WO2024059686A2 - Squelettes d'arpine et échafaudages d'imagerie par microscopie électronique rigidifiée - Google Patents

Squelettes d'arpine et échafaudages d'imagerie par microscopie électronique rigidifiée Download PDF

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WO2024059686A2
WO2024059686A2 PCT/US2023/074156 US2023074156W WO2024059686A2 WO 2024059686 A2 WO2024059686 A2 WO 2024059686A2 US 2023074156 W US2023074156 W US 2023074156W WO 2024059686 A2 WO2024059686 A2 WO 2024059686A2
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seq
protein
subunit
amino acid
darpin
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PCT/US2023/074156
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WO2024059686A3 (fr
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Todd Yeates
Roger CASTELLS GRAELLS
Kyle MEADOR
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The Regents Of The University Of California
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
    • G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
    • G01N23/2251—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion using incident electron beams, e.g. scanning electron microscopy [SEM]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
    • G01N23/2202—Preparing specimens therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2223/00—Investigating materials by wave or particle radiation
    • G01N2223/60—Specific applications or type of materials
    • G01N2223/612—Specific applications or type of materials biological material

Definitions

  • Cryo-electron microscopy is a rapidly expanding method for determining the atomic structures of large molecular assemblies. It is, however, not suitable for determining the structures of small-to-medium sized molecules. A size of about 40-50 kDa. For comparison, the average eukaryotic protein chain is about 35 kDa in mass, while bacterial proteins are generally smaller.
  • Nanobodies, antibody fragments, and DARPins have been employed as scaffolds with limited resolution, e.g., 3.8 ⁇ for a 27 kDa cargo protein; 2.49 ⁇ for a cargo protein of about 250 kDa; 3.03 ⁇ for a 57 kDa cargo protein; about 2.7 ⁇ for a cargo protein of about 52 kDa.
  • the finest resolution achieved thus far in the prior art is about 3.5 ⁇ for a 22 kDa cargo protein.
  • the invention is directed to a protein comprising (1) X1-X2-X3-X4-V-X5-X6-L-L-A-X7-G-A-D-V-N (SEQ ID NO: 1) wherein X1 is Y, Q, I or D, preferably Y, Q, or I, more preferably, Y or Q, most preferably Y; X2 is D or K, preferably D; X3 is D or Y, preferably D; X4 is Q, E, or A, preferably Q or E, more preferably Q; X5 is A or D, preferably A; X6 is A or W, preferably A; X7 is K or L, preferably K; and wherein when X5 is D then X6 is A, and when X6 is W
  • the invention is directed to a protein comprising or consisting of K-K-L-L-E-A-X-X-X-X-X-X1-X2-X3-X4-V-X5-X6-L-L-A-X7-G-A-D-V-N- X-X-X-X-X-X-P-L-X-L-A-X-X-X-X-H-X8-E-I-V-X9-V-L-L-X10-R-G-X11-D- X12-X13-X-X-X-X-X-X-P-L-H-L-A-X-X-X-X-X-H-L-E-I-X14-E-X15-L-L-X16- X17-G-A-D-V-N (SEQ ID NO: 4) wherein X1 is Y, Q, I or D, preferably Y, Q,
  • the protein comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 51 to SEQ ID NO: 71. In some embodiments, the protein comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 17. [0017] In some embodiments, the invention is directed to a fusion protein comprising or consisting of the protein as described above, e.g., having SEQ ID NO: 4, linked to a subunit protein of a self-assembling protein cage.
  • the fusion protein comprises a protein having SEQ ID NO: 4 fused to a subunit protein of a self- assembling protein cage, said subunit protein comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18.
  • the protein is fused to the subunit protein via a peptide linker.
  • the peptide linker is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid residues long.
  • the peptide linker is not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long.
  • the peptide linker (a) is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid residues long, and (b) not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long.
  • the peptide linker joins the N-terminus of the protein to the C-terminus of the subunit protein.
  • the peptide linker forms an alpha-helical structure.
  • the peptide linker has an amino acid sequence comprising KEELD (SEQ ID NO: 37), PAPAP (SEQ ID NO: 38), KEELD (SEQ ID NO: 39), KDELD (SEQ ID NO: 40), RDERN (SEQ ID NO: 41), an (E(A)3K)n motif, an (E4(R/K)4)n motif, or an (AP)n motif.
  • the subunit protein comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a subunit sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 36.
  • the subunit protein comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18.
  • the fusion protein comprises or consists of a sequence selected from the group consisting of SEQ NO: 42 to SEQ ID NO: 50.
  • the invention is directed to a protein particle comprising multiple copies of a fusion protein as described herein, e.g., a fusion protein comprising or consisting of the protein as described above, e.g., having SEQ ID NO: 4, linked to a subunit protein of a self-assembling protein cage.
  • the fusion protein comprises a protein having SEQ ID NO: 4 fused to a subunit protein of a self- assembling protein cage, said subunit protein comprises or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18.
  • the protein is fused to the subunit protein via a peptide linker.
  • the peptide linker (a) is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid residues long.
  • the peptide linker is not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long. In some embodiments, the peptide linker joins the N-terminus of the protein to the C-terminus of the subunit protein. In some embodiments, the peptide linker forms an alpha-helical structure.
  • the peptide linker has an amino acid sequence comprising KEELD (SEQ ID NO: 37), PAPAP (SEQ ID NO: 38), KEELD (SEQ ID NO: 39), KDELD (SEQ ID NO: 40), RDERN (SEQ ID NO: 41), an (E(A) 3 K)n motif, an (E 4 (R/K) 4 )n motif, or an (AP)n motif.
  • the subunit protein comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a subunit sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 36.
  • the subunit protein comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18.
  • the fusion protein comprises or consists of a sequence selected from the group consisting of SEQ NO: 42 to SEQ ID NO: 50.
  • the protein particle further comprises one or more additional subunit proteins of the self-assembling protein cage, wherein the one or more additional subunit proteins lack a DARPin backbone having SEQ ID NO: 4.
  • the one or more additional subunit proteins comprise or consist of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a subunit sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 36.
  • the one or more additional subunit proteins comprise or consist of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the one or more additional subunit proteins have an amino acid sequence that differs from subunit protein that is fused to a DARPin backbone having SEQ ID NO: 4.
  • the present invention is directed to a method of assaying the structure of a target protein of interest which comprises binding the target protein to the protein particle as described herein to result in a complex, and obtaining an image of the complex.
  • the target protein is bound to a moiety such as a solid substrate (e.g., a bead), another protein, or a chemical (e.g., a drug).
  • the image is obtained by cryo-electron microscopy.
  • the methods comprise assaying the structure of the target protein of interest when bound to a given ligand (e.g., another protein or chemical such as a drug of interest), which comprise forming a complex comprising the given ligand bound to the target protein and the fusion protein bound to the given ligand and/or the target protein; and obtaining a cryo-electron micrograph of the complex.
  • the methods further comprise comparing the cryo-electron micrograph to a reference cryo- electron micrograph, said reference cryo-electron micrograph is of (a) a complex comprising the fusion protein bound to the target protein, said complex excludes the presence of the given ligand or another ligand bound thereto; (b) a complex comprising (i) the given ligand bound to a mutant of the target protein and (ii) the fusion protein bound to the given ligand and/or the mutant, said mutant having at least one amino acid substitution, deletion, or addition as compared to the target protein; or (c) a complex comprising (i) a second ligand bound to the target protein and (ii) the fusion protein bound to the given ligand and/or the target protein, wherein said second ligand is different the given ligand.
  • the fusion protein is provided in the form of a REMIS cage as described herein.
  • Figure 1 Schematically shows the structure of a REMIS cage.
  • FIG. 2 Schematically shows the structure of a prior art cage protein having DARPins attached thereto, which DARPins lack a DARPin backbone comprising SEQ ID NO: 4.
  • Figure 3 Cryo-EM micrograph of imaging scaffold bound to KRas (left), 2D classes from the selected particles (right).
  • Figure 4 Cryo-EM imaging of KRas bound to the drug molecule AMG510 while bound to the REMIS cage RCG-33. The molecular model is shown on the left. A surface rendering of the 3-D cryo-EM density map that formed the basis for the molecular model is shown on the right.
  • DARPin backbones Disclosed herein are DARPin backbones. DARPins having DARPin backbones as described herein are capable of forming multimers when in close proximity. Also disclosed herein are self-assembling protein cages which comprise the DARPin backbones. Adjacent DARPin backbones on the protein cages form multimers and thereby “rigidify” the protein cage structure.
  • the rigidified protein cages when used as a scaffold for Cyro-EM imaging, the rigidified protein cages enable 3 ⁇ resolution imaging of proteins smaller than 20 kDa.
  • the rigidified protein cages are referred to herein as Rigid Electron Microscopy Imaging Scaffold cages (“REMIS cages”) and the fusion proteins that comprise a DARPin backbone fused to a subunit of a self- assembling protein cage are referred to herein as “REMIS subunits”.
  • REMIS cages Rigid Electron Microscopy Imaging Scaffold cages
  • REMIS subunits fusion proteins that comprise a DARPin backbone fused to a subunit of a self- assembling protein cage.
  • the exemplified REMIS cages present 12 copies of the target cargo, thereby tending to provide EM images with very large numbers of individual views of the cargo protein.
  • the high symmetry of the REMIS cage means that an individual particle (REMIS cage plus its bound target cargo) presents the target cargo in 12 different orientations, thereby strongly mitigating preferred orientation effects that challenge many cryo-EM studies.
  • DARPIN BACKBONES have an amino acid sequence that comprises (1) X1-X2-X3-X4-V-X5-X6-L-L-A-X7-G-A-D-V-N (SEQ ID NO: 1) wherein X1 is Y, Q, I or D, preferably Y, Q, or I, more preferably, Y or Q, most preferably Y; X2 is D or K, preferably D; X3 is D or Y, preferably D; X4 is Q, E, or A, preferably Q or E, more preferably Q; X5 is A or D, preferably A; X6 is A or W, preferably A; X7 is K or L, preferably K; and wherein when X5 is D then X6 is A, and when X6 is W then X7 is L; (2) H-X8-E-I-V-X9-V-L-L-X10-R-
  • amino acid residues X5, X6, 9L of SEQ ID NO: 1, X7, 11R of SEQ ID NO: 2, X15, X16, and X17 are key in the formation of multimers
  • amino acid residues 1Y and 4Q of SEQ ID NO: 1, and 15I of SEQ ID NO: 2 improve multimer formation and/or multimer stability or rigidity
  • amino acid residues X8 and X14 are based on variability at corresponding locations in known DARPins, and are not expected to disrupt multimer formation, stability, or rigidity.
  • the DARPin backbones have an amino acid sequence that comprises or consists of K-K-L-L-E-A-X-X-X-X-X1-X2-X3-X4-V-X5-X6-L-L-A-X7-G-A-D-V-N-X-X-X-X-X- X-X-X-P-L-X-L-A-X-X-X-X-X-H-X8-E-I-V-X9-V-L-L-X10-R-G-X11-D-X12-X13-X-X- X-X-X-X-X-P-L-H-L-A-X-X-X-X-X-X-H-L-E-I-X14-E-X15-L-L-X16-X17-G-A-D-V-N (SEQ ID NO: 4) wherein X1 is
  • amino acid residues X5, X6, 19L, X7, 54R, X15, X16, and X17 of SEQ ID NO: 4 are key in the formation of multimers
  • amino acid residues 11Y, 14Q, and 58I of SEQ ID NO: 4 improve multimer formation and/or multimer stability or rigidity
  • amino acid residues X8 and X14 of SEQ ID NO: 4 are based on variability at corresponding locations in known DARPins, and are not expected to disrupt multimer formation, stability, or rigidity.
  • Exemplary DARPins having a DARPin backbone include DARPin against GFP: KKLLEAARAGYDDQVAALLAKGADVNAADDVGVTPLHLAAQRGHLEIVEVLLKRGADINAADLWG QTPLHLAATAGHLEIVELLLRWGADVNARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKF GKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 5) DARPin against GFP: KKLLEAARAGDKYAVDALLAKGADVNAADDVGVTPLHLAAQRGHLEIVEVLLKRGWDINAADLWG QTPLHLAATAGHLEIVELLLWYGADVNARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKF GKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO: 6) DARPin against GFP: KKLLEAARAGYDDQVA
  • Proteins comprising a DARPin backbone will typically comprise between 4 and 6 tandem copies of the 33 amino acid motif (“repeat unit”) that identifies the protein as belonging to the DARPin protein family. In each repeat unit, the amino acid residues of the loop regions are variable and may be selected based on the intended cargo protein to be bound thereby. See, e.g., Plückthun (2015). Proteins comprising a DARPin backbone may comprise “capping repeats”, i.e., the first and last repeat units that are typical of DARPins.
  • a protein having a DARPin backbone may be recombinantly linked to a subunit of a self-assembling protein cage (“protein cage”) to result in a REMIS subunit.
  • Subunits of protein cages include those described in US8969521, US9066870, US9630994, US10248758, US10501733, US20200397886, US20210163540, and WO2020/220044 and the following: Example, Subunit B: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKR (SEQ ID NO: 18) Cage Protein T33-51, Subunit A: MRITTKVGDKGSTRLFGGEEVWKDDPI
  • the peptide linker will typically be a sequence that is predicted to favor an alpha-helical conformation in accordance with rules and patterns in the art. See, e.g., Padilla et al. (2001). [0043] In some embodiments, the peptide linker is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 and not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long. In some embodiments, the peptide linker comprises an amino acid sequence or amino acid motif that forms an alpha-helical structure.
  • the amino acid sequence of the peptide linker comprises KEELD (SEQ ID NO: 37), PAPAP (SEQ ID NO: 38), KEELD (SEQ ID NO: 39), KDELD (SEQ ID NO: 40), RDERN (SEQ ID NO: 41), (E(A) 3 K)n motif, (E4(R/K)4)n motif, or (AP)n motif.
  • the amino acid sequence of the subunit has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 18 to SEQ ID NO: 36.
  • REMIS subunits comprise (A) a protein having a DARPin backbone that (1) comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, or (2) comprises or consists of SEQ ID NO: 4, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, and each X is independently any amino acid; (B) a peptide linker that is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 and not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long, said peptide linker preferably forms an alpha-helical structure and/or has an amino acid sequence comprising KEELD (SEQ ID NO: 37), PAP
  • the peptide linker joins the N- terminus of the DARPin backbone to the C-terminus of the subunit.
  • the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17.
  • REMIS subunits comprise (A) a protein having a DARPin backbone that (1) comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, or (2) comprises or consists of SEQ ID NO: 4, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, and each X is independently any amino acid; (B) a peptide linker that comprises or consists of KEELD (SEQ ID NO: 32), and (C) a subunit having an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, wherein the peptide linker joins the N-terminus of the DARPin back
  • the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17.
  • REMIS subunits comprise (A) a protein having a DARPin backbone that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71; (B) a protein having a DARPin backbone that comprises
  • the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17.
  • the REMIS subunit comprises or consists of one of the following: RCG-10, Subunit B, which binds GFP: MFTRRGDQGETDLANRARVGKDSPVVEVQGTIDELNSFIGYALVLSRWDDIRNDLFRIQNDLFVL GEDVSTGGKGRTVTMDMIIYLIKRSVEMKAEIGKIELFVVPGGSVESASLHMARAVSRRLERRIK AASELTEINANVLLYANMLSNILFMHALISNKRKEELDKKLLEAARAGYDDQVAALLAKGADVNA ADDVGVTPLHLAAQRGHLEIVEVLLKRGADINAADLWGQTPLHLAATAGHLEIVELLLRWGADVN ARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKFGKTPFDLAIDNGNEDIAEVL
  • REMIS cages also comprise multiple copies of an additional subunit, which additional subunit does not contain a protein having a DARPin backbone linked thereto.
  • the additional subunit comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 18 to SEQ ID NO: 36.
  • the additional subunit comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17.
  • the REMIS cages comprise or consist of (I) a REMIS subunit that comprises or consists of (A) a protein having a DARPin backbone that (1) comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, or (2) comprises or consists of SEQ ID NO: 4, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, and each X is independently any amino acid; (B) a peptide linker that is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 and not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues long, said peptide linker preferably forms an alpha-helical structure and/or has an
  • the sequence of the additional subunit is different from that of the subunit of the REMIS subunit.
  • the subunit of the REMIS subunit and the additional subunit are counterparts, i.e., subunit A and subunit B, of a known self-assembling protein cage.
  • the additional subunit comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17.
  • the REMIS cages comprise or consist of (I) a REMIS subunit that comprises or consists of (A) a protein having a DARPin backbone that (1) comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, or (2) comprises or consists of SEQ ID NO: 4, wherein X1 to X17 are the same as that indicated for the DARPin Backbone Formula set forth above, and each X is independently any amino acid; (B) a peptide linker that comprises or consists of KEELD (SEQ ID NO: 32), and (C) a subunit having an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, wherein the
  • the sequence of the additional subunit is different from that of the subunit of the REMIS subunit.
  • the subunit of the REMIS subunit and the additional subunit are counterparts, i.e., subunit A and subunit B, of a known self-assembling protein cage.
  • the additional subunit comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17.
  • the REMIS cages comprise or consist of (I) a REMIS subunit that comprises or consists of (A) a protein having a DARPin backbone that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71; (B) a peptide linker that comprises or consists of KEELD (SEQ ID NO: 32
  • the sequence of the additional subunit is different from that of the subunit of the REMIS subunit.
  • the subunit of the REMIS subunit and the additional subunit are counterparts, i.e., subunit A and subunit B, of a known self- assembling protein cage.
  • the additional subunit comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the protein having the DARPin backbone comprises or consists of a sequence selected from SEQ ID NO: 5 to SEQ ID NO: 17.
  • the subunits (the REMIS subunit and, if present, the additional subunit) are typically present in 12, 24, or 60 copies in the assembled REMIS cage, according to the symmetries of Platonic solids. In its assembled form, a REMIS cage would typically be in the range of about 400 kDa up to about 3 MDa.
  • the DARPin backbones of REMIS subunits protrude from REMIS cages like “arms”.
  • a REMIS cage is schematically shown in Figure 1 as solid shapes and ribbon diagrams.
  • Figure 2 schematically shows the “prior art”, i.e., protein cages having DARpins that lack a DARPin backbone as described herein. As shown in Figure 2, the DARpins do not bind each other.
  • DARPin backbones, REMIS subunits, and REMIS cages described herein may be made using methods known in the art including chemical synthesis, biosynthesis or in vitro synthesis using recombinant DNA methods, and solid phase synthesis.
  • DARPin backbones, REMIS subunits, and REMIS cages may be purified using protein purification techniques known in the art such as reverse phase high- performance liquid chromatography (HPLC), ion-exchange or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis. See, e.g., Olsnes and Pihl (1973) Biochem 12(16):3121-3126; and Scopes (1982) Protein Purification, Springer-Verlag, NY, which are herein incorporated by reference. Polynucleotides that encode the DARPin backbones, REMIS subunits, and REMIS cages described herein are also contemplated herein.
  • the DARPin backbones, REMIS subunits, and REMIS cages are isolated.
  • the DARPin backbones and REMIS subunits are substantially purified.
  • a “substantially purified” compound refers to a compound that is removed from its natural environment and/or is at least about 60% free, preferably about 75% free, and more preferably about 90% free, and most preferably about 95-100% free from other macromolecular components or compounds with which the compound is associated with in nature or from its synthesis.
  • a protein comprising a DARPin backbone specifically binds the given target it was designed to bind.
  • a protein comprising a DARPin backbone “specifically binds” its given target if it reacts or associates more frequently, more rapidly, with greater duration, and/or with greater binding affinity with the given target than it does with a given alternative, and/or indiscriminate binding that gives rise to non-specific binding and/or background binding.
  • non-specific binding and background binding refer to an interaction that is not dependent on the presence of a specific structure (e.g., a given epitope).
  • the proteins were expressed in E. coli BL21(DE3) cells (New England Biolabs) in Terrific Broth at 18 °C overnight upon 1 mM IPTG induction at OD 1.0.
  • pellets were resuspended in resuspension buffer (50 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0) supplemented with benzonase nuclease, 1 mM PMSF, protease inhibitor cocktail EDTA-free (Thermo Scientific) and 0.1% LDAO and lysed using an EmulsiFlex C3 homogenizer (Avestin).
  • resuspension buffer 50 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0
  • benzonase nuclease 1 mM PMSF
  • protease inhibitor cocktail EDTA-free Thermo Scientific
  • 0.1% LDAO EmulsiFlex
  • the cell lysate was cleared by centrifugation at 20,000 xg for 20 minutes at 4 °C, the resulting supernatant was recovered and centrifuged at 10,000 xg for 10 minutes at 4 °C and then loaded onto a HisTrap column (GE Healthcare) pre-equilibrated with the same resuspension buffer.
  • the imaging scaffold was eluted with a linear gradient to 300 mM imidazole. Upon elution, 5 mM EDTA and 5 mM BME were added immediately. The eluted protein was concentrated using Amicon Ultra-15100 kDa MWCO for the imaging scaffold and 3 kDa MWCO for the GFP.
  • the concentrated protein was further purified by size exclusion chromatography using a Superose 6 Increase column, eluted with 20 mM Tris pH 8.0, 100 mM NaCl, 5 mM BME, 5 mM EDTA. Fractions were analyzed by SDS-PAGE and negative stain EM for the presence of the imaging scaffold. [0063] KRas G12V and KRas G13C proteins were prepared using methods in the art. [0064] Negative Stain EM [0065] The concentration of a 3.5 ⁇ l sample of fresh Supersoe 6 Increase eluent was adjusted to about 100 ⁇ g/ml, applied to glow-discharged for one minute and blotted away.
  • Cryo-EM Data Processing and Model Building [0069] Motion correction, CTF correction, particle picking and 2D classification were performed in cryoSPARC v.3.2 using methods in the art. [0070] An initial set of particles was automatically picked using a blob-picker. The extracted particles were 2D classified and then an ab initio reconstruction was generated. The model was used to obtain a 3D reconstruction enforcing T symmetry. The 3D structure was used to generate 2D projections of the particles and then used to repick the particles from the images using a template picker. The picked particles were extracted from the micrographs and then 3D reconstructed enforcing T symmetry. These were further refined using focused 3D classification and local refinements.
  • the models were subjected to multiple rounds of refinement with Coot (Emsley et al., 2010) and PHENIX (Afonine et al., 2018) [0072] CYRO-EM IMAGING Five different REMIS cages were made based on the original T33-51 protein cage.
  • the original T33-51 protein cage comprises subunit A (SEQ ID NO: 19) and subunit B (SEQ ID NO: 20) in a stoichiometry of A 12 B 12 .
  • the REMIS cages contained a REMIS subunit comprising SEQ ID NO: 18 (instead of SEQ ID NO: 20) and a DARPin backbone (i.e., SEQ ID NO: 4) linked thereto via a peptide linker (i.e., SEQ ID NO: 37) in the same stoichiometry.
  • the REMIS cages were RCG-10, RCG-5, RCG- 8, RCG-13, and RCG-14, and their REMIS subunits were SEQ ID NO: 42 to SEQ ID NO: 46, respectively.
  • Each REMIS cage assembled according to their elution volumes in size exclusion chromatography and bound their intended target (“target cargo”).
  • RCG- 10 was used as the reference scaffold upon which DARPin sequence changes could be made to present other proteins of interest.
  • the oncogenic protein KRas (19.4 kDa) and MBP were selected as the target cargo and the loop regions of SEQ ID NO: 4 were substituted accordingly.
  • target cargo was added to REMIS cages and then purified by SEC prior to cryo-EM analysis.
  • Three KRas mutants were studied: G12V, G12C, and G13C.
  • the KRas mutants contained GDP as a bound ligand and were bound by RCG-33 (SEQ ID NO: 50).
  • the DARPin represented by SEQ ID NO: 49 binds KRas including the KRas mutants.
  • cyro-EM imaging using the REMIS cages can be used to characterize the structures of proteins as small as about 19.4 kDa and also identify and/or distinguish proteins having single amino acid differences from one another.
  • cryo-EM imaging revealed the conformation of the drug molecule while bound to KRas. See Figure 4.
  • the resolution of REMIS cage plus MBP cargo was similar. See Figure 5. Therefore, cryo-EM imaging using the REMIS cages can be used to elucidate the conformation of protein- ligand interactions, i.e., the structure of the target protein when bound to a given ligand of interest.
  • protein-ligand e.g., drug interactions between target protein and a given ligand of interest can be assayed and/or characterized by comparing the cryo-EM image of the complex comprising (i) the given ligand bound to the target protein and (ii) the REMIS cage bound to the target protein and/or the given ligand.
  • the reference cryo-electron micrograph is of (a) a complex comprising the fusion protein bound to the target protein, said complex excludes the presence of the given ligand or another ligand bound thereto; (b) a complex comprising (i) the given ligand bound to a mutant of the target protein and (ii) the fusion protein bound to the given ligand and/or the mutant, said mutant having at least one amino acid substitution, deletion, or addition as compared to the target protein; or (c) a complex comprising (i) a second ligand bound to the target protein and (ii) the fusion protein bound to the given ligand and/or the target protein, wherein said second ligand is different the given ligand.
  • Differences between the cryo-EM image and one or more reference cryo-electron micrographs can then be used to characterize the protein-ligand interaction between a target protein and given ligand, identify the target protein or a ligand that binds the target protein, distinguish the target protein from other proteins, or distinguish a ligand bound to the target protein from other ligands that also bind the target protein.
  • Structural analysis indicates that the key amino acid residues for multimer formation between DARPins comprising a DARPin backbone are at positions 16, 17, 19, 21, 54, 83, 86, and 87 of SEQ ID NO: 4 and amino acid residues a positions 11Y, 14Q, and 58I of SEQ ID NO: 4 improve the formation and/or structural integrity of multimers.
  • REMIS cages having SEQ ID NO: 4, wherein X5 is A, X6 is A, X7 is K, X15 is L, X16 is R, and X17 is W were found to be more rigid than those where: ⁇ X5 is D, X6 is A, X7 is K, X15 is L, X16 is W, and X17 is Y; ⁇ X5 is A, X6 is W, X7 is L, X15 is K, X16 is R, and X17 is C; ⁇ X5 is A, X6 is A, X7 is K, X15 is L, X16 is R, and X17 is C; and ⁇ X5 is D, X6 is W, X7 is L, X15 is L, X16 is R, and X17 is C.
  • DARPin backbones, REMIS subunits, and REMIS cages comprise or consist of: SEQ ID NO: 51: KKLLEAXXXXX1X2X3X4VAALLAKGADVNXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX1 1DX12X13XXXXXXXXPLHLAXXXXHLEIX14ELLLRWGADVN; SEQ ID NO: 52: KKLLEAXXXXX1X2X3X4VDALLAKGADVNXXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX1 1DX12X13XXXXXXXXXPLHLAXXXXXHLEIX14ELLLWYGADVN; SEQ ID NO: 53: KKLLEAXXXXX1X2X3X4VAWLLALGADVNXXXXX
  • REMIS cages having SEQ ID NO: 4, wherein X1 is Y, X4 is Q, X5 is A, X6 is A, X7 is K, X12 is I, X15 is L, X16 is R, and X17 is W were found to be more rigid than those where: ⁇ X1 is D, X4 is A, X5 is D, X6 is A, X7 is K, X12 is I, X15 is L, X16 is W, and X17 is Y; ⁇ X1 is Y, X4 is K, X5 is A, X6 is W, X7 is L, X12 is I, X15 is K, X16 is R, and X17 is C; ⁇ X1 is I, X4 is A, X5 is A, X6 is A, X7 is K, X12 is I, X15 is L, X16 is R, and X17 is C;
  • DARPin backbones, REMIS subunits, and REMIS cages comprise or consist of: SEQ ID NO: 56: KKLLEAXXXXYX2X3QVAALLAKGADVNXXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX11D IX13XXXXXXXXPLHLAXXXXHLEIX14ELLLRWGADVN; SEQ ID NO: 57: KKLLEAXXXXDX2X3AVDALLAKGADVNXXXXXXXXXPLXLAXXXXHX8EIVX9VLLX10RGX11D IX13XXXXXXXXXPLHLAXXXXXHLEIX14ELLLWYGADVN; SEQ ID NO: 58: KKLLEAXXXYX2X3KVAWLLALGADVNXXXXXXXXPLXLAX
  • DARPin backbones, REMIS subunits, and REMIS cages comprise or consist of: SEQ ID NO: 63: KKLLEAXXXXYDDQVAALLAKGADVNXXXXXXXXPLXLAXXXXHLEIVEVLLKRGADINXXXXXX XXPLHLAXXXXHLEIVELLLRWGADVN; SEQ ID NO: 64: KKLLEAXXXXDKYAVDALLAKGADVNXXXXXXXXXXPLXLAXXXXHLEIVEVLLKRGWDINXXXXXXX XPLHLAXXXXHLEIVELLLWYGADVN; SEQ ID NO: 65: KKLLEAXXXYDDKVAWLLALGADVNXXXXXXXXPLXLAXXXXHLEIVEVLLKRGADINXXXXXXX XPLHLAXXXXXHLEIVEVLLKRGAD
  • REMIS cages having SEQ ID NO: 63 were found to be the most rigid.
  • preferred DARPin backbones, REMIS subunits, and REMIS cages comprise or consist of SEQ ID NO: 63.
  • REFERENCES The following references are herein incorporated by reference in their entirety with the exception that, should the scope and meaning of a term conflict with a definition explicitly set forth herein, the definition explicitly set forth herein controls: Afonine et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D Struct Biol.2018 Jun 1;74(Pt 6):531-544.
  • a and/or B means “A, B, or both A and B” and “A, B, C, and/or D” means “A, B, C, D, or a combination thereof” and said “A, B, C, D, or a combination thereof” means any subset of A, B, C, and D, for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D).
  • a single member subset e.g., A or B or C or D
  • a two-member subset e.g., A and B; A and C; etc.
  • a three-member subset e.g., A, B, and C; or A, B, and D; etc.
  • the phrase “one or more of”, e.g., “one or more of A, B, and/or C” means “one or more of A”, “one or more of B”, “one or more of C”, “one or more of A and one or more of B”, “one or more of B and one or more of C”, “one or more of A and one or more of C” and “one or more of A, one or more of B, and one or more of C”.
  • the phrase “comprises or consists of A” is used as a tool to avoid excess page and translation fees and means that in some embodiments the given thing at issue: comprises A or consists of A.
  • the sentence “In some embodiments, the composition comprises or consists of A” is to be interpreted as if written as the following two separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition consists of A.”
  • a sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself.
  • the sentence “In some embodiments, the composition comprises A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition comprises B.
  • the composition comprises C.”
  • the sentence “In some embodiments, the composition comprises at least A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises at least A. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C.”
  • sample is used in its broadest sense and includes specimens and cultures obtained from any source, as well as biological samples and environmental samples.
  • an “isolated” compound refers to a compound that is isolated from its native environment.
  • an isolated polynucleotide is a one which does not have the bases normally flanking the 5’ end and/or the 3’ end of the polynucleotide as it is found in nature.
  • an isolated polypeptide is a one which does not have its native amino acids, which correspond to the full-length polypeptide, flanking the N-terminus, C-terminus, or both.
  • protein protein
  • polypeptide and “peptide” are used interchangeably to refer to two or more amino acids linked together. Groups or strings of amino acid abbreviations are used to represent peptides.
  • nucleic acid sequences are indicated with the 5’ end on the left and the sequences are written from 5’ to 3’.
  • sequence identity refers to the percentage of nucleotides or amino acid residues that are the same between sequences, when compared and optimally aligned for maximum correspondence over a given comparison window, as measured by visual inspection or by a sequence comparison algorithm in the art, such as the BLAST algorithm, which is described in Altschul et al., (1990) J Mol Biol 215:403-410.
  • Software for performing BLAST (e.g., BLASTP and BLASTN) analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov).
  • the comparison window can exist over a given portion, e.g., a functional domain, or an arbitrarily selection a given number of contiguous nucleotides or amino acid residues of one or both sequences.
  • the comparison window can exist over the full length of the sequences being compared.
  • a given comparison window e.g., over 80% of the given sequence
  • the recited sequence identity is over 100% of the given sequence.
  • the percentages are determined using BLASTP 2.8.0+, scoring matrix BLOSUM62, and the default parameters available at blast.ncbi.nlm.nih.gov/Blast.cgi.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv Appl Math 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol Biol 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection.

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Abstract

L'invention concerne des squelettes de DARPin qui forment des cages de protéines multimères et à auto-assemblage qui comprennent des protéines, qui comprennent des squelettes de DARPin, fusionnés à des protéines de sous-unités des cages de protéines à auto-assemblage et des procédés de fabrication et d'utilisation de ceux-ci.
PCT/US2023/074156 2022-09-15 2023-09-14 Squelettes d'arpine et échafaudages d'imagerie par microscopie électronique rigidifiée WO2024059686A2 (fr)

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