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WO2024100450A2 - Peptides for affinity purification of adeno-associated virus - Google Patents

Peptides for affinity purification of adeno-associated virus Download PDF

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Publication number
WO2024100450A2
WO2024100450A2 PCT/IB2023/000676 IB2023000676W WO2024100450A2 WO 2024100450 A2 WO2024100450 A2 WO 2024100450A2 IB 2023000676 W IB2023000676 W IB 2023000676W WO 2024100450 A2 WO2024100450 A2 WO 2024100450A2
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WO
WIPO (PCT)
Prior art keywords
seq
peptide
amino acid
aav
acid sequence
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PCT/IB2023/000676
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French (fr)
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WO2024100450A3 (en
Inventor
Wouter POS
Anthony Newcombe
Stefan MARSDEN
Original Assignee
Vectory B.V.
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Publication of WO2024100450A2 publication Critical patent/WO2024100450A2/en
Publication of WO2024100450A3 publication Critical patent/WO2024100450A3/en

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    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material

Definitions

  • Adeno-associated virus is an attractive gene therapy vector that has been widely used for delivery of therapeutic genes to treat a plethora of genetic diseases.
  • AAV vectors are of particular interest due to their high safety profile, broad tropism, and relative ease of genetic engineering.
  • AAV serotypes there is a wide variety of AAV serotypes, each with a distinct transduction profile which makes it possible to develop AAV-based gene therapies for a wide range of clinical applications.
  • AAV-based gene therapies requires large-scale manufacture of highly purified preparations of AAV vectors.
  • a challenge in the downstream process of AAV vector manufacturing is that a relatively high amount of AAV capsid particles produced do not contain packaged vector genomes or contain an incomplete genome (e.g., empty capsids). Such empty capsids may not have therapeutic benefit but can sometimes represent up to 90% of a clinical product (Hebben et al., Cell Gene Therapy Insights 2018; 4(2), 131-146).
  • many reported purification systems are serotype dependent and different methods need to be applied for downstream processing of different AAV serotypes.
  • peptides that bind to AAV capsids are highly useful for purification of parvovirus products (e. , AAV capsids). Furthermore, provided herein are methods of screening the peptides for binding to parvovirus capsids, such as AAV capsids.
  • the peptides disclosed herein can bind to AAV capsids of more than one AAV serotype and can differentiate between empty and full AAV capsids.
  • the peptides provided herein are peptides derived from polycystic kidney disease- 1 (PKD1) amino acid sequence of the AAVR. These peptides are modified for increased binding to parvoviruses such as AAV. In an embodiment, the peptides may be useful for purification of AAV. In an embodiment, the peptides may be useful for binding AAV.
  • PPD1 polycystic kidney disease- 1
  • a peptide comprising an amino acid sequence of at least 5 amino acids in length derived from SEQ ID NO: 1, wherein (a) the peptide is 50 amino acids or less in length; and/or (b) the amino acid sequence derived from SEQ ID NO: 1 has at least one amino acid insertion, deletion, or substitution compared to the corresponding amino acid in SEQ ID NO: 1.
  • SEQ ID NO: 1 is derived from a PKD1 sequence (SEQ ID NO: 14). Providing a sequence longer than the length of 14 amino acids SEQ ID NO: 1 may include sequences corresponding with the further sequence of SEQ ID NO: 14 at the N-terminus and/or C-terminus. Providing a sequence longer than the length of 14 amino acids may include additional sequences not derived from PKD1, which are useful, e.g., for attachment to a support, such as described herein.
  • the peptide is 50 amino acids or less in length. In an embodiment, the peptide is 40 amino acids or less in length. In an embodiment, the peptide is 20 amino acids or less in length. In an embodiment, the peptide is 14 amino acids or less in length.
  • the peptide comprises 1-10 amino acid substitution(s) relative to the corresponding amino acids of SEQ ID NO: 1. In an embodiment, at least one amino acid substitution is a conservative substitution. In an embodiment, the amino acid sequence derived from SEQ ID NO: 1 comprises 1-10 amino acid insertion(s) relative to the corresponding amino acids of SEQ ID NO: 1. In an embodiment, the amino acid sequence derived from SEQ ID NO: 1 comprises 1, 2, or 3 amino acid deletions relative to the corresponding amino acids of SEQ ID NO: 1.
  • a peptide comprising an amino acid sequence of 14 contiguous amino acids, wherein the amino acids at positions 3, 6, 7, 9 and 13 are I, P, R, Y and M, respectively.
  • the amino acids at positions 1, 3, 4, 6, 7, 8, 9, 12, and 13 are R, I, Y, P, R, W, Y, R, and M, respectively.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 15.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 16.
  • the peptide comprises one of the following amino acid sequences: QKI, QRI, RLI, RKI, ITH, IYY, IYH, ITHPR (SEQ ID NO: 17), IYHPR (SEQ ID NO: 18), IYYPR (SEQ ID NO: 19), RWY, RDY, DYS, WYS, WYM, WYS, DYM, YSG, YMK, YMG, GEM, KRM, GRM, MGE, MGR, MKR, SGR, SGE, RMF, EME, or RME.
  • the peptide comprises: (a) QKI, QRI, RLI, or RKI at amino acid position 1-3, or (b) ITH, IYY, or IYH at amino acid position 3-5, or (c) ITHPR (SEQ ID NO: 17) or IYHPR (SEQ ID NO: 18), or IYYPR (SEQ ID NO: 19) at amino acid position 3-7, or (d) RWY or RDY at amino acid position 7-9, or (e) DYS, WYS, WYM, WYS, or DYM at amino acid position 8-10, or (f) YSG, or YMK, or YMG at amino acid position 9-11, or (g) GEM, or KRM, or GRM at amino acid position 11-13, or (h) MGE, MGR, MKR, SGR, or SGE at amino acid position 10-12, or (i) RMF, EME, or RME at amino acid position 12-14.
  • a peptide comprising an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, wherein: (a) the amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is I; (b) the amino acid in the peptide corresponding to amino acid 6 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is I; (
  • a peptide comprising an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 7, wherein the amino acids in the peptide corresponding to amino acids 3, 6, 7, 9, and 13 of SEQ ID NO: 7 are I, P, R, Y, and M, respectively.
  • the amino acid in the peptide corresponding to amino acid 1 of SEQ ID NO: 7 is Q or R;
  • the amino acid in the peptide corresponding to amino acid 2 of SEQ ID NO: 7 is K, R, or L;
  • the amino acid in the peptide corresponding to amino acid 4 of SEQ ID NO: 7 is T or Y;
  • the amino acid in the peptide corresponding to amino acid 5 of SEQ ID NO: 7 is H or Y;
  • the amino acid in the peptide corresponding to amino acid 8 of SEQ ID NO: 7 is D or W;
  • the amino acid in the peptide corresponding to amino acid 10 of SEQ ID NO: 7 is S or M;
  • the amino acid in the peptide corresponding to amino acid 11 of SEQ ID NO: 7 is G or K;
  • the amino acid in the peptide corresponding to amino acid 12 of SEQ ID NO: 7 is E or R; and/or (i) the amino acid in
  • amino acids in the peptide corresponding to amino acids 1, 3, 4, 6, 7, 8, 9, 12, and 13 of SEQ ID NO: 7 are R, I, Y, P, R, W, Y, R, and M, respectively.
  • the peptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
  • a peptide comprising an amino acid sequence that comprises at least 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
  • the peptide comprises amino acids 5-10 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 4-11 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 3-12 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 2-13 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • a peptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 1-7 amino acid changes.
  • the peptide comprises 1-7 amino acid substitution(s) relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • at least one amino acid substitution is a conservative substitution.
  • the peptide comprises 1-7 amino acid insertion(s) relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 1-7 amino acid deletion(s) relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • At least one amino acid deletion is at the N-terminus of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • At least one amino acid deletion is at the C-terminus of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide consists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide is connected to a linker and/or a tag.
  • the linker or tag is at the N-terminus of the peptide.
  • the linker or tag is at the C-terminus of the peptide.
  • the linker is selected from the group consisting of beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5- aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), and polyethylene glycol (PEG) linkers, or a combination thereof.
  • the tag is selected from the group consisting of biotin, 6His, lOHis, FLAG, V5, HA, His, GST (glutathione S-transferase), maltose binding protein (MBP), GFP, and cMYC.
  • the isoelectric point (pl) value of the peptide is at least 6. In an embodiment, the pl is in the range of 6-12. In an embodiment, the pl is in the range of 9-12. In an embodiment, the peptide is soluble at a concentration of 1.5 mM up to 15 mM.
  • the solubility is determined in a physiological buffer.
  • the peptide binds to an adeno-associated virus (AAV) capsid protein.
  • AAV capsid protein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or an engineered variant thereof.
  • the AAV capsid protein is a chimeric AAV capsid protein.
  • the chimeric AAV capsid protein is AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9.
  • a solid support comprising a peptide disclosed herein.
  • the solid support is selected from the group consisting of a membrane, a fdter, a biosensor, a chip, a slide, a wafer, a fiber, a magnetic or non-magnetic bead, a gel, tubing, a strip, a plate, a rod, a polymer, a particle, a microparticle, a capillary, a column, and a resin.
  • the solid support further comprises a binding moiety.
  • the binding moiety is an antibody, streptavidin, avidin, neutravidin, or a fragment thereof.
  • the solid support is a biosensor and the peptide comprises a biotin tag.
  • nucleic acid encoding a peptide disclosed herein.
  • a vector comprising a nucleic acid disclosed herein.
  • the vector is a viral vector.
  • the vector is a retrovirus vector, a herpes virus vector, a baculovirus vector, or an adenovirus vector.
  • composition comprising a peptide disclosed herein.
  • a method of detecting an adeno-associated virus (AAV) particle in a sample comprising: (a) contacting the sample with a peptide disclosed herein, under conditions such that the peptide can form a complex with an AAV particle; and (b) detecting the peptide/ AAV particle complex.
  • AAV adeno-associated virus
  • a method for purifying an AAV particle from a sample comprising the AAV particle and at least one contaminant comprising contacting the composition with a peptide disclosed herein or a solid support disclosed herein under conditions such that the peptide or solid support binds to the AAV particle.
  • the method further comprises washing the peptide or solid support with a wash solution under conditions such that the peptide or solid support remains bound to the AAV particle. In an embodiment, the method further comprises eluting the AAV particle from the peptide or solid support. [0045] In an embodiment, at least one contaminant is an AAV particle that lacks a complete genome.
  • the method further comprises formulating the eluted AAV particle in a formulation buffer suitable for administration to a human subject.
  • the AAV particle is a recombinant AAV (rAAV) comprising an rAAV genome comprising a transgene.
  • the transgene encodes a polypeptide.
  • the transgene encodes an antibody or a fragment thereof, fusion protein, miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, IncRNA, ribozyme, or mRNA.
  • the rAAV comprises an AAV capsid comprising an AAV capsid protein.
  • the AAV capsid protein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or an engineered variant thereof.
  • the AAV capsid protein is a chimeric AAV capsid protein.
  • the chimeric AAV capsid protein is AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9.
  • composition obtained by a method disclosed herein comprising less than 30% of AAV particles that lack a complete genome.
  • composition obtained by a method disclosed herein comprising less than 15% of AAV particles that lack a complete genome.
  • composition obtained by a method disclosed herein comprising less than 10% of AAV particles that lack a complete genome.
  • a pharmaceutical composition comprising a purified AAV particle obtained by a method disclosed herein.
  • a method of screening a peptide for binding to an adeno-associated virus comprising the steps of (a) providing a peptide disclosed herein; (b) providing an AAV; and (c) determining the binding of said peptide with said AAV.
  • the method further comprises (a) providing a reference peptide known for binding to said AAV; (b) determining the binding of said reference peptide with said AAV; and (c) comparing the binding of said peptide and said reference peptide with said AAV.
  • the peptide is immobilized on a solid support.
  • the solid support is as defined herein.
  • the solid support comprises a biosensor coated with streptavidin and wherein said peptide comprises a biotin tag.
  • the method of screening comprises label free technology such as bio-layer interferometry or surface plasmon resonance (SPR).
  • the AAV is determined for full and/or empty capsids of the AAV.
  • the dissociation rate constant (kd (s' 1 )) and/or association rate constant (k a (M' 1 s' 1 )) is determined based on 1 : 1 binding of the AAV with the peptide.
  • the dissociation equilibrium constant KD is determined.
  • the peptides are selected that have a dissociation rate constant kd in the range of 10' 1 - 10' 7 s' 1 .
  • the peptides are selected that have an association rate constant (k a (M' 1 s' 1 )) in the range of 10 3 - 10 7 M ⁇ s' 1 .
  • the peptides are selected that differentiate between binding of empty and full capsids.
  • a plurality of peptides is screened for and peptides capable of binding AAV are identified.
  • a peptide as identified herein for the purification of an AAV particle.
  • a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, and variants thereof.
  • a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.
  • a method of processing a composition comprising an AAV comprising the steps of: (a) providing a composition comprising an AAV; (b) contacting said composition with a solid support comprising a peptide disclosed herein; (c) allowing binding of the parvovirus to the solid support comprising the peptide, (d) optionally, washing the solid support comprising the peptide and the bound AAV with a buffer, and (e) eluting the bound AAV particles from the solid support with an elution buffer.
  • composition comprising a purified AAV using a method disclosed herein.
  • FIG. 1 is a graph showing the response curves for the ligand load optimization of Super-Streptavidin (SSA) Biosensors with anti-AAV5 PRAAV antibodies.
  • SSA Super-Streptavidin
  • FIG. 2 is a graph showing the association and dissociation of different AAV5.2- CAG-GFP (full capsid) analyte concentrations using biosensors prepared from the undiluted PRAAV5 antibody stock to determine the k L! , kd, and KD.
  • FIG. 3 is a graph showing the association and dissociation of different AAV5.2 (empty capsid) analyte concentrations using biosensors prepared from the undiluted PRAAV5 antibody stock to determine k a kd, and KD.
  • FIG. 4 is a graph showing the loading of WT PKD1 (peptide 1) onto the biosensor to determine the optimal loading conditions.
  • FIG. 5 is a graph showing the analyte association of AAV5.2-CAG-GFP to custom biosensors prepared from peptides 1-13.
  • FIG. 6 is a graph showing the response curves for the binding of AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, and AAV9 to peptide 7.
  • FIG. 7 is a graph showing the response curves for the binding of AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, and AAV9 to peptide 9.
  • FTG. 8 is a graph showing the response curves for the binding of AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, and AAV9 to peptide 11.
  • FIG. 9 is a graph showing the response curves for the binding of AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, and AAV9 to peptide 13.
  • FIG. 10A is a graph showing the response curves for binding of AAV5.2 (empty capsid) to peptide 7.
  • FIG. 10B is a graph showing the response curve for binding of AAV5.2-CAG-GFP (full capsid) to peptide 7.
  • FIG. 11A is a graph showing the response curves for binding of AAV5.2 (empty capsid) to peptide 9.
  • FIG. 11B is a graph showing the response curve for binding of AAV5.2-CAG-GFP (full capsid) to peptide 9.
  • FIG. 12A is a graph showing the response curves for binding of AAV5.2 (empty capsid) to peptide 11.
  • FIG. 12B is a graph showing the response curve for binding of AAV5.2-CAG-GFP (full capsid) to peptide 11.
  • FIG. 13A is a graph showing the response curves for binding of AAV5.2 (empty capsid) to peptide 13.
  • FIG. 13B is a graph showing the response curve for binding of AAV5.2-CAG-GFP (full capsid) to peptide 13.
  • peptides that bind to AAV capsids are highly useful for purification of parvovirus products (e.g., AAV capsids). Furthermore, provided herein are methods of screening the peptides for binding to parvovirus capsids, such as AAV capsids.
  • the peptides disclosed herein can bind to AAV capsids of more than one AAV serotype and can differentiate between empty and full capsids.
  • a peptide derived from refers to obtaining a non-naturally occurring peptide from a particular naturally occurring amino acid sequence.
  • a peptide derived from a naturally occurring amino acid sequence may be a fragment of naturally occurring amino acid sequence such as SEQ ID NO: 1.
  • a peptide derived from a naturally occurring amino acid sequence may comprise one or more amino acid substitutions, insertions, or deletions compared to the naturally occurring amino acid sequence, when aligned herewith.
  • a peptide derived from a naturally occurring amino acid sequence is obtained by using predictive computational modelling and/or mutational analysis.
  • the term “conservative substitution” refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine (K), arginine (R), histidine (H)); acidic side chains (e.g., aspartic acid (D), glutamic acid (E)); uncharged polar side chains (e.g., glycine (G); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C)); nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), beta-branched side chains (e.g.,
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative amino acid substitutions in the sequence of a peptide confer or improve specific binding of the peptide to a target of interest.
  • conservative amino acid substitutions in the sequences of a peptide do not reduce or abrogate the binding of the peptide to a target of interest.
  • conservative amino acid substitutions do not significantly affect specific binding of a peptide to a target of interest.
  • rAAV recombinant adeno-associated virus
  • a “vector” refers to a nucleic acid molecule that is a vehicle for introducing a nucleic acid molecule (e.g., a polynucleotide disclosed herein) into a cell.
  • an “expression vector” refers to a vector comprising transcriptional regulatory elements operably linked to a gene of interest (e.g., a polynucleotide disclosed herein) that facilitate the expression of the gene of interest in a cell and/or a cell free expression system.
  • transgene refers to a non-AAV nucleic acid sequence that encodes a polypeptide (e.g., an antibody or antibody derivative, such as scFv) or non-coding RNA (e.g., an miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, or RNA aptamer).
  • a polypeptide e.g., an antibody or antibody derivative, such as scFv
  • non-coding RNA e.g., an miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, or RNA aptamer.
  • the “percentage identity” between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between the pair of aligned sequences by 100, and dividing by the length of the aligned region, including internal gaps. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another. When a sequence is described herein as being a certain percentage identical to a reference sequence, the percentage identity to the reference sequence is determined across the full length of the reference sequence.
  • the term “contaminant” refers to any material present at any stage of a method disclosed herein that is not the desired intact AAV particle. Contaminants include, without limitation, viral and cellular proteins or nucleic acids, or byproducts thereof, that arise in the production process of the desired intact AAV particle, or any undesired AAV particle or byproduct thereof, including, for example, residual medium components, anti-foam, a chemical lysis agent, endonucleases, or an AAV particle that lacks a complete vector genome (also referred to herein as an “empty capsid”).
  • a contaminant also includes any host cell proteins, host cell nucleic acids, or host cell fragment that results from any stage of an AAV production process.
  • the terms “host cell protein,” “host cell nucleic acid,” and “host cell fragment” are used herein to refer to any unwanted protein, nucleic acid, or cell fragment that originates from the cell used to produce an AAV particle.
  • peptides derived from SEQ ID NO: 1 amino acids 347-360 ofthe PKDl domain, a.k.a., KIAA1837, SEQ ID NO: 14, see Table 1; SEQ ID NO: 1 is underlined and in bold).
  • the peptides disclosed herein are capable of binding wild-type AAV capsids, chimeric AAV capsids, and engineered AAV capsids. Table 1.
  • Amino acid sequence of PKD1 amino acids 347-360 ofthe PKDl domain, a.k.a., KIAA1837, SEQ ID NO: 14, see Table 1; SEQ ID NO: 1 is underlined and in bold.
  • the peptides disclosed herein are capable of binding wild-type AAV capsids, chimeric AAV capsids, and engineered AAV capsids. Table 1.
  • Amino acid sequence of PKD1 amino acids 347-360 ofthe PKDl domain, a.k.a.
  • a peptide comprising an amino acid sequence derived from SEQ ID NO: 1.
  • the peptide is at least 5 amino acids in length.
  • the peptide is 59, 58, 57, 56, 55, 54, 53, 52, 51, or 50 amino acids or less in length.
  • the peptide is 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40 amino acids or less in length.
  • the peptide is 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 amino acids or less in length.
  • the peptide is 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 amino acids or less in length.
  • the peptide is 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids or less in length.
  • the peptide is 14 amino acids or less in length.
  • the peptide comprises an amino acid sequence that has at least one amino acid insertion, deletion, or substitution compared to the corresponding amino acid in SEQ ID NO: 1.
  • the amino insertion, deletion or substitution can be incorporated at any place within the peptide compared to the corresponding amino acid in SEQ ID NO: 1.
  • the amino insertion, deletion, or substitution can be incorporated randomly, or at the N-terminal region or at the C-terminal region.
  • the insertion(s), deletion(s), or substitution(s) can be incorporated manually or based on computational predictions, for example by using Schrodinger software or other software known in the art.
  • he peptide may be a linear and comprises different amino acid combinations as shown in the following embodiments.
  • the peptide comprises 1-10 amino acid substitution(s) relative to the corresponding amino acids of SEQ ID NO: 1.
  • At least one amino acid substitution is a conservative substitution.
  • the peptide comprises an amino acid sequence derived from SEQ ID NO: 1, wherein the amino acid sequence derived from SEQ ID NO: 1 comprises 1-10 amino acid insertion(s) relative to the corresponding amino acids of SEQ ID NO: 1.
  • the peptide comprises an amino acid sequence derived from SEQ ID NO: 1, wherein the amino acid sequence derived from SEQ ID NO: 1 comprises 1, 2, or 3 amino acid deletions relative to the corresponding amino acids of SEQ ID NO: 1.
  • the peptide comprises an amino acid sequence of 14 contiguous amino acids, wherein the amino acids at positions 3, 6, 7, 9, and 13 are I, P, R, Y, and M, respectively.
  • the amino acids at positions 1, 3, 4, 6, 7, 8, 9, 12, and 13 are R, I, Y, P, R, W, Y, R, and M, respectively.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 15.
  • the sequence of SEQ ID NO: 15 represents the consensus sequence of all designed peptide sequences as described in the examples.
  • this peptide comprises the amino acid sequence of SEQ ID NO: 16.
  • the sequence of SEQ ID NO: 16 represents the consensus sequence of designed peptide sequences as described in the examples selected therefrom that were shown to be capable of binding parvoviruses such as AAV.
  • the peptide comprises one of the following amino acid sequences: QKI, QRI, RLI, RKI, ITH, IYY, IYH, ITHPR (SEQ ID NO: 17), IYHPR (SEQ ID NO: 18), IYYPR (SEQ ID NO: 19), RWY, RDY, DYS, WYS, WYM, WYS, DYM, YSG, YMK, YMG, GEM, KRM, GRM, MGE, MGR, MKR, SGR, SGE, RMF, EME, or RME.
  • the peptide comprises one or more of the following amino acid sequences: QKI, QRI, RLI, RKI, ITH, IYY, IYH, ITHPR (SEQ ID NO: 17), IYHPR (SEQ ID NO: 18), IYYPR (SEQ ID NO: 19), RWY, RDY, DYS, WYS, WYM, WYS, DYM, YSG, YMK, YMG, GEM, KRM, GRM, MGE, MGR, MKR, SGR, SGE, RMF, EME, or RME.
  • the peptide derived from SEQ ID NO: 1 comprises at positions of said peptide relative to SEQ ID NO: 1:
  • the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises an amino acid sequence that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises an amino acid sequence that comprises at least 6, 7, 8, 9, 10, 11, 12, 13 or 14 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises an amino acid sequence that comprises 6 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises an amino acid sequence that comprises 7 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises an amino acid sequence that comprises 8 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises an amino acid sequence that comprises 9 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises an amino acid sequence that comprises
  • SEQ ID NO: 4 10 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises an amino acid sequence that comprises
  • the peptide comprises an amino acid sequence that comprises
  • SEQ ID NO: 4 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises an amino acid sequence that comprises
  • the peptide comprises an amino acid sequence that comprises
  • SEQ ID NO: 4 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 9-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 8-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 8-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 7-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 7-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 7-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 6-11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 6-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 6-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 6- 14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 5-10 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 5-11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 5-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 5- 13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 5-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 4-9 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 4-10 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 4-11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 4- 12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 4-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 4-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 3-8 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 3-9 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 3-10 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 3- 11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 3-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 3-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 3-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 2-7 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 2-8 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 2-9 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 2-10 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 2-11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 2-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 2-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 2- 14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 1-6 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 1-7 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 1-8 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 1-9 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 1-10 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 1-11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 1-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 1- 13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises amino acids 1-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 1-7 amino acid changes.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 1 amino acid change.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 2 amino acid changes.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 3 amino acid changes.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 4 amino acid changes.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 5 amino acid changes.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 6 amino acid changes.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 7 amino acid changes.
  • the peptide comprises 1-7 amino acid substitution(s) relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 1 amino acid substitution relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 2 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 3 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 4 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 5 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 7 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • at least one amino acid substitution is a conservative substitution.
  • the peptide comprises 1-7 amino acid insertion(s) relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 1 amino acid insertion relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 2 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 3 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 4 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 5 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 6 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 7 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 1-7 amino acid deletion(s) relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 1 amino acid deletion relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 2 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ IDNO: 13.
  • the peptide comprises 3 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 4 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 5 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 6 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 7 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises at least 1 amino acid deletion at the N- terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 1 amino acid deletion at the N-terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 2 amino acid deletions at the N-terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 3 amino acid deletions at the N- terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises at least 1 amino acid deletion at the C- terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 1 amino acid deletion at the C-terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10 SEQ IDNO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 2 amino acid deletions at the C-terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises 3 amino acid deletions at the C-terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide consists of the amino acid sequence of SEQ ID NO:
  • SEQ ID NO: 3 SEQ ID NO: 4
  • SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 13. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 9. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 11. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 13.
  • the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 7. In a further embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 7, wherein the amino acids in the peptide corresponding to amino acids
  • ID NO: 7 are R, I, Y, P, R, W, Y, R, and M, respectively.
  • the amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 7 is I or A.
  • the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 9. In a further embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 9, wherein the amino acids in the peptide corresponding to amino acids 3, 6, 7, 9, and 13 of SEQ ID NO: 9 are I, P, R, Y, and M, respectively.
  • amino acids in the peptide corresponding to amino acids 1, 3, 4, 6, 7, 8, 9, 12, and 13 of SEQ ID NO: 9 are R, I, Y, P, R, W, Y, R, and M, respectively.
  • amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 9 is I or A.
  • the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 11. In a further embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 11, wherein the amino acids in the peptide corresponding to amino acids 3, 6, 7, 9, and 13 of SEQ ID NO: 11 are I, P, R, Y, and M, respectively.
  • amino acids in the peptide corresponding to amino acids 1, 3, 4, 6, 7, 8, 9, 12, and 13 of SEQ ID NO: 11 are R, I, Y, P, R, W, Y, R, and M, respectively.
  • amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 11 is I or A.
  • the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 13. In a further embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 13, wherein the amino acids in the peptide corresponding to amino acids 3, 6, 7, 9, and 13 of SEQ ID NO: 13 are I, P, R, Y, and M, respectively.
  • amino acids in the peptide corresponding to amino acids 1, 3, 4, 6, 7, 8, 9, 12, and 13 of SEQ ID NO: 13 are R, I, Y, P, R, W, Y, R, and M, respectively.
  • amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 13 is I or A.
  • the peptide further comprises a linker and/or a tag.
  • the peptide comprises a linker and/or tag so that the peptide can be coupled to a solid support.
  • linkers, tags and the solid support include, but are not limited to, examples provided herein.
  • the linker or tag is at the N-terminus of the peptide. In another embodiment, the linker or tag is at the C-terminus of the peptide.
  • the linker is selected from the group consisting of Beta-alanine, 4-aminobutyric acid (GABA), (2- aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), a 6-aminohexanoic acid (Ahx), and polyethylene glycol (PEG) linkers, or a combination thereof.
  • the tag is selected from the group consisting of biotin, 6His, lOHis, FLAG, V5, HA, His, GST (glutathione S-transferase), maltose binding protein (MBP), GFP, and cMYC.
  • the peptide comprising a linker and/or tag is used for labelling virus particles.
  • the peptides disclosed herein are useful for downstream processing, e.g., by attaching a peptide disclosed herein to a column material or matrix, or the like for capturing parvovirus product.
  • the linker allows for covalent attachment of the peptide to a matrix and/or column material for a robust processing step.
  • a linker and/or tag is selected for the peptide that preserves the functional properties of the peptide.
  • a linker and/or tag is selected that preserves binding of the peptide to a parvovirus capsid, for example an AAV capsid.
  • the peptide may be provided with a linker and/or tag which is a peptide and the rigidity and/or flexibility of the linker and/or tag may be modified by the inclusion of one or more amino acids.
  • the peptide comprises a linker which is a chemical linker selected from the group consisting of a carbohydrate linker, a lipid linker, a fatty acid linker, and a polyether linker.
  • the peptide comprises a tag that binds to a binding moiety that has been coated on the solid support.
  • a peptide disclosed herein is covalently bound to a solid support.
  • the peptide is covalently bound to a solid support via the N-terminus of the peptide through the formation of a chemical bond with a primary amine.
  • bonds may include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters.
  • the isoelectric point (pl) value of the peptide is determined.
  • the pl is the pH at which the net charge of the peptide is zero.
  • the pl can be calculated from the amino acid sequence based on amino acid residue properties which can be done, e.g., using online tools such as described in the examples herein.
  • the pl value of the peptide is at least 6.
  • the pl value of the peptide is in the range of 6-12.
  • the pl value of the peptide is in the range of 9-12.
  • the solubility of the peptide is determined.
  • the solubility may be determined by visual observation with an unaided eye. For example, by observing a change in opaqueness, color, clarity and/or viscosity, a change in solubility may be observed. In some embodiments, a person of skill in the art may measure the solubility by known methods in the art.
  • the peptide is soluble at a concentration of 1.5 mM up to 15mM. Such soluble peptides are useful for coating peptides to a surface.
  • the peptide is soluble in a buffer suitable for preparation of a solid support comprising said peptide.
  • said solubility of the peptide is determined in a physiological buffer.
  • suitable buffers can be readily determined by a person skilled in the art based on the desired pH of the final solution.
  • Low pH solutions ⁇ pH 5.5
  • High pH solutions can be made, for example, in Tris-HCl, phosphate buffers, or sodium bicarbonate buffers.
  • a number of conditions may be used to determine KD and off-rates for the purpose of determining, for example, optimal pH and/or salt concentrations.
  • the buffer comprises Phosphate-buffered saline (PBS) and Pluronic F-68.
  • solubility ++
  • such properties hence may be indicative of the suitability of peptides for attachment of a solid support and/or efficient binding to parvovirus capsids, such as AAV.
  • a peptide is selected for screening for binding with parvovirus capsids and/or AAV capsids based on the pl of the peptide.
  • the peptides disclosed herein bind to an adeno-associated virus (AAV) capsid.
  • AAV capsid is an AAV capsid from clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I.
  • the AAV capsid is an AAV capsid from clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, or an engineered variant thereof.
  • said AAV capsid is selected from, AAVgo.l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid, or an engineered variant thereof.
  • the AAV capsid is selected from AAVgo. l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid, or a chimeric or engineered capsid variant thereof.
  • the AAV capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid, or an engineered variant thereof.
  • the AAV capsid is a chimeric AAV capsid.
  • the AAV capsid is an engineered variant capsid comprising amino acid sequences from at least two different AAV capsid serotypes.
  • the chimeric AAV capsid is an AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9 chimeric capsid protein.
  • the peptides disclosed herein bind to an adeno-associated virus (AAV) capsid protein.
  • AAV capsid protein is a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid protein, or an engineered variant thereof.
  • the AAV capsid protein is a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid protein, or an engineered variant thereof.
  • the AAV capsid protein is a chimeric AAV capsid protein.
  • the AAV capsid protein is an engineered variant capsid protein comprising amino acid sequences from at least two different AAV capsid proteins.
  • the chimeric AAV capsid is a AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9 chimeric capsid protein.
  • the AAV capsid protein comprises a VP1 amino acid sequence from AAV2 and a VP2 and VP3 amino acid sequence from AAV5. It is understood that such a chimeric AAV capsid comprises one or more of the viral proteins from a different serotype.
  • the chimeric AAV capsid protein comprises a VP1 amino acid sequence from a first AAV serotype and a VP2 and VP3 amino acid sequence from a second AAV serotype.
  • the chimeric AAV capsid protein comprises a VP1 and VP2 amino acid sequence from a first AAV serotype and a VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP3 amino acid sequence from a first AAV serotype and a VP2 amino acid sequence from a second AAV serotype.
  • the peptide comprises a sequence selected from SEQ ID NOs: 7, 9, 11, and 13, and the peptide binds to a parvovirus capsid, such as AAV capsids, including AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9 capsids.
  • a solid support comprising a peptide disclosed herein.
  • the solid support is selected from the group consisting of a membrane, a filter, a biosensor, a chip, a slide, a wafer, a fiber, a magnetic or non-magnetic bead, a gel, tubing, a strip, a plate, a rod, a polymer, a particle, a microparticle, a capillary, a column, and a resin.
  • Suitable solid supports include a chromatographic resin or matrix (e.g. , SEPHAROSE-4 FF agarose beads), the wall or floor of a well in a plastic microtiter dish, a silica-based biochip, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof.
  • a peptide disclosed herein may be attached on such a support material by a non-covalent association or by covalent bonding, using reagents and techniques known in the art.
  • a binding moiety may be coated onto the solid support to facilitate binding or immobilization of the peptide on said solid support.
  • the solid support further comprises a binding moiety.
  • the binding moiety of the solid support is an antibody, streptavidin, avidin, neutravidin, or a fragment thereof.
  • the binding moiety of the solid support is an antibody, streptavidin, avidin, neutravidin, or a fragment thereof, and the peptide comprises a biotin tag.
  • the solid support is a biosensor, wherein the biosensor is provided with said binding moiety and the peptide comprises a biotin tag.
  • nucleic acid encoding a peptide disclosed herein. It is understood that encoding means that the peptide may be expressed (comprised, e.g., in a propeptide or the like with appropriate cleavage and secretory signal peptide, if needed, and the like).
  • vector comprising a nucleic acid encoding a peptide disclosed herein.
  • Such vector may be a viral vector.
  • the vector can be a retrovirus vector, a herpes virus vector, a baculovirus vector, or an adenovirus vector.
  • a peptide disclosed herein may be chemically synthesized, such as described in the examples, and other methods known in the art.
  • a composition comprising a peptide disclosed herein.
  • a method of detecting an adeno-associated virus (AAV) particle in a sample comprising: (a) contacting the sample with a peptide disclosed herein, under conditions such that the peptide can form a complex with an AAV particle; and (b) detecting the peptide/ AAV particle complex.
  • the peptide comprises a detectable label (e.g, a fluorescent label) to allow such detection.
  • a method for purifying an AAV particle from a sample comprising the AAV particle and at least one contaminant comprising contacting the composition with a peptide disclosed herein or a solid support comprising a peptide disclosed herein under conditions such that the peptide or solid support binds to the AAV particle.
  • the method further comprises washing the peptide or solid support with a wash solution under conditions such that the peptide or solid support remains bound to the AAV particle.
  • the method further comprises eluting the AAV particle from the peptide or solid support.
  • At least one contaminant is an AAV particle that lacks a complete vector genome. It is understood that in a typical production process, c.g, utilizing baculovirus vectors and insect cells, or mammalian cell culture in which AAV production plasmids may be transfected, full and empty capsids are produced, and it may be desirable to remove empty capsids and/or separate empty from full capsids.
  • contaminants can include bulk cellular material, obtained via lysis and/or enzymatic treatments (e.g., DNase) and the like.
  • the method comprises further formulating the eluted AAV particle in a formulation buffer suitable for administration to a human subject.
  • such methods for purifying include use of a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, and variants thereof.
  • a method for processing a composition comprising a parvovirus or an engineered parvovirus comprising the steps of:
  • composition comprising a parvovirus
  • composition comprising a parvovirus
  • solid support comprising a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, and variants thereof;
  • a method for processing a composition comprising a parvovirus or an engineered parvovirus comprising the steps of:
  • composition contacting said composition with a solid support comprising a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13;
  • solid supports comprising said peptides are useful for processing of AAV capsids, such as AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9, and/or chimeric and/or engineered variants thereof. Such processing methods are highly useful in manufacturing of AAV gene therapy products and the like. As shown in the example section, the peptides corresponding with or comprising SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, are highly suitable for such methods.
  • the AAV is an AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9 capsid.
  • the AAV is an engineered AAV capsid comprising AAV capsid proteins from one or more of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9.
  • a peptide disclosed herein is for use in binding to a parvovirus, such as AAV.
  • the AAV is a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo. l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, AAV5.2, or AAV5 capsid.
  • the AAV is an engineered AAV capsid comprising AAV capsid proteins from one or more of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9.
  • the AAV capsid protein is an engineered variant capsid protein comprising amino acid sequences from at least two different AAV capsid proteins.
  • the chimeric AAV capsid is an AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9 chimeric capsid protein.
  • the AAV capsid protein comprises a VP1 amino acid sequence from AAV2 and a VP2 and VP3 amino acid sequence from AAV5.
  • the chimeric AAV capsid protein comprises a VP1 amino acid sequence from a first AAV serotype and a VP2 and VP3 amino acid sequence from a second AAV serotype.
  • the chimeric AAV capsid protein comprises a VP1 and VP2 amino acid sequence from a first AAV serotype and a VP3 amino acid sequence from a second AAV serotype.
  • the chimeric AAV capsid protein comprises a VP1 and VP3 amino acid sequence from a first AAV serotype and a VP2 amino acid sequence from a second AAV serotype.
  • a peptide comprising the amino acid sequence of SEQ ID NO : 7, SEQ ID NO : 9, SEQ ID NO : 11 , or SEQ ID NO : 13 , for use in binding to a parvovirus, such as AAV.
  • a peptide comprising the amino acid sequence of SEQ ID NO: 7 for use in binding to a parvovirus, such as AAV.
  • a peptide comprising the amino acid sequence of SEQ ID NO: 9, for use in binding to a parvovirus, such as AAV.
  • the AAV is an AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9 capsid.
  • the AAV is an engineered AAV capsid comprising AAV capsid proteins from one or more of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9.
  • the AAV particle or product is a recombinant AAV (rAAV) comprising an rAAV genome comprising a transgene.
  • rAAV recombinant AAV
  • the transgene encodes a polypeptide.
  • the transgene encodes an antibody or a fragment thereof (e.g., an scFv), fusion protein, miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, IncRNA, ribozyme, or mRNA.
  • the rAAV comprises an AAV capsid comprising an AAV capsid protein.
  • the AAV capsid protein is from a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, AAV5.2, or AAV5 capsid protein, or an engineered variant thereof.
  • the AAV capsid protein is a chimeric AAV capsid protein.
  • the chimeric AAV capsid protein comprises a VP1 amino acid sequence from a first AAV serotype and a VP2 and VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP2 amino acid sequence from a first AAV serotype and a VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP3 amino acid sequence from a first AAV serotype and a VP2 amino acid sequence from a second AAV serotype.
  • the chimeric AAV capsid protein is an AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9 chimeric capsid protein.
  • the AAV capsid protein comprises a VP1 amino acid sequence from AAV2 and a VP2 and VP3 amino acid sequence from AAV5.
  • an AAV gene therapy product may be highly purified yet comprises a substantial amount of empty capsids and/or capsid proteins.
  • a peptide disclosed herein is for use in purifying an AAV viral vector product after a first purification.
  • the composition comprises less than 30% of AAV particles that lack a complete genome.
  • the percentage of AAV particles that lack a complete genome is determined by Analytical ultracentrifugation or cryo-TEM. [00181]
  • the composition comprises less than 15% of AAV particles that lack a complete genome.
  • the composition comprises less than 10% of AAV particles that lack a complete genome.
  • composition comprising purified AAV particles obtained by a method disclosed herein.
  • a method of screening a peptide for binding to an adeno-associated virus comprising the steps of: (a) providing a peptide disclosed herein; (b) providing an AAV; and (c) determining the binding of said peptide with said AAV.
  • peptides may be designed based on SEQ ID NO: 1 and screened for binding to a capsid.
  • the method of screening may further comprise: (a) providing a reference peptide known for binding to said AAV; (b) determining the binding of said reference peptide with said AAV; and (c) comparing the binding of said peptide and said reference peptide with said AAV.
  • Suitable reference peptides that are provided herein include a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7, 9, 11, or 13.
  • the method of screening comprises the step of immobilizing the provided peptide on a solid support.
  • a plurality of peptides may thus be screened for.
  • the solid support is a biosensor coated with streptavidin and the peptide comprises a biotin tag.
  • Other suitable tags and solid supports are known in the art.
  • the method of screening comprises label free technology such as bio-layer interferometry or surface plasmon resonance (SPR).
  • the method of screening may comprise determining the dissociation rate constant kd (s' 1 )) or association rate constant (k a (M' 1 s' 1 )) based on 1 : 1 binding of the AAV with the peptide, or the method of screening comprises determining the dissociation rate constant (kd (s' 1 )) or association rate constant (k a (M' 1 s' 1 )), which constants are determined based on an assumed one-to-one binding of an AAV capsid with a peptide disclosed herein. Based on the determined constants, the dissociation equilibrium constant KD can be calculated.
  • the methods of screening may comprise determining binding of the AAV to the peptide, including of full and/or empty capsids of the AAV.
  • peptides may be identified that allow to differentiate between the two (e.g., either have highly reduced association or have highly reduced dissociation of the empty capsids relative to full capsids (or vice versa)).
  • the dissociation and association rate constants were determined, and the KD was calculated.
  • the dissociation rate constant for SEQ ID NOs: 7 and 13 was much lower for the analyte comprising full AAV5.2 capsids as compared with the analyte comprising capsids without vector genome, resulting in differentiating affinity.
  • peptides comprising SEQ ID NOs: 9 and 11
  • SEQ ID NOs: 9 and 11 no such or much less differentiating constants were found.
  • Either type of peptide is of use in accordance with the invention.
  • Further peptides that may be contemplated and which can be useful may include peptides that more strongly bind empty capsids as compared with full capsids.
  • peptides as identified in the methods of screening disclosed herein may allow for purification of empty and/or full capsids.
  • peptides as identified in the methods of screening disclosed herein may allow for purification of full capsids.
  • peptides as identified in the methods of screening disclosed herein may allow for purification of empty and full capsids. For example, one may identify a first peptide to which both full and empty capsids strongly bind. The empty capsids, showing lower affinity, are washed by subjecting the solid support with a suitable buffer, for example pH 2.8.
  • useful methods for purification of AAV capsids and/or AAV products or the like may comprise different consecutive binding and elution strategies utilizing different peptides that have different properties with regard to binding profiles of the AAV capsid or product for which a purification strategy is to be determined, which may also depend on the outcome of screening strategies.
  • a peptide that is identified as having desirable properties can be modified e.g., by amino acid substitution) in order to obtain desirable properties.
  • the method of screening comprises the selection of peptides that have a dissociation rate constant kd in the range of 10' 1 to IO' 10 s' 1 .
  • the method of screening comprises the selection of peptides that have an association rate constant (k a (M' 1 s' 1 )) in the range of 10 3 to 10 7 M' 1 .
  • the method of screening comprises the selection of peptides that differentiate between binding of empty and full capsids, wherein the association rate constants are similar, and the dissociation rate constants differ when comparing the rate constants for empty and full parvovirus, e.g., AAV capsids.
  • the method of screening comprises the identification of a plurality of peptides that are capable of binding AAV.
  • a peptide as identified in a method of screening disclosed herein for the purification of a parvovirus product, e.g., an AAV vector product for gene therapy.
  • a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, including variants thereof.
  • variants of such peptides have similar solubility and/or isoelectric point properties.
  • such variants may have similar association rate constants for empty and full capsids, while dissociation rate constants for empty and full capsids differ by at least 100-fold.
  • the dissociation rate of one of the empty and full capsids is in the range of 10' 1 to 10' 5 s' 1 , whereas the other is at least 100-fold lower.
  • a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.
  • the peptide is chemically or recombinantly synthesized.
  • the peptide is 50, 40, 30, or 20 amino acids in length or less. Such peptides are highly advantageous for binding, processing, and/or purification of AAV gene therapy products.
  • a peptide disclosed herein for binding an AAV comprising a capsid of serotype clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo. l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, AAV5.2, or AAV5, or a derivative thereof.
  • the AAV capsid protein is an engineered variant capsid protein comprising amino acid sequences from at least two different AAV capsid proteins.
  • the chimeric AAV capsid is a AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9 chimeric capsid protein.
  • the AAV capsid protein comprises a VP1 amino acid sequence from AAV2 and a VP2 and VP3 amino acid sequence from AAV5.
  • the chimeric AAV capsid protein comprises a VP1 amino acid sequence from a first AAV serotype and a VP2 and VP3 amino acid sequence from a second AAV serotype.
  • the chimeric AAV capsid protein comprises a VP1 and VP2 amino acid sequence from a first AAV serotype and a VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP3 amino acid sequence from a first AAV serotype and a VP2 amino acid sequence from a second AAV serotype. In another aspect as provided herein is a use of a peptide disclosed herein for purification or binding of AAV.
  • a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13, and variants thereof.
  • the peptide is chemically or recombinantly synthesized.
  • the peptide is 50, 40, 30, or 20 amino acids in length or less.
  • Such peptides are highly advantageous for binding, processing, and/or purification of AAV gene therapy products and may allow to differentiate between empty and full capsids.
  • a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 11.
  • the peptide is chemically or recombinantly synthesized.
  • the peptide is 50, 40, 30, or 20 amino acids in length or less. Such peptides are highly advantageous for binding, processing, and/or purification of AAV gene therapy products.
  • the peptide is used in purification of said AAV.
  • a peptide disclosed herein wherein said use comprises separating full and empty capsids of said AAV.
  • Adeno-associated virus AAV
  • AAVR adeno-associated virus receptor
  • AAVR adeno-associated virus receptor
  • KIAA0319L a proteinaceous receptor
  • AAVR is an essential cellular receptor required for the transduction of cells by multiple AAV serotypes.
  • AAVR is a glycosylated membrane protein that recycles from the plasma membrane to the trans-Golgi network using the cellular endosomal network.
  • AAVR comprises an ectodomain of a MANEC (motif at the N terminus with eight cysteines) domain and five Ig-like domains known as PKD (polycystic kidney disease) domains.
  • PKD polycystic kidney disease
  • AAV is capable of utilizing these PKD domains to facilitate transduction of a cell.
  • PKD domains 1 (PKD1), 2 (PKD2) and 3 (PKD3) within the ectodomain of AAVR are capable of interacting with AAV (Pillay S et al., J Virol. 2017 Aug 24; 91(18): e00391-17).
  • a cryo-EM structure (2.5 A) of AAV5 in complex with AAVR demonstrated that AAV5 binds to the first polycystic kidney disease (PKD1) domain (SEQ ID NO: 14) of the AAV receptor (AAVR). It was found that the PKD1 residues I349-G357 and L372-P374 contain most of the binding interactions and a minimal amino acid sequence in the PKD1 loop (SEQ ID NO: 1) binds to AAV5.
  • PPD1 domain SEQ ID NO: 14
  • the WT sequence of the PKD1 peptide (SEQ ID NO: 2) was used as input for a systematic mutational analysis in silico for optimal binding to AAV5.2 (Schrodinger software). Based on the mutational analysis, 10 additional PKD1 peptide variants (SEQ ID NOs: 4-13) were generated. These peptides lacked the N-terminal W amino acid residue and were 14 amino acids in length (the 14 amino acid wild-type sequence corresponding therewith is SEQ ID NO: 1) [00201] The optimized peptides where synthesized (Genscript) and a 6-aminohexanoic acid (Ahx) linker and biotinylation were included as N-terminal modifications to allow for immobilization onto a solid support.
  • Genscript synthesized
  • Ahx 6-aminohexanoic acid
  • Peptide molecular weights were calculated from the molecular weight of biotin (244 g/mol), 6-aminohexanoic acid (131 g/mol), and the individual peptide molecular weight, while taking the elimination of two water molecules into account during the coupling reaction (-2x 18 g/mol).
  • Peptide MW and isoelectric point (pl) values were calculated using the online tool at https://web.expasy.org/computej3i/. Solubility was assessed qualitatively by inspection of visible, undissolved particles during the preparation of stock solutions. Peptides that were readily soluble are indicated with ++ and peptides that required mixing by vortex are indicated with + in Table 3.
  • Ligand load optimization Anti-AAV5 antibody (PRAAV5 ELISA kit, Progen) was reconstituted in 750 pL of buffer (lx DPBS + 0.001% Pluronic F-68), and 1- to 64-fold serial dilutions were prepared.
  • AAV5.2 CAG-GFP was used as analyte at a concentration of 5xl0 13 cp/mL (83 nM) based on Stunner measurements.
  • the kinetics experiment was executed using the following steps: Initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed.
  • the kinetics experiment was executed using the following steps: initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed.
  • Data analysis step corrections were applied for “Start of association” and “Start of dissociation”, and curve fitting was performed using a “1 : 1 binding model” and “Global” fitting.
  • results shown in FIG. 2 demonstrate the association (420-720 s) and dissociation (720-1020 s) of different AAV5.2-CAG-GFP analyte concentrations (12-93 nM) using biosensors prepared from the undiluted PRAAV5 antibody stock to determine k a , kd, and KD.
  • the results show that the custom biosensors were successfully prepared and used for determining the kinetic constants that characterize the interactions between the PRAAV5 antibody and AAV5.2-CAG-GFP as a benchmark (see Table 3).
  • results shown in FIG. 3 demonstrate the association (420-720 s) & dissociation (720-1020 s) of different AAV5.2 (empty capsid) analyte concentrations (0.6-33 nM) using biosensors prepared from the undiluted PRAAV5 antibody stock to determine k a , kd, and KD.
  • the results show that the custom biosensors were successfully prepared and used for determining the kinetic constants that characterize the interactions between the PRAAV5 antibody and AAV5.2 (empty capsid) as a benchmark (see Table 3).
  • Example 3 Optimization of the ligand loading conditions for the biosensors.
  • WT PKD1 (SEQ ID NO: 2) ligand scout loading: A 5mM stock solution of WT PKD1 (SEQ ID NO: 2) was prepared in lx DPBS + 0.001% Pluronic F-68, from which additional dilutions were prepared in the same buffer (lOOx, l’000x, 2’000x). These ligand solutions were used for studying the preparation of biosensors, with AAV5.2-GFP as analyte at 2xl0 13 cp/mL to subsequently confirm functionality of the prepared biosensors.
  • the kinetics experiment was executed using the following steps: Initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed. Custom biosensors were then prepared for all 12 peptides (SEQ ID NOs: 2 - 13) under these conditions. The results shown in FIG.4 demonstrate successful loading of WT PKD1 (SEQ ID NO: 2) onto the biosensor.
  • Example 4 Screening of the peptides for binding to AAV5.2-CAG-GFP
  • the kinetics experiment was executed using the following steps: Initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed.
  • the top four peptides that showed the highest responses were subjected to a more detailed ligand loading scout, and their affinity constants were determined towards both AAV5.2-CAG-GFP (full capsid) and AAV5.2 (empty capsid).
  • Example 5 Screening of the peptides for binding to different AAV Serotype
  • AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, and AAV9 (all including the transgene: CAG-GFP).
  • AAV serotypes were commercially produced, and purification was done by two rounds of gradient density centrifugation (CsCl) followed by buffer exchange into lx PBS + 0.001% Pluronic F-68 for AAV1, AAV3, AAV4, AAV6, AAV8, and AAV9, while AAV2 additionally contained 100 mM of sodium citrate.
  • All the serotypes were titered and diluted to a final titer of 2xl0 13 vg/mL.
  • Biosensors were prepared from peptide stock solutions of peptide 7, peptide 9, peptide 11, and peptide 13 at 25-100 pM in lx DPBS + 0.001% Pluronic F-68.
  • the AAV serotypes were resuspended by pipetting and used undiluted (2xl0 13 vg/mL) as analyte.
  • the kinetics experiment was executed using the following steps: initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed.
  • results shown in FIG. 6 demonstrate that peptide 7 strongly binds to AAV3, AAV4, AAV6, AAV8, and AAV9 and moderately binds to AAV1.
  • results shown in FIG. 7 demonstrate that peptide 9 strongly binds to AAV3, AAV4, AAV6, AAV8, and AAV9.
  • results shown in FIG. 8 demonstrate that peptide 11 strongly binds to AAV3, AAV4, AAV6, AAV8, and AAV9.
  • results shown in FIG. 9 demonstrate that peptide 13 strongly binds to AAV3, AAV4, AAV6, AAV8, and AAV9.
  • Example 6 Screening of the peptides for bindins to AAV5.2 (empty capsid) and AAV5.2-CAG- GFP (full capsid)
  • a challenge in AAV manufacturing for clinical use is purifying the AAV capsids containing a complete genome from the AAV capsids containing no genome or an incomplete genome (empty capsids).
  • peptide 7, peptide 9, peptide 11, and peptide 13 were analyzed for their ability to distinguish between full and empty capsids.
  • Biosensors were prepared from peptide stock solutions of peptide 7, peptide 9, peptide 11, and peptide 13 at 25 pM in lx DPBS + 0.001% Pluronic F-68.
  • AAV5.2 empty capsid
  • AAV5.2-CAG-GFP full capsid
  • the kinetics experiment was executed using the following steps: Initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed. Data analysis', step corrections were applied for “Start of association” and “Start of dissociation”, and curve fitting was performed using a “1:1 binding model” and “Global” fitting. The results shown in FIG. 10, FIG. 11, FIG. 12, and FIG.
  • affinity constants primarily originated from lower dissociation rate constants kd of AAV5.2-CAG-GFP (full capsids).
  • peptides 7 and 13 are able to differentiate between empty AAV capsids and full AAV capsids.

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Abstract

Provided herein are peptides that bind to AAV capsids and methods of using the same.

Description

PEPTIDES FOR AFFINITY PURIFICATION OF ADENO-ASSOCIATED VIRUS
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 63/382,660, filed November 7, 2022, the content of which is incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in ST.26 format and is hereby incorporated by reference in its entirety. Said ST.26 copy created on October 24, 2023, is named 20551 l_SL.xml and is 23,490 bytes in size.
INTRODUCTION
[0003] Adeno-associated virus (AAV) is an attractive gene therapy vector that has been widely used for delivery of therapeutic genes to treat a plethora of genetic diseases. AAV vectors are of particular interest due to their high safety profile, broad tropism, and relative ease of genetic engineering. Moreover, there is a wide variety of AAV serotypes, each with a distinct transduction profile which makes it possible to develop AAV-based gene therapies for a wide range of clinical applications.
[0004] Commercial use of AAV-based gene therapies requires large-scale manufacture of highly purified preparations of AAV vectors. A challenge in the downstream process of AAV vector manufacturing is that a relatively high amount of AAV capsid particles produced do not contain packaged vector genomes or contain an incomplete genome (e.g., empty capsids). Such empty capsids may not have therapeutic benefit but can sometimes represent up to 90% of a clinical product (Hebben et al., Cell Gene Therapy Insights 2018; 4(2), 131-146). Furthermore, many reported purification systems are serotype dependent and different methods need to be applied for downstream processing of different AAV serotypes.
[0005] Therefore, there is a need in the art for further and/or improved methods for downstream processing of AAV products for clinical use that can differentiate between empty and full capsids, and/or can be used for multiple AAV serotypes. SUMMARY
[0006] Provided herein are peptides that bind to AAV capsids and methods of using the same. In an aspect, the peptides disclosed herein are highly useful for purification of parvovirus products (e. , AAV capsids). Furthermore, provided herein are methods of screening the peptides for binding to parvovirus capsids, such as AAV capsids. Advantageously, the peptides disclosed herein can bind to AAV capsids of more than one AAV serotype and can differentiate between empty and full AAV capsids.
[0007] In an aspect, the peptides provided herein are peptides derived from polycystic kidney disease- 1 (PKD1) amino acid sequence of the AAVR. These peptides are modified for increased binding to parvoviruses such as AAV. In an embodiment, the peptides may be useful for purification of AAV. In an embodiment, the peptides may be useful for binding AAV.
[0008] In an aspect, provided herein is a peptide comprising an amino acid sequence of at least 5 amino acids in length derived from SEQ ID NO: 1, wherein (a) the peptide is 50 amino acids or less in length; and/or (b) the amino acid sequence derived from SEQ ID NO: 1 has at least one amino acid insertion, deletion, or substitution compared to the corresponding amino acid in SEQ ID NO: 1.
[0009] It is understood that SEQ ID NO: 1 is derived from a PKD1 sequence (SEQ ID NO: 14). Providing a sequence longer than the length of 14 amino acids SEQ ID NO: 1 may include sequences corresponding with the further sequence of SEQ ID NO: 14 at the N-terminus and/or C-terminus. Providing a sequence longer than the length of 14 amino acids may include additional sequences not derived from PKD1, which are useful, e.g., for attachment to a support, such as described herein.
[0010] In an embodiment, the peptide is 50 amino acids or less in length. In an embodiment, the peptide is 40 amino acids or less in length. In an embodiment, the peptide is 20 amino acids or less in length. In an embodiment, the peptide is 14 amino acids or less in length.
[0011] In an embodiment, the peptide comprises 1-10 amino acid substitution(s) relative to the corresponding amino acids of SEQ ID NO: 1. In an embodiment, at least one amino acid substitution is a conservative substitution. In an embodiment, the amino acid sequence derived from SEQ ID NO: 1 comprises 1-10 amino acid insertion(s) relative to the corresponding amino acids of SEQ ID NO: 1. In an embodiment, the amino acid sequence derived from SEQ ID NO: 1 comprises 1, 2, or 3 amino acid deletions relative to the corresponding amino acids of SEQ ID NO: 1.
[0012] In an aspect, provided herein is a peptide comprising an amino acid sequence of 14 contiguous amino acids, wherein the amino acids at positions 3, 6, 7, 9 and 13 are I, P, R, Y and M, respectively.
[0013] In an embodiment, the amino acids at positions 1, 3, 4, 6, 7, 8, 9, 12, and 13 are R, I, Y, P, R, W, Y, R, and M, respectively. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 15. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 16.
[0014] In an embodiment, the peptide comprises one of the following amino acid sequences: QKI, QRI, RLI, RKI, ITH, IYY, IYH, ITHPR (SEQ ID NO: 17), IYHPR (SEQ ID NO: 18), IYYPR (SEQ ID NO: 19), RWY, RDY, DYS, WYS, WYM, WYS, DYM, YSG, YMK, YMG, GEM, KRM, GRM, MGE, MGR, MKR, SGR, SGE, RMF, EME, or RME.
[0015] In an embodiment, the peptide comprises: (a) QKI, QRI, RLI, or RKI at amino acid position 1-3, or (b) ITH, IYY, or IYH at amino acid position 3-5, or (c) ITHPR (SEQ ID NO: 17) or IYHPR (SEQ ID NO: 18), or IYYPR (SEQ ID NO: 19) at amino acid position 3-7, or (d) RWY or RDY at amino acid position 7-9, or (e) DYS, WYS, WYM, WYS, or DYM at amino acid position 8-10, or (f) YSG, or YMK, or YMG at amino acid position 9-11, or (g) GEM, or KRM, or GRM at amino acid position 11-13, or (h) MGE, MGR, MKR, SGR, or SGE at amino acid position 10-12, or (i) RMF, EME, or RME at amino acid position 12-14.
[0016] In an aspect, provided herein is a peptide comprising an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, wherein: (a) the amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is I; (b) the amino acid in the peptide corresponding to amino acid 6 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is P; (c) the amino acid in the peptide corresponding to amino acid 7 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is R; (d) the amino acid in the peptide corresponding to amino acid 9 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is Y; and/or (e) the amino acid in the peptide corresponding to amino acid 13 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is M.
[0017] In an aspect, provided herein is a peptide comprising an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 7, wherein the amino acids in the peptide corresponding to amino acids 3, 6, 7, 9, and 13 of SEQ ID NO: 7 are I, P, R, Y, and M, respectively.
[0018] In an embodiment, (a) the amino acid in the peptide corresponding to amino acid 1 of SEQ ID NO: 7 is Q or R; (b) the amino acid in the peptide corresponding to amino acid 2 of SEQ ID NO: 7 is K, R, or L; (c) the amino acid in the peptide corresponding to amino acid 4 of SEQ ID NO: 7 is T or Y; (d) the amino acid in the peptide corresponding to amino acid 5 of SEQ ID NO: 7 is H or Y; (e) the amino acid in the peptide corresponding to amino acid 8 of SEQ ID NO: 7 is D or W; (f) the amino acid in the peptide corresponding to amino acid 10 of SEQ ID NO: 7 is S or M; (g) the amino acid in the peptide corresponding to amino acid 11 of SEQ ID NO: 7 is G or K; (h) the amino acid in the peptide corresponding to amino acid 12 of SEQ ID NO: 7 is E or R; and/or (i) the amino acid in the peptide corresponding to amino acid 14 of SEQ ID NO: 7 is E or F.
[0019] In an embodiment, the amino acids in the peptide corresponding to amino acids 1, 3, 4, 6, 7, 8, 9, 12, and 13 of SEQ ID NO: 7 are R, I, Y, P, R, W, Y, R, and M, respectively.
[0020] In an embodiment, the peptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[0021] In an aspect, provided herein is a peptide comprising an amino acid sequence that comprises at least 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. [0022] In an embodiment, the peptide comprises amino acids 5-10 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[0023] In an embodiment, the peptide comprises amino acids 4-11 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[0024] In an embodiment, the peptide comprises amino acids 3-12 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[0025] In an embodiment, the peptide comprises amino acids 2-13 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[0026] In an aspect, provided herein is a peptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 1-7 amino acid changes.
[0027] In an embodiment, the peptide comprises 1-7 amino acid substitution(s) relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, at least one amino acid substitution is a conservative substitution.
[0028] In an embodiment, the peptide comprises 1-7 amino acid insertion(s) relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[0029] In an embodiment, the peptide comprises 1-7 amino acid deletion(s) relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[0030] In an embodiment, at least one amino acid deletion is at the N-terminus of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[0031] In an embodiment, at least one amino acid deletion is at the C-terminus of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[0032] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[0033] In an embodiment, the peptide consists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[0034] In an embodiment, the peptide is connected to a linker and/or a tag. In an embodiment, the linker or tag is at the N-terminus of the peptide. In an embodiment, the linker or tag is at the C-terminus of the peptide. In an embodiment, the linker is selected from the group consisting of beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5- aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), and polyethylene glycol (PEG) linkers, or a combination thereof. In an embodiment, the tag is selected from the group consisting of biotin, 6His, lOHis, FLAG, V5, HA, His, GST (glutathione S-transferase), maltose binding protein (MBP), GFP, and cMYC.
[0035] In an embodiment, the isoelectric point (pl) value of the peptide is at least 6. In an embodiment, the pl is in the range of 6-12. In an embodiment, the pl is in the range of 9-12. In an embodiment, the peptide is soluble at a concentration of 1.5 mM up to 15 mM.
[0036] In an embodiment, the solubility is determined in a physiological buffer. [0037] In an embodiment, the peptide binds to an adeno-associated virus (AAV) capsid protein. In an embodiment, the AAV capsid protein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or an engineered variant thereof. In an embodiment, the AAV capsid protein is a chimeric AAV capsid protein. In an embodiment, the chimeric AAV capsid protein is AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9.
[0038] In an aspect, provided herein is a solid support comprising a peptide disclosed herein. In an embodiment, the solid support is selected from the group consisting of a membrane, a fdter, a biosensor, a chip, a slide, a wafer, a fiber, a magnetic or non-magnetic bead, a gel, tubing, a strip, a plate, a rod, a polymer, a particle, a microparticle, a capillary, a column, and a resin. In an embodiment, the solid support further comprises a binding moiety. In an embodiment, the binding moiety is an antibody, streptavidin, avidin, neutravidin, or a fragment thereof. In an embodiment, the solid support is a biosensor and the peptide comprises a biotin tag.
[0039] In an aspect, provided herein is a nucleic acid encoding a peptide disclosed herein.
[0040] In an aspect, provided herein is a vector comprising a nucleic acid disclosed herein.
In an embodiment, the vector is a viral vector. In an embodiment, the vector is a retrovirus vector, a herpes virus vector, a baculovirus vector, or an adenovirus vector.
[0041] In an aspect, provided herein is a composition comprising a peptide disclosed herein.
[0042] In an aspect, provided herein is a method of detecting an adeno-associated virus (AAV) particle in a sample, the method comprising: (a) contacting the sample with a peptide disclosed herein, under conditions such that the peptide can form a complex with an AAV particle; and (b) detecting the peptide/ AAV particle complex.
[0043] In an aspect, provided herein is a method for purifying an AAV particle from a sample comprising the AAV particle and at least one contaminant, the method comprising contacting the composition with a peptide disclosed herein or a solid support disclosed herein under conditions such that the peptide or solid support binds to the AAV particle.
[0044] In an embodiment, the method further comprises washing the peptide or solid support with a wash solution under conditions such that the peptide or solid support remains bound to the AAV particle. In an embodiment, the method further comprises eluting the AAV particle from the peptide or solid support. [0045] In an embodiment, at least one contaminant is an AAV particle that lacks a complete genome.
[0046] In an embodiment, the method further comprises formulating the eluted AAV particle in a formulation buffer suitable for administration to a human subject.
[0047] In an embodiment, the AAV particle is a recombinant AAV (rAAV) comprising an rAAV genome comprising a transgene. In an embodiment, the transgene encodes a polypeptide.
[0048] In an embodiment, the transgene encodes an antibody or a fragment thereof, fusion protein, miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, IncRNA, ribozyme, or mRNA.
[0049] In an embodiment, the rAAV comprises an AAV capsid comprising an AAV capsid protein. In an embodiment, the AAV capsid protein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or an engineered variant thereof. In an embodiment, the AAV capsid protein is a chimeric AAV capsid protein. In an embodiment, the chimeric AAV capsid protein is AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9.
[0050] In an aspect, provided herein is a composition obtained by a method disclosed herein, comprising less than 30% of AAV particles that lack a complete genome.
[0051] In an aspect, provided herein is a composition obtained by a method disclosed herein, comprising less than 15% of AAV particles that lack a complete genome.
[0052] In an aspect, provided herein is a composition obtained by a method disclosed herein, comprising less than 10% of AAV particles that lack a complete genome.
[0053] In an aspect, provided herein is a pharmaceutical composition comprising a purified AAV particle obtained by a method disclosed herein.
[0054] In an aspect, provided herein is a method of screening a peptide for binding to an adeno-associated virus (AAV), comprising the steps of (a) providing a peptide disclosed herein; (b) providing an AAV; and (c) determining the binding of said peptide with said AAV.
[0055] In an embodiment, the method further comprises (a) providing a reference peptide known for binding to said AAV; (b) determining the binding of said reference peptide with said AAV; and (c) comparing the binding of said peptide and said reference peptide with said AAV. [0056] In an embodiment, the peptide is immobilized on a solid support. In an embodiment, the solid support is as defined herein. In an embodiment, the solid support comprises a biosensor coated with streptavidin and wherein said peptide comprises a biotin tag.
[0057] In an embodiment, the method of screening comprises label free technology such as bio-layer interferometry or surface plasmon resonance (SPR). In an embodiment, the AAV is determined for full and/or empty capsids of the AAV.
[0058] In an embodiment, the dissociation rate constant (kd (s'1)) and/or association rate constant (ka (M'1 s'1)) is determined based on 1 : 1 binding of the AAV with the peptide.
[0059] In an embodiment, the dissociation equilibrium constant KD is determined.
[0060] In an embodiment, the peptides are selected that have a dissociation rate constant kd in the range of 10'1 - 10'7 s'1.
[0061] In an embodiment, the peptides are selected that have an association rate constant (ka (M'1 s'1)) in the range of 103 - 107 M^s'1.
[0062] In an embodiment, the peptides are selected that differentiate between binding of empty and full capsids.
[0063] In an embodiment, a plurality of peptides is screened for and peptides capable of binding AAV are identified.
[0064] In an aspect, provided herein is a use of a peptide as identified herein, for the purification of an AAV particle.
[0065] In an aspect, provided herein is a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, and variants thereof.
[0066] In an aspect, provided herein is a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.
[0067] In an aspect, provided herein is a use of a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13 for binding an AAV capsid of serotype AAV3, AAV4, AAV5, AAV5.2, AAV6, AAV8, AAV9, or a derivative thereof. [0068] In an aspect, provided herein is a use of a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13 for purification of an AAV particle.
[0069] In an aspect, provided herein is a use of a peptide comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 13 for use in separating full and empty AAV capsids. [0070] In an aspect, provided herein is a method of processing a composition comprising an AAV, comprising the steps of: (a) providing a composition comprising an AAV; (b) contacting said composition with a solid support comprising a peptide disclosed herein; (c) allowing binding of the parvovirus to the solid support comprising the peptide, (d) optionally, washing the solid support comprising the peptide and the bound AAV with a buffer, and (e) eluting the bound AAV particles from the solid support with an elution buffer.
[0071] In an aspect, provided herein is a composition comprising a purified AAV using a method disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] FIG. 1 is a graph showing the response curves for the ligand load optimization of Super-Streptavidin (SSA) Biosensors with anti-AAV5 PRAAV antibodies.
[0073] FIG. 2 is a graph showing the association and dissociation of different AAV5.2- CAG-GFP (full capsid) analyte concentrations using biosensors prepared from the undiluted PRAAV5 antibody stock to determine the kL!, kd, and KD.
[0074] FIG. 3 is a graph showing the association and dissociation of different AAV5.2 (empty capsid) analyte concentrations using biosensors prepared from the undiluted PRAAV5 antibody stock to determine ka kd, and KD.
[0075] FIG. 4 is a graph showing the loading of WT PKD1 (peptide 1) onto the biosensor to determine the optimal loading conditions.
[0076] FIG. 5 is a graph showing the analyte association of AAV5.2-CAG-GFP to custom biosensors prepared from peptides 1-13.
[0077] FIG. 6 is a graph showing the response curves for the binding of AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, and AAV9 to peptide 7.
[0078] FIG. 7 is a graph showing the response curves for the binding of AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, and AAV9 to peptide 9. [0079] FTG. 8 is a graph showing the response curves for the binding of AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, and AAV9 to peptide 11.
[0080] FIG. 9 is a graph showing the response curves for the binding of AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, and AAV9 to peptide 13.
[0081] FIG. 10A is a graph showing the response curves for binding of AAV5.2 (empty capsid) to peptide 7.
[0082] FIG. 10B is a graph showing the response curve for binding of AAV5.2-CAG-GFP (full capsid) to peptide 7.
[0083] FIG. 11A is a graph showing the response curves for binding of AAV5.2 (empty capsid) to peptide 9.
[0084] FIG. 11B is a graph showing the response curve for binding of AAV5.2-CAG-GFP (full capsid) to peptide 9.
[0085] FIG. 12A is a graph showing the response curves for binding of AAV5.2 (empty capsid) to peptide 11.
[0086] FIG. 12B is a graph showing the response curve for binding of AAV5.2-CAG-GFP (full capsid) to peptide 11.
[0087] FIG. 13A is a graph showing the response curves for binding of AAV5.2 (empty capsid) to peptide 13.
[0088] FIG. 13B is a graph showing the response curve for binding of AAV5.2-CAG-GFP (full capsid) to peptide 13.
DETAILED DESCRIPTION
[0089] Provided herein are peptides that bind to AAV capsids and methods of using the same. In an aspect, the peptides disclosed herein are highly useful for purification of parvovirus products (e.g., AAV capsids). Furthermore, provided herein are methods of screening the peptides for binding to parvovirus capsids, such as AAV capsids. Advantageously, the peptides disclosed herein can bind to AAV capsids of more than one AAV serotype and can differentiate between empty and full capsids.
Definitions
[0090] As used herein, the term “derived from” refers to obtaining a non-naturally occurring peptide from a particular naturally occurring amino acid sequence. A peptide derived from a naturally occurring amino acid sequence may be a fragment of naturally occurring amino acid sequence such as SEQ ID NO: 1. A peptide derived from a naturally occurring amino acid sequence may comprise one or more amino acid substitutions, insertions, or deletions compared to the naturally occurring amino acid sequence, when aligned herewith. In an embodiment, a peptide derived from a naturally occurring amino acid sequence is obtained by using predictive computational modelling and/or mutational analysis.
[0091] As used herein, the term “conservative substitution” refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine (K), arginine (R), histidine (H)); acidic side chains (e.g., aspartic acid (D), glutamic acid (E)); uncharged polar side chains (e.g., glycine (G); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C)); nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), beta-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)); and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In some embodiments, conservative amino acid substitutions in the sequence of a peptide confer or improve specific binding of the peptide to a target of interest. In some embodiments, conservative amino acid substitutions in the sequences of a peptide do not reduce or abrogate the binding of the peptide to a target of interest. In some embodiments, conservative amino acid substitutions do not significantly affect specific binding of a peptide to a target of interest. Methods of identifying nucleotide and amino acid conservative substitutions and nonconservative substitutions which confer, alter, or maintain selective binding affinity are known in the art (see, e.g., Brummell, Biochem. 32: 1180-1187 (1993); Kobayashi, Protein Eng. 12(10):879- 884 (1999); and Burks, PNAS 94:412-417(1997)).
[0092] As used herein, the term “recombinant adeno-associated virus” or “rAAV” refers to an AAV comprising a genome lacking functional rep and cap genes.
[0093] As used herein, a “vector” refers to a nucleic acid molecule that is a vehicle for introducing a nucleic acid molecule (e.g., a polynucleotide disclosed herein) into a cell. [0094] As used herein, an “expression vector” refers to a vector comprising transcriptional regulatory elements operably linked to a gene of interest (e.g., a polynucleotide disclosed herein) that facilitate the expression of the gene of interest in a cell and/or a cell free expression system.
[0095] As used herein, the term “transgene” refers to a non-AAV nucleic acid sequence that encodes a polypeptide (e.g., an antibody or antibody derivative, such as scFv) or non-coding RNA (e.g., an miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, or RNA aptamer).
[0096] As used herein, the “percentage identity” between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between the pair of aligned sequences by 100, and dividing by the length of the aligned region, including internal gaps. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another. When a sequence is described herein as being a certain percentage identical to a reference sequence, the percentage identity to the reference sequence is determined across the full length of the reference sequence.
[0097] As used herein, the term “contaminant” refers to any material present at any stage of a method disclosed herein that is not the desired intact AAV particle. Contaminants include, without limitation, viral and cellular proteins or nucleic acids, or byproducts thereof, that arise in the production process of the desired intact AAV particle, or any undesired AAV particle or byproduct thereof, including, for example, residual medium components, anti-foam, a chemical lysis agent, endonucleases, or an AAV particle that lacks a complete vector genome (also referred to herein as an “empty capsid”). A contaminant also includes any host cell proteins, host cell nucleic acids, or host cell fragment that results from any stage of an AAV production process. The terms “host cell protein,” “host cell nucleic acid,” and “host cell fragment” are used herein to refer to any unwanted protein, nucleic acid, or cell fragment that originates from the cell used to produce an AAV particle.
Peptides
[0098] Disclosed herein are peptides derived from SEQ ID NO: 1 (amino acids 347-360 ofthe PKDl domain, a.k.a., KIAA1837, SEQ ID NO: 14, see Table 1; SEQ ID NO: 1 is underlined and in bold). The peptides disclosed herein are capable of binding wild-type AAV capsids, chimeric AAV capsids, and engineered AAV capsids. Table 1. Amino acid sequence of PKD1
Figure imgf000015_0001
[0099] Provided herein is, in one aspect, a peptide comprising an amino acid sequence derived from SEQ ID NO: 1. In an embodiment, the peptide is at least 5 amino acids in length. In an embodiment, the peptide is 59, 58, 57, 56, 55, 54, 53, 52, 51, or 50 amino acids or less in length. In an embodiment, the peptide is 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40 amino acids or less in length. In an embodiment, the peptide is 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 amino acids or less in length. In an embodiment, the peptide is 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 amino acids or less in length. In an embodiment, the peptide is 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids or less in length. In an embodiment, the peptide is 14 amino acids or less in length.
[00100] In an embodiment, the peptide comprises an amino acid sequence that has at least one amino acid insertion, deletion, or substitution compared to the corresponding amino acid in SEQ ID NO: 1.
[00101] The amino insertion, deletion or substitution can be incorporated at any place within the peptide compared to the corresponding amino acid in SEQ ID NO: 1. For example, the amino insertion, deletion, or substitution can be incorporated randomly, or at the N-terminal region or at the C-terminal region. In an embodiment, the insertion(s), deletion(s), or substitution(s) can be incorporated manually or based on computational predictions, for example by using Schrodinger software or other software known in the art. In an embodiment, he peptide may be a linear and comprises different amino acid combinations as shown in the following embodiments.
[00102] In an embodiment, the peptide comprises 1-10 amino acid substitution(s) relative to the corresponding amino acids of SEQ ID NO: 1.
[00103] In an embodiment, at least one amino acid substitution is a conservative substitution.
[00104] In an embodiment, the peptide comprises an amino acid sequence derived from SEQ ID NO: 1, wherein the amino acid sequence derived from SEQ ID NO: 1 comprises 1-10 amino acid insertion(s) relative to the corresponding amino acids of SEQ ID NO: 1.
[00105] In an embodiment, the peptide comprises an amino acid sequence derived from SEQ ID NO: 1, wherein the amino acid sequence derived from SEQ ID NO: 1 comprises 1, 2, or 3 amino acid deletions relative to the corresponding amino acids of SEQ ID NO: 1.
[00106] Exemplary peptide amino acid sequences derived from SEQ ID NO: 1 are disclosed in Table 2 below.
Table 2. Exemplary peptide sequences derived from SEQ ID NO: 1
Figure imgf000016_0001
Figure imgf000017_0001
[00107] In an embodiment, the peptide comprises an amino acid sequence of 14 contiguous amino acids, wherein the amino acids at positions 3, 6, 7, 9, and 13 are I, P, R, Y, and M, respectively. In an embodiment, the amino acids at positions 1, 3, 4, 6, 7, 8, 9, 12, and 13 are R, I, Y, P, R, W, Y, R, and M, respectively.
[00108] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 15. The sequence of SEQ ID NO: 15 represents the consensus sequence of all designed peptide sequences as described in the examples. In another embodiment, this peptide comprises the amino acid sequence of SEQ ID NO: 16. The sequence of SEQ ID NO: 16 represents the consensus sequence of designed peptide sequences as described in the examples selected therefrom that were shown to be capable of binding parvoviruses such as AAV.
[00109] In an embodiment, the peptide comprises one of the following amino acid sequences: QKI, QRI, RLI, RKI, ITH, IYY, IYH, ITHPR (SEQ ID NO: 17), IYHPR (SEQ ID NO: 18), IYYPR (SEQ ID NO: 19), RWY, RDY, DYS, WYS, WYM, WYS, DYM, YSG, YMK, YMG, GEM, KRM, GRM, MGE, MGR, MKR, SGR, SGE, RMF, EME, or RME. In an embodiment, the peptide comprises one or more of the following amino acid sequences: QKI, QRI, RLI, RKI, ITH, IYY, IYH, ITHPR (SEQ ID NO: 17), IYHPR (SEQ ID NO: 18), IYYPR (SEQ ID NO: 19), RWY, RDY, DYS, WYS, WYM, WYS, DYM, YSG, YMK, YMG, GEM, KRM, GRM, MGE, MGR, MKR, SGR, SGE, RMF, EME, or RME.
[00110] In an embodiment, the peptide derived from SEQ ID NO: 1 comprises at positions of said peptide relative to SEQ ID NO: 1:
(a) QKI, QRI, RLI, or RKI at amino acid position 1-3, or
(b) ITH, IYY, or IYH at amino acid position 3-5, or
(c) ITHPR (SEQ ID NO: 17) or IYHPR (SEQ ID NO: 18) or IYYPR (SEQ ID NO: 19) at amino acid position 3-7, or
(d) RWY or RDY at amino acid position 7-9, or
(e) DYS, WYS, WYM, WYS, or DYM at amino acid position 8-10, or
(f) YSG, or YMK, or YMG at amino acid position 9-11, or
(g) GEM, or KRM, or GRM at amino acid position 11-13, or
(h) MGE, MGR, MKR, SGR, or SGE at amino acid position 10-12, or
(i) RMF, EME, or RME at amino acid position 12-14.
[00111] In an embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is I; the amino acid in the peptide corresponding to amino acid 6 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is P; the amino acid in the peptide corresponding to amino acid 7 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is R; the amino acid in the peptide corresponding to amino acid 9 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is Y; and/or the amino acid in the peptide corresponding to amino acid 13 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is M. [00112] In an embodiment, the peptide comprises an amino acid sequence that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00113] In an embodiment, the peptide comprises an amino acid sequence that comprises at least 6, 7, 8, 9, 10, 11, 12, 13 or 14 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00114] In an embodiment, the peptide comprises an amino acid sequence that comprises 6 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00115] In an embodiment, the peptide comprises an amino acid sequence that comprises 7 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00116] In an embodiment, the peptide comprises an amino acid sequence that comprises 8 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00117] In an embodiment, the peptide comprises an amino acid sequence that comprises 9 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00118] In an embodiment, the peptide comprises an amino acid sequence that comprises
10 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00119] In an embodiment, the peptide comprises an amino acid sequence that comprises
11 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, or SEQ ID NO: 13.
[00120] In an embodiment, the peptide comprises an amino acid sequence that comprises
12 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00121] In an embodiment, the peptide comprises an amino acid sequence that comprises
13 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00122] In an embodiment, the peptide comprises an amino acid sequence that comprises
14 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00123] In an embodiment, the peptide comprises amino acids 9-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00124] In an embodiment, the peptide comprises amino acids 8-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 8-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00125] In an embodiment, the peptide comprises amino acids 7-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 7-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 7-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00126] In an embodiment, the peptide comprises amino acids 6-11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 6-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 6-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 6- 14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00127] In an embodiment, the peptide comprises amino acids 5-10 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 5-11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 5-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 5- 13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 5-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00128] In an embodiment, the peptide comprises amino acids 4-9 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 4-10 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 4-11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 4- 12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 4-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 4-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00129] In an embodiment, the peptide comprises amino acids 3-8 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 3-9 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 3-10 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 3- 11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 3-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 3-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 3-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00130] In an embodiment, the peptide comprises amino acids 2-7 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 2-8 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 2-9 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 2-10 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 2-11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 2-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 2-13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 2- 14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00131] In an embodiment, the peptide comprises amino acids 1-6 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 1-7 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 1-8 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 1-9 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 1-10 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 1-11 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 1-12 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 1- 13 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises amino acids 1-14 of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13
[00132] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 1-7 amino acid changes.
[00133] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 1 amino acid change.
[00134] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 2 amino acid changes.
[00135] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 3 amino acid changes.
[00136] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 4 amino acid changes.
[00137] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 5 amino acid changes.
[00138] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 6 amino acid changes.
[00139] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 7 amino acid changes.
[00140] In an embodiment, the peptide comprises 1-7 amino acid substitution(s) relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 1 amino acid substitution relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 2 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 3 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 4 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 5 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 7 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, at least one amino acid substitution is a conservative substitution.
[00141] In an embodiment, the peptide comprises 1-7 amino acid insertion(s) relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 1 amino acid insertion relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 2 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 3 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 4 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 5 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 6 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 7 amino acid insertions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00142] In an embodiment, the peptide comprises 1-7 amino acid deletion(s) relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 1 amino acid deletion relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 2 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ IDNO: 13. In an embodiment, the peptide comprises 3 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 4 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 5 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 6 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 7 amino acid deletions relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00143] In an embodiment, the peptide comprises at least 1 amino acid deletion at the N- terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 1 amino acid deletion at the N-terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 2 amino acid deletions at the N-terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 3 amino acid deletions at the N- terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00144] In an embodiment, the peptide comprises at least 1 amino acid deletion at the C- terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 1 amino acid deletion at the C-terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10 SEQ IDNO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 2 amino acid deletions at the C-terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, or SEQ ID NO: 13. In an embodiment, the peptide comprises 3 amino acid deletions at the C-terminus relative to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00145] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00146] In an embodiment, the peptide consists of the amino acid sequence of SEQ ID NO:
2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
[00147] In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 13. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 9. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 11. In one embodiment, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 13.
[00148] In one embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 7. In a further embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 7, wherein the amino acids in the peptide corresponding to amino acids
3, 6, 7, 9, and 13 of SEQ ID NO: 7 are I, P, R, Y, and M, respectively. In a further embodiment, the amino acid in the peptide corresponding to amino acid 1 of SEQ ID NO: 7 is Q orR; the amino acid in the peptide corresponding to amino acid 2 of SEQ ID NO: 7 is K, R, or L; the amino acid in the peptide corresponding to amino acid 4 of SEQ ID NO: 7 is T or Y; the amino acid in the peptide corresponding to amino acid 5 of SEQ ID NO: 7 is H or Y; the amino acid in the peptide corresponding to amino acid 8 of SEQ ID NO: 7 is D or W; the amino acid in the peptide corresponding to amino acid 10 of SEQ ID NO: 7 is S or M; the amino acid in the peptide corresponding to amino acid 11 of SEQ ID NO: 7 is G or K; the amino acid in the peptide corresponding to amino acid 12 of SEQ ID NO: 7 is E or R; and/or the amino acid in the peptide corresponding to amino acid 14 of SEQ ID NO: 7 is E or F. In yet another further embodiment, the amino acids in the peptide corresponding to amino acids 1, 3, 4, 6, 7, 8, 9, 12, and 13 of SEQ
ID NO: 7 are R, I, Y, P, R, W, Y, R, and M, respectively. In one further embodiment, the amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 7 is I or A.
[00149] In one embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 9. In a further embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 9, wherein the amino acids in the peptide corresponding to amino acids 3, 6, 7, 9, and 13 of SEQ ID NO: 9 are I, P, R, Y, and M, respectively. In a further embodiment, the amino acid in the peptide corresponding to amino acid 1 of SEQ ID NO: 9 is Q or R; the amino acid in the peptide corresponding to amino acid 2 of SEQ ID NO: 9 is K, R, or L; the amino acid in the peptide corresponding to amino acid 4 of SEQ ID NO: 9 is T or Y; the amino acid in the peptide corresponding to amino acid 5 of SEQ ID NO: 9 is H or Y; the amino acid in the peptide corresponding to amino acid 8 of SEQ ID NO: 9 is D or W; the amino acid in the peptide corresponding to amino acid 10 of SEQ ID NO: 9 is S or M; the amino acid in the peptide corresponding to amino acid 11 of SEQ ID NO: 9 is G or K; the amino acid in the peptide corresponding to amino acid 12 of SEQ ID NO: 9 is E or R; and/or the amino acid in the peptide corresponding to amino acid 14 of SEQ ID NO: 9 is E or F. In yet another further embodiment, the amino acids in the peptide corresponding to amino acids 1, 3, 4, 6, 7, 8, 9, 12, and 13 of SEQ ID NO: 9 are R, I, Y, P, R, W, Y, R, and M, respectively. In one further embodiment, the amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 9 is I or A.
[00150] In one embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 11. In a further embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 11, wherein the amino acids in the peptide corresponding to amino acids 3, 6, 7, 9, and 13 of SEQ ID NO: 11 are I, P, R, Y, and M, respectively. In a further embodiment, the amino acid in the peptide corresponding to amino acid 1 of SEQ ID NO: 11 is Q or R; the amino acid in the peptide corresponding to amino acid 2 of SEQ ID NO: 11 is K, R, or L; the amino acid in the peptide corresponding to amino acid 4 of SEQ ID NO: 11 is T or Y; the amino acid in the peptide corresponding to amino acid 5 of SEQ ID NO: 1 1 is H or Y; the amino acid in the peptide corresponding to amino acid 8 of SEQ ID NO: 11 is D or W; the amino acid in the peptide corresponding to amino acid 10 of SEQ ID NO: 11 is S or M; the amino acid in the peptide corresponding to amino acid 11 of SEQ ID NO: 11 is G or K; the amino acid in the peptide corresponding to amino acid 12 of SEQ ID NO: 11 is E or R; and/or the amino acid in the peptide corresponding to amino acid 14 of SEQ ID NO: 11 is E or F. In yet another further embodiment, the amino acids in the peptide corresponding to amino acids 1, 3, 4, 6, 7, 8, 9, 12, and 13 of SEQ ID NO: 11 are R, I, Y, P, R, W, Y, R, and M, respectively. In one further embodiment, the amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 11 is I or A.
[00151] In one embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 13. In a further embodiment, the peptide comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 13, wherein the amino acids in the peptide corresponding to amino acids 3, 6, 7, 9, and 13 of SEQ ID NO: 13 are I, P, R, Y, and M, respectively. In a further embodiment, the amino acid in the peptide corresponding to amino acid 1 of SEQ ID NO: 13 is Q or R; the amino acid in the peptide corresponding to amino acid 2 of SEQ ID NO: 13 is K, R, or L; the amino acid in the peptide corresponding to amino acid 4 of SEQ ID NO: 13 is T or Y; the amino acid in the peptide corresponding to amino acid 5 of SEQ ID NO: 13 is H or Y; the amino acid in the peptide corresponding to amino acid 8 of SEQ ID NO: 13 is D or W; the amino acid in the peptide corresponding to amino acid 10 of SEQ ID NO: 13 is S or M; the amino acid in the peptide corresponding to amino acid 11 of SEQ ID NO: 13 is G or K; the amino acid in the peptide corresponding to amino acid 12 of SEQ ID NO: 13 is E or R; and/or the amino acid in the peptide corresponding to amino acid 14 of SEQ ID NO: 13 is E or F. In yet another further embodiment, the amino acids in the peptide corresponding to amino acids 1, 3, 4, 6, 7, 8, 9, 12, and 13 of SEQ ID NO: 13 are R, I, Y, P, R, W, Y, R, and M, respectively. In one further embodiment, the amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 13 is I or A.
[00152] In an embodiment, the peptide further comprises a linker and/or a tag. In an embodiment, the peptide comprises a linker and/or tag so that the peptide can be coupled to a solid support. Examples of linkers, tags and the solid support include, but are not limited to, examples provided herein. [00153] In one embodiment, the linker or tag is at the N-terminus of the peptide. In another embodiment, the linker or tag is at the C-terminus of the peptide. In an embodiment, the linker is selected from the group consisting of Beta-alanine, 4-aminobutyric acid (GABA), (2- aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), a 6-aminohexanoic acid (Ahx), and polyethylene glycol (PEG) linkers, or a combination thereof. In another embodiment, the tag is selected from the group consisting of biotin, 6His, lOHis, FLAG, V5, HA, His, GST (glutathione S-transferase), maltose binding protein (MBP), GFP, and cMYC.
[00154] In an embodiment, the peptide comprising a linker and/or tag is used for labelling virus particles. In an embodiment, the peptides disclosed herein are useful for downstream processing, e.g., by attaching a peptide disclosed herein to a column material or matrix, or the like for capturing parvovirus product. In such embodiments, the linker allows for covalent attachment of the peptide to a matrix and/or column material for a robust processing step. In one embodiment, a linker and/or tag is selected for the peptide that preserves the functional properties of the peptide. In an embodiment, a linker and/or tag is selected that preserves binding of the peptide to a parvovirus capsid, for example an AAV capsid. In another embodiment, the peptide may be provided with a linker and/or tag which is a peptide and the rigidity and/or flexibility of the linker and/or tag may be modified by the inclusion of one or more amino acids. In another embodiment, the peptide comprises a linker which is a chemical linker selected from the group consisting of a carbohydrate linker, a lipid linker, a fatty acid linker, and a polyether linker. In another embodiment, the peptide comprises a tag that binds to a binding moiety that has been coated on the solid support.
[00155] In an embodiment, a peptide disclosed herein is covalently bound to a solid support. In an embodiment, the peptide is covalently bound to a solid support via the N-terminus of the peptide through the formation of a chemical bond with a primary amine. Such bonds may include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters.
[00156] In an embodiment, the isoelectric point (pl) value of the peptide is determined. The pl is the pH at which the net charge of the peptide is zero. The pl can be calculated from the amino acid sequence based on amino acid residue properties which can be done, e.g., using online tools such as described in the examples herein. In one embodiment, the pl value of the peptide is at least 6. Tn another embodiment, the pl value of the peptide is in the range of 6-12. In a further embodiment, the pl value of the peptide is in the range of 9-12.
[00157] In an embodiment, the solubility of the peptide is determined. The solubility may be determined by visual observation with an unaided eye. For example, by observing a change in opaqueness, color, clarity and/or viscosity, a change in solubility may be observed. In some embodiments, a person of skill in the art may measure the solubility by known methods in the art. [00158] In one embodiment, the peptide is soluble at a concentration of 1.5 mM up to 15mM. Such soluble peptides are useful for coating peptides to a surface. In another embodiment, the peptide is soluble in a buffer suitable for preparation of a solid support comprising said peptide. In a further embodiment, said solubility of the peptide is determined in a physiological buffer. Such suitable buffers can be readily determined by a person skilled in the art based on the desired pH of the final solution. Low pH solutions (<pH 5.5) can be made, for example, in citrate buffer, glycine-HCl buffer, or in succinic acid buffer. High pH solutions can be made, for example, in Tris-HCl, phosphate buffers, or sodium bicarbonate buffers. A number of conditions may be used to determine KD and off-rates for the purpose of determining, for example, optimal pH and/or salt concentrations. In one embodiment, the buffer comprises Phosphate-buffered saline (PBS) and Pluronic F-68.
[00159] As shown in the example section, peptides that were shown to be effective in binding AAV5, and further AAV serotypes, were shown to have a pl in the range of 6-12 and/or have good solubility (++) in a physiological buffer. Without being bound by theory, such properties hence may be indicative of the suitability of peptides for attachment of a solid support and/or efficient binding to parvovirus capsids, such as AAV. In an embodiment, a peptide is selected for screening for binding with parvovirus capsids and/or AAV capsids based on the pl of the peptide.
[00160] In another embodiment, the peptides disclosed herein bind to an adeno-associated virus (AAV) capsid. In an embodiment, the AAV capsid is an AAV capsid from clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I. In another embodiment, the AAV capsid is an AAV capsid from clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, or an engineered variant thereof. In another embodiment, said AAV capsid is selected from, AAVgo.l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid, or an engineered variant thereof. In yet another embodiment, the AAV capsid is selected from AAVgo. l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid, or a chimeric or engineered capsid variant thereof. In an embodiment, the AAV capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid, or an engineered variant thereof. In another embodiment, the AAV capsid is a chimeric AAV capsid. In an embodiment, the AAV capsid is an engineered variant capsid comprising amino acid sequences from at least two different AAV capsid serotypes. In an embodiment, the chimeric AAV capsid is an AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9 chimeric capsid protein.
[00161] In one embodiment, the peptides disclosed herein bind to an adeno-associated virus (AAV) capsid protein. In one embodiment, the AAV capsid protein is a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid protein, or an engineered variant thereof. In an embodiment, the AAV capsid protein is a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid protein, or an engineered variant thereof. In another embodiment, the AAV capsid protein is a chimeric AAV capsid protein. In an embodiment, the AAV capsid protein is an engineered variant capsid protein comprising amino acid sequences from at least two different AAV capsid proteins. In an embodiment, the chimeric AAV capsid is a AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9 chimeric capsid protein. In an embodiment, the AAV capsid protein comprises a VP1 amino acid sequence from AAV2 and a VP2 and VP3 amino acid sequence from AAV5. It is understood that such a chimeric AAV capsid comprises one or more of the viral proteins from a different serotype. For example, the VP1 amino acid sequence from AAV2 and a VP2 and VP3 amino acid sequence from AAV5. In an embodiment, the chimeric AAV capsid protein comprises a VP1 amino acid sequence from a first AAV serotype and a VP2 and VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP2 amino acid sequence from a first AAV serotype and a VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP3 amino acid sequence from a first AAV serotype and a VP2 amino acid sequence from a second AAV serotype. [00162] In an embodiment, the peptide comprises a sequence selected from SEQ ID NOs: 7, 9, 11, and 13, and the peptide binds to a parvovirus capsid, such as AAV capsids, including AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9 capsids.
[00163] In another aspect, provided herein is a solid support comprising a peptide disclosed herein. A person skilled in the art would understand that any column (or column material or the like), to which a peptide, ligand, antibody, or other protein may be attached to may be suitable. In a further embodiment, the solid support is selected from the group consisting of a membrane, a filter, a biosensor, a chip, a slide, a wafer, a fiber, a magnetic or non-magnetic bead, a gel, tubing, a strip, a plate, a rod, a polymer, a particle, a microparticle, a capillary, a column, and a resin. Other suitable solid supports include a chromatographic resin or matrix (e.g. , SEPHAROSE-4 FF agarose beads), the wall or floor of a well in a plastic microtiter dish, a silica-based biochip, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof. In an embodiment, a peptide disclosed herein may be attached on such a support material by a non-covalent association or by covalent bonding, using reagents and techniques known in the art. In a further embodiment, a binding moiety may be coated onto the solid support to facilitate binding or immobilization of the peptide on said solid support.
[00164] In one embodiment, the solid support further comprises a binding moiety. In a further embodiment, the binding moiety of the solid support is an antibody, streptavidin, avidin, neutravidin, or a fragment thereof. In a further embodiment, the binding moiety of the solid support is an antibody, streptavidin, avidin, neutravidin, or a fragment thereof, and the peptide comprises a biotin tag. In yet a further embodiment, the solid support is a biosensor, wherein the biosensor is provided with said binding moiety and the peptide comprises a biotin tag.
[00165] In another aspect, provided herein is a nucleic acid encoding a peptide disclosed herein. It is understood that encoding means that the peptide may be expressed (comprised, e.g., in a propeptide or the like with appropriate cleavage and secretory signal peptide, if needed, and the like). In yet a further aspect, provided herein is vector comprising a nucleic acid encoding a peptide disclosed herein. Such vector may be a viral vector. For example, the vector can be a retrovirus vector, a herpes virus vector, a baculovirus vector, or an adenovirus vector. In an embodiment, a peptide disclosed herein may be chemically synthesized, such as described in the examples, and other methods known in the art. [00166] In a further embodiment provided herein is a composition comprising a peptide disclosed herein.
Methods o f Use
[00167] In another aspect, provided herein is a method of detecting an adeno-associated virus (AAV) particle in a sample, the method comprising: (a) contacting the sample with a peptide disclosed herein, under conditions such that the peptide can form a complex with an AAV particle; and (b) detecting the peptide/ AAV particle complex. In an embodiment, the peptide comprises a detectable label (e.g, a fluorescent label) to allow such detection.
[00168] In an aspect, provided herein is a method for purifying an AAV particle from a sample comprising the AAV particle and at least one contaminant, the method comprising contacting the composition with a peptide disclosed herein or a solid support comprising a peptide disclosed herein under conditions such that the peptide or solid support binds to the AAV particle. [00169] In a further embodiment, the method further comprises washing the peptide or solid support with a wash solution under conditions such that the peptide or solid support remains bound to the AAV particle. In a further embodiment, the method further comprises eluting the AAV particle from the peptide or solid support.
[00170] In an embodiment, at least one contaminant is an AAV particle that lacks a complete vector genome. It is understood that in a typical production process, c.g, utilizing baculovirus vectors and insect cells, or mammalian cell culture in which AAV production plasmids may be transfected, full and empty capsids are produced, and it may be desirable to remove empty capsids and/or separate empty from full capsids. In an embodiment, contaminants can include bulk cellular material, obtained via lysis and/or enzymatic treatments (e.g., DNase) and the like. In yet a further embodiment, the method comprises further formulating the eluted AAV particle in a formulation buffer suitable for administration to a human subject.
[00171] In an embodiment, such methods for purifying include use of a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, and variants thereof. In an embodiment, provided herein is a method for processing a composition comprising a parvovirus or an engineered parvovirus, comprising the steps of:
(i) providing a composition comprising a parvovirus; (ii) contacting said composition with a solid support comprising a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, and variants thereof;
(iii) allowing binding of the parvovirus to the solid support comprising the peptide;
(iv) optionally, washing the solid support comprising the peptide and the bound parvovirus with a buffer; and
(v) eluting the bound parvovirus particles from the solid support with an elution buffer.
[00172] In another embodiment, a method is provided for processing a composition comprising a parvovirus or an engineered parvovirus, comprising the steps of:
(i) providing a composition comprising a parvovirus;
(ii) contacting said composition with a solid support comprising a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13;
(iii) allowing binding of the parvovirus to the solid support comprising the peptide;
(iv) optionally, washing the solid support comprising the peptide and the bound parvovirus with a buffer; and
(v) eluting the bound parvovirus particles from the solid support with an elution buffer. [00173] It is understood that solid supports comprising said peptides are useful for processing of AAV capsids, such as AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9, and/or chimeric and/or engineered variants thereof. Such processing methods are highly useful in manufacturing of AAV gene therapy products and the like. As shown in the example section, the peptides corresponding with or comprising SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, are highly suitable for such methods.
[00174] In one embodiment, provided herein is a peptide comprising the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, for use in isolating a parvovirus, such as AAV. In a further embodiment, provided herein is a peptide comprising the amino acid sequence of SEQ ID NO: 7 for use in isolating a parvovirus, such as AAV. In a further embodiment, provided herein is a peptide comprising the amino acid sequence of SEQ ID NO: 9, for use in isolating a parvovirus, such as AAV. In a further embodiment, provided herein is a peptide comprising the amino acid sequence of SEQ ID NO: 11, for use in isolating a parvovirus, such as AAV. In a further embodiment, provided herein is a peptide comprising the amino acid sequence of SEQ ID NO: 13, for use in isolating a parvovirus, such as AAV. In a further embodiment, the AAV is an AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9 capsid. In an embodiment, the AAV is an engineered AAV capsid comprising AAV capsid proteins from one or more of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9.
[00175] In an aspect, a peptide disclosed herein is for use in binding to a parvovirus, such as AAV. In an embodiment, the AAV is a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo. l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, AAV5.2, or AAV5 capsid. In an embodiment, the AAV is an engineered AAV capsid comprising AAV capsid proteins from one or more of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9. In an embodiment, the AAV capsid protein is an engineered variant capsid protein comprising amino acid sequences from at least two different AAV capsid proteins. In an embodiment, the chimeric AAV capsid is an AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9 chimeric capsid protein. In an embodiment, the AAV capsid protein comprises a VP1 amino acid sequence from AAV2 and a VP2 and VP3 amino acid sequence from AAV5. In an embodiment, the chimeric AAV capsid protein comprises a VP1 amino acid sequence from a first AAV serotype and a VP2 and VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP2 amino acid sequence from a first AAV serotype and a VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP3 amino acid sequence from a first AAV serotype and a VP2 amino acid sequence from a second AAV serotype.
[00176] In one embodiment, provided herein is a peptide comprising the amino acid sequence of SEQ ID NO : 7, SEQ ID NO : 9, SEQ ID NO : 11 , or SEQ ID NO : 13 , for use in binding to a parvovirus, such as AAV. In a further embodiment, provided herein is a peptide comprising the amino acid sequence of SEQ ID NO: 7 for use in binding to a parvovirus, such as AAV. In a further embodiment, provided herein is a peptide comprising the amino acid sequence of SEQ ID NO: 9, for use in binding to a parvovirus, such as AAV. In a further embodiment, provided herein is a peptide comprising the amino acid sequence of SEQ ID NO: 11, for use in binding to a parvovirus, such as AAV. In a further embodiment, provided herein is a peptide comprising the amino acid sequence of SEQ ID NO: 13, for use in binding to a parvovirus, such as AAV. In a further embodiment, the AAV is an AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9 capsid. In an embodiment, the AAV is an engineered AAV capsid comprising AAV capsid proteins from one or more of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, or AAV9.
[00177] In another embodiment, the AAV particle or product is a recombinant AAV (rAAV) comprising an rAAV genome comprising a transgene.
[00178] In one embodiment, the transgene encodes a polypeptide. In another embodiment, the transgene encodes an antibody or a fragment thereof (e.g., an scFv), fusion protein, miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, IncRNA, ribozyme, or mRNA.
[00179] In an embodiment, the rAAV comprises an AAV capsid comprising an AAV capsid protein. In a further embodiment, the AAV capsid protein is from a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, AAV5.2, or AAV5 capsid protein, or an engineered variant thereof. In a further embodiment, the AAV capsid protein is a chimeric AAV capsid protein. In an embodiment, the chimeric AAV capsid protein comprises a VP1 amino acid sequence from a first AAV serotype and a VP2 and VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP2 amino acid sequence from a first AAV serotype and a VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP3 amino acid sequence from a first AAV serotype and a VP2 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein is an AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9 chimeric capsid protein. In an embodiment, the AAV capsid protein comprises a VP1 amino acid sequence from AAV2 and a VP2 and VP3 amino acid sequence from AAV5.
[00180] In one aspect, provided herein is a composition obtained by a method disclosed herein. In an embodiment, an AAV gene therapy product may be highly purified yet comprises a substantial amount of empty capsids and/or capsid proteins. In an embodiment, a peptide disclosed herein is for use in purifying an AAV viral vector product after a first purification. In one embodiment, the composition comprises less than 30% of AAV particles that lack a complete genome. In an embodiment, the percentage of AAV particles that lack a complete genome is determined by Analytical ultracentrifugation or cryo-TEM. [00181] In one embodiment, the composition comprises less than 15% of AAV particles that lack a complete genome. In another embodiment, the composition comprises less than 10% of AAV particles that lack a complete genome.
[00182] In another aspect, provided herein is a pharmaceutical composition comprising purified AAV particles obtained by a method disclosed herein.
[00183] In an aspect, provided herein is a method of screening a peptide for binding to an adeno-associated virus (AAV), comprising the steps of: (a) providing a peptide disclosed herein; (b) providing an AAV; and (c) determining the binding of said peptide with said AAV. In an embodiment, peptides may be designed based on SEQ ID NO: 1 and screened for binding to a capsid. In a further embodiment, the method of screening may further comprise: (a) providing a reference peptide known for binding to said AAV; (b) determining the binding of said reference peptide with said AAV; and (c) comparing the binding of said peptide and said reference peptide with said AAV. Suitable reference peptides that are provided herein include a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7, 9, 11, or 13. In a further embodiment, the method of screening comprises the step of immobilizing the provided peptide on a solid support. In an embodiment, a plurality of peptides may thus be screened for. In one embodiment, the solid support is a biosensor coated with streptavidin and the peptide comprises a biotin tag. Other suitable tags and solid supports are known in the art.
[00184] In a further embodiment, the method of screening comprises label free technology such as bio-layer interferometry or surface plasmon resonance (SPR). The method of screening may comprise determining the dissociation rate constant kd (s'1)) or association rate constant (ka (M'1 s'1)) based on 1 : 1 binding of the AAV with the peptide, or the method of screening comprises determining the dissociation rate constant (kd (s'1)) or association rate constant (ka (M'1 s'1)), which constants are determined based on an assumed one-to-one binding of an AAV capsid with a peptide disclosed herein. Based on the determined constants, the dissociation equilibrium constant KD can be calculated.
[00185] As shown in the example section, the methods of screening may comprise determining binding of the AAV to the peptide, including of full and/or empty capsids of the AAV. By determining the dissociation rate constant and association rate constants for both empty and full capsids, peptides may be identified that allow to differentiate between the two (e.g., either have highly reduced association or have highly reduced dissociation of the empty capsids relative to full capsids (or vice versa)). For example, as shown in the example section herein, for peptides capable of binding AAV5.2 capsids, i.e., SEQ ID NOs: 7, 9, 11, and 13, the dissociation and association rate constants were determined, and the KD was calculated. As can be observed, the dissociation rate constant for SEQ ID NOs: 7 and 13 was much lower for the analyte comprising full AAV5.2 capsids as compared with the analyte comprising capsids without vector genome, resulting in differentiating affinity. This indicates that, from capsid compositions comprising both empty and full capsids, under the conditions tested and without being bound by theory, full capsids will predominantly remain strongly bound to the peptide whereas empty capsids will not substantially. Without being bound by theory, this will allow for capturing, e.g., predominantly full capsids while separating empty capsids therefrom. In contrast, for peptides comprising SEQ ID NOs: 9 and 11, no such or much less differentiating constants were found. Either type of peptide is of use in accordance with the invention. Further peptides that may be contemplated and which can be useful may include peptides that more strongly bind empty capsids as compared with full capsids.
[00186] In an embodiment, peptides as identified in the methods of screening disclosed herein may allow for purification of empty and/or full capsids. In an embodiment, peptides as identified in the methods of screening disclosed herein may allow for purification of full capsids. In yet another embodiment, peptides as identified in the methods of screening disclosed herein may allow for purification of empty and full capsids. For example, one may identify a first peptide to which both full and empty capsids strongly bind. The empty capsids, showing lower affinity, are washed by subjecting the solid support with a suitable buffer, for example pH 2.8. After removing the empty capsids, the full capsids could be removed by further lowering of the pH or modifying the buffer conditions, for example pH 2.0, resulting in a substantially pure fraction. Of course, iterations of such processes may be contemplated as well as variations thereof. In an embodiment, useful methods for purification of AAV capsids and/or AAV products or the like may comprise different consecutive binding and elution strategies utilizing different peptides that have different properties with regard to binding profiles of the AAV capsid or product for which a purification strategy is to be determined, which may also depend on the outcome of screening strategies. In an embodiment, a peptide that is identified as having desirable properties can be modified e.g., by amino acid substitution) in order to obtain desirable properties. [00187] In one embodiment, the method of screening comprises the selection of peptides that have a dissociation rate constant kd in the range of 10'1 to IO'10 s'1.
[00188] In an embodiment, the method of screening comprises the selection of peptides that have an association rate constant (ka (M'1 s'1)) in the range of 103 to 107 M' 1.
[00189] In an embodiment, the method of screening comprises the selection of peptides that differentiate between binding of empty and full capsids, wherein the association rate constants are similar, and the dissociation rate constants differ when comparing the rate constants for empty and full parvovirus, e.g., AAV capsids.
[00190] In one embodiment, the method of screening comprises the identification of a plurality of peptides that are capable of binding AAV. In an embodiment, provided herein is the use of a peptide as identified in a method of screening disclosed herein, for the purification of a parvovirus product, e.g., an AAV vector product for gene therapy.
[00191] In one embodiment, provided herein is a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, including variants thereof. In an embodiment, variants of such peptides have similar solubility and/or isoelectric point properties. In an embodiment, such variants may have similar association rate constants for empty and full capsids, while dissociation rate constants for empty and full capsids differ by at least 100-fold. In an embodiment, the dissociation rate of one of the empty and full capsids is in the range of 10'1 to 10'5 s'1, whereas the other is at least 100-fold lower.
[00192] In one aspect, provided herein is a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13. In an embodiment, the peptide is chemically or recombinantly synthesized. In an embodiment, the peptide is 50, 40, 30, or 20 amino acids in length or less. Such peptides are highly advantageous for binding, processing, and/or purification of AAV gene therapy products.
[00193] In one aspect, provided herein is use of a peptide disclosed herein for binding an AAV, comprising a capsid of serotype clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo. l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, AAV5.2, or AAV5, or a derivative thereof. In an embodiment, the AAV capsid protein is an engineered variant capsid protein comprising amino acid sequences from at least two different AAV capsid proteins. In an embodiment, the chimeric AAV capsid is a AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9 chimeric capsid protein. Tn an embodiment, the AAV capsid protein comprises a VP1 amino acid sequence from AAV2 and a VP2 and VP3 amino acid sequence from AAV5. In an embodiment, the chimeric AAV capsid protein comprises a VP1 amino acid sequence from a first AAV serotype and a VP2 and VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP2 amino acid sequence from a first AAV serotype and a VP3 amino acid sequence from a second AAV serotype. In an embodiment, the chimeric AAV capsid protein comprises a VP1 and VP3 amino acid sequence from a first AAV serotype and a VP2 amino acid sequence from a second AAV serotype. In another aspect as provided herein is a use of a peptide disclosed herein for purification or binding of AAV.
[00194] In another aspect, provided herein is the use of a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13, and variants thereof. In an embodiment, the peptide is chemically or recombinantly synthesized. In an embodiment, the peptide is 50, 40, 30, or 20 amino acids in length or less. Such peptides are highly advantageous for binding, processing, and/or purification of AAV gene therapy products and may allow to differentiate between empty and full capsids.
[00195] In an aspect, provided herein is a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 11. In an embodiment, the peptide is chemically or recombinantly synthesized. In an embodiment, the peptide is 50, 40, 30, or 20 amino acids in length or less. Such peptides are highly advantageous for binding, processing, and/or purification of AAV gene therapy products.
[00196] In an embodiment, the peptide is used in purification of said AAV.
[00197] In an aspect, provided herein is use of a peptide disclosed herein, wherein said use comprises separating full and empty capsids of said AAV.
EXAMPLES
Example 1: Generation of optimized PKD1 -derived peptide
[00198] The entry of adeno-associated virus (AAV) into a cell is determined by the interactions of the AAV with specific surface glycans and a proteinaceous receptor referred to as Adeno-associated virus receptor (AAVR) (also known as KIAA0319L). Thus, AAVR is an essential cellular receptor required for the transduction of cells by multiple AAV serotypes. AAVR is a glycosylated membrane protein that recycles from the plasma membrane to the trans-Golgi network using the cellular endosomal network. AAVR comprises an ectodomain of a MANEC (motif at the N terminus with eight cysteines) domain and five Ig-like domains known as PKD (polycystic kidney disease) domains. AAV is capable of utilizing these PKD domains to facilitate transduction of a cell. Without being bound by theory, it is believed that specifically the PKD domains 1 (PKD1), 2 (PKD2) and 3 (PKD3) within the ectodomain of AAVR are capable of interacting with AAV (Pillay S et al., J Virol. 2017 Aug 24; 91(18): e00391-17).
[00199] A cryo-EM structure (2.5 A) of AAV5 in complex with AAVR (Silveira et al., (2020) Viruses 12(11): 1326) demonstrated that AAV5 binds to the first polycystic kidney disease (PKD1) domain (SEQ ID NO: 14) of the AAV receptor (AAVR). It was found that the PKD1 residues I349-G357 and L372-P374 contain most of the binding interactions and a minimal amino acid sequence in the PKD1 loop (SEQ ID NO: 1) binds to AAV5. In a different study (Zhang et al. 2019, Nature Communications, 10(1), 3760. https://doi.org/10.1038/s41467-019-11668-x), an affinity constant (KD) of 287 nM was determined for WT AAVR in complex with AAV5, and a KD of 7 nM for the AAVR mutant I349A (sequence numbering relative to SEQ ID NO: 14). Based on these data, biotinylated WT PKD1 (SEQ ID NO: 2) and mutant I349A PKD1 (SEQ ID NO: 3) peptides (derived from amino acids 346-360 of PKD1 amino acid sequence (SEQ ID NO: 14)) with a N-terminal 6-aminohexanoic acid (Ahx) linker were synthesized by Genscript. The affinities of these peptides towards AAV5.2 was determined by bio-layer interferometry (BLI) using streptavidin-coated biosensors on the Octet Nl.
[00200] The WT sequence of the PKD1 peptide (SEQ ID NO: 2) was used as input for a systematic mutational analysis in silico for optimal binding to AAV5.2 (Schrodinger software). Based on the mutational analysis, 10 additional PKD1 peptide variants (SEQ ID NOs: 4-13) were generated. These peptides lacked the N-terminal W amino acid residue and were 14 amino acids in length (the 14 amino acid wild-type sequence corresponding therewith is SEQ ID NO: 1) [00201] The optimized peptides where synthesized (Genscript) and a 6-aminohexanoic acid (Ahx) linker and biotinylation were included as N-terminal modifications to allow for immobilization onto a solid support. Peptide molecular weights (MW) were calculated from the molecular weight of biotin (244 g/mol), 6-aminohexanoic acid (131 g/mol), and the individual peptide molecular weight, while taking the elimination of two water molecules into account during the coupling reaction (-2x 18 g/mol). Peptide MW and isoelectric point (pl) values were calculated using the online tool at https://web.expasy.org/computej3i/. Solubility was assessed qualitatively by inspection of visible, undissolved particles during the preparation of stock solutions. Peptides that were readily soluble are indicated with ++ and peptides that required mixing by vortex are indicated with + in Table 3.
[00202] Amino acid sequences of peptides with their respective MW, pl and solubility are set forth in Table 3 below.
Table 3. List of the peptides
Figure imgf000045_0001
Example 2: Preparation o f custom biosensors
[00203] Initial method development for the preparation of custom biosensors and the determination of affinity constants in a kinetics experiment were performed on an Octet N1 using a biotinylated anti-AAV5 antibody as ligand (PRAAV5, ELISA kit, Progen) and AAV5.2 CAG- GFP as analyte.
[00204] Ligand load optimization: Anti-AAV5 antibody (PRAAV5 ELISA kit, Progen) was reconstituted in 750 pL of buffer (lx DPBS + 0.001% Pluronic F-68), and 1- to 64-fold serial dilutions were prepared. AAV5.2 CAG-GFP was used as analyte at a concentration of 5xl013 cp/mL (83 nM) based on Stunner measurements. The kinetics experiment was executed using the following steps: Initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed.
[002051 Kinetics experiment PRAAV5: The anti-AAV5 antibody stock solution (PRAAV5 ELISA kit, Progen) was used undiluted as “ligand” during ligand association based on results from the above ligand load optimization. 1- to 64-fold serial dilutions of 2.4xlOlj cp/mL of AAV5.2- empty (empty capsid) and 5xl013 cp/mL of AAV5.2-CAG-GFP (full capsid) were prepared as “analyte” in lx DPBS + 0.001% Pluronic F-68. The kinetics experiment was executed using the following steps: initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed. Data analysis: step corrections were applied for “Start of association” and “Start of dissociation”, and curve fitting was performed using a “1 : 1 binding model” and “Global” fitting.
[00206] The results shown in FIG. 1 demonstrate that the biotinylated ligand was successfully immobilized onto a streptavidin-coated biosensor, based on the increase in optical signal (nm) during ligand loading (60-360 s), with only minimal decrease during a subsequent wash step (360-420 s). Equilibrium conditions could not be reached within the maximum association time of 300 seconds (due to evaporation) for the lx ligand stock. Therefore, the undiluted antibody stock was used with an association time of 300 s for the preparation of biosensors. At low ligand loadings (e.g., run 3), a low association of AAV5.2-CAG-GFP was observed (420-720 s), excluding any relevant levels of AAV that could be bound to the biosensor surface in an unspecific manner.
[00207] The results shown in FIG. 2 demonstrate the association (420-720 s) and dissociation (720-1020 s) of different AAV5.2-CAG-GFP analyte concentrations (12-93 nM) using biosensors prepared from the undiluted PRAAV5 antibody stock to determine ka, kd, and KD. The results show that the custom biosensors were successfully prepared and used for determining the kinetic constants that characterize the interactions between the PRAAV5 antibody and AAV5.2-CAG-GFP as a benchmark (see Table 3).
[00208] The results shown in FIG. 3 demonstrate the association (420-720 s) & dissociation (720-1020 s) of different AAV5.2 (empty capsid) analyte concentrations (0.6-33 nM) using biosensors prepared from the undiluted PRAAV5 antibody stock to determine ka, kd, and KD. The results show that the custom biosensors were successfully prepared and used for determining the kinetic constants that characterize the interactions between the PRAAV5 antibody and AAV5.2 (empty capsid) as a benchmark (see Table 3).
Example 3: Optimization of the ligand loading conditions for the biosensors.
[00209] The above method was subsequently followed to determine suitable conditions for the immobilization of biotinylated WT PKD1 (SEQ ID NO: 2) onto the biosensor surface in a ligand loading scout.
[00210] WT PKD1 (SEQ ID NO: 2) ligand scout loading: A 5mM stock solution of WT PKD1 (SEQ ID NO: 2) was prepared in lx DPBS + 0.001% Pluronic F-68, from which additional dilutions were prepared in the same buffer (lOOx, l’000x, 2’000x). These ligand solutions were used for studying the preparation of biosensors, with AAV5.2-GFP as analyte at 2xl013 cp/mL to subsequently confirm functionality of the prepared biosensors. The kinetics experiment was executed using the following steps: Initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed. Custom biosensors were then prepared for all 12 peptides (SEQ ID NOs: 2 - 13) under these conditions. The results shown in FIG.4 demonstrate successful loading of WT PKD1 (SEQ ID NO: 2) onto the biosensor. Overloading of the biosensor with ligand resulted in additional (unspecific) binding of the peptide ligand (e.g., run 1, c = 5 mM), which was washed off during the “baseline 2” step between 360 to 420 seconds. Non-specific binding of the ligand should be kept to a minimum to minimize any possible interference from residual free ligand during the experiment. Further, equilibrium conditions (i.e., reaching the plateau) should be established during ligand loading to increase the reproducibility of biosensor preparation. Run 2 (c = 50 pM) satisfied both criteria and was therefore chosen as a suitable target concentration range for the preparation of biosensors to screen binding interactions between AAV5.2-CAG-GFP and peptides 1-13.
Example 4: Screening of the peptides for binding to AAV5.2-CAG-GFP
[00211] All 12 peptides (SEQ ID NOs: 2 - 13) were loaded onto biosensors and subjected to an initial screening of binding interactions with AAV5.2-CAG-GFP.
[00212] Initial screening of SEQ ID NOs: 2 - 13 for AAV5.2-CAG-GFP binding: Peptide stock solutions (5 mM) were prepared in lx DPBS + 0.001% Pluronic F-68. For less soluble peptides (see Table 2), peptide stocks were prepared as saturated solutions by vortexing for 5 minutes. The stock solutions were then diluted 100-fold in lx DPBS + 0.001% Pluronic F-68 and used for ligand loading. AAV5.2-CAG-GFP was used as analyte at 5xl013 cp/mL to identify binding interactions. The kinetics experiment was executed using the following steps: Initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed. The top four peptides that showed the highest responses were subjected to a more detailed ligand loading scout, and their affinity constants were determined towards both AAV5.2-CAG-GFP (full capsid) and AAV5.2 (empty capsid). The results shown in FIG. 5 demonstrate the response in optical shift (1- 5.5 nm), confirming successful immobilization of all peptide ligands onto the biosensor surface, and equilibrium conditions were achieved in all cases during the ligand loading phase (60 - 120 s). Minor amounts of non-specifically bound ligand were removed during the wash phase (120 - 180 s). These results demonstrate that peptide 7, peptide 9, peptide 11, and peptide 13 have a high affinity for AAV5.2-CAG-GFP.
Example 5: Screening of the peptides for binding to different AAV Serotype
[00213] The four peptides that showed the best results from the initial screening (peptide 7, peptide 9, peptide 11, and peptide 13) were subsequently screened for binding interactions with additional AAV serotypes, AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, and AAV9 (all including the transgene: CAG-GFP). The AAV serotypes were commercially produced, and purification was done by two rounds of gradient density centrifugation (CsCl) followed by buffer exchange into lx PBS + 0.001% Pluronic F-68 for AAV1, AAV3, AAV4, AAV6, AAV8, and AAV9, while AAV2 additionally contained 100 mM of sodium citrate. All the serotypes were titered and diluted to a final titer of 2xl013 vg/mL. Biosensors were prepared from peptide stock solutions of peptide 7, peptide 9, peptide 11, and peptide 13 at 25-100 pM in lx DPBS + 0.001% Pluronic F-68. The AAV serotypes were resuspended by pipetting and used undiluted (2xl013 vg/mL) as analyte. The kinetics experiment was executed using the following steps: initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed.
[00214] The results shown in FIG. 6 demonstrate that peptide 7 strongly binds to AAV3, AAV4, AAV6, AAV8, and AAV9 and moderately binds to AAV1. The results shown in FIG. 7 demonstrate that peptide 9 strongly binds to AAV3, AAV4, AAV6, AAV8, and AAV9. The results shown in FIG. 8 demonstrate that peptide 11 strongly binds to AAV3, AAV4, AAV6, AAV8, and AAV9. The results shown in FIG. 9 demonstrate that peptide 13 strongly binds to AAV3, AAV4, AAV6, AAV8, and AAV9.
[00215] These results demonstrate that these peptides not only bind to AAV5.2 capsids but are also highly efficient in binding capsids of other serotypes, such as AAV3, AAV4, AAV6, AAV8, and AAV9. Thus, these peptides can be useful for binding to and purifying AAV capsids from various AAV serotypes.
Example 6: Screening of the peptides for bindins to AAV5.2 (empty capsid) and AAV5.2-CAG- GFP (full capsid)
[00216] A challenge in AAV manufacturing for clinical use is purifying the AAV capsids containing a complete genome from the AAV capsids containing no genome or an incomplete genome (empty capsids). Thus peptide 7, peptide 9, peptide 11, and peptide 13 were analyzed for their ability to distinguish between full and empty capsids.
[00217] Determination of kinetic rate constants. Biosensors were prepared from peptide stock solutions of peptide 7, peptide 9, peptide 11, and peptide 13 at 25 pM in lx DPBS + 0.001% Pluronic F-68. AAV5.2 (empty capsid) and AAV5.2-CAG-GFP (full capsid) were commercially produced and resuspended by pipetting after thawing. Serial dilutions of lx, 2x, 4x, 8x, 16x, 32x, and 64x were prepared in the same buffer, and the titers where calculated based on Stunner measurements. The kinetics experiment was executed using the following steps: Initial baseline (60 s), ligand loading (300 s), baseline (60 s), analyte association (300 s), dissociation (300 s) using Super-Streptavidin Biosensors (SSA), and 2’200 rpm as shaker speed. Data analysis', step corrections were applied for “Start of association” and “Start of dissociation”, and curve fitting was performed using a “1:1 binding model” and “Global” fitting. The results shown in FIG. 10, FIG. 11, FIG. 12, and FIG. 13 demonstrate the response curves for binding of AAV5.2 (empty capsid) and AAV5-CAG-GFP (full capsid) to peptide 7, peptide 9, peptide 11, and peptide 13 respectively. This was used for determining the kinetic constants (Table 4). Global curve fitting was applied during data analysis, and curve fits are shown for each curve. The calculated kinetic constants (Table 4) show that peptide 7 and peptide 13 have significantly higher affinity (lower affinity constant (KD) value) for AAV5.2-CAG-GFP (full capsids) as compared to the binding of these peptides to AAV5.2 (empty capsid). The difference in affinity constants (KD) primarily originated from lower dissociation rate constants kd of AAV5.2-CAG-GFP (full capsids). Thus, peptides 7 and 13 are able to differentiate between empty AAV capsids and full AAV capsids.
Table 4. Kinetic rate constants obtained with different ligands and AAV analytes.
Figure imgf000050_0001
* * *
[00218] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
[00219J All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
[00220] Other embodiments are within the following claims.

Claims

1. A peptide comprising an amino acid sequence of at least 5 amino acids in length derived from SEQ ID NO: 1, wherein
(a) the peptide is 50 amino acids or less in length; and/or
(b) the amino acid sequence derived from SEQ ID NO: 1 has at least one amino acid insertion, deletion, or substitution compared to the corresponding amino acid in SEQ ID NO: 1.
2. The peptide of claim 1, wherein the peptide is 50 amino acids or less in length.
3. The peptide of claim 1, wherein the peptide is 40 amino acids or less in length.
4. The peptide of claim 1, wherein the peptide is 20 amino acids or less in length.
5. The peptide of claim 1, wherein the peptide is 14 amino acids or less in length.
6. The peptide of any one of claims 1-5, wherein the peptide comprises 1-10 amino acid substitution(s) relative to the corresponding amino acids of SEQ ID NO: 1.
7. The peptide of claim 6, wherein at least one amino acid substitution is a conservative substitution.
8. The peptide of any one of claims 1-7, wherein the amino acid sequence derived from SEQ ID NO: 1 comprises 1-10 amino acid insertion(s) relative to the corresponding amino acids of SEQ ID NO: 1.
9. The peptide of any one of claims 1-8, wherein the amino acid sequence derived from SEQ ID NO: 1 comprises 1, 2, or 3 amino acid deletions relative to the corresponding amino acids of SEQ ID NO: 1.
10. A peptide comprising an amino acid sequence of 14 contiguous amino acids, wherein the amino acids at positions 3, 6, 7, 9, and 13 are I, P, R, Y, and M, respectively.
11. The peptide of claim 10, wherein the amino acids at positions 1, 3, 4, 6, 7, 8, 9, 12, and 13 are R, I, Y, P, R, W, Y, R, and M, respectively.
12. The peptide of claim 10, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 15.
13. The peptide of claim 10, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 16.
14. The peptide of any one of claims 1-13, wherein the peptide comprises one of the following amino acid sequences: QKI, QRI, RLI, RKI, ITH, IYY, IYH, ITHPR (SEQ ID NO: 17), IYHPR (SEQ ID NO: 18), IYYPR (SEQ ID NO: 19), RWY, RDY, DYS, WYS, WYM, WYS, DYM, YSG, YMK, YMG, GEM, KRM, GRM, MGE, MGR, MKR, SGR, SGE, RMF, EME, or RME.
15. The peptide of any one of claims 10-14, wherein the peptide comprises:
(a) QKI, QRI, RLI, or RKI at amino acid position 1-3, or
(b) ITH, IYY, or IYH at amino acid position 3-5, or
(c) ITHPR (SEQ ID NO: 17) or IYHPR (SEQ ID NO: 18), or IYYPR (SEQ ID NO: 19) at amino acid position 3-7, or
(d) RWY or RDY at amino acid position 7-9, or
(e) DYS, WYS, WYM, WYS, or DYM at amino acid position 8-10, or
(f) YSG, YMK, or YMG at amino acid position 9-11, or
(g) GEM, KRM, or GRM at amino acid position 11-13, or
(h) MGE, MGR, MKR, SGR, or SGE at amino acid position 10-12, or
(i) RMF, EME, or RME at amino acid position 12-14.
16. A peptide comprising an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, wherein:
(a) the amino acid in the peptide corresponding to amino acid 3 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is I;
(b) the amino acid in the peptide corresponding to amino acid 6 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is P;
(c) the amino acid in the peptide corresponding to amino acid 7 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is R;
(d) the amino acid in the peptide corresponding to amino acid 9 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is Y; and/or
(e) the amino acid in the peptide corresponding to amino acid 13 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 is M.
17. A peptide comprising an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 7, wherein the amino acids in the peptide corresponding to amino acids 3, 6, 7, 9, and 13 of SEQ ID NO: 7 are I, P, R, Y, and M, respectively.
18. The peptide of claim 17, wherein:
(a) the amino acid in the peptide corresponding to amino acid 1 of SEQ ID NO: 7 is Q or R;
(b) the amino acid in the peptide corresponding to amino acid 2 of SEQ ID NO: 7 is K, R, or L;
(c) the amino acid in the peptide corresponding to amino acid 4 of SEQ ID NO: 7 is T or Y; (d) the amino acid in the peptide corresponding to amino acid 5 of SEQ ID NO: 7 is H or Y;
(e) the amino acid in the peptide corresponding to amino acid 8 of SEQ ID NO: 7 is D or W; f) the amino acid in the peptide corresponding to amino acid 10 of SEQ ID NO: 7 is S or M; g) the amino acid in the peptide corresponding to amino acid 11 of SEQ ID NO: 7 is G or K; h) the amino acid in the peptide corresponding to amino acid 12 of SEQ ID NO: 7 is E or R; and/or i) the amino acid in the peptide corresponding to amino acid 14 of SEQ ID NO: 7 is E or F.
19. The peptide of claim 17 or 18, wherein the amino acids in the peptide corresponding to amino acids 1, 3, 4, 6, 7, 8, 9, 12, and 13 of SEQ ID NO: 7 are R, I, Y, P, R, W, Y, R, and M, respectively.
20. The peptide of claim 17, wherein the peptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
21. A peptide comprising an amino acid sequence that comprises at least 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
22. The peptide of claim 21, wherein the peptide comprises amino acids 5-10 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
23. The peptide of claim 21, wherein the peptide comprises amino acids 4-11 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
24. The peptide of claim 21, wherein the peptide comprises amino acids 3-12 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
25. The peptide of claim 21, wherein the peptide comprises amino acids 2-13 of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
26. A peptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a variant thereof comprising 1-7 amino acid changes.
27. The peptide of claim 26, wherein the peptide comprises 1-7 amino acid substitution(s) relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
28. The peptide of claim 27, wherein at least one amino acid substitution is a conservative substitution.
29. The peptide of claim 26, wherein the peptide comprises 1-7 amino acid insertion(s) relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
30. The peptide of claim 26, wherein the peptide comprises 1-7 amino acid deletion(s) relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
31. The peptide of claim 30, wherein at least one amino acid deletion is at the N-terminus of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
32. The peptide of claim 30, wherein at least one amino acid deletion is at the C-terminus of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
33. The peptide of claim 26, comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
34. The peptide of claim 26, consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.
35. The peptide of any one of the preceding claims, wherein the peptide is connected to a linker and/or a tag.
36. The peptide of claim 35, wherein the linker or tag is at the N-terminus of the peptide.
37. The peptide of claim 35, wherein the linker or tag is at the C-terminus of the peptide.
38. The peptide of any one of claims 35-37, wherein the linker is selected from the group consisting of Beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5- aminovaleric acid (Ava), a 6-aminohexanoic acid (Ahx), and polyethylene glycol (PEG) linkers, or a combination thereof.
39. The peptide of any one of claims 35-37, wherein the tag is selected from the group consisting of biotin, 6His, lOHis, FLAG, V5, HA, His, GST (glutathione S-transferase), maltose binding protein (MBP), GFP, and cMYC.
40. The peptide of any one of the preceding claims, wherein the isoelectric point (pl) value of the peptide is at least 6.
41. The peptide of claim 40, wherein the pl is in the range of 6-12.
42. The peptide of claim 40, wherein the pl is in the range of 9-12.
43. The peptide of any one of the preceding claims, wherein the peptide is soluble at a concentration of 1.5 mM up to 15mM.
44. The peptide of claim 43, wherein the solubility is determined in a physiological buffer.
45. The peptide of any one of the preceding claims, wherein the peptide binds to an adeno- associated virus (AAV) capsid protein.
46. The peptide of claim 45, wherein the AAV capsid protein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, or an engineered variant thereof.
47. The peptide of claim 45, wherein the AAV capsid protein is a chimeric AAV capsid protein.
48. The peptide of claim 47, wherein the chimeric AAV capsid protein is AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9.
49. A solid support comprising the peptide of any one of the preceding claims.
50. The solid support of claim 49, wherein the solid support is selected from the group consisting of a membrane, a fdter, a biosensor, a chip, a slide, a wafer, a fiber, a magnetic or non-magnetic bead, a gel, tubing, a strip, a plate, a rod, a polymer, a particle, a microparticle, a capillary, a column, and a resin.
51. The solid support of claim 49 or 50, wherein the solid support further comprises a binding moiety.
52. The solid support of claim 51, wherein the binding moiety is an antibody, streptavidin, avidin, neutravidin, or a fragment thereof.
53. The solid support of claim 50, wherein the solid support is a biosensor and the peptide comprises a biotin tag.
54. A nucleic acid encoding the peptide of any one of claims 1-48.
55. A vector comprising the nucleic acid of claim 54.
56. The vector of claim 55, wherein the vector is a viral vector.
57. The vector of claim 56, wherein the vector is a retrovirus vector, a herpes virus vector, a baculovirus vector, or an adenovirus vector.
58. A composition comprising the peptide of any one of claims 1-48.
59. A method of detecting an adeno-associated virus (AAV) particle in a sample, the method comprising:
(a) contacting the sample with the peptide of any of one of claims 1-48, under conditions such that the peptide can form a complex with an AAV particle; and
(b) detecting the peptide/ AAV particle complex.
60. A method for purifying an AAV particle from a sample comprising the AAV particle and at least one contaminant, the method comprising contacting the sample with the peptide of any one of claims 1-48 or the solid support of any one of claims 49-53 under conditions such that the peptide or solid support binds to the AAV particle.
61. The method of claim 60, wherein the method further comprises: washing the peptide or solid support with a wash solution under conditions such that the peptide or solid support remains bound to the AAV particle.
62. The method of claim 60 or 61, wherein the method further comprises eluting the AAV particle from the peptide or solid support.
63. The method of any one of claims 60-62, wherein at least one contaminant is an AAV particle that lacks a complete genome.
64. The method of claim 62, wherein the method further comprises formulating the eluted AAV particle in a formulation buffer suitable for administration to a human subject.
65. The method of any one of claims 60-64, wherein the AAV particle is a recombinant AAV (rAAV) comprising an rAAV genome comprising a transgene.
66. The method of claim 65, wherein the transgene encodes a polypeptide.
67. The method of claim 66, wherein the transgene encodes an antibody or a fragment thereof, fusion protein, miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, IncRNA, ribozyme, or mRNA.
68. The method of any one of claims 65-67, wherein the rAAV comprises an AAV capsid comprising an AAV capsid protein.
69. The method of claim 68, wherein the AAV capsid protein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or an engineered variant thereof.
70. The method of claim 68, wherein the AAV capsid protein is a chimeric AAV capsid protein.
71. The method of claim 70, wherein the chimeric AAV capsid protein is AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8, or AAV2/9.
72. A composition obtained by the method of any one of claims 60-71, comprising less than 30% of AAV particles that lack a complete genome.
73. A composition obtained by the method of any one of claims 60-71, comprising less than 15% of AAV particles that lack a complete genome.
74. A composition obtained by the method of any one of claims 60-71, comprising less than 10% of AAV particles that lack a complete genome.
75. A pharmaceutical composition comprising a purified AAV particle obtained by the method of any one of claims 60-71.
76. A method of screening a peptide for binding to an adeno-associated virus (AAV), comprising the steps of:
(a) providing a peptide as defined in any of claims 1-48; (b) providing an AAV; and
(c) determining the binding of said peptide with said AAV.
77. The method of claim 76, wherein the method further comprises:
(a) providing a reference peptide known for binding to said AAV;
(b) determining the binding of said reference peptide with said AAV; and
(c) comparing the binding of said peptide and said reference peptide with said AAV.
78. The method of claim 76 or 77, wherein said peptide is immobilized on a solid support.
79. The method of claim 78, wherein said solid support is as defined in claims 49-53.
80. The method of claim 78, wherein said solid support comprises a biosensor coated with streptavidin and wherein said peptide comprises a biotin tag.
81. The method of any one of claims 76-80, wherein said method of screening comprises label free technology such as bio-layer interferometry or surface plasmon resonance (SPR).
82. The method of claim 76, wherein binding of the AAV is determined for full and/or empty capsids of the AAV.
83. The method of any one of claims 76-82, wherein the dissociation rate constant ka (s'1)) and/or association rate constant (ka (M'1 s'1)) is determined based on 1 : 1 binding of the AAV with the peptide.
84. The method of any one of claims 76-83, wherein the dissociation equilibrium constant KD is determined.
85. The method of any one of claims 83-84, wherein peptides are selected that have a dissociation rate constant ka in the range of 10'1 - 10'7 s'1.
86. The method of any one of claims 83-85, wherein peptides are selected that have an association rate constant ka (M'1 s'1)) in the range of 103 - 107 M^s'1.
87. The method of any one of claims 76-86, wherein peptides are selected that differentiate between binding of empty and full capsids.
88. The method of any one of claims 76-87, wherein a plurality of peptides are screened for and peptides capable of binding AAV are identified.
89. Use of a peptide as identified in any of one of claims 76-88, for the purification of an AAV particle.
90. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, and variants thereof.
91. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.
92. Use of a peptide as defined in claim 90 or claim 91, for binding an AAV capsid of serotype AAV3, AAV4, AAV5, AAV5.2, AAV6, AAV8, or AAV9, or a derivative thereof.
93. Use of a peptide in accordance with claim 92, in purification of said AAV.
94. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13, and variants thereof.
95. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 11.
96. Use of a peptide as defined in claims 94 and 95 for binding an AAV, comprising a capsid of serotype AAV5, or a derivative thereof.
97. Use of a peptide as defined in claim 96, in purification of said AAV.
98. Use of a peptide as defined in claim 97, wherein said use comprises separating full and empty capsids of said AAV.
99. A method of processing a composition comprising an AAV, comprising the steps of
(a) providing a composition comprising an AAV;
(b) contacting said composition with a solid support comprising a peptide as defined in claim 94 or claim 95;
(c) allowing binding of the parvovirus to the solid support comprising the peptide;
(d) optionally, washing the solid support comprising the peptide and the bound parvovirus with a buffer; and
(e) eluting the bound parvovirus particles from the solid support with an elution buffer.
100. A composition comprising a purified AAV using the method according to claim 99.
PCT/IB2023/000676 2022-11-07 2023-11-07 Peptides for affinity purification of adeno-associated virus WO2024100450A2 (en)

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