2 (en)

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