WO2015183986A1 - Procédés de sélection de transpeptidases évoluées et leur utilisation pour créer des conjugués de protéines - Google Patents

Procédés de sélection de transpeptidases évoluées et leur utilisation pour créer des conjugués de protéines Download PDF

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WO2015183986A1
WO2015183986A1 PCT/US2015/032717 US2015032717W WO2015183986A1 WO 2015183986 A1 WO2015183986 A1 WO 2015183986A1 US 2015032717 W US2015032717 W US 2015032717W WO 2015183986 A1 WO2015183986 A1 WO 2015183986A1
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tpase
sequence
seq
variant
substrate
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PCT/US2015/032717
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Ramesh Baliga
Dean Ng
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Extend Biopharma, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)

Definitions

  • ADCs Antibody-drug conjugates
  • ADCs There are currently three FDA-approved ADCs on the market: Mylotarg, Adcetris, and Kadcyla. The remarkable clinical success of ADCs has prompted development of more than 40 other ADCs, which are currently in various stages of clinical testing (Burris et al, Clin Breast Cancer 2011, 11 :275-282; Senter et al, Nat Biotechnol 2012, 30:631-637).
  • a key problem that has impeded the progress in the development of ADCs is the lack of an approach to easily and efficiently conjugate the small molecule warhead to specific sites on the antibody.
  • the present invention provides selection and screening methods which take advantage of techniques in protein engineering and directed evolution to identify transpeptidase (TPase) variants that have a modified catalytic activity for a native recognition site in a peptide, polypeptide, or protein of interest compared to a corresponding wildtype TPase or that recognize new sequence motifs present in the endogenous peptide, polypeptide, or protein sequence that differ from the native recognition site.
  • TPase transpeptidase
  • the selection and screening approaches for TPases as described herein enable the construction of a tool box of these enzymes for the conjugation of the original sequence of peptides, polypeptides, or proteins of interest with other moieties such as peptides, polypeptides, proteins, labels, or small molecules.
  • the present invention further provides compositions and kits comprising the evolved TPase variants or the conjugates produced by the use of the variants identified by the selection and/or screening methods described herein.
  • the present invention provides a method for isolating a TPase variant that cleaves a recognition sequence at the C-terminus of a first substrate with a desired catalytic activity than a corresponding wildtype TPase, the method comprising:
  • the desired catalytic activity is increased catalytic activity compared to the corresponding wildtype TPase. In other embodiments, the desired catalytic activity is decreased (attenuated) catalytic activity compared to the corresponding wildtype TPase. In some cases, increased catalytic activity includes increased specificity for a substrate or recognition motif and/or increased selectivity for a substrate or recognition motif.
  • the substrate can be a native (original or wildtype) substrate that is recognized by the corresponding wildtype TPase.
  • the recognition motif can be a native (original or wildtype) recognition motif that is recognized by the corresponding wildtype TPase.
  • the desired catalytic activity is decreased dependence of a cofactor for enzymatic activity, compared to the wildtype enzyme.
  • the cofactor is an organic cofactor, an inorganic cofactor, an ion, e.g., a metal ion, and the like.
  • the desired catalytic activity is a change in thermostability.
  • the desired catalytic activity is elimination of the reverse traspeptidase reaction.
  • the corresponding wildtype TPase does not cleave the recognition sequence.
  • the TPase is a transamidase such as a sortase, e.g., Sortase A (SEQ ID NO: l).
  • the recognition sequence comprises or is LPXTG (SEQ ID NO: 2, wherein the cleavage site is between the T and G).
  • the recognition sequence does not comprise or is not LPXTG (SEQ ID NO: 2, wherein the cleavage site is between the T and G).
  • the recognition sequence is LSPGK (SEQ ID NO: 3, wherein the cleavage site is between the G and K).
  • the first substrate is an antibody light or heavy chain.
  • the antibody light or heavy chain contains the original (wildtype) amino acid sequence.
  • the second substrate is a peptide that comprises a tag.
  • the tag is biotin.
  • the N-terminal acceptor sequence comprises one, two, three, four, five, or more contiguous glycine residues.
  • the library of genetic elements is a randomized protein library generated by oligonucleotide-directed randomization.
  • the microcompartment is an emulsion. In particular instances, the emulsion is a water-in-oil emulsion.
  • the microcompartment is a well in a microtiter plate.
  • each microcompartment contains an individual genetic element expressing only one particular TPase variant.
  • a plurality of microcompartments e.g., each containing a unique genetic element expressing a single type of TPase variant
  • the TPase variant has an alteration in one or more amino acid residues that contact the first substrate.
  • the present invention provides a method for isolating a TPase variant that targets an acceptor sequence at the N-terminus of a substrate that is not a native acceptor sequence at the N-terminus targeted by a corresponding wildtype TPase, the method comprising:
  • the corresponding wildtype TPase does not target the N- terminal acceptor sequence.
  • a native acceptor sequence at the N-terminus can be 1, 2, 3, 4, 5, or more glycine residues that are recognized by the wildtype TPase.
  • the TPase is a transamidase such as a sortase, e.g., Sortase A.
  • the recognition sequence is LPXTG (SEQ ID NO:2, wherein the cleavage site is between the T and G).
  • the N-terminal acceptor sequence does not comprise five contiguous glycine residues.
  • the substrate is an antibody light or heavy chain. In particular instances, the antibody light or heavy chain contains the original (wildtype) amino acid sequence.
  • the test substrate is a peptide that comprises a tag.
  • the tag is biotin.
  • the N-terminal acceptor sequence is DIQXT, wherein X is any amino acid (SEQ ID NO:4).
  • the N- terminal acceptor sequence is DIQMT (SEQ ID NO:5) or DIQLT (SEQ ID NO:6).
  • the N-terminal acceptor sequence is XiVQLX 2 , wherein Xi and X 2 are independently any amino acid (SEQ ID NO:7).
  • the N-terminal acceptor sequence is EVQLV (SEQ ID NO:8), QVQLV(SEQ ID NO:9), or QVQLQ (SEQ ID NO: 10).
  • the library of genetic elements is a randomized protein library generated by oligonucleotide-directed randomization.
  • the microcompartment is an emulsion.
  • the emulsion is a water-in-oil emulsion.
  • the microcompartment is a well in a microtiter plate.
  • each microcompartment contains an individual genetic element expressing only one particular TPase variant.
  • a plurality of microcompartments ⁇ e.g., each containing a unique genetic element expressing a single type of TPase variant) can be arranged on an array in addressable locations.
  • the TPase variant has an alteration in one or more amino acid residues that contact the first substrate.
  • the present invention provides an isolated and/or purified TPase variant identified by the methods described herein.
  • the present invention provides an isolated and/or purified nucleic acid encoding a TPase variant identified by the methods described herein.
  • the present invention provides an isolated and/or purified vector comprising the nucleic acid.
  • the vector further comprises a regulatory sequence operably linked to the nucleic acid.
  • the vector comprises an expression vector.
  • the present invention provides an isolated host such as an isolated cell transformed or transfected or transduced with the nucleic acid or a vector comprising the nucleic acid.
  • the present invention provides a kit comprising a TPase variant identified by the methods described herein.
  • the present invention provides a conjugate produced by a TPase variant identified by the methods described herein.
  • the conjugate comprises an antibody light or heavy chain containing the recognition sequence attached to a molecule containing the N-terminal acceptor sequence.
  • the antibody light or heavy chain is attached to a linker, e.g., a peptide linker, located between the antibody light or heavy chain and the recognition sequence.
  • the molecule is a label, an anticancer drug, or combinations thereof.
  • the molecule is attached to a linker containing the N-terminal acceptor sequence.
  • the conjugate comprises a molecule containing the recognition sequence attached to an antibody light or heavy chain containing the N-terminal acceptor sequence.
  • the molecule is a label, an anticancer drug, or combinations thereof.
  • the molecule is attached to a linker containing the recognition sequence.
  • the present invention provides a pharmaceutical composition comprising the conjugate and a pharmaceutically acceptable carrier.
  • the present invention provides a kit comprising a conjugate described herein.
  • the present invention provides a method for treating cancer in a subject comprising administering a therapeutically effective amount of a conjugate to the subject.
  • FIG. 1 provides a schematic diagram of certain exemplary embodiments.
  • Libraries of a transpeptidase (TPase) of interest are subjected to directed evolution to create catalysts for site-specific labeling of proteins such as antibodies with a small molecule, peptide, or protein of interest.
  • TPase transpeptidase
  • FIGS. 2A and 2B depict the biochemical characterization of wildtype S. aureus srtA and the srtA mutant using a FRET based proteolysis assay and the Dab-LPETG-EDANS substrate (SEQ ID NO: l 1).
  • FIG. 2A shows RFUs vs time.
  • FIG. 2B shows a linear fit of k obs vs concentration of TPase to obtain kcat/K m .
  • FIGS. 3 A and 3B depict the biochemical characterization using a FRET based proteolysis assay and the Dab-LPETG-EDANS substrate (SEQ ID NO: 11) of wildtype S.
  • FIG. 3A shows Dab-LPETG- EDANS substrate (SEQ ID NO: l 1).
  • FIG. 3B shows RFUs vs time of an in vitro transcription and translation reaction with the PCR templates for the indicated TPases.
  • FIG. 4 provides a schematic diagram of an exemplary selection scheme for directed evolution of transpeptidases (TPases).
  • TPases transpeptidases
  • the DNA template is encapsulated in an in vitro transcription translation mix, along with the tracer peptide (e.g., target substrate) containing biotin.
  • Active TPase mutants are able to tether the relevant DNA sequences to the biotin-containing acceptor peptide, which enables the isolation of those DNA sequences by streptavidin bead-based pulldowns for further amplification and enrichment.
  • FIG. 5 shows the duplex DNA PCR product coding the TPase mutant. It was produced using a primer tagged at its 5 '-end with an LPETGG peptide sequence (SMPH- CLPETGG) (SEQ ID NO: 12). The tagged duplex was incubated with 1 ⁇ the TPase mutant for 2 hrs at room temperature in the presence of the nucleophile GGGGSK-FAM (SEQ ID NO: 13).
  • FIGS. 6A and 6B show the results of the quantitative PCR assay for detection the TPase mutant templates, e.g., the e-srtA and C184A templates.
  • FIG. 6A shows qPCR assay with primer corresponding to e-srtA enables detection of e-srtA template DNA from 1000 to 10 million copies.
  • FIG. 7 shows the data of magnetic bead based pulldown of DNA templates.
  • qPCR assay with primer sets corresponding to either e-srtA or C184A enables detection of template DNA after bead based pulldown.
  • Biotinylated e-srtA template (positive control) and non biotinylated CI 84 A DNA (negative control) were used in mock pulldown experiments. S/B of greater than 1000 is achievable when the target template is fully biotinylated, under our optimized conditions for binding, washing and release.
  • FIGS. 8A and 8B show quantitative PCR assay for detection of e-srtA and C184A templates recovered after IVTT/IVC.
  • qPCR assay with primer corresponding to e-srtA enables detection of e-srtA template DNA from 1000 to 10 million copies (FIG. 8A).
  • FIG. 9 shows the construction of 2XNNK library around srtA residues P94 and D165.
  • FIGS. 10A and 10B show data from the real-time fluorescence assay for detection of enzyme activity produced by PCR templates at various concentrations.
  • FIG. 10A shows titration of the PCR template of a TPase mutant (e-srtA).
  • FIG. 10B shows the enzyme activity of various TPase mutants identified in an exemplary embodiment of the screening method provided herein.
  • FIG. 11 provides a schematic diagram of the antibody constructs for TPase mediated conjugation.
  • the TPase recognition sequence is incorporated as the last six residues of the heavy chain, adjacent to the last residue of the HC or connected via a short linker.
  • FIG. 12 provides a synthesis scheme for making a derivative of Monomethyl Auristatin E (MMAE) having a cleavable linker Val-Cit and an N-terminal 5xGlycine sequence that can be used for conjugation with TPases variants described herein.
  • FIGS. 13A and 13B depict coomassie (FIG. 13A) and UV (FIG. 13B) images of a Herceptin derivative carrying a wildtype TPase recognition site separated from the C- terminus by a linker.
  • FIG. 14 shows hydrophobic interation chromatography (HIC) analysis of the Herceptin tagged with the TPase sequence conjugated with 5xGlycine-MMAE.
  • FIG. 15 shows that the Herceptin conjugate (Herc-HC-V-MMAE) effectively killed HER2-expressing cells (SKBR-3 cells). Controls experiments with Herceptin alone in the presence of a secondary anti-human Fc antibody tagged with cleavable linker conjugated MMAE (Moradec Inc) provide a comparison of the activity of directly and indirectly labeled Herceptin.
  • FIG. 16 shows the stability of the human Fc carrying the G4SFAM tag in rodent plasma.
  • FIGS. 17A and 17B provide exemplary TPase variants.
  • a set of discrete variants of a focused library is arrayed in a 96-well plate (FIG. 17A).
  • the parent sequences (scaffolds) are shown in FIG. 17B.
  • FIG. 18 shows the increased and decreased enzymatic activity (compared to wildtype TPase) of TPase variants identified in an exemplary embodiment of the screening method provided herein.
  • FIG. 19 shows the increased enzyme activity (compared to wildtype TPase) of TPase variants identified in an exemplary embodiment of the screening method provided herein.
  • the P94t and P94E mutants shows increased activity of about 5-1 OX compared to the wt enzyme.
  • FIG. 20 shows the decreased enzyme activity (compared to wildtype TPase) of a TPase mutant (W192A) identified in an exemplary embodiment of the screening method provided herein.
  • the present invention provides selection and screening methods which take advantage of techniques in protein engineering and directed evolution to start with a transpeptidase (TPase) scaffold that recognizes a sequence of interest such as the sequence LPXTG (SEQ ID NO: 2, , wherein the cleavage site is between the T and G, Popp et ah, Angew Chem Int Ed Engl 2011, 50:5024-5032) and modify the recognition site to make it compatible with the terminus of a protein of interest, e.g., the C- or N-terminus of an antibody light or heavy chain.
  • TPase transpeptidase
  • FIG. 1 illustrates certain exemplary embodiments of the present invention for selecting TPases identified from a transpeptidase library subjected to directed evolution and for using these TPases as catalysts for site-specific labeling of proteins such as antibodies with a small molecule (or other protein) of interest.
  • the present invention also provides compositions comprising the TPases identified by the selection and screening methods described herein.
  • the present invention further provides compositions comprising the conjugates produced by the use of the TPases described herein.
  • these methods all suffer from severe drawbacks that render them unsuitable for the site-specific conjugation of molecules of interest to each other See, e.g., Bentley et ah, J Biol Chem 2008, 283:14762-71 (recognition specificity from the native Sortase A sequence LPXTG (SEQ ID NO: 2, wherein the cleavage site is between the T and G) changed to the Sortase B recognition sequence NPQTN (SEQ ID NO: 14, wherein the cleavage site is between the T and N)), Piotukh et ah, J Am Chem Soc 2011, 133: 17536-39 (phage display- based approach to identify mutations that changed the recognition specificity of Sortase A from LPXTG (SEQ ID NO: 2, wherein the
  • either substrate in cis is not required and thus enables full exploration of conditions for optimization of TPase ⁇ e.g., sortase) activity under various concentrations of both substrates, e.g., the Sortase A recognition sequence LPXTG (SEQ ID NO: 2, wherein the cleavage site is between the T and G) on the first substrate or other site-containing substrate, and/or the target recognition sequence H 2 N-GGGGG (SEQ ID NO: 15) on the second substrate.
  • the Sortase A recognition sequence LPXTG SEQ ID NO: 2 wherein the cleavage site is between the T and G
  • the target recognition sequence H 2 N-GGGGG SEQ ID NO: 15
  • the selection process is significantly faster than any in vivo approach because cell free expression, activity and selection can all be performed in the same day, allowing rapid iteration of selection and mutagenesis cycles.
  • the selection conditions described herein can be applied to generate TPases with either substantially improved catalytic efficiency on the native recognition sequence LPXTG (SEQ ID NO: 2, wherein the cleavage site is between the T and G) present on a first substrate or directed to a slightly different sequence such as LPXTA (SEQ ID NO: 16, wherein the cleavage site is between the T and A) present on the first substrate.
  • the in vitro selection technology described herein can be used to identify a target (second) substrate other than (G)5-K-Biotin (SEQ ID NO: 17) so that N-terminal labeling with alternate N-termini present on the target (second) substrate of interest can be performed.
  • the selection conditions described herein can be used to generate TPases that can bind a target polypeptide of interest containing the amino acid sequence DIQMT (SEQ ID NO:5) at the N-terminus ⁇ e.g., antibodies containing the amino acid sequence DIQMT (SEQ ID NO:5) at the N-terminus of their light chains).
  • nucleic acid or “polynucleotide” includes deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form such as, for example, DNA and RNA.
  • Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-0-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof and complementary sequences as well as the sequence explicitly indicated.
  • a "genetic element" as used herein includes a molecule or construct such as a DNA molecule, an RNA molecule, a partially or wholly artificial nucleic acid molecule consisting of exclusively synthetic or a mixture of naturally-occurring and synthetic bases, any one of the foregoing linked to a peptide or polypeptide, and any one of the foregoing linked to any other molecular group or construct.
  • the other molecular group or construct may include nucleic acids, polymeric substances, particularly beads such as polystyrene beads, and magnetic or paramagnetic substances such as magnetic or paramagnetic beads.
  • the nucleic acid portion of the genetic element may comprise regulatory sequences such as those sequences that are required for efficient expression of the gene product, including but not limited to, promoters, enhancers, translational initiation sequences, polyadenylation sequences, splice sites and the like.
  • native or endogenous in the context of a sequence such as an amino acid sequence includes an original, wildtype, and/or naturally-occurring sequence in which changes to the sequence itself are not introduced.
  • a native or endogenous sequence does not contain any modifications within the sequence, e.g., the sequence lacks any amino acid substitutions, insertions, or deletions, non-natural amino acids and/or chemical linkages.
  • protein include a polymer of amino acid residues linked together by peptide (amide) bonds.
  • the terms include a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long.
  • a protein, peptide, or polypeptide may include an individual protein or a collection of proteins.
  • One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification.
  • a protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex.
  • a protein, peptide, or polypeptide may also comprise a fragment of a naturally occurring protein or peptide.
  • a protein, peptide, or polypeptide may be naturally occurring, recombinant, synthetic, or any combination thereof.
  • conjugated includes an association of two molecules ⁇ e.g., two proteins, one protein and one label, one protein and one small molecule drug, etc.), with one another in a way that they are linked by a direct or indirect covalent or non-covalent interaction.
  • the association is covalent, and the entities are said to be “conjugated” to one another.
  • a protein is post-translationally conjugated to another molecule, for example, a second protein, a small molecule, a detectable label, a cytotoxin, or a binding agent, by forming a covalent bond between the protein and the other molecule after the protein has been formed.
  • a "variant" of a particular polynucleotide or polypeptide has one or more alterations ⁇ e.g., additions, substitutions, and/or deletions) with respect to a corresponding original, wildtype, and/or naturally occurring polynucleotide or polypeptide sequence.
  • a variant can be the same length as the original polynucleotide or polypeptide, or may be shorter or longer.
  • the sequence of a variant is typically at least about 70% identical to the sequence of the original polynucleotide or polypeptide over a region of at least about 50% of the original polynucleotide or polypeptide.
  • a variant is at least about 50%>, 60%>, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the original polynucleotide or polypeptide over a substantial portion of the length of the original polypeptide, e.g., a region at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% of the original polynucleotide or polypeptide.
  • a transpeptidase (TPase) variant has a sequence with one or more alterations ⁇ e.g., additions, substitutions, and/or deletions) in the domain implicated in the specificity of recognition by TPases ⁇ e.g., ⁇ 6/ ⁇ 7 loop for sortases).
  • a TPase variant cleaves a recognition sequence on a first substrate with a catalytic activity that is greater than the corresponding original or wildtype TPase, or cleaves a recognition sequence on a first substrate that is usually not cleaved by the corresponding original or wildtype TPase.
  • a TPase variant targets an acceptor sequence on a second substrate that is usually not recognized or targeted by the corresponding original or wildtype TPase.
  • label refers to a moiety that has at least one element, isotope, or functional group incorporated into the moiety which enables detection of the molecule, e.g., a protein, polypeptide, or peptide, or other entity, to which the label is attached.
  • Labels can be directly attached ⁇ i.e., via a bond) or attached by a tether or linker.
  • a label is attached to a protein of interest such as an antibody using the TPase variants identified by the methods of the present invention.
  • a label can fall into any one (or more) of five classes: (a) a label which contains isotopic moieties, which may be radioactive or heavy isotopes, including, but not limited to, 2 H, 3 H, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 67 Ga, 76 Br, 99m Tc (Tc-99m), m In, 123 I, 125 I, 131 I, 153 Gd, 169 Yb, and 186 Re; (b) a label which contains an immune moiety, which may be antibodies or antigens, which may be bound to enzymes ⁇ e.g., such as horseradish peroxidase); (c) a label which is a colored, luminescent, phosphorescent, or fluorescent moieties ⁇ e.g., such as the fluorescent label fluoresceinisothiocyanate (FITC); (d) a label which has one or more photo-affinity moieties; and
  • a label comprises a radioactive isotope, preferably an isotope which emits detectable particles, such as ⁇ particles.
  • the label comprises a fluorescent moiety.
  • the label is the fluorescent label FITC.
  • the label comprises a ligand moiety with one or more known binding partners.
  • the label comprises biotin.
  • the label is a fluorescent polypeptide (e.g., GFP or a derivative thereof such as enhanced GFP (EGFP)) or a luciferase (e.g., a firefly, Renilla, or Gaussia luciferase).
  • a label may react with a suitable substrate (e.g., a luciferin) to generate a detectable signal.
  • a suitable substrate e.g., a luciferin
  • fluorescent proteins include GFP and derivatives thereof, proteins comprising chromophores that emit light of different colors such as red, yellow, and cyan fluorescent proteins, etc.
  • Exemplary fluorescent proteins include, e.g., Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP, mTFPl, mUkGl, mAGl, AcGFPl, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mK02, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, niNeptune, T-Sapphire, mAmetrine, mKeima.
  • a label comprises a dark quencher, e.g., a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat.
  • an antibody includes a glycoprotein belonging to the immunoglobulin superfamily. With some exceptions, mammalian antibodies are typically made of basic structural units each with two large heavy chains and two small light chains. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, IgG, IgA, IgE, IgD, and IgM, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter. In some embodiments, an antibody is an IgG antibody, e.g., an antibody of the IgGl, 2, 3, or 4 human subclass.
  • linker refers to a chemical group or molecule covalently linked to a molecule, for example, a protein, and a chemical group or moiety.
  • the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two.
  • the linker is an amino acid or a plurality of amino acids (e.g., a peptide, polypeptide or protein).
  • the linker is an organic molecule, group, polymer (e.g., PEG), or chemical moiety.
  • k cat refers to the turnover rate of an enzyme, e.g., the number of substrate molecules that the respective enzyme converts to product per time unit. Generally, kcat designates the turnover of an enzyme working at maximum efficiency.
  • sortase refers to an enzyme able to carry out a transpeptidation reaction conjugating the C-terminus of a protein to the N-terminus of a protein via transamidation. Sortases are also referred to as transamidases, and typically exhibit both a protease and a transpeptidation activity.
  • sortase substrate refers to a molecule or entity that can be utilized in a sortase-mediated transpeptidation reaction.
  • a sortase utilizes two substrates, such as a first substrate comprising a C-terminal sortase recognition motif, and a second substrate comprising an N-terminal sortase recognition motif (N-terminal acceptor sequence), and the transpeptidation reaction results in a conjugation of both substrates via a covalent bond.
  • Sortase-mediated conjugation of the substrates in such cases results in the formation of an intramolecular bond, e.g., a circularization of a single amino acid sequence, or, if multiple polypeptides of a protein complex are involved, the formation of an intra- complex bond.
  • the C-terminal and N-terminal recognition motifs are comprised in different amino acid sequences, for example, in separate proteins, peptides, and the like.
  • a second substrate for a wildtype sortase may comprise an LPXTG motif (SEQ ID NO:2), the N-terminus of which is conjugated to any agent, e.g., a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label.
  • a second substrate for a sortase variant of the present disclosure may comprise a recognition motif that is not an LPXTG motif (SEQ ID NO:2), the N-terminus of which is conjugated to any agent, e.g., a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label.
  • the second substrate may comprise a GGG motif or a variant thereof, the C-terminus of which is conjugated to any agent, e.g., a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label.
  • agent e.g., a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label.
  • cancer includes any member of a class of diseases characterized by the uncontrolled growth of aberrant cells. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre- and post-metastatic cancers.
  • cancers include, but are not limited to, breast cancer; lung cancer (e.g., non-small cell lung cancer); digestive and gastrointestinal cancers such as colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, and stomach (gastric) cancer; esophageal cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; ovarian cancer; renal cancer (e.g., renal cell carcinoma); cancer of the central nervous system; skin cancer; lymphomas; choriocarcinomas; head and neck cancers; osteogenic sarcomas; and blood cancers.
  • lung cancer e.g., non-small cell lung cancer
  • digestive and gastrointestinal cancers such as colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, and stomach (gas
  • subject typically includes humans, but can also include other animals such as, e.g., other primates, rodents, canines, felines, equines, o vines, porcines, and the like.
  • the present invention provides selection and screening methods for identifying transpeptidase (TPase) variants that have a modified (e.g., increased or decreased) catalytic activity for a native recognition site in a peptide, polypeptide, or protein of interest compared to a corresponding wildtype TPase or that recognize new sequence motifs present in the peptide, polypeptide, or protein of interest that differ from the native recognition site.
  • the new sequence motif may also differ from the native recognition sequence that is present in the original sequence (or parental sequence) of the peptide, polypeptide, or protein of interest.
  • the present invention provides isolated TPase variants, nucleic acids, and vectors, as well as conjugates produced by the use of the TPase variants and pharmaceutical compositions and kits comprising such conjugates. Methods for treating cancer in a subject in need thereof using the conjugates described herein are also provided.
  • the conjugates of the invention comprising a label such as a detectable label are useful for imaging or localizing a cell, tissue, organ, or tumor, e.g., in a subject.
  • the present invention provides selection and screening methods for the directed evolution of transpeptidases (TPases) to generate mutant TPases that have modified (e.g., improved or diminished) catalytic properties over the wildtype (wt) sequence, that can bind and cleave new sequences such as new recognition sequences on a first substrate (e.g., at the C-terminus), and/or that can target alternative N-terminal acceptor sequences on a target (second) substrate to which the first substrate is conjugated by the mutant TPases.
  • TPases transpeptidases
  • the methods of the invention are based on in vitro compartmentalization (IVC) and utilize a cell-free transcription and translation system within microcompartments such as emulsions (e.g., droplets), microplate wells, and combinations thereof.
  • IVC in vitro compartmentalization
  • each genetic element e.g., each DNA sequence encoding a test TPase in a library of genetic elements is linked (e.g., covalently attached such as tethered) to a TPase recognition sequence of interest and incubated with a second substrate containing a target acceptor sequence for the gene product encoded by the genetic element (e.g., the test TPase).
  • each linked genetic element is compartmentalized with the second substrate within the same microcompartment.
  • the gene product encoded by the genetic element is also compartmentalized within the same microcompartment such that the encoded gene product cannot interact with genetic elements in any other microcompartments.
  • the microcompartments used in the methods of the present invention are capable of being produced in very large numbers, and can thereby compartmentalize a library of genetic elements which encodes a repertoire of gene products (e.g., library of DNA sequences encoding a plurality of test TPases).
  • the genetic elements in the library are produced by limiting dilution followed by polymerase chain reaction (PCR) such as, e.g., by single molecule PCR, to generate individual, isolated genetic elements in distinct microcompartments such as the wells of a microtiter plate or emulsions. These isolated genetic elements are then used as templates for the generation of the endoded protein. See, e.g., Stapleton and Swartz, PLOSOne 5(12) 2010.
  • PCR polymerase chain reaction
  • each genetic element in a library of genetic elements encodes a different test variant TPase that is linked to a recognition sequence that is not a substrate for the corresponding wildtype TPase.
  • each of these linked genetic elements is compartmentalized with a second substrate containing a target acceptor sequence for the test variant TPase that is also recognized by the corresponding wildtype TPase, e.g., a sequence with one or more glycine residues.
  • each genetic element in a library of genetic elements encodes a different test variant TPase that is linked to a recognition sequence that is also a substrate for the corresponding wildtype TPase.
  • each of these linked genetic elements is compartmentalized with a different second substrate containing a target acceptor sequence for the test variant TPase that is not recognized by the corresponding wildtype TPase, e.g., a sequence present at the N-terminus of a peptide, polypeptide, or protein of interest that is targeted by the test variant TPase and conjugated to the substrate containing the recognition sequence for the test variant TPase.
  • a target acceptor sequence for the test variant TPase that is not recognized by the corresponding wildtype TPase, e.g., a sequence present at the N-terminus of a peptide, polypeptide, or protein of interest that is targeted by the test variant TPase and conjugated to the substrate containing the recognition sequence for the test variant TPase.
  • the N-terminus of a therapeutic antibody of interest which is not recognized by a wildtype TPase, is recognized by the corresponding test variant TPase selected by
  • each genetic element in a library of genetic elements encodes a different test variant TPase that is linked to a recognition sequence that is also a substrate for the corresponding wildtype TPase, and each of these linked genetic elements is compartmentalized with a second substrate containing a target acceptor sequence for the test variant TPase that is also recognized by the corresponding wildtype TPase, e.g., a sequence with one or more glycine residues.
  • each genetic element in a library of genetic elements encodes a different test variant TPase that is linked to a recognition sequence that is not a substrate for the corresponding wildtype TPase, and each of these linked genetic elements is compartmentalized with a different second substrate containing a target acceptor sequence for the test variant TPase that is not recognized by the corresponding wildtype TPase.
  • microcompartment includes an artificial compartment whose delimiting borders restrict the exchange of the components compartmentalized therein such that the components do not diffuse between microcompartments.
  • microcompartments include, but are not limited to, emulsions ⁇ e.g., droplets), wells in a microplate, and combinations thereof.
  • emulsions ⁇ e.g., droplets
  • microplate wells in a microplate
  • microencapsulation procedures are available ⁇ see, Benita, S., Ed. (1996). Microencapsulation: methods and industrial applications. Drugs and pharmaceutical sciences. Edited by Swarbrick, J. New York: Marcel Dekker) and may be used to create the microcompartments used in accordance with the invention. Examples of microencapsulation methods are described in, e.g., Finch (1993) Spec. Publ.-R. Soc. Chem. 138, 35.
  • Non-limiting examples of microencapsulation methods include membrane enveloped aqueous vesicles such as lipid vesicles (liposomes) (New, R. R. C, Ed. (1990). Liposomes: a practical approach. The practical approach series. Edited by Rickwood, D. & Hames, B. D. Oxford: Oxford University Press) and non-ionic surfactant vesicles (van Hal et al. (1996) Nonionic surfactant vesicles containing estradiol for topical application. In Microencapsulation: methods and industrial applications (Benita, S., ed.), pp. 329-347. Marcel Dekker, New York).
  • lipid molecules are usually phospholipids, but sterols such as cholesterol may also be incorporated into the membranes (New, 1990).
  • enzyme-catalyzed biochemical reactions can be performed within liposomes (Chakrabarti et al. (1994) J Mol Evol 39(6), 555-9; Oberholzer et al. (1995) Chemistry and Biology 2, 677-682; Oberholzer et al.
  • aqueous phase is outside the vesicles and is therefore non-compartmentalized. In certain instances, this continuous, aqueous phase is removed or the biological systems in it inhibited or destroyed (e.g., by digestion of nucleic acids with DNase or RNase) such that the reactions are limited to the microcompartments (Luisi et al. (1987) Methods Enzymol 136(188), 188-216).
  • Enzyme-catalyzed biochemical reactions have also been demonstrated in microcompartments generated by a variety of other methods. Many enzymes are active in reverse micellar solutions (Bru et al. (1991) Eur J Biochem 199(1), 95-103; Bru et al. (1993) Biochem Mol Biol Int 31(4), 685-92; Creagh et al. (1993) Enzyme Microb Technol 15(5), 383-92; Haber et al. (1993) Eur J Biochem 217(2), 567-73; Kumar et al. (1989) Biochim Biophys Acta 996(1-2), 1-6; Luisi et al.
  • Microcompartments can also be generated by interfacial polymerization and interfacial complexation (Whateley (1996) Microcapsules: preparation by interfacial polymerisation and interfacial complexation and their applications. In Microencapsulation: methods and industrial applications (Benita, S., ed.), pp. 349-375. Marcel Dekker, New York). Microcompartments of this sort can have rigid nonpermeable membranes or semipermeable membranes.
  • Non-membranous microencapsulation systems based on phase partitioning of an aqueous environment in a colloidal system, such as an emulsion, may also be used.
  • the microcompartments of the present invention are formed from emulsions.
  • Emulsions are generally heterogeneous systems of two immiscible liquid phases with one of the phases dispersed in the other as droplets of microscopic or colloidal size ⁇ see, Becher (1957) Emulsions: theory and practice. Reinhold, New York; Sherman (1968) Emulsion science. Academic Press, London; Lissant, ed. (1974) Emulsions and emulsion technology.
  • Emulsions may be produced from any suitable combination of immiscible liquids.
  • the emulsion has water (containing the biochemical components) as the phase present in the form of finely divided droplets ⁇ e.g., the disperse, internal or discontinuous phase) and a hydrophobic, immiscible liquid (an oil) as the matrix in which these droplets are suspended ⁇ e.g., the nondisperse, continuous or external phase).
  • water-in-oil W/O
  • This has the advantage that the entire aqueous phase containing the biochemical components is compartmentalized in discrete droplets ⁇ e.g., the internal phase).
  • the external phase being a hydrophobic oil, generally contains none of the biochemical components and hence is inert.
  • the emulsion may be stabilized by the addition of one or more surface-active agents ⁇ e.g., surfactants).
  • surface-active agents such as surfactants are termed emulsifying agents and act at the water/oil interface to prevent (or at least delay) separation of the phases.
  • Many oils and emulsifiers are suitable for use to generate water-in-oil emulsions; an exemplary compilation lists over 16,000 surfactants, many of which are used as emulsifying agents (Ash and Ash (1993) Handbook of industrial surfactants. Gower, Aldershot).
  • Suitable oils include light white mineral oil and non-ionic surfactants (Schick (1966) Nonionic surfactants. Marcel Dekker, New York) such as sorbitan monooleate (SpanTM 80) and polyoxyethylenesorbitan monooleate (TweenTM 80).
  • anionic surfactants may also be beneficial.
  • Suitable surfactants include, but are not limited to, sodium cholate and/or sodium taurocholate. Particularly preferred is sodium deoxycholate, preferably at a concentration of 0.5% w/v, or below. Inclusion of such surfactants can in some cases increase the expression of the genetic elements and/or the activity of the gene products. Addition of some anionic surfactants to a non-emulsified reaction mixture completely abolishes translation. During emulsification, however, the surfactant is transferred from the aqueous phase into the interface and activity is restored. Addition of an anionic surfactant to the mixtures to be emulsified ensures that reactions proceed only after compartmentalization.
  • Aqueous microcompartments formed in water-in-oil emulsions are generally stable with little if any exchange of genetic elements and/or gene products between microcompartments. Biochemical reactions can also proceed in emulsion microcompartments. Complicated biochemical processes such as gene transcription and translation are also active in emulsion microcompartments.
  • the preferred microcompartment size will vary depending upon the precise requirements of any individual selection process that is to be performed according to the present invention. In all cases, there will be an optimal balance between gene library size, the required enrichment, and the required concentration of components in the individual microcompartments to achieve efficient expression and reactivity of the gene products.
  • the processes of in vitro transcription and translation can occur within each individual microcompartment.
  • the mean volume of the microcompartments is less than about 5.2 x 10 "16 m 3 , (corresponding to a spherical microcompartment of diameter less than about 10 ⁇ ), less than about 6.5 x 10 "17 m 3 (less than about 5 ⁇ diameter), about 4.2 x 10 " 18 m 3 (about 2 ⁇ diameter) or about 9 x 10 " 18 m 3 (about 2.6 ⁇ diameter).
  • the microcompartment size is preferably sufficiently large to accommodate all of the required components of the biochemical reactions that are needed to occur within the microcompartment.
  • the microcompartment size is preferably sufficiently large to accommodate all of the required components of the biochemical reactions that are needed to occur within the microcompartment.
  • the microcompartment size is preferably sufficiently large to accommodate all of the required components of the biochemical reactions that are needed to occur within the microcompartment.
  • the microcompartment size is preferably sufficiently large to accommodate
  • volume is between about 5.2 x 10 " m and about 5.2 x 10 " m , corresponding to a sphere of diameter between about 0.1 ⁇ and about 10 ⁇ , more preferably of between about 5.2 x 10 "
  • the size of emulsion microcompartments may be varied by tailoring the emulsion conditions used to form the emulsion according to the requirements of the selection system. The larger the microcompartment size, the larger is the volume that will be required to encapsulate a given genetic element library, since the limiting factor will be the size of the microcompartment and thus the number of microcompartments possible per unit volume.
  • IVC in vitro compartmentalization
  • the transpeptidase is a transamidase.
  • the transamidase is a sortase. Enzymes identified as "sortases” from Gram- positive bacteria cleave and translocate proteins to proteoglycan moieties in intact cell walls. Sortase A (Srt A) and Sortase B (Srt B) are among the sortases that have been isolated from Staphylococcus aureus.
  • the TPase used in accordance with the present invention comprises a Sortase A and/or a Sortase B, e.g., from S. aureus, as well as mutants and variants thereof, e.g., evolved TPases with the ability to cleave, bind, recognize, and/or target new recognition sequence motifs and/or acceptor sites on substrates of interest.
  • Sortases have been classified into 4 classes, designated A, B, C, and D, based on sequence alignment and phylogenetic analysis of 61 sortases from Gram-positive bacterial genomes (Dramsi et ah, Res. Microbiol, 156(3):289-97, (2005)). These classes correspond to the following subfamilies, into which sortases have also been classified (Comfort et ah, Infect Immun., 72(5):2710-22, (2004)): Class A (Subfamily 1), Class B (Subfamily 2), Class C (Subfamily 3), Class D (Subfamilies 4 and 5).
  • amino acid sequences of Srt A and Srt B and the nucleotide sequences that encode them are known to those of skill in the art.
  • the amino acid sequences of S. aureus SrtA and SrtB are homologous, sharing, for example, 22% sequence identity and 37% sequence similarity.
  • the sortase is from S. pyogenes, A. naeslundii, S. mutans, E.faecalis, or B. subtilis.
  • S. aureus Sortase A polypeptide sequence is set forth in, e.g., Uniprot Accession No.Q9S446 or NCBI Ref. Sequence No. WP 000759361. Sortase A is also referred to as IUBMB enzyme EC 3.4.22.70.
  • transpeptidases having novel or altered properties for cleaving a recognition sequence on a first substrate that is the same or different from the native recognition sequence and/or targeting an N-terminal acceptor sequence motif on a second substrate that is the same or different from the native motif can be identified by performing directed evolution on randomized protein libraries.
  • structural information and predictive computational tools can be used in guiding the design of randomized libraries.
  • web servers such as ConSurf-HSSP and SCHEMA allow the prediction of sites to target for producing functional variants, and algorithms such as GLUE, PEDEL, and DRIVeR are useful for estimating library completeness and diversity.
  • Any technique known in the art for the construction of randomized protein libraries can be used in the methods of the present invention.
  • Non-limiting examples include oligonucleotide-directed randomization, error-prone PCR, in vitro recombination, and combinations thereof.
  • NNN A/C/G/T
  • B C/G/T
  • K G/T
  • S G/C
  • the crystal/NMR structures of a number of TPase variants in the presence and absence of their substrates can be used to design mutant TPase libraries using oligonucleotide-directed randomization.
  • 6XNNK libraries covering residues in contact with the substrate as well as C-terminal to the cleavage site of TPases such as sortases can be designed based on the structural information for the ⁇ 6/ ⁇ 7 loop, which has been implicated in the specificity of recognition by TPases.
  • Error-prone PCR is commonly used to generate diversity at any position in a target gene, and the original protocol (Cadwell et al, PCR Methods Appl (1992), 2:28-33) has been modified extensively to afford control over the nucleotide substitution rate.
  • polymerases such as Mutazyme ® (Strategene), which in combination with Taq DNA polymerase displays a near-uniform mutational spectrum, can be used in error-prone PCR methods.
  • site-directed mutants of the Pyrococcus furiosus DNA polymerase can be used.
  • Non-limiting examples of methods for in vitro recombination include DNA shuffling and the staggered extension process (StEP PCR).
  • Other in vitro recombination methods include, without limitation, degenerate homoduplex recombination (Coco et al, Nat Biotechnol (2002), 20: 1246-50), synthetic shuffling (Ness et al, Nat Biotechnol (2002), 20: 1251-5), and assembly of designed oligonucleotides (Zha et al, ChemBioChem (2003), 4:34-9).
  • Additional in vitro recombination methods include, without limitation, homo logy- independent recombination protocols such as incremental truncation (Ostermeier et al, Nat Biotechnol (1999), 17: 1205-9; Lutz et al, Nucleic Acids Res (2001), 29:el6), sequence homology-independent protein recombination (Sieber et al, Nat Biotechnol (2001), 19:456- 60), SCRATCHY (Lutz et al, Proc Natl Acad Sci USA (2001), 98: 11248-53).
  • homo logy- independent recombination protocols such as incremental truncation (Ostermeier et al, Nat Biotechnol (1999), 17: 1205-9; Lutz et al, Nucleic Acids Res (2001), 29:el6), sequence homology-independent protein recombination (Sieber et al, Nat Biotechnol (2001), 19:456- 60),
  • a desired recognition motif may be longer or shorter than the corresponding wildtype recognition motif, may comprise one or more amino acid substitutions, insertions, or deletions as compared to the corresponding wildtype transpeptidase recognition motif, or may be designed de novo, e.g., not based on any naturally occurring transpeptidase recognition motif.
  • the TPase variants exhibit a different substrate preference with respect to the corresponding wildtype TPase.
  • the variant may recognize a modified recognition motif.
  • the variant binds to a specific substrate or recognition motif with higher or lower affinity and/or higher or lower specificity compared to the wildtype TPase.
  • the methods provided herein can be used to generate a TPase mutant that recognizes a recognition sequence that the wildtype TPase does not recognize or bind.
  • the TPase variants generated according to the selection and screening methods of the present disclosure can have improved or increased catalytic activity compared to the corresponding wildtype (wt) TPase (e.g., wt S. aureus Sortase A or SEQ ID NO: 1) for a specific substrate, e.g., a specific recognition sequence.
  • wt wildtype
  • TPase e.g., wt S. aureus Sortase A or SEQ ID NO: 1
  • the increased catalytic activity may be an at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold or greater increase in catalytic activity.
  • the TPase variant exhibits enhanced reaction kinetics, for example, in that it catalyzes a transpeptidation reaction at a greater speed or turnover rate than the corresponding wildtype TPase.
  • the TPase variant exhibits a modified substrate preference, for example, in that it utilizes a different substrate ⁇ e.g., a polypeptide comprising an altered TPase recognition motif) or binds a given substrate with higher or lower affinity, or with higher or lower specificity than the wildtype TPase.
  • the TPase variant recognizes a recognition motif that the wildtype TPase does not recognize or bind.
  • the TPase variant provided herein exhibits enhanced reaction kinetics, for example, in that it can achieve a greater maximum turnover per time unit (k cat ) or a greater turnover per time at physiological conditions, compared to the corresponding wildtype (wt) TPase ⁇ e.g., wt S. aureus Sortase A or SEQ ID NO: 1) for a specific substrate.
  • k cat maximum turnover per time unit
  • wt S. aureus Sortase A or SEQ ID NO: 1 for a specific substrate.
  • the TPase variant exhibits a k cat that is at least 1.5-fold, at least 2-fold, at least 2.5 -fold, at least 3 -fold, at least 3.5 -fold, at least 4-fold, at least 5 -fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or at least 100-fold greater than the k cat of the corresponding wildtype TPase.
  • the TPase variant provided herein exhibits enhanced reaction specificities, e.g., in that it can bind a substrate with higher affinity or with higher selectivity, or in that it binds a substrate that is not bound or not efficiently bound by the respective wildtype (wt) TPase ⁇ e.g., wt S. aureus Sortase A or SEQ ID NO: 1).
  • some TPase variants provided herein exhibit a K m for a substrate bound by a corresponding wildtype TPase that is at least 2-fold less, at least 3-fold less, at least 4-fold less, at least 5- fold less, at least 6-fold less, at least 7-fold less, at least 8-fold less, at least 9-fold less, at least 10-fold less, at least 15-fold less, at least 20-fold less, at least 25-fold less, at least 50- fold less, at least 60-fold less, at least 70-fold less, at least 80-fold less, at least 90-fold less, or at least 100-fold less than the K m of the corresponding wildtype TPase for that substrate.
  • TPase variants provided herein exhibit a K m for a substrate comprising a C-terminal recognition motif that is not bound by a wildtype TPase that is at least 2-fold less, at least 3-fold less, at least 4-fold less, at least 5-fold less, at least 6-fold less, at least 7-fold less, at least 8-fold less, at least 9-fold less, at least 10-fold less than the K m of the corresponding wildtype TPase for a substrate comprising a C-terminal recognition sequence of LPXTG (SEQ ID NO:2).
  • the TPase variants provided herein bind to a first substrate (e.g., a substrate with a C-terminal recognition motif) with a decreased K m while exhibiting no or only a slight decrease in the K m for the second substrate (e.g. , a substrate with an N- terminal acceptor sequence).
  • a first substrate e.g., a substrate with a C-terminal recognition motif
  • the second substrate e.g. , a substrate with an N- terminal acceptor sequence
  • some TPase variants exhibit a K m for a substrate comprising a C-terminal recognition sequence (e.g.
  • aureus Sortase A or SEQ ID NO: 1) for that substrate and also exhibit a K m for a substrate comprising an N-terminal acceptor sequence (e.g., GGG, an oligoglycine and the like) that is not more than 2-fold greater, not more than 5 -fold greater, not more than 10-fold greater, or not more than 20-fold greater than the K m of the corresponding wildtype TPase for that substrate.
  • N-terminal acceptor sequence e.g., GGG, an oligoglycine and the like
  • the TPase variants provided herein exhibit a ratio of k cat /K m for a substrate that can bind to the corresponding wildtype TPase that is least 2-fold greater, at least 5-fold greater, at least 10-fold greater, at least 20-fold greater, at least 50-fold greater, at least 100-fold greater, or at least 120-fold greater than the k cat /K m ratio of the corresponding wildtype TPase.
  • the TPase variants generated according to the selection and screening methods of the present disclosure can have decreased or attentuated catalytic activity compared to the corresponding wildtype (wt) TPase (e.g., wt S. aureus Sortase A or SEQ ID NO: 1) for a specific substrate, e.g., a specific recognition sequence.
  • wt wildtype
  • TPase e.g., wt S. aureus Sortase A or SEQ ID NO: 1
  • the attentuated catalytic activity may be an at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3 -fold, at least 3.5 -fold, at least 4-fold, at least 5 -fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold less in catalytic activity compared to the corresponding wildtype TPase.
  • the TPase variant exhibits diminished reaction kinetics, for example, in that it catalyzes a transpeptidation reaction at a slower speed or turnover rate than the corresponding wildtype (wt) TPase (e.g., wt S. aureus Sortase A or SEQ ID NO: 1) for a specific substrate, e.g., a specific recognition sequence.
  • wt wildtype
  • TPase e.g., wt S. aureus Sortase A or SEQ ID NO: 1
  • the TPase variant exhibits a modified substrate preference, for example, in that it utilizes a different substrate ⁇ e.g., a polypeptide comprising an altered TPase recognition motif) or binds a given substrate with higher or lower affinity, or with higher or lower specificity than the wildtype TPase.
  • the TPase variant recognizes a recognition motif that the wildtype TPase does not recognize or bind.
  • the TPase variant provided herein exhibits diminished reaction kinetics, for example, in that it can achieve a lower maximum turnover per time unit (kcat) or a lower turnover per time at physiological conditions, compared to the corresponding wildtype (wt) TPase ⁇ e.g., wt S. aureus Sortase A or SEQ ID NO: 1) for a specific substrate, e.g., a specific recognition sequence.
  • kcat maximum turnover per time unit
  • wt S. aureus Sortase A or SEQ ID NO: 1 for a specific substrate, e.g., a specific recognition sequence.
  • the TPase variant exhibits a k cat that is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or at least 100-fold less than the k cat of the corresponding wildtype TPase.
  • the TPase variants provided herein exhibit a ratio of k cat /K m for a substrate that can bind to the corresponding wildtype TPase that is least 2-fold lower, at least 5-fold lower, at least 10-fold lower, at least 20-fold lower, at least 50-fold lower, at least 100-fold lower, or at least 120-fold lower than the k cat /K m ratio of the corresponding wildtype TPase.
  • the transpeptidase reaction described herein is a reversible reaction.
  • a TPase variant of the invention has diminished activity and optionally, catalyzes an irreversible ligation (labeling) reaction.
  • the TPase variant comprises an amino acid sequence that is homologous to the amino acid sequence of wildtype TPase ⁇ e.g., to the amino acid sequence of S. aureus Sortase A set forth as SEQ ID NO: 1), or a fragment thereof.
  • the amino acid sequence of the TPase variant comprises one or more mutations as compared to the sequence of the wildtype TPase (SEQ ID NO: 1).
  • the amino acid sequence of the TPase variant identified according to the methods disclosed herein may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more mutations.
  • the sequence of the TPase variant is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to a wildtype TPase (SEQ ID NO: 1).
  • the TPase variant can have one or more amino acid substitutions at any position of the polypeptide.
  • the TPase variant can carry an amino acid substitution at position 59, 86, 94, 98, 104, 105, 106, 108, 118, 122, 124, 127, 134, 154, 160, 165, 172, 173, 174, 177, 182, 184, 190, 192 and/or 196 of the wildtype TPase sequence (SEQ ID NO: l).
  • the TPase variant includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions.
  • One or more amino acid substitutions can be located in the catalytic domain, the sortase domain, the calcium binding domain, or any combination thereof.
  • the TPase variant is a calcium-independent variant.
  • the TPase variant identified using the methods provided herein is a cofactor- independent.
  • TPases are identified from randomized protein libraries in accordance with the selection and screening methods of the present invention ⁇ e.g., using in vitro compartmentalization) that specifically cleave a recognition sequence on a first substrate that is different from the recognition sequence for the corresponding wildtype TPase.
  • Sortase A variants can be identified that recognize and cleave a new sequence motif at the C- or N-terminus of an antibody light or heavy chain instead of its native recognition sequence LPXTG (SEQ ID NO:2).
  • the evolved Sortase A variants selected for and identified by the methods described herein are able to recognize the amino acid sequence motif LSPGK (SEQ ID NO:3) that is present, e.g., at the C-terminus of the heavy chains of therapeutic antibodies such as adalimumab (Humira ® ), bevacizumab (Avastin ® ), alemtuxumab (Campath ® ), trastuzumab (Herceptin ® ), efalizumab (Raptiva ® ), omalizumab (Xolair ® ), daclizumab (Zenapax ® ), cetuximab (Erbitux ® ), rituximab (Rituxan ® ), basiliximab (Simulect ® ), panitumumab (Vectibix ® ), and muromonab-CD3 (Orthoclone ® ).
  • the evolved TPase further recognizes one, two, three, four, five, six, or more amino acid residues on either or both sides of the new recognition sequence.
  • the new recognition sequence motif recognized by the evolved TPase is present within the antibody light or heavy chain polypeptide sequence and not at the N- or C-terminus thereof.
  • TPases are identified from randomized protein libraries in accordance with the selection and screening methods of the present invention (e.g., using in vitro compartmentalization) that specifically target an acceptor sequence on a second substrate that is different from the acceptor sequence motif for the corresponding wildtype TPase.
  • the evolved TPases cleave a wildtype recognition sequence present on a first substrate (e.g., the Sortase A native recognition sequence LPXTG (SEQ ID NO:2)) and catalyze the conjugation of the first substrate to a second substrate by recognizing an acceptor sequence on the second substrate that is different from the wildtype acceptor sequence.
  • Sortase A variants can be identified that recognize a new sequence motif at the N-terminus of an antibody light or heavy chain instead of the wildtype Sortase A acceptor pentaglycine sequence.
  • the evolved Sortase A variants selected for and identified by the present methods are able to recognize the acceptor sequence motif DIQMT that is present, e.g., at the N-terminus of the light chains of therapeutic antibodies such as, e.g., adalimumab (Humira ® ), bevacizumab (Avastin ® ), alemtuxumab (Campath ® ), certolizumab pegol (Cimzia ® ), trastuzumab (Herceptin ® ), efalizumab (Raptiva ® ), eculizumab (Soliris ® ), palivizumab (Synagis ® ), natalizumab (Rysabri ®
  • the evolved Sortase A variants selected for and identified by the methods described herein are able to recognize the acceptor sequence motif DIQLT that is present, e.g., at the N- terminus of the light chains of therapeutic antibodies such as Lucentis and Xolair.
  • the evolved Sortase A variants are able to recognize the acceptor sequence motif DIQXT, wherein X is any amino acid, and wherein X is preferably M or L.
  • the evolved Sortase A variants are able to recognize one or more acceptor sequence motifs such as, e.g., DIQMT (SEQ ID NO:5), DIQLT (SEQ ID NO:6), DILLT (SEQ ID NO: 18), QIVST (SEQ ID NO: 19), QIVLS (SEQ ID NO:20), QIVLT (SEQ ID NO:21), DVVMT (SEQ ID NO:22), DVQLT (SEQ ID NO:23), and combinations thereof.
  • DIQMT SEQ ID NO:5
  • DIQLT SEQ ID NO:6
  • DILLT SEQ ID NO: 18
  • QIVST SEQ ID NO: 19
  • QIVLS SEQ ID NO:20
  • QIVLT SEQ ID NO:21
  • DVVMT SEQ ID NO:22
  • DVQLT SEQ ID NO:23
  • the evolved Sortase A variants selected for and identified by the methods described herein are able to recognize the acceptor sequence motif EVQLV (SEQ ID NO:8), QVQLV (SEQ ID NO:9), or QVQLQ (SEQ ID NO: 10) that is present, e.g., at the N-terminus of the heavy chains of therapeutic antibodies.
  • the evolved Sortase A variants are able to recognize the acceptor sequence motif X 1 VQLX 2 , wherein Xi and X 2 are independently any amino acid (SEQ ID NO: 7), and wherein Xi is preferably E or Q and X 2 is preferably V or Q.
  • the present invention provides protein conjugates that are created by the evolved TPase variants described herein. These TPase variants are particularly useful for conjugating an original sequence from a peptide, polypeptide, or protein of interest ⁇ e.g., any antibody light or heavy chain sequence) with any other molecule of interest (other peptides, polypeptides, proteins, labels, or small molecules such as anticancer drugs).
  • the protein conjugate comprises an antibody-drug conjugate (ADC), wherein a therapeutic antibody is conjugated to a cytotoxic small molecule drug by an evolved TPase variant identified using the methods of the present invention.
  • ADC antibody-drug conjugate
  • FIG. 1 illustrates one embodiment for site-specific labeling of antibodies with a small molecule of interest.
  • a monoclonal antibody having a sequence motif at the C-terminus of one of its light or heavy chains is recognized by a TPase variant, which then catalyzes the site-specific conjugation of the antibody to a linker attached to an anticancer drug such as a cytotoxin, wherein the linker contains the native target N-terminal sequence that is recognized by the wildtype TPase ⁇ e.g., a pentaglycine motif located at the N-terminus of the linker for evolved sortase variants).
  • the native N-terminal acceptor sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous glycine residues.
  • the N-terminal acceptor sequence may include an oligoglycine sequence, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous glycine residues.
  • the peptide, polypeptide, or protein of interest corresponds to the first substrate and contains a recognition sequence for the evolved TPase variant that differs from the recognition sequence for the corresponding wildtype TPase.
  • the second substrate may contain a peptide linker at its N-terminus comprising a sequence such as a pentaglycine that accepts the recognition sequence on the first substrate and is conjugated thereto by the TPase variant.
  • the linker can comprise, e.g., from about 5 to about 50 amino acids, from about 5 to about 25 amino acids, from about 5 to about 20 amino acids, from about 5 to about 15 amino acids, from about 5 to about 10 amino acids, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • the peptide, polypeptide, or protein of interest corresponds to the second substrate and contains an N-terminal acceptor sequence for the evolved TPase variant that differs from the N-terminal acceptor sequence for the corresponding wildtype TPase.
  • the first substrate may contain a peptide linker at its C-terminus comprising a recognition sequence that can be recognized by both the wildtype and evolved TPase variant (e.g., a peptide linker comprising the Sortase A native recognition sequence LPXTG (SEQ ID NO: 2)).
  • the evolved TPase variant is thus able to catalyze the site-specific conjugation of the peptide linker present on the first substrate to the N-terminal acceptor sequence present on the second substrate.
  • the peptide linker can comprise, e.g., from about 5 to about 50 amino acids, from about 5 to about 25 amino acids, from about 5 to about 20 amino acids, from about 5 to about 15 amino acids, from about 5 to about 10 amino acids, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • any antibody or fragment thereof is suitable for use in the protein conjugates of the present invention.
  • commercial antibodies include adalimumab (Humira ® ), bevacizumab (Avastin ® ), alemtuxumab (Campath ® ), certolizumab pegol (Cimzia ® ), trastuzumab (Herceptin ® ), efalizumab (Raptiva ® ), eculizumab (Soliris ® ), palivizumab (Synagis ® ), natalizumab (Rysabri ® ), daclizumab (Zenapax ® ), gemtuxumab (Mylotarg ® ) omalizumab (Xolair ® ), daclizumab (Zenapax ® ), cetuximab (
  • the small molecule comprises an anticancer drug.
  • anticancer drugs include chemotherapeutic agents (i.e., cytotoxins), tyrosine kinase inhibitors, anti-proliferative agents, hormonal therapeutic agents, radiotherapeutic agents, vaccines, and/or any other compound with the ability to reduce or abrogate the uncontrolled growth of aberrant cells such as cancerous cells.
  • the label comprises any of the labels described herein such as a detectable label.
  • Non-limiting examples of chemotherapeutic agents include platinum-based drugs (e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin, satraplatin, etc.), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, etc.), anti-metabolites (e.g., 5- fluorouracil, azathioprine, 6-mercaptopurine, methotrexate, leucovorin, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine (Gemzar ® ), pemetrexed (ALIMTA ® ), raltitrexed, etc.), plant alkaloids (e.g., vincristine, vinblastine
  • tyrosine kinase inhibitors include, but are not limited to, gefitinib (Iressa ® ), sunitinib (Sutent ® ), erlotinib (Tarceva ® ), lapatinib (GW-572016; Tykerb ® ), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006; Nexavar ® ), imatinib mesylate (Gleevec ® ), leflunomide (SU101), vandetanib (ZACTIMATM; ZD6474), and combinations thereof.
  • Exemplary anti-pro liferative agents include mTOR inhibitors such as sirolimus (rapamycin), temsirolimus (CCI-779), and everolimus (RAD001); Akt inhibitors such as lL6-hydroxymethyl-chiro-inositol-2-(R)-2-0-methyl-3-0-octadecyl-5/7-glycerocarbonate, 9- methoxy-2-methylellipticinium acetate, l,3-dihydro-l-(l-((4-(6-phenyl-lH-imidazo[4,5- g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one, 10-(4'-(N- diethylamino)butyl)-2-chlorophenoxazine, 3-formylchromone thiosemicarbazone (Cu(II)Cl 2 complex), API-2, a 15
  • hormonal therapeutic agents include, without limitation, aromatase inhibitors (e.g., aminoglutethimide, anastrozole (Arimidex ® ), letrozole (Femara ® ), vorozole, exemestane (Aromasin ® ), 4-androstene-3,6,17-trione (6-OXO), l,4,6-androstatrien-3,17- dione (ATD), formestane (Lentaron ® ), etc.), selective estrogen receptor modulators (e.g., apeledoxifene, clomifene, fulvestrant, lasofoxifene, raloxifene, tamoxifen, toremifene, etc.), steroids (e.g., dexamethasone), finasteride, and gonadotropin-releasing hormone agonists (GnPvH) such as goserelin, pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof
  • Non-limiting examples of cancer vaccines include ANYARA from Active Biotech, DCVax-LB from Northwest Biotherapeutics, EP-2101 from IDM Pharma, GV1001 from Pharmexa, IO-2055 from Idera Pharmaceuticals, INGN 225 from Introgen Therapeutics and Stimuvax from Biomira/Merck.
  • radiotherapeutic agents include, but are not limited to, radionuclides such as 47 Sc, 64 Cu, 67 Cu, 89 Sr, 86 Y, 87 Y, 90 Y, 105 Rh, m Ag, m In, 117m Sn, 149 Pm, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 211 At, 212 Bi, and combinations thereof.
  • radionuclides such as 47 Sc, 64 Cu, 67 Cu, 89 Sr, 86 Y, 87 Y, 90 Y, 105 Rh, m Ag, m In, 117m Sn, 149 Pm, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 211 At, 212 Bi, and combinations thereof.
  • D. Peptide Linkers such as 47 Sc, 64 Cu, 67 Cu, 89 Sr, 86 Y, 87 Y, 90 Y, 105 Rh, m Ag, m In, 117m S
  • the peptide, polypeptide, or protein of interest that contains a C-terminal recognition motif also contains a peptide linker, e.g., between the C-terminal end of the peptide, polypeptide, or protein of interest and the C-terminal recognition motif.
  • the peptide linker in the conjugate of the present invention is located between the peptide, polypeptide, or protein of interest and a C-terminal recognition sequence that is attached to a molecule, e.g., small molecule, containing an N-terminal acceptor sequence.
  • the peptide linker prior to the linkage reaction with a TPase described herein, is located between the C-terminal end of the peptide, polypeptide, or protein of interest and a C-terminal recognition sequence.
  • the peptide linker may comprise about 1 to about 20 amino acids, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In some embodiments, the peptide linker includes more than 20 amino acids.
  • the linker may be about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90 or about 100 amino acids.
  • the linker may comprise a sequence based on (GGGGS) n , wherein n is at least 1 (SEQ ID NO: 24); (GGGS) connect, wherein n is at least 1 (SEQ ID NO: 25); (GGS) connect, wherein n is at least 1 (SEQ ID NO: 26); (GS) connector, wherein n is at least 1 (SEQ ID NO: 27); (G m S) connector, wherein n is at least 1 and m is at least 1 (SEQ ID NO: 28); or (X m S) n , wherein n is at least 1, m is at least 1, and X is any amino acid (SEQ ID NO: 29).
  • the linker is a G4S linker (SEQ ID NO: 30). IV. Examples
  • Example 1 A Selection and Screening Platform for Generating Transpeptidase (TPase) Variants for Use for Bio-Conjugations.
  • TPase Transpeptidase
  • TPase transpeptidase
  • the TPase variants can be used to append cytotoxics, peptides or proteins to any antibody.
  • the linkages used are native peptide bonds. No non-natural amino acids or non-natural chemical linkages are introduced, so there are fewer concerns about immunogenicity.
  • the reaction is carried out with bio-therapeutics manufactured using established processes, the method is a flexible and modular process can be used with any number of different combinations of biologies, half- life extension (HLE) handles and cytotoxics.
  • HLE half- life extension
  • ADC's Antibody drug conjugates
  • Bertozzi and coworkers have described an approach where a small tag sequence is incorporated into a desired site within a protein and then chemoenzymatically converted to a chemical handle that can be derivatized using standard aldehyde chemistry (Rabuka et ah, Nat Protoc, 2012, 7: 1052-1067). More recently Strop and colleagues have described an approach where microbial transglutaminase which recognizes a specific tag sequence, is used to attach a cytotoxic small molecule mono-methyl Auristatin D (MMAD) to a modified antibody (Strop et al, Chem Biol, 2013, 20: 161-167). While these approaches have been deployed with some success, each suffers from its unique drawbacks.
  • MMAD mono-methyl Auristatin D
  • the non-natural amino acid incorporation used by Ambrx requires the incorporation of potentially immunogenic non-natural amino-acids and linkages and requires a specialized manufacturing process.
  • the formyl glycine enzyme (FGE) based approach used by Redwood Biosciences also requires a specialized cell line and introduces a chemical linkage not found in nature.
  • FGE formyl glycine enzyme
  • Pfizer the microbial transglutaminase based approach used by Pfizer, is limited in the types of chemical linkages it can introduce and the size of the partners used for conjugation.
  • Transpeptidases are enzymes that can cleave a specific peptide sequence to form an acyl enzyme intermediate that can then be reacted with a different small molecule, peptide or protein donor resulting in the ligation of the two components with a native peptide bond. Since their discovery in the late 90 's transpeptidases have been applied to a variety of bio-conjugation reactions with varying amount of success. A key limitation in their use has been due to the fact that they are poor catalysts (k cat /K m of 10-100 M ' V 1 ) and require the sequences LPXTG (SEQ ID NO:2) and GGGG (SEQ ID NO: 34) for the acyl donor and acceptor.
  • Sortase A recognizes the motif LPXTG (SEQ ID NO:2), cleaves to the N-terminal side of the glycine residue, forming an acyl enzyme intermediate that can then be reacted with any protein carrying free glycines at their N-terminus.
  • C- or N- termini of mAb heavy or light chains can be sequence specifically derivatized provided the transpeptidase is able to recognize the termini.
  • TPases transpeptidases
  • TPases from bacteria have previously been expressed in E. coli (Ton-That et al., Proc Natl Acad Sci U S A 1999, 96: 12424-12429), on the surface of phage as a fusion protein with phage coat protein (Piotukh et al., J Am Chem Soc 2011, 133: 17536-17539), and also on the surface of yeast as a fusion protein with Aga2p (Chen et al, Proc Natl Acad Sci U S A 2011, 108: 11399-11404).
  • TPases were expressed in an in vitro transcription and translation system in order to access larger libraries of TPase variants as well as to enable subsequent selections carried out using the selection schemes of the present invention.
  • the e-srtA variant contains the following amino acid substitutions: P94A, D160N, K190E, K196T, and a 6xHis tag.
  • IVTT in vitro transcription and translation
  • the post-PCR purified DNA template is encapsulated in in vitro transcription translation mix, along with the tracer peptide containing biotin (the second substate (nucleophile); (G) 5 -K-biotin, SEQ ID NO: 17).
  • DNA sequences coding for active transpeptidases are able to cleave the target peptide attached to the template DNA within the individual compartment in the emulsion, and ligate the DNA to the biotinylated peptide tag. These "winner" sequences can then be captured using streptavidin magnetic beads (Dynal) and amplified for the next round of mutagenesis.
  • the isolated sequences are further mutagenized to create more diversity and fine tune the catalytic properties for a given recognition sequence. Because this system is performed entirely in vitro, a far greater amount of flexibility is possible with regard to concentrations of the IVC components, thereby allowing the isolation of more efficient catalysts.
  • the first step in setting up this selection system was to synthesize a duplex template coding for the TPase variants carrying a covalently linked peptide tag that can be recognized and ligated to the GGGK-Bio tag using the IVTT produced TPase.
  • the primer carrying a long chain alkyl amine was covalently linked to a small peptide recognition site of wt sortase (CLPETG, SEQ ID NO:31) using an N-terminal cysteine as a handle and a hetero- bifunctional linker (SMPH, Pierce Biotechnologies).
  • the purified primer was used to produce the tagged duplex and the duplex purified away from the primer using a membrane based protocol (PCR purification kit, Qiagen).
  • the purified duplex was then tested for its ability to be tagged with an appropriate nucleophile in the presence of the evolved sortase (e- srtA) variant using a GGGGSK-FAM tag (SEQ ID NO: 13).
  • the tagged duplex was incubated with 1 ⁇ e-srtA for 2 hrs at room temperature in the presence of the nucleophile GGGGSK-FAM (SEQ ID NO: 13).
  • the reaction products were run on an agarose gel and visualized under UV light before and after staining with ethidium bromide. As shown in FIG.
  • the most critical step in the selection process is the ability to segregate the template DNA duplexes into individual compartments by emulsification in an oil-water-surfactant mix (Miller et ah, Nat Methods, 2006, 3:561-570). Emulsification was performed under conditions expected to achieve at most one duplex per compartment. This parameter impedes active enzymes produced in the micro-droplets from tagging the peptide on templates coding for inactive enzymes. Exhaustive testing of a series of conditions for emulsification based on several published reports (Levy and Ellington, Chem Biol, 2008, 15:979-989) was performed.
  • a 2XNNK library was constructed around srtA residues P94 and D165 ⁇ e.g., P94NNK and D165NNK).
  • the mutant P94S/D165A has been shown to have about 15X improved k cat /K m relative to wildtype.
  • a three part extension reaction was set up with oligos containing NNK mutations at the appropriate positions followed by PCR amplification using primers at the termini (FIG. 9).
  • the amplified sequence was purified using Qiaquick PCR purification kit and sequenced to verify degeneracy at the positions targeted for mutagenesis.
  • the final product of the extension reactions was the correct expected length and was sequence verified.
  • the IVC based selection system described herein can be performing using the (G) 5 - K-biotin as probe and the 2XNNK library described above or a focused library.
  • the focused library can include one or more sortase mutants with known activity, such as those described in Chen et al, Proc Natl Acad Sci USA, 2011, 108: 11399-11404.
  • the enzyme activity of TPases produced by PCR templates at various concentration were measured using a real-time fluorescence assay (FIG. 10A). IVTT reactions in the NEB PURE system were set up with the sortase 520 FRET substrate to read out production and activity of TPases in real-time.
  • trastuzumab as described above and in FIG. 11 were constructed using standard molecular biology techniques. The constructs were transfected into HEK293 cells and antibodies were harvested from cell culture conditioned serum free media after 1 week of growth using Protein A affinity chromatography. Antibody preparations were analyzed using gel electrophoresis and quantitated using UV absorbance.
  • Conjugation reactions with each antibody construct were carried out in standard assay buffer (50 mM HEPES pH7.5, 150 mM NaCl, 5 mM CaCl 2 ) using 5-10 ⁇ antibody, 1-10 ⁇ TPase and 100-500 ⁇ of appropriately tagged peptide substrate. Reactions with each antibody construct were optimized with respect to time, temperature and solution conditions. Analysis of extent of conjugation was done using gel based assays (G4SFAM, FIGS. 13A and 13B) or using hydrophobic interaction chromatography (HIC) on an HPLC system (FIG. 14).
  • G4SFAM gel based assays
  • HIC hydrophobic interaction chromatography
  • a key parameter for successful use of ADC's in the clinic is their ability to selectively kill cells overexpressing the target antigen.
  • a second important parameter is stability of these molecules in human plasma and the ability to release the conjugated toxin only inside the lysosomes of target cells after internalization.
  • a surrogate G4S-FAM conjugate
  • the optimal ADC was tested for ability to kill cells using a panel of cell lines expressing different levels of the Her2 receptor.
  • the cell lines SKBR-3 and MCF-7 were used for Her2 sensitive and resistant cell lines, respectively.
  • the conjugate was able to kill Her2 overexpressing SKBR-3 cells with a potency (0.2 nM). This result is comparable to what has been published for KadcylaTM.
  • this example illustrates the use of a TPase variant to efficiently label the C-terminus of an antibody carrying an appropriate tag and linker sequence. Also, the example demonstrates the ability of the resulting ADC to kill cells expressing the target antigen. Moreover, the example shows that a linker, such as a G4S linker, can be placed prior to the recognition site at the C-terminus of an antibody or a fragment thereof. The linker was shown to be stable for at least 1 week, which corresponds to the expected half-life of an ADC.
  • a linker such as a G4S linker
  • Example 3 Discovery of TPase Variants with Modified (Increased or Decreased) Catalytic Activity.
  • This example describes several TPase variants identified using the selection and screening method described in Example 1. Such a method can be used to generate and identify TPase variants with specificity for new recognition motifs and/or better specificity.
  • the variants can also have higher or reduced catalytic activity compared to the wildtype TPase. In some cases, the variant may have lower metal ion dependence and/or increased thermostability. In other cases, the TPase variant with decreased activity may have a slower turnover rate than the wildtype enzyme.
  • the TPase variant may carry one or more mutations that prevent the enzyme from catalyzing the reverse reaction.
  • NEB IVTT reaction mix
  • FRET substrate such as Sensolyte 520 Sortase Substrate (Anapsec)
  • FIG. 17A shows a panel of selected variants included in a focused library of TPase mutants.
  • FIG. 17B shows the mutations in the scaffolds (e.g., the parental TPase variants) used for making the focused library.
  • the selection and screening method described herein can be used to identify TPases with a desired catalytic activity and carrying an amino acid substitution at a known position of the Sortase A protein, such as those described in Chen et al., Proc Natl Acad Sci USA,, 2011, 108: 11399-11404, and Hirakawa et al, Biotechnol Bioeng, 2012, 109(12):2955-61.
  • FIG. 18 shows how the IVTT realtime fluorescence method allows for the identification of variants with both increased and decreased activity relative to the scaffolds (e.g., the parental variants of FIG. 17B).
  • FIG. 19 and 20 show TPase variants with increased activity compared to wildtype Sortase A (SEQ ID NO: 1).
  • the TPase variants with either P94T; P94E; and P94R, D160N and D165A (e-srtA) mutations showed increased catalytic activity (FIGS. 18 and 19).
  • FIG. 20 shows a TPase variant generated according to the selection and screening method provided herein. This TPase has diminished, but not eliminated activity (FIG. 20).
  • the variant W192A shows about 10X diminished enzymatic activity compared to wt Sortase A for the same substrate.
  • XiVQLX 2 wherein X 1 and X 2 are independently any amino acid
  • NPQTN wherein the cleavage site is between T and N SEQ ID NO: 15
  • LPXTA wherein the cleavage site is between T and A SEQ ID NO: 17

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Abstract

La présente invention concerne des procédés de sélection et de criblage pour identifier des variants de transpeptidase (TPase) qui ont une activité catalytique modifiée pour un site de reconnaissance natif dans un peptide, un polypeptide ou une protéine d'intérêt par rapport à une TPase sauvage correspondante ou qui reconnaissent de nouveaux motifs de séquence présents dans le peptide, le polypeptide ou la protéine d'intérêt qui diffèrent du site de reconnaissance natif. La présente invention concerne également des variants de TPase, des acides nucléiques et des vecteurs isolés, ainsi que des conjugués produits en utilisant les variants de TPase, et des compositions pharmaceutiques et des kits comprenant ces conjugués. L'invention concerne également des procédés de traitement du cancer chez un sujet le nécessitant au moyen des conjugués décrits dans la présente invention.
PCT/US2015/032717 2014-05-27 2015-05-27 Procédés de sélection de transpeptidases évoluées et leur utilisation pour créer des conjugués de protéines WO2015183986A1 (fr)

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CN108949787A (zh) * 2018-07-05 2018-12-07 上海海洋大学 一种金鱼Tgf2转座酶及其制备与保存方法

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US20140057317A1 (en) * 2012-06-21 2014-02-27 President And Fellows Of Harvard College Evolution of bond-forming enzymes
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US20030153020A1 (en) * 1999-04-15 2003-08-14 Olaf Schneewind Identification of sortase gene
US20140057317A1 (en) * 2012-06-21 2014-02-27 President And Fellows Of Harvard College Evolution of bond-forming enzymes
WO2014070865A1 (fr) * 2012-10-30 2014-05-08 President And Fellows Of Harvard College Immobilisation, libération et remplacement catalysés par sortase de molécules fonctionnelles sur des surfaces solides

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