WO2015138711A1 - Polypeptides to inhibit epstein barr viral protein bhrf1 and b cell lymphoma family proteins - Google Patents

Polypeptides to inhibit epstein barr viral protein bhrf1 and b cell lymphoma family proteins Download PDF

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WO2015138711A1
WO2015138711A1 PCT/US2015/020155 US2015020155W WO2015138711A1 WO 2015138711 A1 WO2015138711 A1 WO 2015138711A1 US 2015020155 W US2015020155 W US 2015020155W WO 2015138711 A1 WO2015138711 A1 WO 2015138711A1
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polypeptide
seq
amino acid
acid sequence
bcl
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PCT/US2015/020155
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French (fr)
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David Baker
Stephanie Ann BERGER
Geoffrey Y. BERGUIG
Erik PROCKO
Yifan SONG
Patrick S. Stayton
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University Of Wahington
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Priority to US15/262,716 priority Critical patent/US9750814B2/en

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    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • EBV Epstein-Barr virus
  • BL Burkitt's lymphoma
  • B cell lymphoma-2 (Bel- 2) family proteins (reviewed in (Martinou and Youle, 201 1)), which fall in to three classes.
  • the 'executioners', Bak and Bax initiate apoptosis by increasing mitochondrial outer membrane permeability and facilitating the release of mitochondrial cytochrome c to the cytosol, which activates downstream signaling.
  • Six human pro-survival Bcf-2 proteins (Bcl-2, BC1-XL, Bcl-B, Mcl-l , Bcl-w and Bfl-l) inhibit this process.
  • pro-survival Bcl-2 proteins dampen apopiotie triggers and enhance chemoresistance by sequestering BH3-only proteins or directly inhibiting Bak and Bax. Increased expression of pro-survival Bcl-2 proteins is a common feature of many cancers.
  • Epstein-Barr viras encodes a pro-survival Bcl-2 homologue, BHRF1 , which prevents lymphocyte apoptosis during initial infection by sequestering pro-apoptotic BH3-only proteins (especially Bim), and interacting directly with the executioner Bak (Desbien et al, 2009; Kvansakul et al, 2010)(Altmann and Hammerschmidt, 2005)(Henderson ei al., 1993).
  • BHRF1 is under the control of an early lytic cycle promoter, low levels of constitutive expression have been observed in some cases of EBV-positive BL when the vims is latent, and it has been speculated that BHRF1 may be a necessary viral factor for lymph omagenesis (Kelly et al, 2009; Leao et al., 2007; Watanabe et al., 2010).
  • the invention provides polypeptides comprising an amino acid sequence having at least 50% amino acid sequence identity over its length relative to the amino acid sequence of SEQ ID NO,: 1, wherein the polypeptide selectively binds to a protein selected from the group consisting of Epstein Ban" protein BHFR1 , and B cell lymphoma family proteins selected from the group consisting of myeloid cell leukemia 1 (Mcl-1), B-cell lymphoma 2 (Bcl-2), Bcl-2-like protein 1 (BCL2L1/Bcl-XL), Bcl-2-like protein 10
  • Mcl-1 myeloid cell leukemia 1
  • Bcl-2 B-cell lymphoma 2
  • Bcl-2-like protein 1 BCL2L1/Bcl-XL
  • the polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity over its length relative to the amino acid sequence selected from the group consisting of SEQ ID NOS:2-6. In various further embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 7- 13, wherein the polypeptide binds to a specific target. In a further embodiment, the polypeptides further comprise a cell-penetrating peptide and/or an antibody or antibody fragment.
  • the invention provides pharmaceutical composition, comprising a polypeptide of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an antibody.
  • the carrier comprises a polymer, such as a polymer comprising a hydrophilic block and an endosomolytic block, or a stimuli-responsive polymer.
  • the invention provides recombinant nucleic acids encoding a polypeptide of the invention, recombinant expression vectors comprising the nucleic acid of the invention operatively linked to a promoter, and recombinant host cells comprising the recombinant expression vectors of the invention.
  • the invention provides methods for treating an Epstein-Barr v i ru s - related diseases comprising administering to a subject in need thereof a therapeutically effective amount of one or more o f the polypeptides of the invention, or salts thereof, pharmaceutical compositions thereof, a recombinant nucleic acid encoding the one or more polypeptides, a recombinant expression vector comprising the recombinant nucleic acids, and/or a recombinant host cells comprising the recombinant expression vector, to treat Epstein-Barr virus related diseases wherein the polypeptide or encoded polypeptide selectively inhibits BHRF i .
  • the invention provides methods for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of one or more of the polypeptides of the invention, salts thereof, a pharmaceutical composition thereof, a recombinant nucleic acid encoding the one or more polypeptides, a recombinant expression vector comprising the recombinant nucleic acid, and/or a recombinant host cell comprising the recombinant expression vector, to treat cancer, wherein the polypeptide or encoded polypeptide selectively inhibits one or more of Mcl- 1 , Bcl-2, BCL2L 1 /Bcl-XL, BCL2L10/Bcl-B, and Al/Bfl- 1.
  • the invention provides methods for determining the Bcl-2 phenotype of a tumor, comprising contacting tumor cells, tumor cell lysates or tumor cellular components with one or more polypeptides selected from the group consisting of SEQ ID NOS: 2-6 or 8- 12, under conditions suitable to promote apoptosis signaling in cells of the tumor that express a bcl-2 homologue targeted by the one or more polypeptides; and
  • FIG. 1 De novo protein assembly protocol.
  • a scaffold grey ribbon
  • the Bim-BH3 motif black
  • the Bim-BH3 peptide is extended on both ends and a new protein structure (black tube) is built using fragment-based assembly (ii), followed by rounds of minimization and sequence design.
  • the newly assembled protein is docked to BHRFl and the surrounding interface is designed (Hi).
  • Many designs are generated that are filtered by multiple criteria (iv).
  • FIG. 3 Predictions of folding probability correlate with designed protein functionality,
  • C-E Properties of the designed interfaces plotted against the experimental enrichment ratios after selection for binding to 100 nM BHRFl .
  • Each data point represents a designed protein (Indexes-Ol to 74). Plotted are the (C) interface buried solvent-accessible surface area, (D) the calculated interface binding energy, and (E) the number of unsatisfied buried polar atoms at the interface. (F-H) As for (C-E), except showing computed metrics for the unbound designed proteins. Plotted are enrichment ratios versus (F) the holes (packing) score of the apo-protein, (G) ca lculated energy, and (H) unsatisfied buried polar atoms.
  • Figure 4 Affinity maturation of designed protein BbpD04.
  • A Computational model of BHRFl (white ribbon) bound to design BbpD04 (surface). The electric field from BHRFl is mapped to the BbpD04 surface; regions experiencing a positive field are shaded dark grey.
  • BbpD04.2 black trace
  • BbpD04.2 L54E black with a mutation in the binding site does not shift (pale grey) when mixed with BHRFl (dark grey).
  • BbpD04.2 has a buried cysteine, which becomes exposed for PEG-maleimide conjugation in the presence of the harsh detergent SDS.
  • Cysteine- linker BbpD04.2 proteins were conjugated to HPDP-biotin for 4 h at room temperature. Biotinylated protein was incubated with streptavidin and aggregation measured by absorbance at 350 nm. Mutation of the internal cysteine (CI 03 A) markedly diminishes aggregation.
  • D DMSO, the solveni used for dissolving HPDP-biotin, did not increase exposure of the internal cysteine for PEG- maleimide modification.
  • BbpD04.2 C 103A and C I 03 V mutations were predicted by the ROSETTA. energy function to be tolerated following minimization.
  • BbpD04.2 CI 03V had reduced specificity by yeast surface display for BHRFl over other prosurvival Bcl-2 proteins, whereas BbpD()4.2 C103A (called BbpD()4.3) had only a minor loss of affinity and specificity.
  • BTND1 has improved bacterial expression and stability.
  • A. All single amino acid substitutions of BbpD04.3 were expressed in a yeast display library and sorted by FACS for high affinity binding to BHRFl . Plotted for each substitution is the log2 enrichment ratio from -3.5 (depleted, black) to +3.5 (enriched, white). Stop codons, *. The region of the incorporated Bim-BH3 motif is boxed with a broken line. Secondary structure and core residues are indicated above. Substitutions to aspartate (depleted for core residues) and to proline (depleted for helical residues) are boxed.
  • B As in (A), except the library was sorted for high affinity and specificity.
  • BbpD04.3 The modeled structure of BbpD04.3 is shaded by sequence Shannon entropy from 2.8 (highly conserved, dark) to 4.3 (variable, white), based on the sequence-fitness landscapes.
  • BbpD04.3 and its derivative BIND! were expressed as C-terminal 6his-tagged proteins in E. coli, precipitated from cleared lysate with NiNTA- agarose and analyzed on a Coomassie-stained SDS-polyacrylaniide electrophoretic gel. An arrow indicates the expected MW of the designed proteins at 15 kD.
  • E CD spectra of BbpD04 and its variants (10 ⁇ in PBS) were collected at 25°C in the presence of guanidinium hydrochloride.
  • BINDI binds BHRF1 with high affinity and specificity.
  • A BINDI or knockout mutant BINDI L54E were mixed with BHRF1 and separated by SEC. A shift in elation volume upon mixing BINDI and BHRFI is abrogated by the knockout mutation.
  • B Biotinylated BHRF 1 was immobilized to a BLI sensor and the interaction with BINDI was measured at the indicated concentrations.
  • C BLI kinetic analysis of BINDI interactions with BHRFI (as in panel B) and human Bel -2 proteins.
  • D BLI kinetic analysis of interactions between the Bim-BH3 motif fused to the C-terminus of maltose-binding protein (MBP) and Bcl-2 proteins immobilized to the sensor surface.
  • MBP maltose-binding protein
  • Figure 9 Structural basis for exceptional affinity and specificity of BINDI.
  • A Slice through the crystal structure of BINDI (black ribbon) bound to BHRF I (white ribbon with surface).
  • the guiding scaffold 3 LHP S (grey) is aligned to BINDI at the Bim-BH3 incorporation site.
  • a direct graft of the BH3 motif into 3LHP_S at this position causes clashes elsewhere with the BHRF ! surface.
  • B Crystal structure of BIND! (black) bound to BHRFI (white).
  • C The surface of BHRFI , with the buried contact surface in
  • BHRFI BINDI shaded black.
  • D The surface of BINDI, with the buried contact surface in BHRFI 'BINDI shaded. Buried residues from the incorporated Bim-BH3 motif are dark grey. Buried residues in the surrounding designed surface are black.
  • E The crystal structure (PDB 2WH6) of Bim-BH3 (black) bound to BHRF I (white).
  • F The surface of BHRFI , with the buried contact surface in BHRF 1 » Bim-BH3 shaded black. (0) The surface of Bim- BH3, with the buried contact surface in BHRF I » Bim-BH3 black.
  • FIG. 1 BINDI triggers apoptosis in an EBV-positive cell line.
  • A Cytochrome c release from mitochondria harvested from Ramos (EBV -negative) or Ramos-- AW ceils (EBV- positive) treated with Bim-BH3 peptide.
  • B As in (A), with mitochondria treated with BINDI protein.
  • BIND! L54E has the equivalent interface mutation as Bim-BH3 L62E.
  • C At left, the crystal structure of BINDI bound to BHRF1 showing the interaction of Asn62 with the N-terminus of helix ⁇ .
  • BINDI mutation N62S is predicted to maintain interface interactions.
  • D BLI kinetic analysis of BINDI N62S interactions with Bcl-2. proteins.
  • E Cytochrome c release from Ramos and Ramos-AW mitochondria treated with BINDI N62S or inactive guide scaffold 3LHP(S).
  • F-H Mitochondria were harvested from four EBV-negaiive and six EBV-positive lines. Cytochrome c release was measured after treatment with 10 ⁇ Bi.m-BH3 peptide (F), guide scaffold 3LHP(S) (G), or BINDI N62S (H).
  • FIG. 12 Intracellular delivery of BINDI induces cell death in an EBV-positive cancer line in vitro.
  • A Cells were incubated with 4 ⁇ antennapedia peptide-fusions of BINDI, BINDI-L54E or 3 LHP chain S. Cell viability after 24 h was assessed by quantifying metabolic activity.
  • FIG. 13 Treatment of EBV-positive B lymphoma xenograft tumors by intracellular delivery of BINDI in vivo.
  • A Schematic representation of the copolymer-based treatment.
  • Pol950 has stabilizing and endosomolytic blocks and forms a micelle at physiological pH. The stabilizing block couples to O.CD19 and BINDI.
  • Nude mice with subcutaneous Ramos- AW xenografts were treated on days 0, 3 and 6 with Pol950 (300 mg/kg) : ⁇ xCD19 (15 mg/kg) : BINDI or 3LHP(S) (105 mg/kg). Mice were injected 30 minutes prior to each treatment with CTX (35 mg ml) and BTZ (0.5 mg/mf).
  • FIG. 1 The docked configuration is computationally designed.
  • BINDI black
  • Mcl- 1 white
  • BLI Designed ionic interactions in MINDI.
  • C Chemical denaturation measured by following loss of CD signal (222 nm)
  • D BLI titration experiment for accurate KB determination. Biotinylated Mcl- l was immobilized to a streptavidin-coated sensor and incubated with the indicated concentrations of soluble MINDI. Raw data is grey, fitted curves are black.
  • E Isoaffinity plot from BLI titrations of MINDI interactions with BCL2 family members (only Mcl- l is labeled).
  • FIG. 16 Qualitative measurements of binding by BLI analysis at a single analyte concentration.
  • the BCL2 proteins are biotinylated and immobilized on streptavidin-sensors.
  • the sensors are dipped for 600 s in 50 iiM of the indicated designed Mcl- l binding proteins, followed by incubation in buffer to monitor dissociation.
  • Mcl- l -specific peptide MB 1 was purified as a MBP fusion and used as a positive control.
  • FIG. 17 Quantitative BLI analysis of optimized designs binding each BCL2 protein.
  • A Sequence alignment of specific BCL2 protein binders (from top to bottom SEQ ID NOs: 1, 5, 2, 6, 3, and 4). Differences from BINDI, the original designed binder targeting viral BHRF I that was repurposed for binding other BCL2 family members, are highlighted. Residues that differ from BINDI in one or two sequences are shaded grey, while residues that differ in three or more of the derived binders are shaded black.
  • B Sequence variation amongst the INDT family is mapped to the structure of BINDI (surface representation) bound to BHRFI (ribbon). Detailed Description of the Invention
  • the present invention provides jjolypeptides comprising or consisting of an amino acid sequence having at least 50% amino acid sequence identity over their length relative to the amino acid sequence of SEQ ID NO.: 1 , wherein the polypeptide selectiv ely binds to a protein selected from the group consisting of Epstein Ban- protein BHFR 1, and B cell lymphoma family proteins selected from the group consisting of myeloid cell leukemia 1 (Mcl- 1), B-cell lymphoma 2 (Bcl-2), Bci-2-like protein 1
  • BCL2L1/Bcl-XL Bcl-2-like protein 10
  • BCL2L10/Bcl-B Bcl-2-like protein Al
  • Al Al
  • the polypeptides of the invention are high- affinity (as low as picomolar affinity), specific protein inhibitors of BHRFl and B cell lymphoma (BCL) family proteins. And can be used, for example, in methods of treating cancer and Epstein-Barr virus-related diseases. Rather than repurposing an existing natural protein of known structure, the polypeptides of the invention were designed de novo for optimum BHRFl or and BCL family protein interactions, and are shown herein to trigger apoptosis in relevant cancer lines and slow BL progression in an animal model in the examples herein. This work therefore represents a major bioengineering accomplishment; the creation of an entirely new class of designer polypeptides and their demonstrated therapeutic potential from the ground up.
  • polypeptides of the invention have at least 50% amino acid sequence identity over their length relative to the amino acid sequence of S ) ID NO,: 1 , which was designed as shown in the examples that follow to selectively and at very high affinity bind to Epstein Barr protein BHFR1.
  • the inventors have carried out saturation mutagenesis on the polypeptide of SEQ ID NO: l to identify modifiable residues.
  • polypeptides of the invention can be modified for selective binding against BCL family proteins.
  • the polypeptides of the invention have at least 55%, 60%, 66%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identity over their length relative to the amino acid sequence of SEQ ID NO: 1.
  • polypeptides may include additional residues at the N-terminus, C- termmus, or both that are not present in SEQ ID NO: 1; these additional residues are not included in determining the percent ideniity of the polypeptides of the invention relative to the reference polypeptide (i.e.: SEQ ID NO: l in this case).
  • the polypeptides selectively bind to a protein selected from the group consisting of Epstein Ban- protein BHFR1, and B cell lymphoma family proteins selected from the group consisting of myeloid cell leukemia 1 (Mci-1), B -eell lymphoma. 2.
  • Bcl-2 Bel-2-iike protein 1 (BCL2L1/Bcl-XL), Bcl-2-like protein 10 (BCL2L10/Bcl-B), and Bcl-2-like protein A .1 (Al/Bfl-I).
  • selectively binds or “specifically binds” refers to the ability of a polypeptide of the invention to bind io its target, such as a BHRFl molecule or BCL family member, with a KD 10 5 M (10000 nM) or less, e.g., 10 6 M, 10 7 M, 10 8 M, 10 9 M, 10 10 M, 10 j l M, 10 , 2 M, or less.
  • Selective binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent.
  • polypeptides described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable ceil binding assay, or as described in the examples that follow.
  • a polypeptide specifically bound to a target is not displaced by a non-similar competitor.
  • a polypeptide is said to selectively bind an antigen when it
  • preferentia lly recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • the polypeptide comprises or consists of an amino acid sequence having at least 50% amino acid sequence identity over its length relative to to the amino acid sequence selected from the group consisting of SEQ ID NOS:2-6.
  • the polypeptide of each of SEQ ID NOS:2-6 shares very high le vels of sequence identity with BIND! (SEQ ID NO: 1), but were designed by the inventors as selective inhibitors of different BCL-family members, as described in detail in the examples that follow. These differing specificities allow use of the polypeptides in methods to treat cancer with different Bel phenotypes, as well as to determine the Bel-2 phenotype of a tumor.
  • BCL-famiiy member target for each of SEQ ID NOS: 2-6 are provided above.
  • the amino acid sequence of the respective targets for each of SEQ ID NOS:l-6 are shown below: BHRF1 (Target for SEQ ID NO: 1)
  • VNVVSVDTARTLF QVMEKEFEDGIINWGMVTIFAFEGIL1KKLLRQQIAPDVDTYK E1SYFVAEFIMNNTGEW1RQNGGWENGFVKKFEPKSG (SEQ ID NO: 72)
  • the inventors have carried out saturation mutagenesis on the polypeptides according to each of SEQ ID NOS:3-6, while the polypeptide of SEQ ID NO:2 shares 84 % identity and
  • the polypeptides of the invention have at least 55%, 60%, 66%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identity over their length relative to the amino acid sequence of SEQ ID NO:2-6.
  • the polypeptides may include additional residues at the N-terminus,
  • the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 7, wherein the polypeptide binds to BHFRI .
  • V/W/Y (A/3 ⁇ 4/G/H/Q/PJS/T)(A''K/L/PJT/V ⁇
  • This embodiment is based on saturation mutagenesis studies described in the examples that follow, in which all residues of SEQ ID NO: 1 were tested to identify allowed sequence variability for the designed proteins that retained function (i.e. : BHFR.1 binding).
  • polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 8, wherein the polypeptide binds to Bel-2.
  • polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO:9, and wherein the polypeptide binds to binds to Bcl-2- like protein 1.
  • This embodiment is based on saturation mutagenesis studies described in the examples that follow, in which all residues of SEQ ID NO:4 were tested to identify allowed sequence variability for the designed proteins that retained function (i.e.: i BCL2L1/Bcl-XL binding).
  • polypeptide comprises an amino acid sequence according to SEQ ID NO: 10, wherein the polypeptide binds to Bcl-2-like protein 10.
  • This embodiment is based on saturation mutagenesis studies described in the examples ihai follow, in which all residues of SEQ ID NO: 5 were tested to identify allowed sequence variability for the designed proteins that retained function (i.e.: BCL2L.10/Bcl-B binding).
  • polypeptide comprises or consists of an amino acid sequence according to SEQ TD NO: 1 1 , wherein the polypeptide binds to Bcl-2-like protein A l (Al Bfl- 1).
  • This embodiment is based on saturation mutagenesis studies described in the examples that follow, in which all residues of SEQ ID NO:6 were tested to identify allowed sequence variability for the designed proteins that retained function (i.e.: Bcl-2-like protein Al (Al/Bfl- l)binding).
  • polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 12, and wherein the polypeptide binds to Bci-2-like protein Mcl- 1.
  • polypeptide comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOS: 1-6.
  • polypeptide comprises or consist of an amino acid sequence having at least 50% identity to the amino acid sequence of SEQ ID NO: 13.
  • polypeptide of SEQ ID NO: 13 is a selective high affinity binder of Epstein Ban protein BHFR1.
  • the inventors have earned out saturation mutagenesis on the polypeptide of SEQ ID NO: 13 to identify modifiable residues.
  • the polypeptides of this embodiment have at least 55%, 60%, 66%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identity over their length relative to the amino acid sequence of SEQ ID NO: 13,
  • the polypeptides may include additional residues at the N-terminus, C- ierminus, or both that are not present in SEQ ID NO: l ; these additional residues are not included in determining the percent identity of the polypeptides of the invention relative to the reference polypeptide (i.e.: SEQ ID NO: 13 in this case).
  • the polypeptide comprises at least one conservative amino acid substitution corresponding to residues 3, 13, 21 , 2.8, 31 , 33, 46, 48, 49, 61, 62, 65, 79, 84, 103, and 104 of the amino acid sequence of SEQ ID NO: 13.
  • conservative amino acid substitution means amino acid or nucleic acid substitutions that do not alter or substantially alter polypeptide or polynucleotide function or other characteristics. A given amino acid can be replaced by a residue having similar physiochemical
  • Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1 ) non-polar: Ala (A), Val (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M); (2.) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (FT).
  • Naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norieueine, Met, Ala, Val, Leu, He; (2.) neutral hydrophiiic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into H is: Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; lie into Leu or into Val; Leu into He or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and'or Phe into Val, into He or into Leu.
  • the polypeptide includes the substitutions K31E, E48R, and E65R relative to SEQ ID NO:13.
  • the polypeptide includes the substitutions 121L, Q79L, L84Q, and H104R relative to SEQ ID NO: 13.
  • the polypeptide includes the substitution C I 03 A relative to SEQ ID NO: 13.
  • the polypeptide includes substitutions W3P, I13Q, F28L, M33R, M46E, W49Y, and F6IY relative to SEQ ID NO: 13.
  • the polypeptide includes the substitution N62S relative to SEQ ID NO: 13.
  • the polypeptides of the invention may include additional residues at the N-terminus, C-terminus, or both. Such residues may be any residues suitable for an intended use, including but not limited to detection tags (i.e.: fluorescent proteins, antibody epitope tags, etc.), linkers, iigands suitable for purposes of purification (His tags, etc.), and peptide domains that add functionality to the polypeptides.
  • the polypeptide of the invention further comprises a cell penetrating peptide.
  • Cell penetrating peptides are useful, for example, to facilitate uptake of the polypeptides by cells, and are known to those of skill i the art.
  • Non-limiting examples of such cell penetrating peptides that can be used with the polypeptides of the invention include:
  • TAT GRKKRRQRRRPPQ (SEQ ID NO: 14);
  • RQIKIWFQNRRMK WKK (SEQ ID NO: 15);
  • MAP KLALKLALKALKAALKLA (SEQ ID NO: 16);
  • transportan/TPIO G WTLN S/AG YLLGKI LKALAAL AKKIL (SEQ ID NO: 17);
  • polyarginine R n , n >7 (SEQ ID NO: 19);
  • MPG GALFLGFLGAAG STMGA (SEQ ID NO:20);
  • Pep-1 KETWWETWWTEWSQPKKKRKV (SEQ ID NO:2.1):
  • pVEC LLHLRRRIRKQAHAHSK (SEQ ID NO:22);
  • YTA2 YTAIAWVKAFIRKLRK (SEQ ID NO:23);
  • YTA4 IAWVKAFIRKLRKGPLG (SEQ ID NO:24): M918: VTVLFRRLRIRRACGPPRVRV (SEQ ID NO:25);
  • CADY GLWRALWRLLRSLWRLLWRA (SEQ ID NO:26).
  • polypeptide is used in its broadest sense to refer to a sequence of subunit amino acids.
  • the polypeptides of the invention may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid- specific proteases in vivo), or a combination of D- and L-amino acids.
  • the polypeptides described herein may be chemically synthesized or recombinantly expressed.
  • the polypeptides may be linked to other compounds to promote an increased half-life in vivo, such as by PEGylation, HESylation, PASylation, glycosylation, or may be produced as an Fc- fusion or in de mmunized variants. Such linkage can be covalent or non-covafent as is understood by those of skill in the art.
  • the invention provides pharmaceutical composition, comprising a polypeptide of any embodiment or combination of embodiments of the invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions of the invention can be used, for example, in the methods of the invention described below.
  • the pharmaceutical composition may comprise in addition to the polypeptide of the invention (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer.
  • the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer.
  • the pharmaceutical composition may also include a lyoprotectant, e.g.
  • the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresoi, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chforocresol, phenylmercuric nitrate, thimerosaf, benzoic acid, and various mixtures thereof.
  • the pharmaceutical composition includes a bulking agent, like glycine.
  • the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate- 85, poloxamer- 1 88, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof.
  • the pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood.
  • Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride.
  • the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form.
  • Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.
  • polypeptides of the invention may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use, including but not limited to anti-HA and anti-NA antibodies.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the pharmaceutical compositions further comprise an antibody, or antibody fragment.
  • the antibody or antibody fragment adds functionality to the composition by, for example, helping target the composition to a cell type that has a cell surface receptor to which the antibody selectively binds.
  • compositions of this embodiment are particularly useful for therapeutic applications.
  • any suitable antibody or fragment thereof can be employed that targets a cell or tissue of interest.
  • the antibody or fragment may be recombinantly expressed as part of the polypeptide, may be linked to the polypeptide directly (such as by a covalent linkage or non-covalent interaction), or may not be directly linked to the polypeptide at all (i.e.: present in the same composition, but unlinked).
  • the pharmaceutical carrier may comprise a polymer. Any- suitable polymer may be used that is pharmaceutically acceptable and which does not interfere with function of the polypeptide.
  • the polymer is a block polymer and comprises a hydrophilic block and an endosomolytic block. Any suitable hydrophiiic block and endosomlytic blocks may be used.
  • the hydrophilic block comprises polyethylene glycol methacrylate.
  • the endosomolytic block comprises a diethylaminoethyl methacrylate-butyl methacrylate copolymer.
  • the polymer is a stimuli-responsive polymer that responds to one or more stimuli selected from the group consisting of pH, temperature, UV-visible light, photo- irradiation, exposure to an electric field, ionic strength, and the concentration of certain chemicals by exhibiting a property change.
  • a "stimuli-responsive polymer” is a polymer that changes its associative properties in response to a stimulus. The stimuli-responsive polymer responds to changes in external stimuli such as the pH, temperature, UV-visible light, photo-irradiation, exposure to an electric field, ionic strength, and the concentration of certain chemicals by exhibiting property change.
  • the chemicals could be poly valent ions such as calcium ion, polyions of either charge, or enzyme substrates such as glucose.
  • a temperature-responsive polymer may be responsive to changes in temperature by exhibiting a LCST in aqueous solution.
  • a stimuli- responsive polymer may be a multi-responsive polymer, where the polymer exhibits property change in response to combined simultaneous or sequential changes in two or more external stimuli.
  • the stimuli-responsive polymers may be synthetic or natural polymers that exhibit reversible conformational or physico-chemical changes such as folding/unfolding transitions, reversible precipitation behavior, or other conformational changes to in response to stimuli, such as to changes in temperature, light, pH, ions, or pressure.
  • Representative stimuli- responsive polymers include temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers.
  • the present invention provides isolated nucleic acids encoding a polypeptide of the present invention.
  • the isolated nucleic acid sequence may comprise R A or DNA.
  • isolated nucleic acids are those that have been removed from their normal surrounding nucleic acid sequences in the genome or in cDNA sequences.
  • Such isolated nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to poly A sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the invention.
  • the present invention provides recombinant expression vectors comprising the isolated nucleic acid of any aspect of the invention operatively linked to a suitable control sequence.
  • Recombinant expression vector includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product.
  • Control sequences operably linked to the nucleic acid sequences of the invention are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operabiy linked" to the coding sequence.
  • Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites.
  • Such expression vectors can be of any type known in the art, including but not limited plasmid and viral-based expression vectors.
  • control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive).
  • promoters including but not limited to, CMV, SV40, RSV, actin, EF
  • inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive.
  • the construction of expression vectors for use in transfecting host cells is well known in the art, and thus can be accomplished via standard techniques. (See, for example, Sambrook, Fritscb, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp
  • the expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA.
  • the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector.
  • the present invention provides host cells that comprise the recombinant expression vectors disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic.
  • the cells can be transiently or stably engineered to incorporate the expression vector of the invention, using standard techniques in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAL dextran mediated-, polycationic mediated-, or viral mediated transfection.
  • standard techniques in the art including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAL dextran mediated-, polycationic mediated-, or viral mediated transfection.
  • a method of producing a polypeptide according to the in v ention is an additional part of the invention.
  • the method comprises the steps of (a) culturing a host according to this aspect of the invention under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed polypeptide.
  • the expressed polypeptide can be recovered from the ceil free extract, but preferably they are recovered from the culture medium. Methods to recover polypeptide from cell free extracts or culture medium are well known to the person skilled in the art.
  • the invention provides methods of treating an Epstein-Barr virus - related disease comprising administering to a subject in need thereof a therapeutically effective amount of one or more of the polypeptides of the invention that selectively inhibits BHRFl, or salts thereof, pharmaceutical compositions thereof, a recombinant nucleic acid encoding the one or more polypeptides, a recombinant expression vector comprising the recombinant nucleic acids, and/or a recombinant host cells comprising the expression vector, to treat and/or limit the Epstein-Barr virus related disease.
  • Epstein-Barr virus encodes a pro-survival Bel-2 homologue, BHRFl , which prevents lymphocyte apoptosis during initial infection by sequestering pro-apoptotic BH3-only proteins (especially Bim), and interacting directly with the executioner Bak (Desbien et al., 2009; Kvansakul et al, 2010)(Altm.ann and Hammerschmidt, 2G05)(Henderson et al, 1993), Even though BHRFl is under the control of an early lytic cycle promoter, low levels of constitutive expression have been observed in some cases of EBV -positive BL when the vims is latent, and it has been speculated that BHRFl may be a necessary viral factor for lymph omagenesis (Kelly et al, 2009; Leao et al., 2007; Watanabe et al, 2010). Thus, inhibitors of BHRFl can be used to treat and/ or limit development of Epstein
  • the Epstein-Barr v i r u s - related disease is selected from the group comprising o f infectious mononucleosis, Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma, nasopharyngeal carcinoma, multiple sclerosis, Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy.
  • the Epstein-Barr v i r u s - related disease is a cancer selected from the group consisting of Burkitt's lymphoma, Hodgkin's lymphoma, non- Hodgkin's lymphoma, mantle cell lymphoma, or nasopharyngeal carcinoma.
  • polypeptides for use in this aspect of the invention are selected from polypeptides comprising or consisting of the amino acid sequence of SEQ ID NOS: 1 and 7, including any embodiments thereof such as, but not limited to, further including cell penetrating peptides or antibodies.
  • the invention provides methods for treati g cancer, comprising administering to a subject in need thereof a therapeutically effective amount of one or more o f th e polypeptides that selectively inhibits one or more of Mcl-1, Bcl-2,
  • Bcl-2 B cell lymphoma- 2.
  • the 'executioners', Bak and Bax initiate apoptosis by increasing mitochondrial outer membrane permeability and facilitating the release of mitochondrial cytochrome c to the cytosol, which activates downstream signaling.
  • Six human pro-survival Bcl-2 proteins (Bcl-2, BC1-XL, Bcl-B, Mcl-L Bcl-w and Bfl- 1) inhibit this process.
  • Cellular stresses activate pro-apoptotic BH3-only proteins, which bind and inhibit pro-survival Bcl-2 members, and directly interact with Bak and Bax to favor mitochondrial permeabilization.
  • pro-survival Bcl-2 proteins dampen apoptotic triggers and enhance chemoressstance by sequestering BH3-only proteins or directly inhibiting Bak and Bax.
  • increased expression of pro-survival Bcl-2 proteins is a common feature of many cancers.
  • the polypeptides of the present invention which bind to and inhibit the pro- survival Bcl-2. proteins, can be used to treat cancer.
  • polypeptides for use in this aspect of the invention are selected from polypeptides comprising or consisting of the amino acid sequence of SEQ ID NOS: 2-6 and 8-12, including any embodiments thereof such as, but not limited to, further including cell penetrating peptides or antibodies.
  • the methods may be used alone or in conjunction with other therapies for treating cancer, such as chemotherapy, radiation therapy, and /or surgical removal of the tumor.
  • the polypeptides permit reduced (sub-therapeutic) dosages of current therapies; in another embodiment, such a combination therapy permits the use of otherwise sub-therapeutic dosages of the polypeptide of the invention; these embodiments can be combined.
  • the methods may be used to overcome tumor resistance to the treatment,
  • an effective dose refers to an amount that provides a therapeutic benefit in the treatment, prevention, or management of Epstein- Barr virus and Epstein-Barr related diseases, or cancer. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject's history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents.
  • the term “treat,” “treatment,” or “treating,” means to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition of the disorder being treated.
  • the term “treating” includes reducing or a lleviating at least one adverse effect or symptom of a condition.
  • Treatment is generally “effective” if one or more symptoms are reduced.
  • treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” may include not just the improvement of symptoms, but also a cessation or slowing of progress or worsening of symptoms that would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishrnent of extent of the deficit, stabilized (i.e., not worsening) state of a tumor or malignancy, delay or slowing of tumor growth and/or metastasis, and an increased lifespan as compared to that expected in the absence of treatment.
  • the ter “administering,” refers to the placement of a therapeutic into a subject by a method or route deemed appropriate.
  • the therapeutic can be administered by any appropriate route which results in an effective treatment in the subject including orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • parenteral as used herein includes, subcutaneous, intravenous, intra-arterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or mtraperitoneally. Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response).
  • a suitable dosage range may, for instance, be 0.1 ug/kg- 100 mg/kg body weight; alternatively, it may be 0.5 ug kg to 50 mg/kg; 1 ug kg to 25 mg/kg, or 5 ug/kg to 10 mg/kg body weight.
  • the polypeptides can be delivered in a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or more times) as determined by an attending physician.
  • the invention provides methods for determining the Bcl-2 phenotype of a tumor, comprising contacting tumor cells, tumor ceil lysates or tumor cellular components with one or more polypeptides selected from the group consisting of SEQ ID NOS: 2-6 and 8- 12, under conditions suitable to promote apoptosis signaling in cells of the tumor that express a bcl-2 homologue targeted by the one or more polypeptides; and determining bcl-2 dependency of the tumor based on the polypeptide that causes apoptosis or apoptotic signaling in the cells of the tumor.
  • the methods of this aspect of the invention can be used, for example, to determine an appropriate polypeptide inhibitor of the invention to treat a tumor, by identifying the bcl-2 dependency of the tumor.
  • the method comprises contacting tumor cells, tumor cell lysates or tumor cellular components with each of polypeptides 2-6, or each of polypeptides 8-12, which permits simultaneously determining the bcl-2 dependency of the tumor for each of the Bcl-2 family proteins.
  • Epstein-Barr virus also called human herpesvirus 4 (HHV-4)
  • HHV-4 human herpesvirus 4
  • Epstein-Barr vims has been implicated in several diseases that include infectious mononucleosis, Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma, nasopharyngeal carcinoma and multiple sclerosis.
  • Epstein-Barr virus has been implicated also in disorders related to alpha-synuclein aggregation, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy.
  • Epstein-Barr related diseases are any diseases related to or caused by Epstein-Barr vims, including those listed immediately above.
  • Pro-survival Bcl-2 proteins share a common domain that resembles a cupped hand, with a characteristic hydrophobic surface groove that clasps one side of an amphipathic BH3 domain helix (Czabotar et aL, 2007; Kvansakul et al., 2.010; Liu et al., 2003).
  • Rigidifying BH3 peptides by use of hydrocarbon staples, disulfides or lactam bridges on the non- interactive back side of the helix can reduce the entropic penalty of a partially- folded peptide acquiring a rigid helical conformation upon binding, and improves BH3 peptide affinity (Azzarito et al., 2013).
  • helical bundle scaffold protein of known structure is used only as a topology guide.
  • the Bim- BH3 helix acts as a folding nucleus, around which protein fragments from the PDB are assembled to build a new protein of matching topology to the guiding scaffold (3 LHP chain S (Correia et al., 2010)).
  • Ca-Ca atom-pair distances from the scaffold constrain the assembling protein to within a defined deviation threshold (3.0 A root mean square deviation, RMSD).
  • Thousands of designed proteins were computationally generated to form a family of structural homologues, all with unique sequences and slightly different backbone structures ( Figure 1).
  • the designed proteins were docked to the BHRF1 surface via alignment of the incorporated Bim-BFB motif, and surrounding interface residues (within 8 A) were then further designed, as the incorporated Bim-BH3 motif provides only a fraction of the interaction surface, and many additional contacts across an expansive interface should be designed. Scaffold residues surrounding the graft site were designed to minimize the energy of the modeled bound complex in the ROSETTA energy function (Kuhlman et al,
  • the proteins were filtered both for stability of the monomer (by computed monomer energy, packing based on RosettaHoles (Sheffler and Baker, 2009) and for the lowest number of buried unsatisfied hydrogen bonding atoms) and for interface quality (high shape complementarity, computed binding energy and a low number of buried unsatisfied hydrogen bonding atoms). From thousands of computer-assembled proteins, a small number of designs were selected for further manual modifications, synthetic E. coii codon-optimized genes were constructed, and those proteins that were expressed and soluble in E. coli were tested by yeast surface display for binding to B IRF l (Table 1 ).
  • ⁇ indicates the region of Bim-BH3 from crystal structure 2WH6 that was
  • Bini-BH3 folding nucleus was located.
  • the library was sorted by a single round ofFACS for cells expressing surface protein (Figure 1C; lane 1 ), for the 2% of cells with highest expression (lane 2), and for cells showing binding signal after incubation with 100 nM (lane 3) or 400 nM BHRF1 (lane 4).
  • DNA from the naive and post- sorted populations was harvested and sequenced by fliumina deep sequencing, and the recovery of each designed sequence determined.
  • a minority of designs (Indexes-00 to 27) were enriched following sorting for expression, and just five designs (Indexes-00 to 04) were highly expressed and enriched after sorting for BHRF1 interaction (Figure 1 C). While the four new functional designs share the same 3 -helix topology, the structural details and sequences differ considerably (Figure 2A-D).
  • BHRF1 binding was validated on clonal yeast populations ( Figure 3A).
  • Lo3 ⁇ 4 Enrichment Ratios Expression 1.60, High Expression 2.73, 100 iiM BHRF1 -2.77, 400 nM
  • GRDGERVV WA NQHENTVDEA DKJ ⁇ 4DNQEDEMR NADDEKLR ETH WIAFALEAIG DVFNDAMQAFELLERF KFGQQEQKKLDEFKEKVERLAREASRKLTYLGKRFALDIESG
  • GKDLDEIIDEAR EMDDDADDG AEKLLKLHAGTNHSQDDFNEAHRRWIAV ' ALEEIGDL FNAALRAWR IEEEIR NQRRKEEAEKAKEKVS EYERASRKAAELG EFEERVEQG (SEQ ID NO: 165)
  • Rim-BH3 12 ⁇ 4 2.02 ⁇ 2.1 ⁇ 0.1 0.6 ⁇ 0. 2 2.1 ⁇ 0.3 3 ⁇ 1 12.2 ⁇
  • the BbpD04.1 gene was diversified by error prone-PCR
  • BbpD04.2 was incompatible with single labeling of an added terminal cysteine residue, due to the presence of a second internal cysteine ( Figure 5).
  • Short peptide linkers containing single cysteines were genetically fused to the BbpD04.2 termini ( Figure 5 A) and found to react in seconds with polyethylene glycol (PEG)- maieimide, producing a. higher MW product with reduced electrophoretic mobility ( Figure 5B).
  • BbpD04.2 has an internal buried cysteine, which becomes exposed for PEG-maJeimide conjugation in the presence of the harsh detergent SDS, indicating the protein is folded and the hydrophobic core is generally shielded from solvent unless chemically denatured.
  • the library was sorted for affinity and specificity (yeast were incubated with 400 pM biotinylated BHRFl and 8 nM of an equimoiar mixture of unlabeled human Bcl-2 proteins as competitors; Figure 6B).
  • DMA was extracted from the naive and post-sorted yeast populations, the BbpD04.3 gene amplified as two fragments to provide full sequencing coverage, and the samples were deep sequenced using iiJumina MiSeq sequencing.
  • the frequency of each protein variant is compared between the naive/pre-sorted and enriched/post-sorted populations to calculate an enrichment ratio, which acts as a proxy for the affinity/specificity fitness of each substitution (Fowler et al, 2010; McLaughlin et al, 2012; Procko et al, 2013; Whitehead et al, 2012).
  • the BbpD04.3 affinity sequence-fitness landscape reveals the critical nature of the incorporated Bim-BH3 motif, with most substitutions of interface residues being depleted (Figure 6A).
  • substitutions to proline which can break regular helical secondary structure, are depleted across the first, second and third helical spans of the designed helical bundle fold (Figure 6A)
  • substitutions to aspartate, a short and charged amino acid are depleted within the hydrophobic core as anticipated ( Figure 6A).
  • the BbpD04.3 affinity- specificity sequence-fitness landscape in which unlabeled Bcl-2. proteins were included as competitors for BHRFl binding, is similar (Figure 6B).
  • BbpD04.3 Bacterial expression of BbpD04.3 was very low, limiting the quantity and purity that could be purified for biochemical applications. Simply combining mutations enriched in the sequence-fitiiess landscapes within libraries, while achieving enhanced BHRFl affinity and specificity by yeast surface display, gave clones with undetectable protein expression in E. coli. Therefore, we sought instead to only combine mutations that improved bacterial expression. Twenty-nine BbpD04.3 point mutants with positive enrichment ratios in either the affinity or affinity-specificity sequence-fitness landscapes were expressed in E. coli and analyzed for increased soluble protein levels by small scale NiNTA-agarose precipitation (Figitre 7A).
  • This variant is named BHRFl -INhibiting Design acting Intracellularly (BINDI).
  • BINDI compared to BbpD04.3 is not due to enhanced protein stability; both BbpD04.3 and BINDI undergo cooperative unfolding at high concentrations (> 3 M) of the chemical denaturant guanidinium hydrochloride measured by circular dicnroism (CD) spectroscopy ( Figure 6E).
  • CD circular dicnroism
  • Figure 6E the original design, BbpD04, has nearly linear loss of CD signal over a 0 to 6 M range of guanidinium hydrochloride ( Figure 6E).
  • the absence of a cooperative melting transition is associated with molten globules that lack a rigid core or single native conformation.
  • BbpD04, BbpD()4.3 and BINDI have high thermostability and retain partly a-helicai CD spectra at 95°C, only the evolved BbpD04.3 and BINDI fully renature when the heated protein solutions are cooled ( Figure 7D-G), Further, the original BbpD04 design is sensitive to rapid hydrolysis by proteases, which require unfolded substrate backbone to access the enzyme active site (Figitre 6F-H and 7H). BbpD04.3 and BINDI are similarly resistant to protease digestion with differences attributable to sequence variation (i.e.
  • the designed BINDI protein has high affinity and specificity
  • BINDI'BHRFl interaction was therefore further characterized by alternative methods, BINDI eluted as a higher molecular weight complex by SEC when mixed with BHRFI in solution, whereas BINDI L54E with a knockout mutation in the designed interface did not ( Figure 8A), Using bio-layer interferometry (BLI) to measure the kinetic rate constants, BINDI*BHRF1 was found to form an extraordinarily tight complex (A , ;.
  • BIND! incorporates the Bim-BH3 motif within a de novo designed fold guided by the topology of PDB 3 LHP chain S.
  • the direct graft of Bim-BH3 interaction residues to the equivalent site within the 3LHP S scaffold (design BbpGT) failed to bind BHRFI .
  • the grafted protein did not bind BHRFI .
  • 3LHP_S is structurally similar to BINDI, it is nonetheless a poor steric fit for the BHRFI binding groove in this design protocol.
  • BINDI triggers apoptosis preferentially in an EBV-infected cell line
  • the affinity for Mci- 1 is diminished six-fold (K 0 230 ⁇ 40 nM).
  • the N62.S variant has enhanced apoptotic activity against EBV-positive Ramos-AW ( Figure 1 IE).
  • BINDI N62S with greater specificity amongst the Bcl-2 family for BHRF1, has even greater discrimination between Ramos and Ramos-AW cells ( Figure 1 IB and 1 IE).
  • the enhanced activity of BINDI to initiate cytochrome c release preferentially in EBV-positive cells is therefore due to BHRFI inhibition.
  • BINDI was genetically fused with a C-terminal antennapedia peptide for non-specific cellular uptake and intracellular delivery in vitro.
  • BINDI-antennapedia applied to the growth medium at 4 ⁇ selectively killed 40% of EBV-positive Ramos-AW cells, with no measurable death of EBV-negative Ramos cells ( Figure 12A).
  • Antennapedia- fused proteins concentrate in endocytic organelles and escape to the cytosol with low efficiency (Duvall et a!., 2010).
  • BINDI-antennapedia was conjugated via a terminal cysteine to a diblock copolymer carrier, PoBOO, containing a hydrophilic first block for stability and a pi i -responsive endosomo lytic second block (Duvall et al, 2010: Mamganiello et al., 2012; Convertine et al., 2010).
  • a lower 2 ⁇ dose of BINDI-antennapedia induced 60% cell death preferentially in Ramos-AW cells when conjugated to the PoBOO polymeric carrier for enhanced cytosolic delivery (Figure 12B).
  • Our data suggest inhibition of BHRF1 can effectively kill EBV-positive BL.
  • the copolymer's hydrophilic first block is composed of polyethylene glycol methacrylate (MA) for stability in the host, pyridyldisulfide MA for cysteine conjugation to BINDI, and biotin-hydroxylethyl MA for coupling to streptavidin-antiCDl 9 (aCDI 9; human monoclonal CAT-I3.1E10-SA).
  • the endosomoiytic second block is composed of diethylaminoethyl MA and butyl MA.
  • the entire complex of copolymer:aCD19:BI DI forms large micelles that disassociate at low pH to expose membrane-destabilizing groups ( Figure 13 A).
  • CD 19 is a rapidly internalizing surface antigen, and bound aCDI 9-complex is endocytosed. Copolymer allows escape from the acidic endosome, and presumably BINDI is then released in the reducing cytosolic environment.
  • Subcutaneous Ramos- AW xenograft tumors were established in nude BALB/c mice.
  • the mice were treated intravenously on days 0, 3 and 6 with antibody-copolymer coupled to the inactive scaffold 3LHP(S) or to BINDI.
  • Thirty minutes prior to each treatment cyclophosphamide (CTX) and bortezomib (BTZ) were injected intraperitoneally at subtherapeutic doses to prime cells for apoptosis (O'Connor et a!., 2006).
  • CTX cyclophosphamide
  • BTZ bortezomib
  • the treatments were nontoxic, with no substantial change in mouse body weight.
  • BCL2 family proteins share similar sequences (> 50% similarity between any two family members) and similar structures ( ⁇ 3 A RMSD). It therefore seemed likely that the BINDI protein, having high complementarity with the binding pocket of BHRFl, could serve as an excellent scaffold for engineering new specificities to other BCL2 proteins. Since earlier variants of BIND! prior to exhaustive optimization bound Mcl- 1 with high affinity, we began by repurposing the BINDI protein as a Mcl-1 binder. First, BINDI (PDB 40YD chain D) was 'docked' into the hydrophobic binding cavity of existing crystaliographic models of Mcl-1. in these models, Mcl-1 is bound to nonspecific BH3 peptides from Bim (PDBID).
  • Mcl-1 binder M-CDP02
  • BINDI (PDB 40YD chain D) was docked into the BH3 binding cavity in the structures of Bax-BH3 -bound Bcl-2 (PDB 2XA0), small molecule inhibitors bound to Bcl-2 (PDBs 4AQ3, 4IEH and 4LVT), Bim-BH3 -bound Bcl-XL (PDB 1 PQ1 ; structure of mouse Bcl-XL, which is 97 % identical to the hitman sequence), modified Bim peptides bound to Bcl-XL (PDBs 2YQ6 and 2YQ7), Bax-bound Bcl-XL (PDB 3PL7), a Puma-derived ⁇ peptide bound to Bcl-XL (PDB 4BPK), Bim-bound Bcl-B (PDB 4B4S), and Bak-bound Bfl-1 (PDB 3 ⁇ ).
  • ADPK.KVLDKAKDQAENRVRELKQKLEELY EARKLDLTQEMRRELRNRATAAILQATGDLL NATQQAKDEADKLKKAGLVNSQQLDELKRRLEELQNEAAEKAADYGEEFWLKLEY (SEQ ID NO: 43)
  • ADPKKILDK ⁇ KDQ ⁇ NRVRELKQELERLYKEARKLDLTQEMRRKLHVRYIAAMLKAIASIL NAIAQAENEADKLKKAGLWSQQLDELRRRLEELTEEAAQKAIIDYGREFQLKLEYG (SEQ ID NO: 59)
  • the pre- and post-sort populations were deep sequenced and enrichment ratios for all single amino acid substitutions calculated. From these sequence-fitness landscapes, mutations were chosen that were highly enriched during selection (Table 7). in the cases of the designed ikl-Xi .. Bcl-B, and Bfl-l binders, these enriching mutations were then combined in a combinatorial library that was selected by five (Bel-XL binder) or three (Bfl-l and Bcl-B binders) rounds of FACS to find variants with significantly improved affinity and/or specificity, each round under more stringent conditions including lower concentrations of target Bcl-2 paralogue and/or higher concentrations of competitors (Tables 7 and 8).
  • 2-INDI binds Bcl-2 with K D 0.839 ⁇ 0.005 nM and > 2,000-fold weaker affinity for the next tightest binding BCL2 family protein
  • XINDI binds Bel-XL with ⁇ ⁇ 5.59 ⁇ 0.03 nM and > 660-fold weaker affinity for other BCL2 proteins
  • 10-INDl binds Bcl-B with 24.7 ⁇ 0.1 nM affinity, and 1000-fold specificity
  • FINDI binds Bfl-1 with K D 0.91 ⁇ 0.01 nM and > 350-fold specificity (Table 10).
  • the different BCL2 inhibitors differ from each other by as many as 39 mutations, yet when any of the sequences is queried against GenBank for homologues by BLAST (E -value threshold 0.1), the proteins are found to be related only to each other, without homologous natural proteins.
  • BLAST E -value threshold 0.1
  • Any modified v ersion of BIND! or its derivatives will similarly belong to our designed protein family but lack homology to any natural protein, and should therefore be covered by the claims in this patent.
  • Our interface can therefore be divided into three regions from the center outwards: (i) a conserved core for binding all BCL2 family members, (si) a region that principally determines specific interactions, and (iii) an extreme periphery that can offer an occasional specificity contact.
  • ROSETTA® software can be downloaded from the Rosetta Commons web site, wherein online documentation and RosettaScripts® syntax can be found.
  • a suitable helical region of the scaffold protein was aligned to the Bim-BH3 motif of PDB 2WH6 (Bim-BH3 » BHRF1 ) using PyMOL (Schrodinger, LLC).
  • the structural alignment was visually inspected for minimal backbone clashes between the scaffold protein and BHRF1 (side chain clashes may be fixed later by sequence design of the scaffold and by retainer repacking on the target).
  • scaffold residues were mutated in PyMol to the corresponding Bim-BH3 residue within the interface core; this 'grafted ' important Bim interaction residues to the scaffold surface by mutation.
  • a new FDB file containing the partially mutated scaffold bound to BHRFl was saved and used as the input for ROSETTA- based design.
  • Design with ROSETTA An example command line to launch ROSETTA (Leaver-Fay et al., 2011) and example recipe/protocol file (Fleishman et ai., 201 l a) was developed. The design run was launched ten times. The consensus sequence was chosen for experimental validation after minor manual modification (e.g. a less-represented amino acid amongst the set of ten designs may be substituted for the consensus residue based on user preference).
  • Proteins that passed the interface design filters were further filtered based on properties of the unbo und designed protein.
  • the lowest scoring 10-20 designs for monomer energy, unsatisfied buried polar atoms, and RosettaHoles score were selected for manual inspection. Designs were human modified to increase packing within the hydrophobic core and increase surface hydrophilicity, using the ROSETTA graphical user interface Foldlt (Cooper et al., 2010). Those designs considered most promising by the human eye were then selected for experimental validation.
  • Predicted binding probabilities for BbpD04 point mutants were calculated using the method of ( Whitehead et al., 2012), with mutations ranked according to specificity
  • Bcl-2 proteins were synthesized (Genscript) and cloned with C-terminal avi-6his tags (GLNDIFEAQ 1EWHEGSHHHHHH (SEQ ID NO: 75)) into plasmid pET29b (Ndel-Xhol sites; Novagen): human Bcl-2 a.a. 1-207 (Accession No. NP_ 000624.2), Bcl-w a.a. 1-182 (AAB09055.1), Bfl-1 a.a. 1-153 (C4S mutation; NP 004040), Bci-B a.a. 1 1 -175
  • E, coli BL21 * (DE3) (Invitrogen) transformed with the relevant plasmid were grown at 37 °C in terrific broth with 50 .ug/ml kanamycin to ODeoo 0.5-0.8, transferred to 21 °C and expression induced overnight with 0.1 mM IPTG. Centrifuged cells were resuspended in lysis buffer (20 mM Tris-Cl pH 8.0, 20 mM imidazole, 300 mM NaCl, 0.5 mM PMSF) supplemented with 0.2 mg/ml lysozyme and 0.06 mg/ml DNase I, and sonicated.
  • lysis buffer (20 mM Tris-Cl pH 8.0, 20 mM imidazole, 300 mM NaCl, 0.5 mM PMSF
  • Proteins were concentrated using a centrifugal ultrafiltration device (Sartorius) and separated from remaining contaminants by SEC using a Sephacryl-100 16/600 column (GE Healthcare) with running buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM DTT). Fractions containing pure protein were pooled, concentrated to 5-20 mg ml based on calculated extinction coefficients for absorbance at 280 nm, and aliquots snap frozen in liquid N 2 for storage at -80 °C. For animal studies, endotoxin was removed with a high-capacity endotoxin removal spin column (Pierce) and reducing agent was removed with a PD-10 desalting column (GE Healthcare).
  • endotoxin was removed with a high-capacity endotoxin removal spin column (Pierce) and reducing agent was removed with a PD-10 desalting column (GE Healthcare).
  • avi-6his-tagged ligands (20 ⁇ ) in reaction buffer (250 mM potassium glutamate, 20 mM Tris-Cl [pH 7.5], 50 mM bicine [pH 8.3], 10 mM ATP, 10 mM MgOAc, 100 ⁇ d-biotin) were enzymaticaily biotinylated with 150 U/ ⁇ BirA (Avidity) at room temperature overnight, followed by purification with NiNTA-agarose and SEC. Biotinylated iigands were stored at 4 °C in 150 mM NaCi, 20 mM Tris-Cl (pH 7.5), 1 mM DTT, 0.02% sodium azide. Yeast surface display
  • Yeast ceils were sorted on a BD Influx cytometer operated by Spigot (BD Biosciences) and recovered in SDCAA media at 30 °C overnight. Yeast were lysed with 125 U/ml Zymolase at 37 °C for 5 h, and DNA was harvested (Zymoprep kit from Zymo Research). Genomic DNA. was digested with 2 U./ ⁇ Exonuclease I and 0.25 U/ ⁇ Lambda exonuclease (New England Biolabs) for 90 min at 30 °C, and plasmid. DNA purified with a QIAquick kit (Qiagen). DNA. was deep sequenced with a MiSeq sequencer (Illumina) and sequences were analyzed with adapted scripts from Enrich (Fowler et ai., 20 1).
  • genes were synthesized (Gen9) with barcodes downstream of the stop codon for easy identification during deep sequencing (Table 3).
  • the genes were PCR amplified using primers that annealed to external regions within the plasmid, followed by a second round of PCR to add flanking sequences for annealing to the Illumina fl ow cell oligonucleotides and a 6-bp sample identification sequence.
  • PCR rounds were 12 cycles each with high-fidelity Phusion polymerase (New England Biolabs).
  • Barcodes were read on a MiSeq sequencer using a 50-cycle reagent kit (Illumina). 257,812 sequences passing the chastity filter were read in the naive population (ranging from 260 to 17,192 reads per gene, with a median of 2,492).
  • the sorted populations had 1 17,720 to 232,195 reads.
  • the BbpD04.3 gene was amplified as two overlapping fragments to provide complete sequencing coverage, and additional flanking DNA for annealing to the Illumina flow cell was added by PCR as described above.
  • Gel-purified DNA was sequenced on a MiSeq sequencer using a 300-cycle paired-end reads reagent kit (lilumina). 3,058,244 sequences passing the chastity filter were read for the naive population. Each single amino acid substitution had 10 to 10,856 reads, with a median of 451 reads per mutant, and only mutation E109F was not represented. Parental protein sequences accounted for -25% of reads. 2,930,499 and 2,548,997 sequences passing the chastity filter were read for the affinity and affinity-specificity sorted populations, respectively.
  • Proteins (20 nmol each) were injected in a 200 ⁇ loop in line with a Superdex-75 10/300 column (GE Healthcare) and separated with running buffer (20 niM Tris-Cl pH 7.5, 150 niM NaCl, 1 mM DTT) at room temperature.
  • Substrates (0.5 mg ml) were incubated at 37°C with protease (0.01 mg/ml) in 50 mM Tris-HCl (pH 8.0), 10 mM CaCl 2 . Reactions were terminated with benzamidine (12.5 mM final), PMSF (1.25 mM final) and 4x load dye. Samples were run on 18% SDS-polyacrylamide gels, stained with Coomassie dye, and the decrease in full-length protein quantified using Image! software (National Institute of Mental Health).
  • CD spectra were recorded with a Model 420 spectrometer (AVIV Biomedical). Unless stated othenvise, proteins were at 10 ⁇ in PBS and data were collected at 25°C.
  • Ceils H0 were equilibrated in 5 ml of homogenization buffer (0.25 M sucrose, 1 mM EGTA, 10 mM HEPES/NaOH, 0.5% BSA, pH 7.4, Roche Complete protease inhibitors) for 5 min. Samples were kept on ice or at 4 °C until assayed. Cells were homogenized under N 2 pressure (400 psi) in a steel disruption vessel (Parr Instrument Company) for 10 min, then centrifuged (750 g) for 10 min to remove intact cells. Supernatant was centrifuged again (12,000 g) for 12 min to collect mitochondria.
  • homogenization buffer 0.25 M sucrose, 1 mM EGTA, 10 mM HEPES/NaOH, 0.5% BSA, pH 7.4, Roche Complete protease inhibitors
  • the pellet was resuspended in 300 ⁇ wash buffer (0.25 M sucrose, 1 niM EDTA, 10 raM Tris/HCi pH 7.4). Proteins at the indicated concentrations were incubated with mitochondria (25 g mitochondrial protein based on BCA assay, Sigma) in 50 id final volume of experimental buffer (125 mM KG, 10 mM Tris-MOPS pH 7.4, 5 mM glutamate, 2.5 mM malate, 1 mM K-PQ 4 , 10 ⁇ EGTA-Tris pH 7.4) for 30 min at room temperature.
  • mitochondria 25 g mitochondrial protein based on BCA assay, Sigma
  • cytochrome c release was quantified using a Cytochrome c ELISA kit (Life Technologies). Complete cytochrome c release was quantified by treatment with 0.5% Triton-XlOO.
  • a 25,000 Da diblock copolymer composed of 95% polyethylene glycol methacrylate (300 Da) for stabilit and 5% pyridyl disulfide methacryate for conjugation in the first block, and 60% diethylaminoethyl methacrylate and 40% butyl methacrylate in the second block, was synthesized by reversible addition-fragmentation chain transfer. Development, and characterization of the diblock copolymer will be published in a separate article. After purification, PoBOO was dissolved in ethanol at 100 mg/ ' ml then diluted into PBS at 1 mg/ml and spin filtered to remove ethanol.
  • Proteins with exposed terminal cysteines were incubated with PoBOO at a molar ratio of 2: 1 (proteinrpolymer) overnight. Protein-polymer conjugation was quantified by measuring pyridyl disulfide release and the absorbance of 2-mercaptopyridine at 343 nm with 8,080 M ⁇ cm "1 as the extinction coefficient. For cell viability studies, protein and protein-polymer conjugates were incubated with Ramos or Ramos-AW cells in a 96 well round bottom plate with 50,000 cells per well in 100 ⁇ .1 media.
  • Cells were cultured in RPM1 1640 containing L-glutamine and 25 mM HEPES supplemented with 1 % penicillin-streptomycin (GIBCO) and 10% fetal bovin serum (Invitrogen) at 37 °C and 5% C0 2 . After 24 h, cell viability was measured using a CeilT iter 96 Aqueous One Solution Cell Proliferation Assay, MTS (Pro mega).
  • Ramos, Ramos-AW, Daudi, Raji, DOHH2, JVM-2, and JVM- 13 were grown in RPMI 1640 containing L-glutamine and 25 mM HEPES supplemented with 1% penicillin-streptomycin (GIBCO) and 10% fetal bovine serum (FBS, nvitrogen).
  • Jeko-1 were grown in similar RPM 1640 media supplemented with 20% FBS.
  • Granta-519 and K562 were grown in Iscove's DMEM supplemented with 10% FBS. All cell lines were maintained in log growth phase at 37 °C and 5% C0 2 .
  • a 44,000 Da diblock copolymer composed of 80% polyethylene glycol methacrylate (950 Da), 10% pyridyl disulfide
  • mice BALB/c nu/nu mice (6 to 8 weeks old) were used from Harian Sprague-Dawley and housed under protocols approved by the FHCRC Institutional Animal Care and Use Committee. Mice were placed on biotin-free diet (Purina Feed) for the duration of study.
  • biotin-free diet Purina Feed
  • mice were injected intraperitoneally with cyclophosphamide (35 mg/kg) and bortezomib (0.5 mg/kg). After 30 rnin, mice were injected via tail vein with conjugates at a dose of 15 mg/kg (aCD19), 300 mg/kg (Pol950) and 105 mg/kg (BINDI or 3LHP). Body weight was monitored for toxicity and tumor sizes were measured while blinded to treatment groups. Measurements were performed in the x, y, and z plane using calipers three times a week. Mice were euthanized when tumors reached a volume of 1250 mm 3 .
  • BINDI should provide a useful tool for characterizing primary isolates of EBV-associated cancers in which the molecular mechanisms of cell transformation remain poorly understood, including EBV-positive BL, Hodgkin's lymphoma, and nasopharyngeal and gastric carcinomas (Young and Murray, 2003).
  • BINDI has a structure and amino acid sequence found after computationally filtering thousands of potential designed conformations for optimum interactions with BHRFl , The ability to custom-tailor the backbone conformation to the challenge at hand helped achieve very high affinity and specificity.
  • BIND! is an artificial polypeptide sequence that folds to a designed structure, with no identifiable homologues in nature.
  • sequence variants of BINDI see Figures 2, 4, 6 and 14
  • Redesigning BINDI to bind other BCL2 family proteins yielded a set of related sequences (MINDI, 2-INDI, XTNDI, 10-INDI and FINDI), with any two differing by as many as 39 mutations (34% of the protein).
  • MINDI, 2-INDI, XTNDI, 10-INDI and FINDI a set of related sequences (MINDI, 2-INDI, XTNDI, 10-INDI and FINDI), with any two differing by as many as 39 mutations (34% of the protein).
  • Saturation mutagenesis of all these designed proteins consistently revealed that significant sequence diversity is tolerated ( Figure 19 and Table 5 5 -20).
  • many sequence homologues can maintain our artificially designed structure and
  • BINDI Intracellular delivery of BINDI, either of encoding nucleic acid or of the polypeptide, is expected to have therapeutic effects in Epstein-Barr related diseases generally. Quantitative analysis of mRNA expression has shown that different cancer lines overexpress different BCL2 family members. The designed proteins described herein can specifically inhibit BCL2 family members at the protein level, thereby demonstrating which BC.L2 proteins are functionally important for preventing apoptosis in different cancers. This will lead to better tumor characterization and future diagnostics, in addition targeted therapies as described for BINDI delivery to EBV- positive cancer.
  • Epstein-Barr virus provides a new paradigm: a requirement for the immediate inhibition of apoptosis.
  • Epstein-Barr virus DNA in human lymphoid cell lines in vitro conversion. Virology 73, 96-105.
  • BH3 domains define selective inhibitory interactions with BHRF-1 and KSHV BCL-2. Cell Death Differ 15, 580-588.
  • RosettaScripts a scripting language interface to the Rosetta macromolecular modeling suite. PLoS One 6, e20161.
  • Epstein-Ba r vims-coded BHRFl protein a viral homologue of Bcl-2, protects human B cells from programmed cell death. Proc Natl Acad Sci U S A 90, 8479-8483.
  • BJA-B Burkitt lymphoma
  • ROSETTA3 an object-oriented software suite for the simulation and design of macrornolecules. Methods Enzymol 487, 545- 574.
  • lymphomagenesis the VVp/BHRFl link.
  • PLoS Pathog 5 el 000341.
  • R.OSETTA3 an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol 487, 545- 574.
  • Epstein- Barr virus-encoded Bcl-2 homologue functions as a survival factor in Wp-restricted Burkitt lymphoma cell line P3HR-1. J Virol 54', 2893-2901.

Abstract

The present invention provides designed polypeptides that selectively bind to and inhibit Epstein Barr protein BHRFl, and B cell lymphoma family proteins, and are thus useful for treating Epstein Barr-related diseases and cancer.

Description

Polypeptides to inhibit Epstein Barr viral protein BHRF1 and B cell lymphoma
family proteins
Cross Reference
This application claims priority to U.S. Provisional Patent Application Serial Number 61/951,988 filed March 12, 2014, incorporated by reference herein in its entirety.
Federal Funding Statement
This invention was made with U.S. government support under P41 GM103533 awarded by the National Institutes of Health and under HDT'RA 1 - 10-0040 awarded by the Defense Threat Reduction Agency. The U.S. Government has certain rights in the invention.
Figure imgf000002_0001
Following virus infection, cells may undergo apoptosis to prevent further virus spread in the host. This has spurred viruses to evolve counteracting mechanisms to prevent host ceil death, and during latent infection these factors may contribute to the development of cancer. This includes multiple cancers associated with Epstein-Barr virus (EBV), in particular Burkitt's lymphoma (BL).
Apoptosis and cell survival are regulated by the homeostaiic balance of B cell lymphoma-2 (Bel- 2) family proteins (reviewed in (Martinou and Youle, 201 1)), which fall in to three classes. The 'executioners', Bak and Bax, initiate apoptosis by increasing mitochondrial outer membrane permeability and facilitating the release of mitochondrial cytochrome c to the cytosol, which activates downstream signaling. Six human pro-survival Bcf-2 proteins (Bcl-2, BC1-XL, Bcl-B, Mcl-l , Bcl-w and Bfl-l) inhibit this process.
Counterbalancing these are numerous pro-apoptotic BH3-only proteins, including Bim. These factors share an approximately 26 residue Bcl-2. homology 3 (BH3) motif, an amphipathic a- helical element which binds a hydrophobic groove on the surface of the canonical Bcl-2 fold. Cellular stresses activate pro-apoptotic BH3-only proteins, which bind and inhibit pro- survival Bcl-2 members, and directly interact with Bak and Bax to favor mitochondrial permeabilization. Conversely, pro-survival Bcl-2 proteins dampen apopiotie triggers and enhance chemoresistance by sequestering BH3-only proteins or directly inhibiting Bak and Bax. Increased expression of pro-survival Bcl-2 proteins is a common feature of many cancers.
Epstein-Barr viras encodes a pro-survival Bcl-2 homologue, BHRF1 , which prevents lymphocyte apoptosis during initial infection by sequestering pro-apoptotic BH3-only proteins (especially Bim), and interacting directly with the executioner Bak (Desbien et al, 2009; Kvansakul et al, 2010)(Altmann and Hammerschmidt, 2005)(Henderson ei al., 1993). Even though BHRF1 is under the control of an early lytic cycle promoter, low levels of constitutive expression have been observed in some cases of EBV-positive BL when the vims is latent, and it has been speculated that BHRF1 may be a necessary viral factor for lymph omagenesis (Kelly et al, 2009; Leao et al., 2007; Watanabe et al., 2010).
Summary of the Invention
In a first aspect, the invention provides polypeptides comprising an amino acid sequence having at least 50% amino acid sequence identity over its length relative to the amino acid sequence of SEQ ID NO,: 1, wherein the polypeptide selectively binds to a protein selected from the group consisting of Epstein Ban" protein BHFR1 , and B cell lymphoma family proteins selected from the group consisting of myeloid cell leukemia 1 (Mcl-1), B-cell lymphoma 2 (Bcl-2), Bcl-2-like protein 1 (BCL2L1/Bcl-XL), Bcl-2-like protein 10
(BCL2L1Q/Bci-B), and Bcl-2-like protein A l (Al/Bfl-ί). In one embodiment, the polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity over its length relative to the amino acid sequence selected from the group consisting of SEQ ID NOS:2-6. In various further embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 7- 13, wherein the polypeptide binds to a specific target. In a further embodiment, the polypeptides further comprise a cell-penetrating peptide and/or an antibody or antibody fragment.
In another aspect, the invention provides pharmaceutical composition, comprising a polypeptide of the invention and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises an antibody. In another embodiment, the carrier comprises a polymer, such as a polymer comprising a hydrophilic block and an endosomolytic block, or a stimuli-responsive polymer.
In various further embodiments, the invention provides recombinant nucleic acids encoding a polypeptide of the invention, recombinant expression vectors comprising the nucleic acid of the invention operatively linked to a promoter, and recombinant host cells comprising the recombinant expression vectors of the invention.
In another aspect, the invention provides methods for treating an Epstein-Barr v i ru s - related diseases comprising administering to a subject in need thereof a therapeutically effective amount of one or more o f the polypeptides of the invention, or salts thereof, pharmaceutical compositions thereof, a recombinant nucleic acid encoding the one or more polypeptides, a recombinant expression vector comprising the recombinant nucleic acids, and/or a recombinant host cells comprising the recombinant expression vector, to treat Epstein-Barr virus related diseases wherein the polypeptide or encoded polypeptide selectively inhibits BHRF i .
In further aspect, the invention provides methods for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of one or more of the polypeptides of the invention, salts thereof, a pharmaceutical composition thereof, a recombinant nucleic acid encoding the one or more polypeptides, a recombinant expression vector comprising the recombinant nucleic acid, and/or a recombinant host cell comprising the recombinant expression vector, to treat cancer, wherein the polypeptide or encoded polypeptide selectively inhibits one or more of Mcl- 1 , Bcl-2, BCL2L 1 /Bcl-XL, BCL2L10/Bcl-B, and Al/Bfl- 1.
In another aspect, the invention provides methods for determining the Bcl-2 phenotype of a tumor, comprising contacting tumor cells, tumor cell lysates or tumor cellular components with one or more polypeptides selected from the group consisting of SEQ ID NOS: 2-6 or 8- 12, under conditions suitable to promote apoptosis signaling in cells of the tumor that express a bcl-2 homologue targeted by the one or more polypeptides; and
determining bcl-2 dependency of the tumor based on the polypeptide that causes apoptosis or apoptotic signaling in the cells of the tumor.
Brief Description of the Drawings
Figure 1. De novo protein assembly protocol. (A) A scaffold (grey ribbon) is aligned to the Bim-BH3 motif (black) bound to BHRF l (white) (i). The Bim-BH3 peptide is extended on both ends and a new protein structure (black tube) is built using fragment-based assembly (ii), followed by rounds of minimization and sequence design. The newly assembled protein is docked to BHRFl and the surrounding interface is designed (Hi). Many designs are generated that are filtered by multiple criteria (iv). (B) Computational models of designed proteins BbpD04 and BbpD07 (black) that bind BHRF l (white). Apparent affinities (mean ± SE, n = 3-6) are from yeast display titrations. (C) Seventy-four computationally designed proteins without human modifications (Indexes-01 to 74) were sorted by FACS for surface expression and BHRF1 binding. BbpD04 (Index- 00) was included as a positive control. The gene frequencies in the sorted population were divided by their frequencies in the naive library to calculate a log? enrichment ratio, plotted from -4 (i.e. depleted, black) to +4 (i.e. enriched, white). See Table 3. (D) Histogram of the mean RMSD between the ten lowest energy structures found in ah initio structure prediction calculations and the intended designed structure for each of the sets of designs included in (C). Designs with computed energy minima near the designed target conformation have a higher probability of binding BHRFl ,
Figure 2. Diversity of designed proteins. (A) Index-21 (black) bound to BHRF 1
(white). Human-made modifications of computationally-designed Index-21 to form derivative BbpD()4 are indicated with labels and side chain spheres. (B) Structures of designs that bound BHRF 1 (Indexes-OO to 04) are aligned via the Bim-BH3 incorporation motif (boxed with broken line). Side view showing structural diversity. (C) As in (B), viewed from N-termini. (D) Sequence alignment of BHR F 1 -binding designs (Indexes-OO to 04 (from top to bottom SEQ ID NOs: 82, 83, 84, 85, 86)) and the guiding scaffold (3 LHP chain S (SEQ ID NO: 74)). Amino acid identity (black shading) or chemical similarity (grey) to design Inde - 00/BbpD04 is shown. The Bim-BH3 incorporation site is marked with a bar above.
Figure 3. Predictions of folding probability correlate with designed protein functionality, (A) Putative binders (Indexes-Ol to 04) were expressed on yeast and validated by titrating BHRF1 to determine apparent binding affinities. Three randomly chosen 'failed' designs did not show interactions with BHRF I . (B) Examples of forward folding landscapes. Proteins index-00, 01 and 04 bind BHRF 1. Protein index- 15, 47 and 67 do not. 1. 30,000- 100,000 decoys were predicted for each query (black points). Cct-Ca RMSD is measured between each decoy and the intended computational model. (C-E) Properties of the designed interfaces plotted against the experimental enrichment ratios after selection for binding to 100 nM BHRFl . Each data point represents a designed protein (Indexes-Ol to 74). Plotted are the (C) interface buried solvent-accessible surface area, (D) the calculated interface binding energy, and (E) the number of unsatisfied buried polar atoms at the interface. (F-H) As for (C-E), except showing computed metrics for the unbound designed proteins. Plotted are enrichment ratios versus (F) the holes (packing) score of the apo-protein, (G) ca lculated energy, and (H) unsatisfied buried polar atoms.
Figure 4. Affinity maturation of designed protein BbpD04. (A) Computational model of BHRFl (white ribbon) bound to design BbpD04 (surface). The electric field from BHRFl is mapped to the BbpD04 surface; regions experiencing a positive field are shaded dark grey.
(B) Based on a computational model of the Mcl-T *BbpD04 complex, the electric field from Mcl- 1 (ribbon) is mapped to the surface of BbpD04. A positive field is shown as dark grey.
(C) Model of BbpD04. Residues rationally mutated to specifically enhance electrostatic complementarity to BHRF1 are shown as spheres and labeled. These mutated sites are located in regions where the electric fields from BHRF1 and Mcl-1 differ. (D) The effect of BbpD04 mutations on specificity. (E) Sorting a randomly mutated library of BbpD04.1 yielded evolved variant BbpD04.2. The four mutations in BbpD()4.2 (white sticks) are shown on the computational model of BHRF 1 -bound BbpD04.1 (black). (B) Purified proteins were analyzed by SEC, In the left panel, BbpD04.2 (black trace) forms a left-shifted higher MW complex (pale grey) when mixed with BHRF l (dark grey), in the right panel, BbpD04.2 L54E (black) with a mutation in the binding site does not shift (pale grey) when mixed with BHRFl (dark grey).
Figure 5. Mutagenesis of an internal cysteine allows site-specific conjugation at the termini. (A) Short peptide linkers were genetically fused to the BbpD04.2 termini. Linker- 3 termini were used in all later experiments where conjugation to a single cysteine was required (from top to bottom SEQ ID NOs: 76, 77, 78, 79, 80 and 81). (B) Cysteme-iinkers reacted with 5kD polyethylene glycol (PEG)-maleimide (Creative PEGWorks), producing higher MW products on Coomassie-stained sodium dodecylsulphate (SDS)-polyacrylamide gels. BbpD04.2 has a buried cysteine, which becomes exposed for PEG-maleimide conjugation in the presence of the harsh detergent SDS. (C) Cysteine- linker BbpD04.2 proteins were conjugated to HPDP-biotin for 4 h at room temperature. Biotinylated protein was incubated with streptavidin and aggregation measured by absorbance at 350 nm. Mutation of the internal cysteine (CI 03 A) markedly diminishes aggregation. (D) DMSO, the solveni used for dissolving HPDP-biotin, did not increase exposure of the internal cysteine for PEG- maleimide modification. (E) PEG-maleimide reacted with a fraction of the BbpD04.2 protein when incubated together overnight at room temperature (RT). (F) Both BbpD04.2 C 103A and C I 03 V mutations were predicted by the ROSETTA. energy function to be tolerated following minimization. BbpD04.2 CI 03V had reduced specificity by yeast surface display for BHRFl over other prosurvival Bcl-2 proteins, whereas BbpD()4.2 C103A (called BbpD()4.3) had only a minor loss of affinity and specificity.
Figure 6. BTND1 has improved bacterial expression and stability. (A.) All single amino acid substitutions of BbpD04.3 were expressed in a yeast display library and sorted by FACS for high affinity binding to BHRFl . Plotted for each substitution is the log2 enrichment ratio from -3.5 (depleted, black) to +3.5 (enriched, white). Stop codons, *. The region of the incorporated Bim-BH3 motif is boxed with a broken line. Secondary structure and core residues are indicated above. Substitutions to aspartate (depleted for core residues) and to proline (depleted for helical residues) are boxed. (B) As in (A), except the library was sorted for high affinity and specificity. (C) The modeled structure of BbpD04.3 is shaded by sequence Shannon entropy from 2.8 (highly conserved, dark) to 4.3 (variable, white), based on the sequence-fitness landscapes. (D) BbpD04.3 and its derivative BIND! were expressed as C-terminal 6his-tagged proteins in E. coli, precipitated from cleared lysate with NiNTA- agarose and analyzed on a Coomassie-stained SDS-polyacrylaniide electrophoretic gel. An arrow indicates the expected MW of the designed proteins at 15 kD. (E) CD spectra of BbpD04 and its variants (10 μΜ in PBS) were collected at 25°C in the presence of guanidinium hydrochloride. The fraction of protein folded was monitored by the change in CD signal at 222. nm. (F-H) BbpD04 and its variants were digested with proteases of different substrate specificities: trypsin (F), chymotrypsin (G) and elastase (H). Shown is mean ± range for 3 repeats. Also see Figure 7H. (I) Summary of all mutations made to BbpD04 during affinity maturation.
Figure 7. BINDI has increased bacterial expression and protein stability. (A)
BbpD04.3 point mutants were expressed overnight at 22 °C in E. coli Rosetta 2 cells. Cells were harvested, the C-terminaliy 6his-tagged proteins precipitated with NiNTA-agarose to partially remove background bands, and analyzed on Coomassie-stained SDS-polyacrylamide electrophoretic gels. White arrows indicate mutations with elevated expression. (B) As in (A), with mutations now combined to provide a large increase in expression. (C)
Computational model of BHRF 1 -bound BbpD()4.3. Combined mutations in variant BINDI are highlighted with dark sticks. (D) Molar ellipticiry at 222 nm as the protein is heated and cooled. Substantial helical structure remains at 95 °C, Evolved variants BbpD04.3 and BINDI fully renature. (E) Molar ellipticitv of original design BhpD04 as a function of wavelength, recorded at 25 °C, 95 °C, and after cooling back to 25 "C, (F) As in (E), measured for variant BbpD04.3. (G) As in (E), measured for variant BINDI. (H) Protease-susceptibility of BbpD04 and affinity-matured variants BbpD04,3 and BINDI. Protein substrates were incubated for 0, 5, 15, 30, 60, and 120 minutes with protease at 37 °C, reactions were terminated with inhibitors, and proteolysis followed on Coomassie-stained SDS- polyacrylamide gels.
Figure 8. BINDI binds BHRF1 with high affinity and specificity. (A) BINDI or knockout mutant BINDI L54E were mixed with BHRF1 and separated by SEC. A shift in elation volume upon mixing BINDI and BHRFI is abrogated by the knockout mutation. (B) Biotinylated BHRF 1 was immobilized to a BLI sensor and the interaction with BINDI was measured at the indicated concentrations. (C) BLI kinetic analysis of BINDI interactions with BHRFI (as in panel B) and human Bel -2 proteins. (D) BLI kinetic analysis of interactions between the Bim-BH3 motif fused to the C-terminus of maltose-binding protein (MBP) and Bcl-2 proteins immobilized to the sensor surface.
Figure 9. Structural basis for exceptional affinity and specificity of BINDI. (A) Slice through the crystal structure of BINDI (black ribbon) bound to BHRF I (white ribbon with surface). The guiding scaffold 3 LHP S (grey) is aligned to BINDI at the Bim-BH3 incorporation site. A direct graft of the BH3 motif into 3LHP_S at this position causes clashes elsewhere with the BHRF ! surface. (B) Crystal structure of BIND! (black) bound to BHRFI (white). (C) The surface of BHRFI , with the buried contact surface in
BHRFI "BINDI shaded black. (D) The surface of BINDI, with the buried contact surface in BHRFI 'BINDI shaded. Buried residues from the incorporated Bim-BH3 motif are dark grey. Buried residues in the surrounding designed surface are black. (E) The crystal structure (PDB 2WH6) of Bim-BH3 (black) bound to BHRF I (white). (F) The surface of BHRFI , with the buried contact surface in BHRF 1 »Bim-BH3 shaded black. (0) The surface of Bim- BH3, with the buried contact surface in BHRF I »Bim-BH3 black.
Figure 10. Mutations within the incorporated Bim-BH3 motif are not the major source of the exceptional specificity of BINDI. (A) Crystal structure of BINDI (surface) bound to BHRFI (black ribbon). The buried contact surface areas are indicated below. (B) The surface of BINDI, with the buried contact surface shaded. Buried residues from the incorporated Bim-BH3 motif are dark grey. Buried residues in the surrounding designed surface are black. (C) Residues of BINDI that changed during affinity maturation are black. Only two residues at the edge of the incorporated Bim-BH3 motif were substituted (W49Y and F61Y). (D)
Sequences of the Bim-BH3 (SEQ ID NO: 87) motif and equivalent regions in BbpD04 (SEQ ID NO: 88) and BINDI (SEQ ID NO: 89). Residues of Bim-BH3 that were fixed in the design of BbpD04 are shaded. Based on these sequences, two 26-residue peptides were fused to maltose-binding protein (MBP): BimBH3-W57Y-F69Y (SEQ ID NO: 90) and BimBH3-5* (SEQ ID NO: 91 ). These have mutations to the Bim-BH3 motif based on changes during affinit maturation of BINDI. (E) MBP-peptide fusions were tested by BLI for binding to Bcl-2 proteins. Neither peptide had the affinity or specificity for BHRFI of BINDI.
Figure 1 1 . BINDI triggers apoptosis in an EBV-positive cell line. (A) Cytochrome c release from mitochondria harvested from Ramos (EBV -negative) or Ramos-- AW ceils (EBV- positive) treated with Bim-BH3 peptide. Bim-BH3 L62E has a knockout mutation in the binding interface. Mean ± SD, n = 4, for all panels. (B) As in (A), with mitochondria treated with BINDI protein. BIND! L54E has the equivalent interface mutation as Bim-BH3 L62E. (C) At left, the crystal structure of BINDI bound to BHRF1 showing the interaction of Asn62 with the N-terminus of helix αό. At right, BINDI mutation N62S is predicted to maintain interface interactions. (D) BLI kinetic analysis of BINDI N62S interactions with Bcl-2. proteins. (E) Cytochrome c release from Ramos and Ramos-AW mitochondria treated with BINDI N62S or inactive guide scaffold 3LHP(S). (F-H) Mitochondria were harvested from four EBV-negaiive and six EBV-positive lines. Cytochrome c release was measured after treatment with 10 μΜ Bi.m-BH3 peptide (F), guide scaffold 3LHP(S) (G), or BINDI N62S (H).
Figure 12. Intracellular delivery of BINDI induces cell death in an EBV-positive cancer line in vitro. (A) Cells were incubated with 4 μΜ antennapedia peptide-fusions of BINDI, BINDI-L54E or 3 LHP chain S. Cell viability after 24 h was assessed by quantifying metabolic activity. (B) Cells were incubated with sub-iethai doses (2 μ,Μ) of antennapedia peptide-fused proteins. Diblock copolymer Pol300 was conjugated to the proteins via a terminal cysteine for enhanced endosomal escape. Cell viability (mean ± SD, n = 3) was measured after 24 hours.
Figure 13. Treatment of EBV-positive B lymphoma xenograft tumors by intracellular delivery of BINDI in vivo. (A) Schematic representation of the copolymer-based treatment. Pol950 has stabilizing and endosomolytic blocks and forms a micelle at physiological pH. The stabilizing block couples to O.CD19 and BINDI. Nude mice with subcutaneous Ramos- AW xenografts were treated on days 0, 3 and 6 with Pol950 (300 mg/kg) : <xCD19 (15 mg/kg) : BINDI or 3LHP(S) (105 mg/kg). Mice were injected 30 minutes prior to each treatment with CTX (35 mg ml) and BTZ (0.5 mg/mf). (B-E) Tumor growth is plotted for each individual mouse until day 1 1 when the first mice are euthanized. (B) PBS control treatment, black, n = 8; (C) chemo-only, grey, n = 9; (D) 3LHP(S)-copolymer treatment, , n = 9; (E) BINDI-copolymer treatment, , n = 10. (F) Kaplan-Meier survival plot. There is a significant increase in survival with treatment (log-rank test χ2 = 46, P < 0.0001 ).
Figure 14. (A) Based on the experimental enrichment ratios for all single amino acid substitutions of BbpD04.3, a conservation score was calculated for all residue positions. (SEQ ID NO: 73) (B) Beginning with a hypothetical population of BbpD04.3 variants that evenly spans all single amino acid substitutions, we applied the experimental enrichment ratios to evolve our population in silico. The probability of finding a particular amino acid at any given position was then calculated This analysis gives an indication of the tolerated sequence diversity in BbpD04.3/BINDI (SEQ ID NO: 73).
Figure 1 5. (A) BINDI (black) was docked to the hydrophobic binding groove of Mcl- 1 (white) by alignment to a bound BH3 peptide (not shown). The docked configuration is computationally designed. (B) Designed ionic interactions in MINDI. (C) Chemical denaturation measured by following loss of CD signal (222 nm), (D) BLI titration experiment for accurate KB determination. Biotinylated Mcl- l was immobilized to a streptavidin-coated sensor and incubated with the indicated concentrations of soluble MINDI. Raw data is grey, fitted curves are black. (E) Isoaffinity plot from BLI titrations of MINDI interactions with BCL2 family members (only Mcl- l is labeled).
Figure 16. Qualitative measurements of binding by BLI analysis at a single analyte concentration. The BCL2 proteins are biotinylated and immobilized on streptavidin-sensors. The sensors are dipped for 600 s in 50 iiM of the indicated designed Mcl- l binding proteins, followed by incubation in buffer to monitor dissociation. Mcl- l -specific peptide MB 1 was purified as a MBP fusion and used as a positive control.
Figure 17. Quantitative BLI analysis of optimized designs binding each BCL2 protein. For a given binding pair, the biotinylated BCL2 protein was immobilized on the surface of streptavidin-coated sensors, incubated with a range of concentrations of soluble designed protein (association), and then placed back in buffer (dissociation). Data were fitted with analysis software. The determined on- and off-rates are plotted, and KDs can be found in Table 10 (mean +/- SD; n=3). Dashed lines indicate where binding was too weak to be accurately measured. Weak interactions that fall below the dashed lines are not plotted.
Figure 18. Computationally designed proteins 2-CDP06 (A), X-CDP07 (B), 10- CDP01 (C) and F-CDPOl (D) and their experimentally optimized derivatives 2-ΓΝΟΙ (A), X I N Dl (B), 10-ECMO 1 and 10-INDI (C), and F-ECM04 and FIND! (D) were denatured with guanidinium hydrochloride. Loss of CD signal at 222 nm was used to calculate the fraction folded.
Figure 19. Beginning with a hypothetical population of diverse protein variants, we applied experimental enrichment ratios for all single amino acid substitutions to evolve our population in silico. The probability of finding a particular amino acid at any given position was then calculated. This analysis gives an indication of the tolerated sequence diversity in the protein. (A) 2-CDP06 (SEQ ID NO: 39) (optimized to 2-lNDI), (B) 10-CDPO l (SEQ ID NO: 52) (optimized to 10-INDI), (C) F-CDP01 (SEQ ID NO: 53) (optimized to FINDI) and (D) X-CDP07 (SEQ ID NO: 47) (optimized to XINDl). Figure 20. (A) Sequence alignment of specific BCL2 protein binders (from top to bottom SEQ ID NOs: 1, 5, 2, 6, 3, and 4). Differences from BINDI, the original designed binder targeting viral BHRF I that was repurposed for binding other BCL2 family members, are highlighted. Residues that differ from BINDI in one or two sequences are shaded grey, while residues that differ in three or more of the derived binders are shaded black. (B) Sequence variation amongst the INDT family is mapped to the structure of BINDI (surface representation) bound to BHRFI (ribbon). Detailed Description of the Invention
Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al, 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2lld Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109- 128, ed. EJ. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX)
As used herein and unless otherwise indicated, the terms "a" and "an" are taken to mean "one", "at least one" or "one or more". Unless otherwise required by context, singular terms used herein shall -include pluralities and plural terms shall include the singular.
All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.
All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise.
In a first aspect, the present invention provides jjolypeptides comprising or consisting of an amino acid sequence having at least 50% amino acid sequence identity over their length relative to the amino acid sequence of SEQ ID NO.: 1 , wherein the polypeptide selectiv ely binds to a protein selected from the group consisting of Epstein Ban- protein BHFR 1, and B cell lymphoma family proteins selected from the group consisting of myeloid cell leukemia 1 (Mcl- 1), B-cell lymphoma 2 (Bcl-2), Bci-2-like protein 1
(BCL2L1/Bcl-XL), Bcl-2-like protein 10 (BCL2L10/Bcl-B), and Bcl-2-like protein Al (Al/Bfl-1 ).
ADPKKVLDKAKDQAENRVRF KQKLEELYKEARKLDLTQFMRRKLELRYIAAM LMAIGDIYNAIRQAKQEADKLKKAGLVNSQQLDELKRRLEELKEEASRKARDYGRE FQLKLEY (BINDI; Target : BHRF 1 ) SEQ ID NO : i
The polypeptides of the invention are high- affinity (as low as picomolar affinity), specific protein inhibitors of BHRFl and B cell lymphoma (BCL) family proteins. And can be used, for example, in methods of treating cancer and Epstein-Barr virus-related diseases. Rather than repurposing an existing natural protein of known structure, the polypeptides of the invention were designed de novo for optimum BHRFl or and BCL family protein interactions, and are shown herein to trigger apoptosis in relevant cancer lines and slow BL progression in an animal model in the examples herein. This work therefore represents a major bioengineering accomplishment; the creation of an entirely new class of designer polypeptides and their demonstrated therapeutic potential from the ground up.
The polypeptides of the invention have at least 50% amino acid sequence identity over their length relative to the amino acid sequence of S ) ID NO,: 1 , which was designed as shown in the examples that follow to selectively and at very high affinity bind to Epstein Barr protein BHFR1. The inventors have carried out saturation mutagenesis on the polypeptide of SEQ ID NO: l to identify modifiable residues. Furthermore, the inventors have demonstrated that polypeptides of the invention can be modified for selective binding against BCL family proteins. In various embodiments, the polypeptides of the invention have at least 55%, 60%, 66%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identity over their length relative to the amino acid sequence of SEQ ID NO: 1. As will be understood by those of skill in the art, the polypeptides may include additional residues at the N-terminus, C- termmus, or both that are not present in SEQ ID NO: 1; these additional residues are not included in determining the percent ideniity of the polypeptides of the invention relative to the reference polypeptide (i.e.: SEQ ID NO: l in this case).
The polypeptides selectively bind to a protein selected from the group consisting of Epstein Ban- protein BHFR1, and B cell lymphoma family proteins selected from the group consisting of myeloid cell leukemia 1 (Mci-1), B -eell lymphoma. 2. (Bcl-2), Bel-2-iike protein 1 (BCL2L1/Bcl-XL), Bcl-2-like protein 10 (BCL2L10/Bcl-B), and Bcl-2-like protein A .1 (Al/Bfl-I). As used herein, "selectively binds" or "specifically binds" refers to the ability of a polypeptide of the invention to bind io its target, such as a BHRFl molecule or BCL family member, with a KD 10 5 M (10000 nM) or less, e.g., 10 6 M, 10 7 M, 10 8 M, 10 9 M, 10 10 M, 10 j l M, 10 , 2 M, or less. Selective binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent. The person of ordinary skill in the art can determine appropriate conditions under which the polypeptides described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable ceil binding assay, or as described in the examples that follow. A polypeptide specifically bound to a target is not displaced by a non-similar competitor. In certain embodiments, a polypeptide is said to selectively bind an antigen when it
preferentia lly recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
In one embodiment, the polypeptide comprises or consists of an amino acid sequence having at least 50% amino acid sequence identity over its length relative to to the amino acid sequence selected from the group consisting of SEQ ID NOS:2-6. ADPKKVLDKAKDQAENRVRELKQVLEELYKEARKLDLTQEMRKKLIERYAA. AJIRAIGDINNAJYQAKQEAEKLKKJ\GLVNSQQLDELLRRLDELQKEASRKANEYGR EFELKLEY (MiNDi; Target: McJ-1) SEQ ID NO:2
ADPKKVLDKAKDEAEm ELKQRLEELYKEARKLDLTQEMRQELVDKAR AASLQANGDIFY^VILRALAEAEKLKK.AGL SQQLDELKRRLEELAEEARRKAEKLR DEFRLKLEY (2-I DI; Target: Bcl-2) SEQ ID NO:3
ADPKKVLDKAKDRAE^ VVRKLKKFXEEXYKEARKLDLTQEMRDRIRRTAIA ARFQAHGDIFHAIKHAKEEARKLKKAGLVNSQQLDELKRRLRELDEEAEQRAEKLG KEFRLKLEY (XINDI; Target: BCL2L1/Bcl-XL) SEQ ID NO:4
ADPKXILDKAKDQVENRVRELKQELERLYKEARKLDLTQEMRRKLHVRYIE AMLKAIAAIMNAIAQAENEADKLKKAGLVNSQQLDELRRRLEELTEEAAQKAHDYG RELQLKLEY (10-INDI; Target: BCL2L10/Bd-B) SEQ ID NO:5
ADPKKVLDKAKDQAE RVRELKQKLEELYKEARKLDLTQEKR KLEVATLG AVLAAHGDILNAIMQAKEEADKLKKAGLVNSQQLDELKRRLEELKEEALRKASDYG NEFHLKRRY (FINDI; Target: Al/Bfl-1) SEQ ID NO:6
The polypeptide of each of SEQ ID NOS:2-6 shares very high le vels of sequence identity with BIND! (SEQ ID NO: 1), but were designed by the inventors as selective inhibitors of different BCL-family members, as described in detail in the examples that follow. These differing specificities allow use of the polypeptides in methods to treat cancer with different Bel phenotypes, as well as to determine the Bel-2 phenotype of a tumor. The BCL-famiiy member target for each of SEQ ID NOS: 2-6 are provided above. The amino acid sequence of the respective targets for each of SEQ ID NOS:l-6 are shown below: BHRF1 (Target for SEQ ID NO: 1)
AYSTREILLALC RDSRVHGNGTLHPVLELAARETPLRLSPEDTVVLRYHVLL EEnERNSETFTETWNRFITHTEHVDLDFNSVFLElFHRGDPSLGRALAWMAWCMHA CRTLCCNQ STPYYWDLS VRGMLEASEGLDG WIHQQGGWSTLIEDNIPGS (SEQ ID NO: 67)
Mcl-1 (Target for SEQ ID NO:l)
GSDELYRQSLEnSRYLRE-QATGAKDTKPMGRSGATSRKALETLRRVGDGVQRNHE TAFQGMLRKLDIKNEDDVKSLSRVMIHV SDGVTNWGRIV LISFGAFVAKHLKTIN QESCIEPLAESITDVLVRTKRDWLVKQRGWDGFVEFFHVEDLEOG (SEQ ID NO: 68)
BcI-2 (Target for SEQ ID NO:3)
AHAGRTGYDNREIVMKYfflYKLSQRGYEWDAGDVGAAPPGAAPAPGlFSSQPGHTP HPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPWHLTLRQAGDDFSRRYRRDFA
EMSSQLIlLTPFTARGRFATVVEELFRDG TSiWGRIVAFFEFGGVMCVES 7NREMSPL VDNLALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMR (SEQ ID NO: 69)
Bel-XL (Target for SEQ ID NO:4)
SQSNRELVVDFLSYKXSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWH
LADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAF'SDLTSQLHIT PGTAYQSFEQVVNELFRDGVTSiWGRrVAFFSFGGALCVESVDKEMQVLVSRIAAWM ATYLNDHLEPWIQENGGWDTFVELYGN AAAESRK (SEQ ID NO: 70)
Bd-B (Target for SEQ ID NO:5)
ADPLRERTELLLADYLGYCAREPGTPEPAPSTPEAAVLRSAAARLRQIHRSFFSAYLG YPGNRFELV\ LMADSVLSDSPGPTWGRV L FAGTLLERGPLVTARWKKWGFQ PRLKEQEGDVARDCQRLVALLSSRLMGQHRAWLQAQGGWDGFCHFFRTPFP (SEQ ID NO: 71 )
Bfl-1 (Target for SEQ ID NO:6) T SEFGYIYRLAQDYLQCVLQIPQPGSGPSKTSRVLQNVAFSVQKEVE LKSCLDN
VNVVSVDTARTLF QVMEKEFEDGIINWGMVTIFAFEGIL1KKLLRQQIAPDVDTYK E1SYFVAEFIMNNTGEW1RQNGGWENGFVKKFEPKSG (SEQ ID NO: 72) The inventors have carried out saturation mutagenesis on the polypeptides according to each of SEQ ID NOS:3-6, while the polypeptide of SEQ ID NO:2 shares 84 % identity and
93 % similarity to the polypeptide of SEQ ID NO: 1, and therefore likely has a similar tolerance for sequence variations, especially at the majority of positions not making interfacial contacts with its t arget. In various embodiments, the polypeptides of the invention have at least 55%, 60%, 66%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identity over their length relative to the amino acid sequence of SEQ ID NO:2-6. As will be understood by those of skill in the art, the polypeptides may include additional residues at the N-terminus,
C-terminus, or both that are not present in SEQ ID NOS:2--6; these additional residues are not included in determining the percent identity of the polypeptides of the invention relative to the reference polypeptide (i.e. : SEQ ID NOS:2-6 in this case).
In one embodiment, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 7, wherein the polypeptide binds to BHFRI .
(A/E/G/H/I/K/M/P^
Figure imgf000015_0001
/W)(A/F/G/I/i P/S/V^
W/YXH/K/L/N/Q/R'WXAH^
R/S/T/V/W/Y)(D/E/G/I/K^
V/W/Y)(A/D/F/G/Hfl/K^
M/T/V)(A/C/D/E/F/^^
Fi/I/L/M/Q/T/Y)(A/C/Fi/I/^
Figure imgf000015_0002
Y)(C/F/H/I/K/L/MM/P/^^
Figure imgf000015_0003
/P/Q/R/TA'/YXC/H/I/K/L/M/
Figure imgf000015_0004
K/N/P/Q/R/S/T)(A/D/^^
H/K/L/M/ /Q/R/T/V/W/Y)(m
(E/F/W/Y)(I)(A/G)(AE/I/Q)(D/Ii/L/M./NA
/M/S/T/V)(G)(D)(I/L/M)(F/M/W^ /I /Y)(R/Y)(A/F/I/K/L^
D/E/F/G/H/I/K/L/M/N/Q^
K/R (A/F/L/M/R/W/Y)(A/F/H/K/^
V/W/Y)(A/¾/G/H/Q/PJS/T)(A''K/L/PJT/V^
Figure imgf000016_0001
/Y)(D/E F/G/I/I^
)(A/G/I/K/L/M/N/^^
N/Q/T)(A/ O/E/F/G^^
A/D/E F/H/K/M/^
N/R)(A/O/E/F/G/H/ yL/M/N/R/T/V/W/Y)
/L/R/S/V/W)(P)(p/E/H ^
)(D/E/G/H/K/Q/R/T/Y) (Target: BHRF1 ) (SEQ ID N0:7)
This embodiment is based on saturation mutagenesis studies described in the examples that follow, in which all residues of SEQ ID NO: 1 were tested to identify allowed sequence variability for the designed proteins that retained function (i.e. : BHFR.1 binding).
In another embodiment, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 8, wherein the polypeptide binds to Bel-2.
(A/E/G/P/SA'A iA/D/E/G/H^
Q)(p/V)(C/D/L/Y)(D/L/N^
E/H/N/P)(E/F/I ^
R)(A/C/E/ G/H/I/K/L/M
R/W"A (E/M )(E/H/I/R' W)(I/L/N)(C/G/HA
(R)(Tq(E/FI/I/L/PA Y)^
/R/Y)(H/K/Q/V)(E/L/W)(K/L/M/V)(A/C/D/E/^
WA (K)(A/G/H/K/N/Q/^
Figure imgf000016_0002
/Q/T)(F)(Y)(A/F/T)(D/I/R)(L^^
/C/F/lJM/W)(E F/S/T/W)(K M)(LX
17Q/R)(C/F/Q/W)Ci/17T)(AT
K/L/N/R/T/V)(D/E/Q)(I^^
L/Q/WV)(K)(A/D/E/F/G/am/E¾/Q/R/S^
W)(D/G/I/ /M/Q/1^/W)(A/D/E H/ / /
/TsVP/Q/R/S)(H/K,'¾/P)(A/C/F/L'LAi P/
) (Target: Bcl-2) (SEQ ID NO:8) This embodiment is based on saturation mutagenesis studies described in the examples ihai follow, in which all residues of SEQ ID NO: 3 were tested to identify allowed sequence variability for the designed proteins that retained function (i.e.: Bcl-2 binding).
In another embodiment, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO:9, and wherein the polypeptide binds to binds to Bcl-2- like protein 1.
(A/E/G/P/R/S/T/V)(A/^^
G/I/M/S/V/W/Y)(C/G/I/L)fD/F/I /M/TsV
M/P/R/S/W/Y)(R/S)(A/D/F/Gffi
YXE/K/N/Q/T/W)(Fm/L/R/YX
W/Y)(L/P)(A/D/E/G/^
H NA (CyK/N R)(A C/D/E F/G H I/^
/R/W^XA/C/D/E H/I/K/lJ yP/Q/R T/^
Figure imgf000017_0001
M/P/R/I (A/I/K/L/Q/R/S/T/V/Y)(A)(F/I/L/W/Y
)(R)(F/I W/Y)(A/G/K P/Q/R/W)(A/F/H/1/^
(A/OD/F/G/H/L/R/S^
A/D/E/G/H/I/K/L/P/Q/R/S/T^^
G/K/L/ /R/S/T/W/^
/R)(K)(A/K/T/W)(G/I/K/L/^
K/L/Q/R/WAO(C L/S/Y (D/E G/ RA (E K/R/W
V/Y)(A/E F/G/H/I/K/L^^
Figure imgf000017_0002
G/K/Q/V)(A/C/E /L/ S)(L/Y)(G/W)(K/R/W)(E/N/
/Q/R/S/T/V/W/Y)(F/L/M/R/T)(K)(K/L/ ^ (Target: BCL2L1/Bcl-XL) (SEQ ID NO:9)
This embodiment is based on saturation mutagenesis studies described in the examples that follow, in which all residues of SEQ ID NO:4 were tested to identify allowed sequence variability for the designed proteins that retained function (i.e.: i BCL2L1/Bcl-XL binding).
In another embodiment, the polypeptide comprises an amino acid sequence according to SEQ ID NO: 10, wherein the polypeptide binds to Bcl-2-like protein 10.
(A/D/E/F/M/S/T/V)(A/D/E/^
WXA/E/F/K/L/TsVP/Q/m F/I/K/L/N/Q/R/T/Y)(^
V)(A/C/E/G/L/S/T/V)(A/C/D/E/K/S/W/Y)(D/I/K/N^
G/H/K/R/S/T)(A/D/E/G/K/Q^
C/D/E/F/G/H/I/K/L/M^
C/D/H N/S/Y)(K/M/N/^^
(0/G/H/RWQ/T)(L Q/ )(A/D/H/K/N/^^^
M/P/Q/R/r/W/Y)(.A/D/E/G/K/Q)(C/F/l/K/L
/N/P/Q/R/V/W/Y)(K)(L)(A/C/D/E/F/Gm ^^^^
W)(D/E G/Q/R)(F/Y^^
K/M/P/S)(A)(l)(A/C/F/G/P/SyS/W)(A/C/^
L/M/P/S/T/V/W)(A/G/M/K/P/Q/S)(A/F/V
R/S/T/V/W/Y)(^(E)(A/K/I )(D^^
A)(D/G)(C/T/G/L/M/P/ )/R/S/V/W)(A/C/D/F/O
T/Y)(E/F/H/K/L/P/Q/R) A/E/H/K/P/Q/R)(
Q)(L/M/P/R/T (A/X:/G/tl/P/Q/R)(C/G/I/I,
Figure imgf000018_0001
P/Q/S/7,A^)(A/D/E/G/H/ yN/Q/R/S/T/Y)(C/D
A D/G/S/T V)(A C/D/E/Gm/1 L/M N/Q/R/S
H /M/ /P/Q R)(A OD/E/G/L/N/Q/S/V/Y)(C^
E/K/P/T)(C/F/I/^^
P/Q/V)(D/E/G/H/K/L/ ^ (Target: BCL2L10/Bcl-B) (SEQ ID
NO: 10)
This embodiment is based on saturation mutagenesis studies described in the examples ihai follow, in which all residues of SEQ ID NO: 5 were tested to identify allowed sequence variability for the designed proteins that retained function (i.e.: BCL2L.10/Bcl-B binding).
In another embodiment the polypeptide comprises or consists of an amino acid sequence according to SEQ TD NO: 1 1 , wherein the polypeptide binds to Bcl-2-like protein A l (Al Bfl- 1).
(A/D/F/^j/H/TL'L/M/P/R S/T/V/W
/I/ /L/P/Q/R/S/T^^
E/F/G/I/V)(A/E/F/H/lvM/P/Q/R/T/V/W/
E/F/G/PI/I/K/L/M^
Q/T/V/W/Y)(A/D/E/G/H/I/K/L/M/N/R/V/W /M/WQ/S/ A/7W/Y)fA/C/D/E/T/G/K/L/M^
L/P/R/S/T/V)(A/D/G/H/^^
S/T/V/W/Y)(A/F/G/H/^
/H/K/M/N/Q/PJS/T/W)(K)(A/C/E/F/G/tl/I/^
MyQ/R'¾/V/W/Y)(A/I)/E/F/G/H/I/K/L/M/N/Q/R^
M/P/R/S/T/V/W/Y)(H^^
F/G/H/l/lvM/P/]¾/S/V/W/Y)(C/H/K/M/ /^
Figure imgf000019_0001
A7W/Y)(A/C/D/E/F/G/H/l/lvM/N/P/Q/¾/SA^/
/Q/R/T)(I/L/P/V)(A/D/E/F/G/tl/K/L/N/P/Q/RyS
/C/D/E/F/G/H/I/K/L/^
Figure imgf000019_0002
Y)(F/l/i W){l/iJV)(Dm/m/S/r)(A/C/D/E/F/G/^
(C/D/E/G/tl/ yL/M/1M/P/Q/R/S/T)(A/D/E/
G/H/I/K/L. N/P/Q/R/S/ /V/W/Y)(A/B/E/I^^^^^
/D/E/F/G/H/I/K/L^
R/S/W)(A/D E F/G/H/I7K/^
Y)(G/K/N/P/Q/R)(F/G/I L^^
I/K L M N/P/R/S/T/V/WA (A/F t^
/K/L/M/P/Q/R/V)(A/C/D/E/F/G/H/1/^
R/S/T/V)(AyC/E/LAl/N/R/S/T/Y)(A/C/D/E/F/G/HA
T)(K/R/W/Y)(A/C/^^
/W/Y)(A/I^/G/H/I/K/L/M^^
/Y)(A/E/F/I/1 (F/L/N/Y)(H/Q/R/S)(A/E/G/H/I/K^
Q/S/T/V/Y)(A/C/D/E/G/FI/K N/P/Q/R/S/ /Y)f
N/P/Q/R/S/WW/Y)(A^^^
F/MyP/V/Y)(A/C/D/E/F/G/H/l/KyL/M/N/P/Q^
/Ry8/T/V/wyY)(A/C/D/E/G/H/K/L^
/I/K/L/M/N/P/Q/^
M N P/Q/R/S/T/W/Y) (Target: Al/Bfl- 1) (SEQ ID Oi l 1)
This embodiment is based on saturation mutagenesis studies described in the examples that follow, in which all residues of SEQ ID NO:6 were tested to identify allowed sequence variability for the designed proteins that retained function (i.e.: Bcl-2-like protein Al (Al/Bfl- l)binding).
In another embodiment, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 12, and wherein the polypeptide binds to Bci-2-like protein Mcl- 1.
SEQ ID NO: 12
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
D72 A/B/E/F/O/H/l/L/MQ/S/TA'VW/Ύ
K73 F/K/R/Y
L74 A/F/L/M/R/W/Ύ
K75 A/F/H/ / /R/S/T/Y
K76 1/K/N/R/W
A77 A/F/GH I/KL'MN/Q R/STA^WY
G78 AD/G/H/Q/R/S/T
L79 A/K/LR/T/V/W/Y
V80 I/L/M/V
N81 A DE N/Q R ST
S82 D/E/G/K M/P/Q/R S/T/V
Q83 A/D/E/F/H/1/L/N/Q/R/SAT/V
Q84 D/EH/M/N/Q/T/Y
L85 A/F/G/H/L/M/R/T/V/W/Y
D86 D/FJF/G I/K/IJN/Q/S TA^/W/Y
E87 A'E/F/I/K/L/MQ/T/W
L88 A/F/I/L/M/T./V
K89 ΑΊ/Κ/Q/R/V/L
R90 A/GI/I LM/N/Q/R S/T/V/WAr
R91 A/CD/E/G/H K/L/N/Q/R/S/T/V/Y
L92 1/1.
E93 A/DE/H/I/ /N/QT
E94 A/C/O/E/F/G/H/l/K/LM/N/Q/R/SiT/V/Y
L95 A/L/T/V
K96 K/QR
E97 A DEG/H/Q/S/ /V E98 A/B/E/F/FI/K/M/¾/P/Q/R./S/W/Y
A99 A/S/V
SI 00 A/G/N/Q/S/T
R101 K/R
K102 K/R
A 103 A/I/M/N/S/T/V
104 D/ N/R
D105 A/D/E^/G/H/i/L M/N/R/T/V/WA^
Y106 A'E/G/H/I/T/Y
G107 D/G/S
R108 K Q R
El 09 A/DEF/G/H/ I. R/S/V/W
F110 F
QUI D/E/H/M/Q
LI 12 A/DF/I/L/P/Q/R
K113 K/Q
LI 14 A/FI/I /L/M/P/R/S/T/V/Y
El 15 D/E/P/R/T
Y116 D/E/G/H/K/Q/R/T/Y
In another embodiment, the polypeptide comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOS: 1-6.
In another embodiment the polypeptide comprises or consist of an amino acid sequence having at least 50% identity to the amino acid sequence of SEQ ID NO: 13.
ADWK VLDKAKDIAENRVREI QKLEEFY KAM LDLTQEMRRKLMLEWIAA MLMAIGDIFNAIEQA QEADKLK AGQVNSQLLDELKRRLEELKEEASRKCHDY GREFQLKLEY SEQ ID NO.: 13 (BbpD04) As shown in the examples that follow, the polypeptide of SEQ ID NO: 13 is a selective high affinity binder of Epstein Ban protein BHFR1. The inventors have earned out saturation mutagenesis on the polypeptide of SEQ ID NO: 13 to identify modifiable residues. In various embodiments, the polypeptides of this embodiment have at least 55%, 60%, 66%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identity over their length relative to the amino acid sequence of SEQ ID NO: 13, As will be understood by those of skill in the art, the polypeptides may include additional residues at the N-terminus, C- ierminus, or both that are not present in SEQ ID NO: l ; these additional residues are not included in determining the percent identity of the polypeptides of the invention relative to the reference polypeptide (i.e.: SEQ ID NO: 13 in this case).
In one embodiment, the polypeptide comprises at least one conservative amino acid substitution corresponding to residues 3, 13, 21 , 2.8, 31 , 33, 46, 48, 49, 61, 62, 65, 79, 84, 103, and 104 of the amino acid sequence of SEQ ID NO: 13. As used herein, "conservative amino acid substitution" means amino acid or nucleic acid substitutions that do not alter or substantially alter polypeptide or polynucleotide function or other characteristics. A given amino acid can be replaced by a residue having similar physiochemical
characteristics, e.g., substituting one aliphatic residue for another (such as He, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. antigen-binding activity and specificity of a nati ve or reference polypeptide is retained,
Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1 ) non-polar: Ala (A), Val (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M); (2.) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (FT). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norieueine, Met, Ala, Val, Leu, He; (2.) neutral hydrophiiic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into H is: Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; lie into Leu or into Val; Leu into He or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and'or Phe into Val, into He or into Leu.
In a further embodiment, the polypeptide includes the substitutions K31E, E48R, and E65R relative to SEQ ID NO:13. In another embodiment, the polypeptide includes the substitutions 121L, Q79L, L84Q, and H104R relative to SEQ ID NO: 13. In a further embodiment, the polypeptide includes the substitution C I 03 A relative to SEQ ID NO: 13. In a still further embodiment, the polypeptide includes substitutions W3P, I13Q, F28L, M33R, M46E, W49Y, and F6IY relative to SEQ ID NO: 13. In another embodiment, the polypeptide includes the substitution N62S relative to SEQ ID NO: 13. These
embodiments may be combined in any suitable combination.
A s noted above, the polypeptides of the invention may include additional residues at the N-terminus, C-terminus, or both. Such residues may be any residues suitable for an intended use, including but not limited to detection tags (i.e.: fluorescent proteins, antibody epitope tags, etc.), linkers, iigands suitable for purposes of purification (His tags, etc.), and peptide domains that add functionality to the polypeptides. In one embodiment, the polypeptide of the invention further comprises a cell penetrating peptide. Cell penetrating peptides are useful, for example, to facilitate uptake of the polypeptides by cells, and are known to those of skill i the art. Non-limiting examples of such cell penetrating peptides that can be used with the polypeptides of the invention include:
TAT: GRKKRRQRRRPPQ (SEQ ID NO: 14);
penetratin: RQIKIWFQNRRMK WKK (SEQ ID NO: 15);
MAP: KLALKLALKALKAALKLA (SEQ ID NO: 16);
transportan/TPIO: G WTLN S/AG YLLGKI LKALAAL AKKIL (SEQ ID NO: 17);
VP22 N AKTRRHERJ J KL AIER (SEQ ID NO: 18);
polyarginine: Rn, n >7 (SEQ ID NO: 19);
MPG: GALFLGFLGAAG STMGA (SEQ ID NO:20);
Pep-1 : KETWWETWWTEWSQPKKKRKV (SEQ ID NO:2.1):
pVEC: LLHLRRRIRKQAHAHSK (SEQ ID NO:22);
YTA2: YTAIAWVKAFIRKLRK (SEQ ID NO:23);
YTA4: IAWVKAFIRKLRKGPLG (SEQ ID NO:24): M918: VTVLFRRLRIRRACGPPRVRV (SEQ ID NO:25); and
CADY: GLWRALWRLLRSLWRLLWRA (SEQ ID NO:26).
As used throughout the present application, the term "polypeptide" is used in its broadest sense to refer to a sequence of subunit amino acids. The polypeptides of the invention may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid- specific proteases in vivo), or a combination of D- and L-amino acids. The polypeptides described herein may be chemically synthesized or recombinantly expressed. The polypeptides may be linked to other compounds to promote an increased half-life in vivo, such as by PEGylation, HESylation, PASylation, glycosylation, or may be produced as an Fc- fusion or in de mmunized variants. Such linkage can be covalent or non-covafent as is understood by those of skill in the art.
In another aspect, the invention provides pharmaceutical composition, comprising a polypeptide of any embodiment or combination of embodiments of the invention, and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention can be used, for example, in the methods of the invention described below. The pharmaceutical composition may comprise in addition to the polypeptide of the invention (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, e.g.
sucrose, sorbitol or trehalose, in certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresoi, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chforocresol, phenylmercuric nitrate, thimerosaf, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate- 85, poloxamer- 1 88, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.
The polypeptides of the invention may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use, including but not limited to anti-HA and anti-NA antibodies. As used herein, the term "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
In one embodiment, the pharmaceutical compositions further comprise an antibody, or antibody fragment. In this embodiment, the antibody or antibody fragment adds functionality to the composition by, for example, helping target the composition to a cell type that has a cell surface receptor to which the antibody selectively binds. As a result, compositions of this embodiment are particularly useful for therapeutic applications. As will be understood by those of skill in the art, any suitable antibody or fragment thereof can be employed that targets a cell or tissue of interest. The antibody or fragment may be recombinantly expressed as part of the polypeptide, may be linked to the polypeptide directly (such as by a covalent linkage or non-covalent interaction), or may not be directly linked to the polypeptide at all (i.e.: present in the same composition, but unlinked).
In another embodiment, the pharmaceutical carrier may comprise a polymer. Any- suitable polymer may be used that is pharmaceutically acceptable and which does not interfere with function of the polypeptide. In one embodiment, the polymer is a block polymer and comprises a hydrophilic block and an endosomolytic block. Any suitable hydrophiiic block and endosomlytic blocks may be used. In one embodiment, the hydrophilic block comprises polyethylene glycol methacrylate. In another embodiment, the endosomolytic block comprises a diethylaminoethyl methacrylate-butyl methacrylate copolymer. In a further embodiment, the polymer is a stimuli-responsive polymer that responds to one or more stimuli selected from the group consisting of pH, temperature, UV-visible light, photo- irradiation, exposure to an electric field, ionic strength, and the concentration of certain chemicals by exhibiting a property change. As used herein, a "stimuli-responsive polymer" is a polymer that changes its associative properties in response to a stimulus. The stimuli-responsive polymer responds to changes in external stimuli such as the pH, temperature, UV-visible light, photo-irradiation, exposure to an electric field, ionic strength, and the concentration of certain chemicals by exhibiting property change. The chemicals could be poly valent ions such as calcium ion, polyions of either charge, or enzyme substrates such as glucose. For example, a temperature-responsive polymer may be responsive to changes in temperature by exhibiting a LCST in aqueous solution. A stimuli- responsive polymer may be a multi-responsive polymer, where the polymer exhibits property change in response to combined simultaneous or sequential changes in two or more external stimuli. The stimuli-responsive polymers may be synthetic or natural polymers that exhibit reversible conformational or physico-chemical changes such as folding/unfolding transitions, reversible precipitation behavior, or other conformational changes to in response to stimuli, such as to changes in temperature, light, pH, ions, or pressure. Representative stimuli- responsive polymers include temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers.
In a further aspect, the present invention provides isolated nucleic acids encoding a polypeptide of the present invention. The isolated nucleic acid sequence may comprise R A or DNA. As used herein, "isolated nucleic acids" are those that have been removed from their normal surrounding nucleic acid sequences in the genome or in cDNA sequences. Such isolated nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to poly A sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the invention.
In another aspect, the present invention provides recombinant expression vectors comprising the isolated nucleic acid of any aspect of the invention operatively linked to a suitable control sequence. "Recombinant expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. "Control sequences" operably linked to the nucleic acid sequences of the invention are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operabiy linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The construction of expression vectors for use in transfecting host cells is well known in the art, and thus can be accomplished via standard techniques. (See, for example, Sambrook, Fritscb, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E.j. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector.
In a further aspect, the present invention provides host cells that comprise the recombinant expression vectors disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of the invention, using standard techniques in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAL dextran mediated-, polycationic mediated-, or viral mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, d Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY). A method of producing a polypeptide according to the in v ention is an additional part of the invention. The method comprises the steps of (a) culturing a host according to this aspect of the invention under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed polypeptide. The expressed polypeptide can be recovered from the ceil free extract, but preferably they are recovered from the culture medium. Methods to recover polypeptide from cell free extracts or culture medium are well known to the person skilled in the art.
In another aspect, the invention provides methods of treating an Epstein-Barr virus - related disease comprising administering to a subject in need thereof a therapeutically effective amount of one or more of the polypeptides of the invention that selectively inhibits BHRFl, or salts thereof, pharmaceutical compositions thereof, a recombinant nucleic acid encoding the one or more polypeptides, a recombinant expression vector comprising the recombinant nucleic acids, and/or a recombinant host cells comprising the expression vector, to treat and/or limit the Epstein-Barr virus related disease.
Epstein-Barr virus encodes a pro-survival Bel-2 homologue, BHRFl , which prevents lymphocyte apoptosis during initial infection by sequestering pro-apoptotic BH3-only proteins (especially Bim), and interacting directly with the executioner Bak (Desbien et al., 2009; Kvansakul et al, 2010)(Altm.ann and Hammerschmidt, 2G05)(Henderson et al, 1993), Even though BHRFl is under the control of an early lytic cycle promoter, low levels of constitutive expression have been observed in some cases of EBV -positive BL when the vims is latent, and it has been speculated that BHRFl may be a necessary viral factor for lymph omagenesis (Kelly et al, 2009; Leao et al., 2007; Watanabe et al, 2010). Thus, inhibitors of BHRFl can be used to treat and/ or limit development of Epstein-Barr virus related disease, as is evidenced by the examples that follow.
In various embodiments, the Epstein-Barr v i r u s - related disease is selected from the group comprising o f infectious mononucleosis, Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma, nasopharyngeal carcinoma, multiple sclerosis, Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In other embodiments, the Epstein-Barr v i r u s - related disease is a cancer selected from the group consisting of Burkitt's lymphoma, Hodgkin's lymphoma, non- Hodgkin's lymphoma, mantle cell lymphoma, or nasopharyngeal carcinoma.
In various embodiments, polypeptides for use in this aspect of the invention are selected from polypeptides comprising or consisting of the amino acid sequence of SEQ ID NOS: 1 and 7, including any embodiments thereof such as, but not limited to, further including cell penetrating peptides or antibodies.
In another aspect, the invention provides methods for treati g cancer, comprising administering to a subject in need thereof a therapeutically effective amount of one or more o f th e polypeptides that selectively inhibits one or more of Mcl-1, Bcl-2,
BCL2L1/Bcl-XL, BCL2L10/Bcl-B, and Al/Bfl- 1, or salts thereof, a pharmaceutical composition thereof, a recombinant nucleic acid encoding the one or more polypeptides, a recombinant expression vector comprising the recombinant nucleic acid, and/or a recombinant host cell comprising the recombinant expression vector, to treat cancer in the subject.
Apoptosis and cell survival are regulated by the homeostatic balance of B cell lymphoma- 2. (Bcl-2) family proteins. The 'executioners', Bak and Bax, initiate apoptosis by increasing mitochondrial outer membrane permeability and facilitating the release of mitochondrial cytochrome c to the cytosol, which activates downstream signaling. Six human pro-survival Bcl-2 proteins (Bcl-2, BC1-XL, Bcl-B, Mcl-L Bcl-w and Bfl- 1) inhibit this process. Cellular stresses activate pro-apoptotic BH3-only proteins, which bind and inhibit pro-survival Bcl-2 members, and directly interact with Bak and Bax to favor mitochondrial permeabilization. Conversely, pro-survival Bcl-2 proteins dampen apoptotic triggers and enhance chemoressstance by sequestering BH3-only proteins or directly inhibiting Bak and Bax. increased expression of pro-survival Bcl-2 proteins is a common feature of many cancers. Thus, the polypeptides of the present invention, which bind to and inhibit the pro- survival Bcl-2. proteins, can be used to treat cancer.
In various embodiments, polypeptides for use in this aspect of the invention are selected from polypeptides comprising or consisting of the amino acid sequence of SEQ ID NOS: 2-6 and 8-12, including any embodiments thereof such as, but not limited to, further including cell penetrating peptides or antibodies.
The methods may be used alone or in conjunction with other therapies for treating cancer, such as chemotherapy, radiation therapy, and /or surgical removal of the tumor. In one embodiment, the polypeptides permit reduced (sub-therapeutic) dosages of current therapies; in another embodiment, such a combination therapy permits the use of otherwise sub-therapeutic dosages of the polypeptide of the invention; these embodiments can be combined. In these various embodiments, the methods may be used to overcome tumor resistance to the treatment,
As used herein, the phrase "therapeutically effective amount", "effective amount" or
"effective dose" refers to an amount that provides a therapeutic benefit in the treatment, prevention, or management of Epstein- Barr virus and Epstein-Barr related diseases, or cancer. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject's history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents.
As used herein, the term "treat," "treatment," or "treating," means to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition of the disorder being treated. The term "treating" includes reducing or a lleviating at least one adverse effect or symptom of a condition. Treatment is generally "effective" if one or more symptoms are reduced. Alternatively, treatment is "effective" if the progression of a condition is reduced or halted. That is, "treatment" may include not just the improvement of symptoms, but also a cessation or slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishrnent of extent of the deficit, stabilized (i.e., not worsening) state of a tumor or malignancy, delay or slowing of tumor growth and/or metastasis, and an increased lifespan as compared to that expected in the absence of treatment.
As used herein, the ter "administering," refers to the placement of a therapeutic into a subject by a method or route deemed appropriate. The therapeutic can be administered by any appropriate route which results in an effective treatment in the subject including orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intra-arterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or mtraperitoneally. Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). A suitable dosage range may, for instance, be 0.1 ug/kg- 100 mg/kg body weight; alternatively, it may be 0.5 ug kg to 50 mg/kg; 1 ug kg to 25 mg/kg, or 5 ug/kg to 10 mg/kg body weight. The polypeptides can be delivered in a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or more times) as determined by an attending physician.
In another aspect, the invention provides methods for determining the Bcl-2 phenotype of a tumor, comprising contacting tumor cells, tumor ceil lysates or tumor cellular components with one or more polypeptides selected from the group consisting of SEQ ID NOS: 2-6 and 8- 12, under conditions suitable to promote apoptosis signaling in cells of the tumor that express a bcl-2 homologue targeted by the one or more polypeptides; and determining bcl-2 dependency of the tumor based on the polypeptide that causes apoptosis or apoptotic signaling in the cells of the tumor.
The methods of this aspect of the invention can be used, for example, to determine an appropriate polypeptide inhibitor of the invention to treat a tumor, by identifying the bcl-2 dependency of the tumor. In one embodiment, the method comprises contacting tumor cells, tumor cell lysates or tumor cellular components with each of polypeptides 2-6, or each of polypeptides 8-12, which permits simultaneously determining the bcl-2 dependency of the tumor for each of the Bcl-2 family proteins.
The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. Tn this regard, no attempt is made to show stmcturai details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
Examples
The Epstein-Barr virus (EBV), also called human herpesvirus 4 (HHV-4), is a vims of the herpes family. Epstein-Barr vims has been implicated in several diseases that include infectious mononucleosis, Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma, nasopharyngeal carcinoma and multiple sclerosis. The Epstein-Barr virus has been implicated also in disorders related to alpha-synuclein aggregation, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. As used herein, "Epstein-Barr related diseases" are any diseases related to or caused by Epstein-Barr vims, including those listed immediately above.
Pro-survival Bcl-2 proteins share a common domain that resembles a cupped hand, with a characteristic hydrophobic surface groove that clasps one side of an amphipathic BH3 domain helix (Czabotar et aL, 2007; Kvansakul et al., 2.010; Liu et al., 2003). Rigidifying BH3 peptides by use of hydrocarbon staples, disulfides or lactam bridges on the non- interactive back side of the helix can reduce the entropic penalty of a partially- folded peptide acquiring a rigid helical conformation upon binding, and improves BH3 peptide affinity (Azzarito et al., 2013). We reasoned that building a folded structure around a BH3 peptide would similarly pre-stabifize the bound helical conformation, Tn previous work, interacting residues of the BH3 domain were grafted to the surface of a minimal stmctured peptide, but after directed evolution these folded peptides displayed only moderate affinity and specificity, and did not always bind to the correct interaction site on the target Bcl-2 protein (Chin and Schepartz, 2001 ; Gemperli et al., 2005). We instead sought to incorporate the interacting residues of the BH3 domain on the exposed surface of a larger 3 -helix bundle, which makes additional contacts extending beyond the ΒΙΊ3 motif. This much larger interaction footprint provides opportunity for making many new contacts to increase affinity and specificity. Creating new proteins for optimized interactions with the BHRF1 iigand-hinding groove.
Current protein design methods nearly always involve the repurposing of an existing protein of known structure from the PDB. This protein of known structure acts as a scaffold on which new side chains can be grafted to an assumed rigid backbone by site-directed mutagenesis. The grafted residues form a ne functional site for binding to a target protein of interest. However, designed proteins from side chain grafting are limited by the rigid backbone of the scaffold, and may have suboptimal steric complementarity for binding to the target surface. To escape this constraint, we used a computational method (Correia et al., 2014) that builds a new de novo protein with an amino acid sequence unseen in nature that incorporates the Bim-BH3 motif. A. helical bundle scaffold protein of known structure is used only as a topology guide. From the crystal structure of Bim-BH3 bound by BHRF1, the Bim- BH3 helix acts as a folding nucleus, around which protein fragments from the PDB are assembled to build a new protein of matching topology to the guiding scaffold (3 LHP chain S (Correia et al., 2010)). Ca-Ca atom-pair distances from the scaffold constrain the assembling protein to within a defined deviation threshold (3.0 A root mean square deviation, RMSD). Thousands of designed proteins were computationally generated to form a family of structural homologues, all with unique sequences and slightly different backbone structures (Figure 1).
The designed proteins were docked to the BHRF1 surface via alignment of the incorporated Bim-BFB motif, and surrounding interface residues (within 8 A) were then further designed, as the incorporated Bim-BH3 motif provides only a fraction of the interaction surface, and many additional contacts across an expansive interface should be designed. Scaffold residues surrounding the graft site were designed to minimize the energy of the modeled bound complex in the ROSETTA energy function (Kuhlman et al,
2003)(Leaver-Fay et al., 201 1). BHRF1 interface residues, which normally reach over the backside of the Bim-BH3 helix, were simultaneously repacked to alternative low energy retainers compatible with the new designed interface.
The proteins were filtered both for stability of the monomer (by computed monomer energy, packing based on RosettaHoles (Sheffler and Baker, 2009) and for the lowest number of buried unsatisfied hydrogen bonding atoms) and for interface quality (high shape complementarity, computed binding energy and a low number of buried unsatisfied hydrogen bonding atoms). From thousands of computer-assembled proteins, a small number of designs were selected for further manual modifications, synthetic E. coii codon-optimized genes were constructed, and those proteins that were expressed and soluble in E. coli were tested by yeast surface display for binding to B IRF l (Table 1 ). Two structural homologues of PDB 3LHP chain S were designed with apparent KDS 58-60 nM (BbpD04 and BbpDOT; Figure IB and Tables 1 and 2), These designs were 'seeded' by a fifteen-residue fragment of the Bim-BH3 motif of which nine side chains contacting the BFIRF l surface were kept fixed. Other residues, primarily on the backside of the motif and buried in the protein core, were designed to minimize the calculated potential energy. The equivalent 3LHP_S fixed backbone graft (i.e. side chain grafting) described in the methods failed (Table 1 ). Thus backbone modification by in silica refolding can be critical for shaping scaffolds to precisely fit against a desired target.
Figure imgf000036_0002
Figure imgf000036_0001
Figure imgf000037_0001
† indicates the region of Bim-BH3 from crystal structure 2WH6 that was
used to nucleate ab initio folding, arsd the site within the topology guide
where the Bini-BH3 folding nucleus was located.
E. coli BL21(DE3) cells cultured in terrific broth to an OD(600 run) ~0.5
were induced with 0.1 niM iPTG overnight at 20 °C and protein expression
investigated by SDS-PAGE.
* Designs were expressed on the yeast surface and incubated with 400 iiM
monomelic BHRFl-bioim, washed, and stained with anti-myc-FITC
(expression) and streptavidin-PE (binding).
§ The native scaffold residue (identity and number) is given first, followed
by the amino acid type it was mutated to.
In siiico folding probability correlates with binding activity
The success rate for designing functional proteins is low, and computational design still requires substantial human intervention to choose and modify the designs prior to experimental validation. For example, working design BbpD()4 contained 15 human- introduced mutations out of 1 16 total residues from its inactive computational 'precursor' (Figure 2A). These mutations increased packing within the hydrophobic core and hydrophilictty of the exposed surface. This motivated us to test a library of designs 'direct from the computer', without any human modifications. Using the Bim-BH3 motif as a seed for ab initio protein assembly, 5,000 proteins were designed as described for BbpD04 and BbpDOT above (i.e. the guiding scaffold was 3 LHP chain S and the Bim-BH3 incorporation site spanned residues 54-67). This was reduced to 74 designs (Indexes-01 to 74) after filtering for strong interface binding energy, low monomer folding energy and a low number of buried unsatisfied hydrogen bonding atoms. Barcoded genes were synthesized (Table 3) and the library transformed in to yeast for surface display (6 x 10" transformants). BbpD04 (Index- 00) was included as a positive control, and the computational precursor for BbpD04 (index- 21; prior to human modification) was also present. The library was sorted by a single round ofFACS for cells expressing surface protein (Figure 1C; lane 1 ), for the 2% of cells with highest expression (lane 2), and for cells showing binding signal after incubation with 100 nM (lane 3) or 400 nM BHRF1 (lane 4). DNA from the naive and post- sorted populations was harvested and sequenced by fliumina deep sequencing, and the recovery of each designed sequence determined. A minority of designs (Indexes-00 to 27) were enriched following sorting for expression, and just five designs (Indexes-00 to 04) were highly expressed and enriched after sorting for BHRF1 interaction (Figure 1 C). While the four new functional designs share the same 3 -helix topology, the structural details and sequences differ considerably (Figure 2A-D). BHRF1 binding was validated on clonal yeast populations (Figure 3A).
Table 3. Sequences of seeded ah initio designs tested by high throughput library sorting.
Enrichment ratios following yeast display and sorting are indicated.
Index 00 (Design BbpD04) DNA Barcode: AGTCATTGC AGTCATTGC (SEQ ID NO: ! 67) GAD \¾KVLDKAKDIAENRVRFJKQKLEEFYKXAMKLDLTQEMRRKLMLEWIAAMLMAIG DIFNAIEQAKQEADKLKKAGQWSQLLDEL RRLEELKEEASRKCHDYGREFQLKLEYG
(SEQ ID NO: 92)
Log* Enrichment Ratios: Expression 1.13, High Expression 2.54, 100 nM BHRF1 3.92, 400 nM
BHRF1 3.19
isidex 01 DNA Barcode: TCAACTGGTTCAACTGGT (SEQ ID NO: 168)
GKRLEETVEETERRLREALREVYLLTLLLAEEAKKKDLKEQNRHEYVTKWIAFMLMAIGDIF NIAEESKR.RI.DI.FAKWGLHDR.NKIDEA KKiDKLALEAiERA YGDWFLNEI.D G (SEQ ID NO: 93)
Log2 Enrichment Ratios: Expression 1.16, High Expression 1.23, 100 nM BHRF1 4.02, 400 nM BHRF 1 3.54
Index 02 DNA Barcode: TTAAGCCTGTTAAGCCTG (SEQ ID NO: 169)
GKSLLGIALEALEEAKRDLEKAKKQMEEMLKKKWKFDTTRDLKARASAEWTAAALKAIGD RFNAKLLIELGLDELFNKGLITQDTKEDIKRRAEEIFE IERLIKQAIKDKDRFE LG (SEQ ID NO: 94)
Lo¾ Enrichment Ratios: Expression 1.13, High Expression 1.08, 100 nM BHRF 3 3.92, 400 nM
BHRF1 3.43 "
Index 03 DNA Barcode: TTAGACCACTTAGACCAC (SEQ ID NO: 170)
GLDHD iVDEARKXMEK IREA DKA Ei 'XKALDNNHDL QFRELAHKWLA.LMLMAIGD AFNiMMEA RKAEWLREQGQQDEDKAEEAKEKLDKAFKEAAERFEEIA iYG QAKNG (SEQ ID NO: 95)
Logi Enrichment Ratios: Expression 1.10, High Expression -0.16, 100 nM BHRF1 2.52, 400 nM BHRF 3 3.17
Index 04 DNA Barcode: GCTATCATCGCTATCATC (SEQ ID NO: 171)
GL1AEEGREQAEERLREARKKAEKAGDKIKDLAKYGQDSDDEKKKFMLKWIAAQLMV1GD
MFTNHAMEALWELLRRLKNNKiSWDAFL
(SEQ ID NO: 96)
Logs Enrichment Ratios: Expression 0.40, High Expression 0.07, 100 nM BHRF1 2.11, 400 nM
BHRF1 1.96
Index 05 DNA Barcode: ACAGCTTCAACAGCTTCA (SEQ ID NO: ! 72)
G DADKK DEAK AEWKEREVFERIF. MEWKKR.KDSVS DDARKFTLKWIADDLEI.IG DLFM EEAREVAEDAARNNQITEEQREEDEKDLEKLAKEHSWRAAYRGKLKAKEF G
(SEQ ID NO: 97)
Log. Enrichment Ratios: Expression 1.07, High Expression 0.31, 100 nM BHRF1 -2.50, 400 nM
BHRF1 -0.83
isidex 06 DNA Barcode: TCCAACATGTCCAACATG (SEQ ID NO: 173)
GRSANDILKQFLEMLQEALRKFDEKKNKIEDEW QFDLSTQRREEATHK.WIAAALMAIGDM FNALRWALEEALKAKLKNLQSSDDLKEAIERMMKLMLEKAQEIQEKGRELADKIEQG (SEQ ID NO: 98) Logs Enrichment Ratios: Expression 0.86, High Expression 2.77, 100 nM BHRF1 -2.35, 400 nM BHRFI -1.45
Index 07 DNA Barcode: CTGAACTGACTGAA.CTGA (SEQ ID NO: ! 74)
GEEFKK L WEEWLIXATNEAENQARNMWQ AEQTDLEDQQRiRAVDFWIAiAI.MAiGD KFNADQEGDEEFEKY KGRASEDKiKEAKDERr)RA KRWEQFVKEAGERAFRGEQLG (SEQ ID NO: 99)
Log Enrichment Ratios: Expression 1.33, High Expression 0.72, 100 nM BHRFI -2.79, 400 nM BHRFI -1.16
Into 08 DNA Barcode: T< :A( X iCATTK l U di 'A l l' ( SKQ I D X< ): ; 7 1 GWDARRALKYVYERMREDLEYARNQIDNMEDRADQYDARTEERKEFT R ALALMLTGD
G.FNAFERAKEW1DDGKNNNQRSSDEADYA DEALKF1FYAAFEARRKGDELDKKAEGG (SEQ ID NO: 100)
Lo¾ Enrichment Ratios: Expression 1.53, High Expression 2.32, 100 nM BHRFI -3.06, 400 nM BHRF I -0.80
Index 09 DNA Barcode: GGAATCGATGGAATCGAT (SEQ ID NO: 176)
GKEAKKJ IQEALEEAKRKAEKLLREHEKKKKEHLLGDKRDREKTEETDKWIAEALML1GDIF NLYMKFEWE EREKKLGLLREEEEKEVEDEAKDAYLKALKLAYLVSKKGHEVAELG (SEQ ID NO: 1 01)
Lo¾ Enrichment Ratios: Expression 0, 18, High Expression 0.86, 100 nM BHRF I -2.55, 400 nM
BHRFI -2.05 "
Index 10 DNA Barcode: GAAGGCTATGAAGGCTAT (SEQ ID NO: 177)
GDSDDDDLKDALLRMLWAAAQAIYHSLENMER E FDMHFEEERRDTLQWIADALRAIGD
-'NEMMRRRRELEKKRENNHSEQRARLYEEFLKRFAEWASRELAKAGKKEANKLNEG (SEQ ID NO: 102)
Logs Enrichment Ratios: Expression 1.30, High Expression 1.47, 100 nM BHRFI -0.72, 400 nM BHRF I 0.62
Index 11 DNA Barcode: GACGTTACAGACGTTACA (SEQ ID NO: 178)
GNILDEAKDEMREEMEKLWKiafKDEVEEERKEAEREEKJiFQERAELTKRVVTARALMAIGD MFNRFREA EKLEKilRELGLISEEDARKALLLLEEFMRRMAEFAf KLGDDLMRDAEKG (SEQ ID NO: 103)
Logs Enrichment Ratios: Expression 0.34, High Expression 2.13, 100 nM BHRFI -2.62, 400 nM
BHRFI -1.55
Index 12 D A Barcode: AGTGGCATAAGTGGC AT A (SEQ ID NO: ! 79)
GEDDDKVLKWALEALRKVLDEAKEKLEKLKKYTDGDGFGEDYRREFFRKWTAIALEAIGDIF MMEALQKADKHKKLNTHDSQKADEAKEKIK.KFADEAEERAKELAKKGEAWT..-LKG (SEQ ID NO: 1 04)
Logs Enrichment Ratios: Expression 1.13, High Expression 1.08, 100 nM BHRFI -2.56, 400 nM BHRFI -1.37
l!Hfex B DNA Bsm.dc:
Figure imgf000039_0001
( SKQ ! i ) X< r. SOi GSKWEEDREKAKREAEKKLDEAKDKLDLYKDFALRFDASDELKTKWTLEWTALALEM1GD VFNYALEAKEFAE KARNNLLLDDLKDLYKLYLALLAKEESKKAIEEGDKLREATEKG (SEQ ID NO: 105)
Logs Enrichment Ratios: Expression 1. 19, High Expression 1.58, 100 nM BHRFI -3.07, 400 nM BHRF I -1.60
Index 14 DNA Barcode: CCTTGAGAACCTTGAGAA (SEQ ID NO: 181)
GLSADDLFDYAEDRMREGWKJ3FEELAGEAEKKAKEHTLSDQERREATEKWIAAALEL1GDA FNAIRWAEELG LYVKLNLDD QKVEEL XLEERAKEEAQKAR RGDKLEDLADSG (SEQ ID NO: 3 06)
Logs Enrichment Ratios: Expression 1 ,32, High Expression 1 .78, 100 nM BHRF I -2.91, 400 nM
BHRFI -0.65 "
Index IS DNA Barcode: CATGTCTCACATGTCTCA (SEQ ID NO: 182)
GNDRDQIEEYHRERMDEELDRAKKRLEELKKLWEKLDGDDLMKFT-AVTFKWIAESLKIIGDL FNRLLRTWEFAEALKKGIGFDEKKAEEA ERAYERAAEAAW AAJ ^SREMREFLLKG (SEQ ID NO: 107)
Lo¾ Enrichment Ratios: Expression 1.60, High Expression 2.73, 100 iiM BHRF1 -2.77, 400 nM
BHRFl -0.71
Index 16 DNA Barcode: CATCTGCTAC ATCTGCTA (SEQ ID NO: ! 83)
GNSADDILDEARDRHE TALWAKDQEDNLKDEAEKGDIGTEQLIRLTMKWIATQLMAIGDAF
NF AMEAKKKI.DLLKKI.NL VQAQKLEEAKERADKFEKKADQLS SKFGREMA RDLAQG (SEQ ID NO: 108)
Log Enrichment Ratios: Expression 0,13, High Expression -2,21, 100 nM BHRF1 1.40, 400 nM BHRF1 1.61
index il DNA Barcode: i 'C A IV I TAGU !\ TTA< · (SKQ ID No: ) S4 i GRSAEJMREILEKQAEDDAKKiRDiAQKWKERRKRYDPRDEEREEEVEKWiAFALMATGDIFN LARWALLQARWERRWNLSHEDEGKNHEENVKDAEDRAHWKAREAAREGAKMSWEG
(SEQ ID NO: 109)
Log2 Enrichment Ratios: Expression 0.20, High Expression -3.24, 100 nM BHRF1 -3.41, 400 nM BHRFl -2.54
Index 18 DNA Barcode: TTCICCGATTTTCJCCGATT (SEQ ID NO: 185)
GGTEDDiKDLAEKWRDDMKKEFLREFLRIKEWTKYWGWREEGRKLATLRWIALSLMHiGD LFNLKELAKKLVDDIKKKGLEHEERAERAREEAEKIMEKAAKLDSILS LAAKLIEEG (SEQ ID NO: 1 10)
Log* Enrichment Ratios: Expression 0,69, High Expression -1,70, 100 nM BHRFl -1.36, 400 nM
BHRFl 0.98 "
Index 19 DNA Barcode: CACGATTCTCACG ATTCT (SEQ ID NO: 186)
GERVEEILRKMLDDALLHFLEHRDDARERKERGERHQPRDEEREELSHDWIAAALMAIGDIF NAKLRAEERAEEFLKWGLRSQDDKKELEERAKEAAKiALKWAEEAGKEADEAEKAG (SEQ ID NO: 111)
Log* Enrichment Ratios: Expression 0.40, High Expression -2.28, 100 nM BHRFl -2.97, 400 nM BHRFl -1.98
Index 20 D A Barcode: AGAATTGCC AGAATTGCC (SEQ ID NO: 187)
GLRFEEIERYAREEADKIADEAKERFEKLK LFLWLTDKDEERLKMTHLWIAGALEAIGDLF NAAELAKELAEKAAJ LTSQDANRRDEARKKIDEAEKEAADKVSKj AKEAAKFFEQG (SEQ ID NO: 112)
Log Enrichment Ratios: Expression 0.54, High Expression -1.99, 100 nM BHRFl -3.87, 400 nM
BHRFl -3.65
Index 21 DNA Barcode: ATTAGTCGGATTAGTCGG (SEQ ID NO: 188)
GFDWKKVLDKAKDIAENDVREAKQKLEEFYKKAMKLDLTQEMRRKLMLEWAAMLMAIG DIFNAIEQGKQEADKLK LGKVLSQLLDELKRRLEELKEEAALKAHDFGREFELKLLFG (SEQ ID NO: 1 13)
Log. Enrichment Ratios: Expression 0,49, High Expression -1,20, 100 nM BHRFl -3.38, 400 nM BHRFl -2.81
¾Hfe 22 DNA Barcode: GATi i.- ill < <A'i'( iA(. ΤΤί ' (SKQ \V> NO: ; 89 i GSSAEDLRDWARDQHEKDVDKMEKRLRLLYFELARKDFNEEELKKATEKWIAAALDA1GD HFNAALKARLLARDAAKKGLIDRNKLDE^KMAELFEELGERKAALKGREFLRWVLm (SEQ ID NO: 134)
Log2 Enrichment Ratios: Expression 0.59, High Expression -3.19, 100 nM BHRF 1 -3.03, 400 nM BHRFl -2.40
Index 23 DNA Barcode: ATCGATCTCATCGATCTC (SEQ ID NO: 190)
GEDEEKDHKDTEEKARRLHERARDMLDKVKDLEEKTDAQDNERRRATHDWIAAALMM1G DAFNSFEDTKRRAEKKRELNLISEDEAKEKIKRAEELRKRIYELLKKAAEFAREAEKGG (SEQ ID NO: 3 15)
Log* Enrichment Ratios: Expression 0,78, High Expression -1,37, 100 nM BHRFl -1.72, 400 nM
BHRFl -0.17 "
Index 24 DNA Barcode: TGTCTAGTGTGTCTAGTG (SEQ ID NO: 191)
GELAREAAEEAHRRVEEDARDAKNRLDEFKKRYKITQLSKSDISRATALWIAAALDAIGDIFN AKQKAEKILGLWYKLGLVQLQEFLEKEDKARYHWQAALERAFEAGRDMLEVAAYG (SEQ ID NO: 116)
Lo¾ Enrichment Ratios: Expression 0.48, High Expression -1.95, 100 nM BHRFl -3.02, 400 nM
BHRFl -2.95
Index 25 DNA Barcode: GGATGTTCTGGATGTTCT (SEQ ID NO: ! 92)
GANHEDAIWEALYKAEDAFKDHLKEIEIYREFSEKPWPLDDY DNI AHWIAAALAAIGDW
F1STVFEEAELKFREAKRKM,RSEDDIKKYRWRLFKALDIAIDLADRVGDEAEKAERLG (SEQ ID NO: 1 17)
Log* Enrichment Ratios: Expression 0.95, High Expression -1.01, 100 nM BHRFl -2.90, 400 nM BHRFl -1.53
Index 26 DNA Bar ode: VK ·( >'!'( >'\X TAT< :( Π'ί Π'ί "!' (SKQ II) Xi r. ; 93 ι GRFAERLFK MLIK.QLLNTQYFRDQLKQLKDRSKKYDASDDDKDEATHRWIAFALMAIGDV FNDKLEIELLTELFAKYGLVHEEER EFRKRLDEFE IFRKWLDEL KLALEALNQG (SEQ ID NO: 118)
Logs Enrichment Ratios: Expression 0.50, High Expression■ 1.87, 100 nM BHRFl -2.74, 400 nM BHRFl -2.00
Index 27 DNA Barcode: CTCAGATCACTCAGATCA (SEQ ID NO: 194)
GLDGD YLMDEAF FIERERERAEEEAK MYELAEKGK Y YEERKTKATKF Wi AL ALEMiGDF FNFEMWFRKYAEKNRENNQRREDLLRRWELLLRFQAWDAAERARELG RLELWFK G
(SEQ ID NO: 3 19)
Logi Enrichment Ratios: Expression 0.71, High Expression -2,21, 100 nM BHRFl -0.72, 400 nM
BHRFl 0.86 "
Index 28 DNA Barcode: CTACGACATCTACGACAT (SEQ ID NO: 195)
GKEGSRLREEAERRGLR LLEVILRWLEDALRMIYGQD DEDR EATHRWiADALELIGDIF NALLEAFiKMELARRFGLLEEQRARDE KXALERAEEFS RARELGE L'rQiLEGG (SEQ ID NO: 120)
Logj Enrichment Ratios: Expression -2.87, High Expression -4.25, 100 nM BHRF l -4.52, 400 nM BHRFl -2.85
Index 29 DNA Barcode: CTAGGTGTACTAGGTGTA (SEQ ID NO: 196)
GEVAKDLAKLAn)LAKi LMLLFWWFFELFKLFAKFTDEWQEWKARGTAFWIALSLAAIGDF FNARRRAELQAREGKQKGLTTEEKEKRWREHLKEAWEKLEKISRLAFLFAQEAENQG (SEQ ID NO: 121)
Lo¾ Enrichment Ratios: Expression -1.14, High Expression -2.44, 100 nM BHRFl -1.66, 400 nM
BHRFl -2.43 ~
Index 30 DNA Barcode: AAGTTGACCAAGTTG ACC (SEQ ID NO: 197)
GSRWFDAEDKMRERKDRAILQLLFMLWIIFYILWYGDDTEEAKRKAMAAWALALIGIGDIF NAEAEFLEELERAIKQGQVSDQLKEELLKRMEDD RDLEKRLYEFLLKALLQWMQG (SEQ ID NO: 122)
Log} Enrichment Ratios: Expression -1.26, High Expression -1.69, 100 nM BHRFl -2.58, 400 nM BHRFl -4.10
Index 31 D\A Barcode: A \G< :(.'(. "ATI A AGGt ί 'ΑΪΤ (SKQ ii) Xi ): ) y¾i GDQADK1KD IKDEAKKKADEFKKRLEQFREYLEKVYSDDLKETYLTTFWIALALMLIGDAF NE MLLEWEFKERKKRNLRHEEELKEEKKKREEAEKALEWASKYASQVGKEAAEEG (SEQ ID NO: 123)
Logz Enrichment Ratios: Expression -2.65, High Expression -3.58, 100 nM BHRF l -3.55, 400 nM BHRFl -4.21
Index 32 DNA Barcode: TCJGCTTCTATGGCTTCTA (SEQ ID NO: 199)
GGDENKLKDYV"K13EIERGLNEIEDLARKiEQLARRFFP DEERMKFlTviWWiAAALMAiGDiF NAKEYARERAEEIRRKGLRREEEARRIEKEIEEEAEKAAKKAAKLGDHLAEELFRG (SEQ ID NO: 124)
Logi Enrichment Ratios: Expression -0.75, High Expression -0.86, 300 nM BHRFl -2.00, 400 nM
BHRFl -2.56
index 33 DNA Barcode: GTCTTCTGAGTCTTCTGA (SEQ ID NO: 200)
GKQWQEAFEEARRRIEEKAREFEDRA KEALLHLFFiPHD EIADNS KWIAWALMLiGDiFN LEEEAAERARRHV RGEISEDDAKQiRKRLQEQAKRAAWWMRYWGEESA FAFlG (SEQ ID NO: 125)
Logs Enrichment Ratios: Expression -2.15, High Expression -3.65, 100 nM BHRF1 -4.18, 400 nM
BHRFl -3.85 ~
Index 34 DNA Barcode: TGCTCACAATGCTCACAA (SEQ ID NO: 201)
GKFKKLFENYAFI,FAR.W 'ADKGKKIAEFJ^RF AEKGLKLQKLWIJFTMIWTAIMLMSIGDA FNi.AIJAFJA QAAKN\ 3WLRDNEADEAEDRVRKFADEASRRALE GLEALRKiLEG (SEQ ID NO: 126)
Log* Enrichment Ratios: Expression -3.29, High Expression -4.31, 100 nM BHRF 1 -3.41, 400 nM BHRF1 -2.21
hnk y. 35 DNA Bsrn.dc ΛΪΛ< ιί ' Κ ΑΊ ΪΛί A TGA ( SKQ !i) NO: 2 2 ) GGDGVKELEELEKRKDEKKNKAEDRI KFKDEAKYADDRTEDKEKLAHRWIALALDITGDA FNLKEEARRRFLRHKFRGELDDSKKEYAEKEMKRFEDDVEKDAEELAQKAKEAFKEG (SEQ ID NO: 127)
Logs Enrichment Ratios: Expression -2.24, High Expression -3.49, 100 nM BHRF 1 -3.51, 400 nM BHRF l -3.29
Index 36 DNA Barcode: AAGTCAGAGAAGTCAGAG (SEQ ID NO: 203)
GYTKEWIRDRAKEELDRFADEAKDKADKIRDDFEKRDDKNQIAAELTKKWIAAELEA1GDA FNRAEEAKERLKKLLKLGLTRKEEAEEAAEKLEKLEKEASEKLSKTAHEVSKHDDQG (SEQ ID NO: 128)
Log. Enrichment Ratios: Expression -2.73, High Expression -3.32, 300 nM BHRFl -3.60, 400 nM
BHRFl -3.65
Index 37 DNA Barcode: TATTGCCTCTATTGCCTC (SEQ ID NO: 204)
GDFWLKAIEIAGGRMLERARESWYRALYFILMVKLFYPSDDLRRIFTLRWIAESLKLIGDAFN LFELARELLELYYKYGWrTLEKALKALWlLL LEEIFSKASKDLGERLAEEIERG (SEQ ID NO: 129)
Logj Enrichment Ratios: Expression -1.72, High Expression -3.52, 100 nM BHRF l -3.13, 400 nM BHRF l -2.14
Index 38 DNA Barcode: GCTTATGGTGCTTATGGT (SEQ ID NO: 205)
GEKLKKLAEELEKKFRK1.FFILKDELDRAYLIALKTQVQRQELARDTKLVVTAVALMIIGDLFN AEIQGKELRDKLIKKNQVEEQKAKEFWKKWEEV QRAEELIKKGGEMVERLADYG (SEQ ID NO: 130)
Lo¾ Enrichment Ratios: Expression -0.73, High Expression 0.48, 100 nM BHRFl -1.71, 400 nM
BHRFl -1.46 ~
Index 39 DNA Barcode: GCTGTATACGCTGTATAC (SEQ ID NO: 206)
GK YLKAARLALYLLWEAYLRGYLNLLLDELEAEFFDPHDERKIRYTLSn^DALMLIGDLF NARLKMEKALWELKKEGKLREEDYEKMERLFRKWMELAFKWLEHFREMAEKAKKKG
(SEQ ID NO: 131)
Log Enrichment Ratios: Expression -1.76, High Expression -2.01, 100 nM BHRF l -2.08, 400 nM BHRFl -2.01
hnk y. 40 DN \ Barcode: GAATU Tt \G.\.\ !'( (. "! i SKQ ! ! > X< r. I'M) GNEAEQRREEFKEIMEKK DEAEK SEKIKRLALAFDLSDDDKTKATDEWTAISLEIIGDAFN FGEGLKDEAKRRKKRGLKRDEEVDKFEKTAEQAIEELR LAEEADERGA HLRDG (SEQ ID NO: 132)
Logi Enrichment Ratios: Expression -2.67, High Expression -3.15, 100 nM BHRF l -3.70, 400 nM BHRF l -4.69
Index 41 DNA Barcode: CATCAGTGTCATCAGTGT (SEQ ID NO: 208)
GEQEDKVK RAKRGALERAREMFEKJVIRKAlYLAELYINNDEGKTKLTDRWIAFALMM iDI FNIALEARLEALKLVLKGLRSQEDAEKVKKLAEEAEREAA RAA LGDKMDE EHEG (SEQ ID NO: 333)
Lo¾ Enrichment Ratios: Expression -0.27, High Expression -1.86, 300 nM BHRFl -2.17, 400 nM
BHRFl -2.39
index 42 DNA Barcode: ACCTGTAACACCTGTAAC (SEQ ID NO: 209)
GQQEEQFiEDFKXEVLRAADDAKDDME RAEEF KXDGDDNEK RKlL WIADALEAiGDL
FNAAQEAKRRAELYF LGLLK ERKEEAEEEAE AKEEASKKLH AAREARI MEKG (SEQ ID NO: 134)
Logs Enrichment Ratios: Expression -3.04, High Expression -3.00, 100 rJvl BHRF1 -3.63, 400 rJvl
BHRFl -3.15 ~
Index 43 DNA Barcode: CCGTAATTGCCGTAATTG (SEQ ID NO: 210)
GKKAEE^KEARKLHEAQLRYAYT MMKDWRF KQQEEKQTQREF WTAWWIALMLMAI GDTF FAEWAKEELDKLREKGLVEKKKAEEA E AEKLAEEASRRASEFAQLFAKWDKEG
(SEQ ID NO: 135)
Logs Enrichment Ratios: Expression -2.29, High Expression -1.97, 100 nM BHRF1 -2.44, 400 nM BHRFl __-_3.04
index 44 DNA Barcode: CCA GCA TCCAAGCAAT (SEQ ID NO: 2 ! Ϊ)
GESGEWILEKTREKIERAIRDAEKKLRLITLLTRLFHPGDDLRALFAAIWIAAELELIGDIFNEKQ DAEEKFKELL KNQFRWEELWRKWLILEWIFQKARR SKELAERAKKAFDFG (SEQ ID NO: 136)
Logs Enrichment Ratios: Expression -0.78, High Expression -3.17, 100 nM BHRF1 -3.29, 400 nM ΒΠΚ Π -3.16
Index 45 DNA Barcode: TAGCGTACTTACJCGTACT (SEQ ID NO: 212)
GYSLDDFLKLA LLAELL RFiR EAERLREL EWLLD'r'T'LGRLiL'T'LEWIAiELMnGDiFNA MLLDKFAKYAEWLGLMKEEEAKQAKKLAKLLLDEV DEARKKADDGEKFAEEG (SEQ ID NO: 137)
Lo¾ Enrichment Ratios: Expression -2.31 , High Expression -2.28, 100 nM BHRF1 -2.44, 400 nM
BHRF1 -3.17
Index 46 DNA Barcode: GCAACTATGGCAACTATG (SEQ ID NO: 213)
GRDGERVV WA NQHENTVDEA DKJ\4DNQEDEMR NADDEKLR ETH WIAFALEAIG DVFNDAMQAFELLERF KFGQQEQKKLDEFKEKVERLAREASRKLTYLGKRFALDIESG
(SEQ ID NO: 138)
Lo s Enrichment Ratios: Expression -0.31, High Expression -3.37, 100 nM BHRF1 -3.41, 400 nM BHRF3 -3.60
Index 47 DNA Barcode: CTGTCGTAACTGTCGTAA (SEQ ID NO: 214)
GWSADWIKDQA ELMLRAAEEMK RADEEEKKFKYKQFTTEFLTKATMRWIALALM IGD NVLMWALEWAKRMAKLNQYRKEELEKAKEEAKKLAEKAARRITEIGREAEQKALKG
(SEQ ID NO: 139)
Lo¾ Enrichment Ratios: Expression -2.07, High Expression -2.42, 100 nM BHRF1 -3.13, 400 nM
BHRFl -2.79 ~
Index 48 DNA Barcode: TTACTGACGTTACTGACG (SEQ ID NO: 215)
GF GKEKAQKFRDITKDILEEAIRLAKDIAEDAKKFDLKLEKLLEATLKWTAAALMAIGDLFN FKDLAEKEVRERHDRGEISSDRRDKYEKEAREGADEAAKELSKLAKIAEKKILEG (SEQ ID NO: 140)
Log2 Enrichment Ratios: Expression -2.24, High Expression -3.39, 100 nM BHRFl -2.80, 400 nM BHRFl -2.33
index 4 DN \ Barcode: ί ΓΛΤί i VK A ί ΓΛΤί i VK · (SKQ \ V> NO: 2 \ ) GWSKDWVLE^TLREKLEEIDREALWKFILIWTE MLGVDDDEQRRKDAAKWIAGSLEAIGDIF NAMMWAKRLLEWLEKANLVRREELEKAKQKAEELAKKAALRAAIYSKIAEEWLWKG
(SEQ ID NO: 141)
Logs Enrichment Ratios: Expression -2.07, High Expression -3.05, 100 nM BHRF l -2.70, 400 nM BHRFl -1.22
Index 50 DNA Barcode: ATCGGTAGTATCGGTAGT (SEQ ID NO: 217)
GKRAEELREEAEERAKEAFKETEQKLREVEERSRQTLARDEELRKAALLWIAAALMGIGDLF NKKEKGKEALEK£EKNGKRRTERAEREKERLEKEVSREAQRFKKKGEEEEKKHKYG (SEQ ID NO: 342)
Lo¾ Enrichment Ratios: Expression -2.93, High Expression -2.75, 300 nM BHRFl -3.79, 400 nM
BHRFl -3.47
index 51 DNA Barcode: GATCAACTGGATCAACTG (SEQ ID NO: 218)
GWTALWLKI FTEQEAR KFREALYYGWMMAMRALEHQLQADELAMWTALWIAAMLEAI
GDMFNDKLRAEKYALLLIWLNLYHKDIAEKWREEHEEKLKEALQEMFEAAEKF'DKFAKF'G (SEQ ID NO: 143)
Lo¾ Enrichment Ratios: Expression -1.53, High Expression -2.24, 100 nM BHRF1 -2.53, 400 nM
BHRFl -2.43 ~
Index 52 DNA Barcode: AGTCT ACCT AGTCT ACCT (SEQ ID NO: 219)
GNDKEKFREDVKK AKYAIAVKIX LADEAKERAL FDPSFJiMKREFTi.EWiAWALEAiGDi FNAWLDGKKYADEAKKQGKARKEEAEETK EATRIA EAHEKASELARKJLYHMLLG (SEQ ID NO: 144)
Log. Enrichment Ratios: Expression -0.35, High Expression -3.17, 100 nM BHRF1 -0.83, 400 nM BHRF1 -0.20
hnk y. 5 i)N \ Barcode: .ATG.YR GGTATGATt GGT (SKQ ii) NO: 22i ) ) GHVAEEEiRRFLRKAE VLQEARRKMEKRRREAEEHDTTTWLLARGTiEWIADAL LlGDAF NFRREAY1RGELYKKFGLIREDDLKDRLKEADQRLDEFAKKMALFGLELHLRLREG (SEQ ID NO: 145)
Lo¾ Enrichment Ratios: Expression -0.27, High Expression -2.43, 100 nM BHRF1 -0.29, 400 nM BHRF l 0.40
lnde 54 DNA Barcode: GTCJCAATGTGTCJCAATGT (SEQ ID NO: 221)
GD HEEAKEEAEKKFEKLRiEARL AEWL KAG YGLQLQELWAKLSDY WiAFALEilGDLF NFLEEHKEKIEKDL KGEALDDRADDTLKDLEKKAKEVSKHAMKLGREAQQF1ELG (SEQ ID NO: 146)
Log. Enrichment Ratios: Expression -1.26, High Expression -1.71 , 100 nM BHRF1 -3.22, 400 nM
BHRF1 -2.24
Index 55 DNA Barcode: TGAATGCCATGAATGCCA (SEQ ID NO: 222)
GEEAE LiKEAKDKFEDLREKAEELLYKJviWLIRYESS DT RGEIYTKKWIAiMLMMlGDAF NMALRARLYLEERRKJ GEKHEEEAEEKERRARWEQEDAYKKAKKGAKRARLYDKLG (SEQ ID NO: 147)
Logi Enrichment Ratios: Expression -1.78, High Expression -1.88, 100 nM BHRF 1 -2.69, 400 nM BHRFl -3.29
Index 56 DNA Barcode: AACAGTCCAAACAGTCCA (SEQ ID NO: 223)
GESAEKWRERLREKAGY AEYAFWLADEAEKRAKIYSASSERRAEWTMRWLAIALAAIGD NEGQK^ ^EKFDELKKQNKRSDDDLDDYKDKIKEEVE j -RKLLKAGDKIADLAEQG (SEQ ID NO: 148)
Log Enrichment Ratios: Expression -2.62, High Expression -3.45, 100 nM BHRFl -3.49, 400 nM
BHRFl -3.81 ~
Index 57 DNA Barcode: TCCTAACGTTCCTAACGT (SEQ ID NO: 224)
GDI. EEIJiERAKKiIRRAI.DEAKDAEDIJ KEAEKRWTTEMATKFVAWWIAGAI.MnGDIF NAAREVKERAEKALKWGVLSQDDIKELLLELENI.,EQEAKERAKEFGEKAE FKKMG (SEQ ID NO: 149)
Log. Enrichment Ratios: Expression -0.83, High Expression -3.04, 100 nM BHRFl -1.97, 400 nM BHRFl -2.85
liide SS DNA Bsm.de: ,\< :( Ά< :ΑΤί .TA< A A< ·ΛΤί ,T (SKQ i ) Xi ): U ? GEKAKKLEEYAREEIERALREGGDLMEEEREFGEKTELTTEWKHRAMAYWIAAALMIIGDG FNALQFIEEEGR FIRKGEFARQKIEEHKERAKERLEKALKQAKKRGDELDRFARLG (SEQ ID NO: 150)
Log2 Enrichment Ratios: Expression -0.99, High Expression -2.52, 100 nM BHRFl -2.36, 400 nM BHRF l -2.11
Index 59 DNA Barcode: GTATCAGTCGTATCAGTC (SEQ ID NO: 226)
GITLEKLWKEAKEKIRKREDEALLKAEWFKKKANNVLDLNDM
IFNYLLE'rEI ARELVRLGLFRQEEAEKK EEA EEAIKSSRNIAKRGEEAAKQMEQG (SEQ ID NO: 351)
Logi Enrichment Ratios: Expression -1.98, High Expression -2.63, 300 nM BHRFl -2.86, 400 nM
BHRFl -2.36
Index 60 DNA Barcode: AATCGTGGAAATCGTGGA (SEQ ID NO: 227)
GRQEDEIKDEATKRALEILQKLEQKVRKAKKFA YGLLLQRWWAWITKVWIAAALDAIGD AFNLGEELKRILEELRRRGLSSEEKAQEIKNW1EWLEKWVAIMA LFGEELEKQFKQG (SEQ ID NO: 152)
Lo¾ Enrichment Ratios: Expression -0.86, High Expression -3.77, 100 nM BHRF1 -2.91, 400 nM
BHRFl -2.63 ~
Index 61 DNA Barcode: CTCGTAATGCTCGTAATG (SEQ ID NO: 228)
GF^LDFJXLKLLWI.AIQFAERAXLTTELWKLWGKITQSYNEWAEKAARD\\n[AAALMIIGDM FNHKQKAEEEAKKFAKKGLKRKEELEEI KKLEEFIKRAKKLIKETAQKHEEASKMG (SEQ ID NO: 153)
Log. Enrichment Ratios: Expression -1.82, High Expression -2.20, 100 nM BHRF 1 -2.79, 400 nM BHRF1 -3.54
hnk y. 62 DN \ Barcode: ΠΧ ',\< :T< :A< · ! '!'( Ά< :T< :A<■ ( SKQ ! i ) X< r. ?: ":.')) GKLGEELREDAEKKGEEDMRRFERRTREI RKLKFGYDFEQRKREATHKWIAFALEMTGDAF NFAQKLERALELFKKWN1YSEDDLRELK RFEEAKEK.LK.KFADR1RDEGLKAVLLG (SEQ ID NO: 154)
Logs Enrichment Ratios: Expression -1.89, High Expression -2.70, 100 nM BHRF 1 -3.21, 400 nM BHRF l -2.65
Index 63 DNA Barcode: GTAAGTCACGTAAGTCAC (SEQ ID NO: 230)
GDDKEKVKDYAKKRALEDVLRAKJiLAEKFlDEAKKSDHSKQNERQYIlAWIAFMLMAIGDV FNAMMEAKRLAELLKRLGLRRWEEAEEVKQKAEELAEEASRLLADLGK.DFAKKJEQG (SEQ ID NO: 155)
Lo¾ Enrichment Ratios: Expression -0.98, High Expression -2.63, 300 nM BHRFl -2.52, 400 nM
BHRFl -2.41
Index 64 DNA Barcode: CTTATCCAGCTTATCCAG (SEQ ID NO: 231)
GLSGDDAEDFARQEIE RAREAEEKAR LIWLASKYDAKREEAL FHLRWlAFALMMiGDA FNAEEIAREMLEIARELGLTREEEAKEKLEKIRKKETEASKKMAERGRRLDNQANNG (SEQ ID NO: 156)
Lo s Enrichment Ratios: Expression -1.81, High Expression -2.68, 100 nM BHRF l -3.51, 400 nM BHRF l -1.68
Index 65 DNA Barcode: AGGACAGTTAGGACAGTT (SEQ ID NO: 232)
GNDLKDIARQlEEQAK ALDDMAKLIRELAEKAEKFYPSKDDIRRLTirYWlAAALMAIGDAF NRLQEARRRAEWLRKWGLRR£EEAEKAKKEAEERI-IER KEL IKMGDEMEEKLKRG (SEQ ID NO: 157)
Lo¾ Enrichment Ratios: Expression -3.11, High Expression -2.45, 100 nM BHRFl -3.20, 400 nM
BHRFl -2.31 ~
Index 66 DNA Barcode: GTCATGCATGTCATGCAT (SEQ ID NO: 233)
GRSKDDAT EAWERLERLLKEFKEKAEKLRDKAQAHYVYKQFALKV ILWIAWALKLIGD AFNFIEEAEKKMRENRERMJSEDDAREEKRKLEEFARRASKKANKJGDDLDRQLELG (SEQ ID NO: 158)
Log Enrichment Ratios: Expression -0.97, High Expression -2.30, 100 nM BHRF l -2.03, 400 nM BHRFl -2.49
hnk y. 67 DNA Barcod : T !X AC ( Ι'Λ'ΠΊ AC < ·Ί ( SKQ li) NO: 23-1 1 GNRSEEVKELMRELAERVLLKFRWRADEMNKEKD KYDKEELKRELTEK AFALDAIGDL FNAAELAKKLADLFKKGTGFLEERLERRKEEIEKLEEKGSRKVSYEGRREAEKIESG (SEQ ID NO: 159)
Log2 Enrichment Ratios: Expression -1.41, High Expression -3.44, 100 nM BHRF l -3.48, 400 nM BHRF l -3.44
Index 68 DNA Barcode: TAGTACGCTTAGTACGCT (SEQ ID NO: 235)
GVSIEWAFDFLENKAEEDAREARRLAQKLAEEFFKHSAREEDRAKLTKKWIAVALMIIGDIF NVEQF KQQGEEFVKRGLRSEDDFKEYLRKMEEKK£EAER1AKRAKDDMLKARDLG (SEQ ID NO: 360)
Log* Enrichment Ratios: Expression -2.49, High Expression -2.65, 300 nM BHRFl -2.57, 400 nM
BHRFl -3.63
index 69 DNA Barcode: TCGTTGAAGTCGTTGAAG (SEQ ID NO: 236)
GEQAEKALRRAKRRA WGLDDAKDILDDIEAEIRWYYPRDEERF'KFVDRWIAAMLMVIGDL FNA REALERAERLMR GLiSQDQF J7ME LEKiiLWGKi7QARKEGRE ESEITQG (SEQ ID NO: 16 i)
Logs Enrichment Ratios: Expression -2.56, High Expression -2.78, 100 nM BHRF1 -3.25, 400 nM
BHRFl -3.99 ~
Index 70 DNA Barcode: CATTAACGCCATTAACGC (SEQ ID NO: 237)
GLLWLAIILKAEELARKKDDEAEERIRRLEDEKRKGDPGTLGEAERTDRWL TMLMATGDAF NVMLEAKEEAEKLEKLGLVHKEI EKVKEEAERLFERSSDNFEEAAKRADDME EG (SEQ ID NO: 1 62)
Log. Enrichment Ratios: Expression -1.16, High Expression -2.58, 100 nM BHRF 1 -3.02, 400 nM BHRF 1 -3.13
Index 71 DNA Bsm.de: ΤΛ< Κ ,< :( ΆΑ Ϊ.Μ Γί ,< A Λ ( SKQ ID Xi r. rs¾i GERAERARDWAKDQMDDELEKAREKLWKLAFIAFKFYLKLELLFKLMFR ATMLEAIGDF
Fm^VAIAKRWLERYKLQNNTRKEEIEKAKERAKKLYEEAADKAAKLGRFYMKLLTSG (SEQ ID NO: 163)
Logs Enrichment Ratios: Expression -2.79, High Expression -3.00, 100 nM BHRF ! -2.58, 400 nM BHRF 1 -3.07
Index 72 DNA Barcode: ACCGTAAGAACCGTAAGA (SEQ ID NO: 239)
GGSYDDiADLA LH JAEEA JDELLKEAFED PYEEEFA KJ\4F WIAL\LMAIGDLF NAAELA RLAEDLKKDNTSIRDENKAEEAKQRAEQFEKEGAEELAKKGEEAAKKLAGG (SEQ ID NO: 1 64)
Log. Enrichment Ratios: Expression -2.19, High Expression -3.48, 100 nM BHRF1 -3.34, 400 nM
BHRF1 -3.47
Index 73 DNA Barcode: GACGAGATTGACGAGATT (SEQ ID NO: 240)
GKDLDEIIDEAR EMDDDADDG AEKLLKLHAGTNHSQDDFNEAHRRWIAV'ALEEIGDL FNAALRAWR IEEEIR NQRRKEEAEKAKEKVS EYERASRKAAELG EFEERVEQG (SEQ ID NO: 165)
Logi Enrichment Ratios: Expression -0.07, High Expression -2.31, 100 nM BHRF 1 -3.14, 400 nM BHRF 1 -2.27
Index 74 DNA Barcode: TACGAAGTCTACGAAGTC (SEQ ID NO: 241)
GTDHQAFDEWARRELERIVEEARERAERLREWIEQKDASREELTKFFAIWL ISLKIAIGDLFN
\^QAKRLAELLEF JLQRKEEIEKSK ^AEKLADEAMKKASKLDAKVEKELMQG (SEQ ID NO: 166)
Logs Enrichment Ratios: Expression -1.92, High Expression -2.09, 100 nM BHRF1 -3.38, 400 nM
BHRFl -2.62
Standard metrics for assessing interface quality (Figure 3C-E) or monomer stability
(Figure 3F-H) did not distinguish the working designs. We hypothesized thai many of the failed designs (Indexes-OS to 74) may simply not fold to the designed conformation. The design calculations find the lowest energy sequence for a given structure, but there is no guarantee that the lowest energy state of that designed sequence is the intended target structure. The likelihood of a protein folding depends on many factors, including the probability of an amino acid stretch adopting the correct secondary structure, the formation of a well-packed hydrophobic core, and a single native conformation of lowest energy amongst a vast assortment of alternative states. We used ROSETTA ab initio structure prediction to assess the likelihood that the designed sequence folds to the designed target conformation. Many folding simulations were carried out to give tens of thousands of possible structures (called decoys) that map out a protein energy landscape. An ideal protein would have an energy funnel from distant high-energy eonformers towards a low energy folded state, and therefore a small mean RMSD between the lowest energy decoys and the intended designed conformation (plotted in Figure ID). Representative energy landscapes are plotted in Figure 3B. A high calculated probability of correct folding is a common attribute of designs that bind BHRF l (Figure ID). Notably, the human-modified BbpD04/Index-()0 control sequence was predicted to fold, but its nonfunctional computational precursor Index-21 was not. This "forward folding" method should be broadly useful in future design efforts.
Enhanced affinity and specificity of a BHRFl -binding protein through improved electrostatic complementarity
To illuminate BHRF l biology, the designed protein should not only bind with high affinity, but do so specifically. Design BbpD04, a de novo designed protein without sequence homologues identified by BLAST (Altschul et al, 1997), bound BHRF l with moderate affinity (apparent KD = 58 + 3 nM) and reasonable specificity, and was therefore chosen for further optimization.
Design BbpD04 binds BHRF l tighter than all human prosurvival Bcl-2 proteins with the exception of Mcl- l (Table 2). Based on a Poisson-Boltzmann electrostatics model (Whitehead et al,), the computed electric field experienced by BbpD04 when bound to BHRFl is markedly more negative than when bound to Mcl- l (Figure 4A-B). W7e therefore introduced nine point substitutions to eight residues of BbpD04 to specifically increase electrostatic complementarity for BHRF l (Figure 4C). Six decreased the
J DCBHRF 1 )//¾(Mcl- 1 ) ratio as predicted (Figure 4D). However, putting many of these beneficial mutations together in combination generally caused a loss in yeast surface expression, possibly indicating poor protein stability. The variant BbpD04.1 containing the best two point mutations (E48R and E65R), together with a third compensatory mutation
(K31E) to preserve a putatively stabilizing salt-bridge, bound BFIRFl slightly tighter
(apparent ΚΏ = 8 ± 4 nM) than any■ of the other human prosurvival Be; [-2 proteins (Table 2).
Table 2. Apparent dissociation constants (nM; mean ± i 5E, n = 3-6) froi n yeast surface display titrations
Protein BHRF l Bcl-2 Bd- Mcl-l. Bfi-1 BckXL Bcl-B
Rim-BH3 12 ± 4 2.02 ± 2.1 ± 0.1 0.6 ± 0. 2 2.1 ± 0.3 3 ± 1 12.2 ±
0.08 0.1
BbpD07 60 ± 10 76 ± 7 - 3.1 ± 0. 3 > 100 > 100
BbpD04 58 ± 3 - - 17 ± 7 > 100 -
Bbp.D04.1 8 * 4 1 10 ± 20 34 ± 5 30 ± 10 > 300 25 ± 1 BhpD04.2 0.6 ± 0.2 33 ± 4 40 ± 10 26 dt 4 70 ± 20 31 ± 2
BbpD04.3 0.54 ± 20 ± 2 34 ± 3 19 ± 1 32 ± 6 34 ± 7
0.01
BIND 0.9 ± 0 .2 45 ± 7 60 ± 10 21.6 ± > 100 > 100
I 0.8
Accurate di ssociatic >n constants (nM ; mean ± 5 !D, is = 4-6) measured by BL1
Bim- 7 3 0.75 ± 20 ± 10 0.17 ± 0.61 : 1.56 ± 7
BH3 0.09 0.02 0.04 0.09
BIND 0.22 ± 2, 1 Q0 ± 870 ± 40 40 ± 10 2,600 dt 810 ± 80 > 10,000
I 0.05 100 800
BIND 0.16 ± 30,000 ± 4,600 ± 230 ± 40 4,000 dt 8,000 ± 50,000 ± ί N62S 0.08 10,000 400 2,000 2,000 10,000
Enhanced affinity and specificity of the designed protein via mutations distant from the interface
To optimize the design, the BbpD04.1 gene was diversified by error prone-PCR
(average error rate 1.3 amino acid substitutions per clone) and a subsequent yeast display library of 2 106 trans formants was sorted by three rounds of fluorescence-activated cell sorting (FACS). During each sort, the librar '- was incubated with 5 nM biotinyiated BHRF1 and 15 nM of each unlabeled human Bcl-2 protein as competitors to favor selectivity. Five mutated sites were identified that increased binding signal in the final sorted population: two mutations at the designed interface (H 104R, predicted to enhance electrostatic
complementarity, and N62S, predicted to improve specificity based on sequence-fitness landscape mapping described below), while three mutations were distal from the interface and might alter protein stability (shown later). 121 V/L slightly alters packing in the hydrophobic core, Q79L increases hydrophobic interactions buttressing the second connecting loop, and L84Q forms a stabilizing hydrogen bond to the loop backbone. The mutations were mixed combinatoriaiiy (72 protein variants) in a yeasi display library with 1 06 transformants that was further sorted for affinity and specificity. Over two rounds of sorting, the library was incubated with 1 nM biotinyiated BHRF 1 and 8 nM of each unlabeled human Bcl-2 protein, and the top one percent of cells based on binding signal intensity relative to surface expression were selected. Of 20 clones sequenced from the final sorted library, there were 12 unique sequences. The poor convergence in such a low complexity library suggests many sequences had similar binding signals under the yeast display conditions.
Screening a number of clones, we identified one (BbpD04.2 with four mutations:
121L, Q79L, I84Q and H104R, see Figure 4E) that was monodisperse and monomelic by size exclusion chromatography (SEC) after protein purification from E. coli. BbpD04.2 eluted as a higher molecular weight (MW) complex by SEC when mixed with BHRFl, indicating their interaction in solution (Figure 4F), A single point mutation of a conserved Bim leucine buried within the hydrophobic interface, L62E, severely diminishes binding of Bim-BH3 to all Bcl-2 family members (data not shown). The equivalent mutation of BbpD04.2, L54E, similarly abolishes the interaction of BbpD04.2 with BHRFl observed by SEC (Figure 4F),
Conjugation of various chemical agents to exposed cysteine residues can allow intracellular delivery, fluorescence detection or surface immobilization for affinity measurements, as described below. BbpD04.2 was incompatible with single labeling of an added terminal cysteine residue, due to the presence of a second internal cysteine (Figure 5). Short peptide linkers containing single cysteines were genetically fused to the BbpD04.2 termini (Figure 5 A) and found to react in seconds with polyethylene glycol (PEG)- maieimide, producing a. higher MW product with reduced electrophoretic mobility (Figure 5B). BbpD04.2 has an internal buried cysteine, which becomes exposed for PEG-maJeimide conjugation in the presence of the harsh detergent SDS, indicating the protein is folded and the hydrophobic core is generally shielded from solvent unless chemically denatured.
However, when cysteine-linker BbpD04.2 proteins were conjugated to HPDP-biotin for longer incubations (4 h) at room temperature, the proteins would subsequently aggregate when mixed with tetrameric streptavidin. We hypothesized that, in addition to the exposed terminal cysteine, the internal cysteine was weakly conjugated under these conditions to form aggregated streptavidin-complexes. Mutation of the internal cysteine (CI 03 A) markedly diminished aggregation (Figure 5C). BbpD04.2 CI 03 A (called BbpD04.3) had only a small loss of affinity and specificity (Figure 5F), and was therefore chosen for further experiments. interface interactions and folded structure are both critical
To probe the sequence-fitness landscape of the designed protein, site-specific saturation mutagenesis according to the protocol of (Procko et al, 2013) was used to independently diversify every codon of the BbpD04.3 gene to NNK (N is any base, K is G or T), producing a library of (1 16 positions) x (20 amino acids + stop codon) = 2,436 protein variants. The variants were expressed by yeast surface display (2.5 χ i06 transformants) and the librar was sorted by a single round of FACS for the 1% of cells with highest binding signal for 400 pM biotinylated BHRFl (Figure 6A). Alternatively, the library was sorted for affinity and specificity (yeast were incubated with 400 pM biotinylated BHRFl and 8 nM of an equimoiar mixture of unlabeled human Bcl-2 proteins as competitors; Figure 6B). DMA was extracted from the naive and post-sorted yeast populations, the BbpD04.3 gene amplified as two fragments to provide full sequencing coverage, and the samples were deep sequenced using iiJumina MiSeq sequencing. The frequency of each protein variant is compared between the naive/pre-sorted and enriched/post-sorted populations to calculate an enrichment ratio, which acts as a proxy for the affinity/specificity fitness of each substitution (Fowler et al, 2010; McLaughlin et al, 2012; Procko et al, 2013; Whitehead et al, 2012).
The BbpD04.3 affinity sequence-fitness landscape reveals the critical nature of the incorporated Bim-BH3 motif, with most substitutions of interface residues being depleted (Figure 6A). In addition, substitutions to proline, which can break regular helical secondary structure, are depleted across the first, second and third helical spans of the designed helical bundle fold (Figure 6A), Substitutions to aspartate, a short and charged amino acid, are depleted within the hydrophobic core as anticipated (Figure 6A). The BbpD04.3 affinity- specificity sequence-fitness landscape, in which unlabeled Bcl-2. proteins were included as competitors for BHRFl binding, is similar (Figure 6B).
Using the sequence-fitness landscape for BHRF 1 affinity, we are able to determine the allowed sequence variation of BbpD04.3. The most conserved residues for BHRFl interaction are found within the second helix of BbpD04.3 and span the incorporated Bim- BH3 motif (Figure 14 A). Residues near the BbpD04.3 C-terminus that also contact BHRFl are similarly conserved. We applied our experimental enrichment ratios to a hypothetical population that evenly covered all single amino acid substitutions at a given residue, and from the evolved population calculated the probability of finding each amino acid. This analysis reveals that significant diversity is tolerated for any single amino acid substitution, except at critically conserved residues (Figure 14B and Table 4). Presumably the tolerance for any two amino acid substitutions would be less, less again for three substitutions and so forth, but it is clear that some positions have little preference for amino acid type. A large number of BbpD04.3 sequence variants can therefore maintain the folded structure and favorable binding to a target BCL2 protein.
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Bacterial expression of BbpD04.3 was very low, limiting the quantity and purity that could be purified for biochemical applications. Simply combining mutations enriched in the sequence-fitiiess landscapes within libraries, while achieving enhanced BHRFl affinity and specificity by yeast surface display, gave clones with undetectable protein expression in E. coli. Therefore, we sought instead to only combine mutations that improved bacterial expression. Twenty-nine BbpD04.3 point mutants with positive enrichment ratios in either the affinity or affinity-specificity sequence-fitness landscapes were expressed in E. coli and analyzed for increased soluble protein levels by small scale NiNTA-agarose precipitation (Figitre 7A). Nine mutations were identified: W3A and W3P increase helical propensity of ihe initiating residue in the starting helix: I13Q, M33R, F61 Y, W49E/Y and M46E decrease surface hydrophobicity; and F28L slightly increases packing in the hydrophobic core while again reducing surface hydrophobicity (the lowest energy Phe rotamer at this position is predicted to point towards solvent, while Leu is directed inwards to the core in the crystal structure described below). Because the mutations are generally surface-exposed at distinct sites on a long helical bundle, we reasoned they could likely be combined without negative interference (Figure 7B). A BbpD()4.3 variant with seven mutations - W3P, II 3Q, F28L, M33R, M46E, W49Y and F61Y (Figure 7C) - had significantly increased bacterial expression and improved specificity with no significant change in BHRF l affinity by yeast surface display (Figure 6D and Table 2). This variant is named BHRFl -INhibiting Design acting Intracellularly (BINDI).
The increased expression of BINDI compared to BbpD04.3 is not due to enhanced protein stability; both BbpD04.3 and BINDI undergo cooperative unfolding at high concentrations (> 3 M) of the chemical denaturant guanidinium hydrochloride measured by circular dicnroism (CD) spectroscopy (Figure 6E). However, the original design, BbpD04, has nearly linear loss of CD signal over a 0 to 6 M range of guanidinium hydrochloride (Figure 6E). 'The absence of a cooperative melting transition is associated with molten globules that lack a rigid core or single native conformation. While BbpD04, BbpD()4.3 and BINDI have high thermostability and retain partly a-helicai CD spectra at 95°C, only the evolved BbpD04.3 and BINDI fully renature when the heated protein solutions are cooled (Figure 7D-G), Further, the original BbpD04 design is sensitive to rapid hydrolysis by proteases, which require unfolded substrate backbone to access the enzyme active site (Figitre 6F-H and 7H). BbpD04.3 and BINDI are similarly resistant to protease digestion with differences attributable to sequence variation (i.e. trypsin cuts after fys or arg residues that are more abundant in BINDI, and chymotrypsin cuts after aromatic residues that are more abundant in BbpDQ4.3). Increased affinity for BHRFl following in vitro evolution correlates with enhanced protein stability. A summary of ail mutations introduced in the original design is provided in Figure 61.
The designed BINDI protein has high affinity and specificity
Apparent dissociation constants by yeast surface display are useful approximations, but may be artificially tight due to avidity effects or ligand rebinding to a dense receptor surface, or may be artificially weak if binding equilibrium is not reached during the incubation time. The BINDI'BHRFl interaction was therefore further characterized by alternative methods, BINDI eluted as a higher molecular weight complex by SEC when mixed with BHRFI in solution, whereas BINDI L54E with a knockout mutation in the designed interface did not (Figure 8A), Using bio-layer interferometry (BLI) to measure the kinetic rate constants, BINDI*BHRF1 was found to form an extraordinarily tight complex (A , ;. 220 ± 50 pM) with a slow dissociation rate (koif = [2.8 ± 0.9] χ 10 " s"1) (Figure 8B-C). BINDI bound human Mel- I with KD 40 ± 10 iiM (180-fold increase compared to BHRF I), Bcl-2 with KYj 2.1 ± 0.1 μΜ (10,000-fold increase), Bcl-w with KD 870 ± 40 nM (4,000-fold increase), Bfl-1 with ΚΏ 2.6 ± 0.8 μΜ (12,000-fold increase), Bcl-B with K > > 10 μΜ (> 45,000-fold increase) and BeJ-XL with KD 810 ± 80 nM (4,000-fold increase). Compared to the measured affinities of Bcl-2 proteins for Bim-BH3 (Figure 8D) and to other published values (Dutta et al., 2013; Dutta et al., 2010; Gemperli et al., 2005; Lessene et al., 2()13)(Tse et al., 2008)(Caria et al., 2012; Flanagan and Lefai, 2008; Kvansakul et al., 2010), the affinity and specificity of BINDI for BHRFI is considerably greater than any previously described BHRFI iigand, and is similar to or exceeds that of any other protein, peptide or drug designed to specifically bind a Bcl-2 family protein.
BIND! incorporates the Bim-BH3 motif within a de novo designed fold guided by the topology of PDB 3 LHP chain S. The direct graft of Bim-BH3 interaction residues to the equivalent site within the 3LHP S scaffold (design BbpGT) failed to bind BHRFI . Even after extensive design of the surrounding interaction surface (design BbpGl .D), the grafted protein did not bind BHRFI . While 3LHP_S is structurally similar to BINDI, it is nonetheless a poor steric fit for the BHRFI binding groove in this design protocol. Aligning the graft site within 3 LHP S to the Bim-BH3 motif of BINDI in the BINDI-BHRFl structure demonstrates how the C-terrninal helix of the grafted design comes too close to the BHRFI surface, such that side chains would clash (Figure 9A), This simple structural alignment demonstrates why building new proteins with unique backbone atom positions can be essential for designing productive interactions. BINDI has an ideal structure and amino acid sequence found after computationally filtering thousands of potential designs for optimum interactions with BHRFI .
Compared to the native Bim-BH3 interaction, BINDI contacts an additional 404 A2 on the surface of BHRFI (Figure 9B-G), Residues from the incorporated Bim-BH3 motif account for just 587 A2 of the BINDI surface buried in the complex, whereas surrounding designed residues account for 839 A2. Only two residues at the periphery of the incorporated Bim-BH3 motif changed during the final round of affinity maturation (the conserv ative W49Y and F61Y substitutions), while all residues in the core of the motif remained unchanged (Figure lOA-C). Introducing these two mutations into a Bim-BH3 peptide, or mutating the Bim-BH3 peptide at all five positions within the BH3 region that distinguish nonspecific BbpD04 from specific HINDI, failed to achieve the high affinity and specificity of BINDI (Figure 10D-E). The extraordinary specificity of BFNDI is therefore accomplished through interactions across an expansive interface, extending well beyond the central Bim- BH3 residues.
BINDI triggers apoptosis preferentially in an EBV-infected cell line
We tested whether inhibition of BHRF l via steric occlusion of the BH3-binding groove with BIND! could induce mitochondrial cytochrome c release in the EBV-positive BL cell line Ramos-AW. Ramos-AW expresses BHRFl at very low levels (Leao et al., 2007), and therefore presents a challenging biological target that likely expresses much higher levels of off- target endogenous Bc!-2. family proteins. BIND! was applied to mitochondria isolated from both Ramos-AW and the EBV-negative parental fine Ramos (Anderssoii and Lindahl, 1976). BFNDI elicited greater cytochrome c release from Ramos-AW mitochondria (Figure 1 IB), indicating an EBV-associated factor is likely a BIND! target. Strikingly, the nonspecific Bim-BH3 peptide had opposite behavior; mitochondria from EBV-negative Ramos cells were more sensitive to Bim-BH3 treatment than those from EBV-positive Ramos-AW cells (Figure 1 1A). Indeed, EBV-positive cell lines are widely reported as more resistant to nonselective apoptotic stimuli (Ishii et al., 1995; Kvansakul et al., 2.010; Leao et al, 2007), making the enhanced activity of BINDI against Ramos-AW cells all the more significant.
While significantly weaker than the picomolar affinity of BINDI for BHRFl, the moderate affinity for Mcl-l is likely the reason BINDI still triggers apoptosis in the EBV- negative Ramos cell line. It is possible that the enhanced toxicity of BINDI towards Ramos- AW reflects increased Mcl-l -dependency in this line, rather than expression of EBV BHRFl . To rule out this possibility, we tested a variant, BINDI N62S, with even greater specificity. During affinity maturation, the N62.S mutation was found to enhance specificity both in the error-prone PCR-based library and in the comprehensive site-specific saturation mutagenesis library (Figure 6). However, the N62S mutation simply wasn't present in clone BbpD04.2 isolated from the combinatorial library , and neither did this mutation improve expression of soluble protein in bacteria, the criterion used for combining mutations to generate BINDI. Asn62 of BINDI (Asn7() in Bim-BH3) hydrogen bonds to the N-terminus of BHRFl helix αό, and serine at this position is predicted to similarly interact at the interface (Figure 1 1 C). BINDI N62S still binds BHRF1 with extraordinarily tight affinity (KD 160 ± 80 pM), but now with even better specificity (Table 2 and Figure 1 ID). Most notably, the affinity for Mci- 1 is diminished six-fold (K0 230 ± 40 nM). Like parental BINDI, the N62.S variant has enhanced apoptotic activity against EBV-positive Ramos-AW (Figure 1 IE). Indeed, BINDI N62S, with greater specificity amongst the Bcl-2 family for BHRF1, has even greater discrimination between Ramos and Ramos-AW cells (Figure 1 IB and 1 IE). The enhanced activity of BINDI to initiate cytochrome c release preferentially in EBV-positive cells is therefore due to BHRFI inhibition.
Expression profiling of EBV-positive BLs has re vealed distinct subgroups (Kelly ei a!., 2013 ; Watanabe et a!,, 2010), and BHRFI may not be important for cell survival in all cases. Mitochondria were isolated from six EBV-positive and four EBV-negative cancer lines. Bim-BH3 peptide triggered cytochrome c release (Figure 1 IF), whereas the inactive guide scaffold 3LHP(S) had no effect (Figure 1 1G; we switched from the L54E knockout mutation to using the scaffold 3LHP(S) as a generic negative control suitable for comparison to any BIND! variant). Incubation with BINDI N62S induced high cytochrome c release in four EBV-positive lines (Figure 1 IFI): BL lines Ramos-AW and Daudi, mantle cell lymphoma line Granta 519, and B-prolymphocytic leukemia JVM- 13. Two of the EBV- positive lines had low levels of cytochrome c release similar to EBV-negative cells: BL line Raji and mantle cell lymphoma line JVM-2. Hence only a subset of EBV-positive cancer lines are dependent on BHRF 1 for survival.
Treatment of EBV-positive B lymphoma in a xenograft mouse model by intracellular delivery of BINDI
BINDI was genetically fused with a C-terminal antennapedia peptide for non-specific cellular uptake and intracellular delivery in vitro. BINDI-antennapedia applied to the growth medium at 4 μΜ selectively killed 40% of EBV-positive Ramos-AW cells, with no measurable death of EBV-negative Ramos cells (Figure 12A). Antennapedia- fused proteins concentrate in endocytic organelles and escape to the cytosol with low efficiency (Duvall et a!., 2010). To enhance endosomal escape, BINDI-antennapedia was conjugated via a terminal cysteine to a diblock copolymer carrier, PoBOO, containing a hydrophilic first block for stability and a pi i -responsive endosomo lytic second block (Duvall et al, 2010: Mamganiello et al., 2012; Convertine et al., 2010). A lower 2 μΜ dose of BINDI-antennapedia induced 60% cell death preferentially in Ramos-AW cells when conjugated to the PoBOO polymeric carrier for enhanced cytosolic delivery (Figure 12B). Our data suggest inhibition of BHRF1 can effectively kill EBV-positive BL.
Intracellular delivery of proteins in vivo is exceptionally challenging, with no efficient artificial methods currently available. Taking inspiration from the entry mechanisms of natural viruses, we developed an antibody -copolymer-based formulation to deliver BINDI to the cytosolic compartaient of B cells within an animal. BINDI is coupled via a C- terminal cysteine to diblock copolymer Po1950 synthesized by reversible addition-fragmentation chain transfer. The copolymer's hydrophilic first block is composed of polyethylene glycol methacrylate (MA) for stability in the host, pyridyldisulfide MA for cysteine conjugation to BINDI, and biotin-hydroxylethyl MA for coupling to streptavidin-antiCDl 9 (aCDI 9; human monoclonal CAT-I3.1E10-SA). The endosomoiytic second block is composed of diethylaminoethyl MA and butyl MA. The entire complex of copolymer:aCD19:BI DI forms large micelles that disassociate at low pH to expose membrane-destabilizing groups (Figure 13 A). CD 19 is a rapidly internalizing surface antigen, and bound aCDI 9-complex is endocytosed. Copolymer allows escape from the acidic endosome, and presumably BINDI is then released in the reducing cytosolic environment.
Subcutaneous Ramos- AW xenograft tumors were established in nude BALB/c mice. The mice were treated intravenously on days 0, 3 and 6 with antibody-copolymer coupled to the inactive scaffold 3LHP(S) or to BINDI. Thirty minutes prior to each treatment, cyclophosphamide (CTX) and bortezomib (BTZ) were injected intraperitoneally at subtherapeutic doses to prime cells for apoptosis (O'Connor et a!., 2006). The treatments were nontoxic, with no substantial change in mouse body weight.
The intracellular delivery of BINDI to the B lymphoma xenograft slowed tumor progression and prolonged survival. Tumors grew rapidly in the untreated PBS and chemo- only control groups (Figure 13B-C), with mean tumor sizes of 1080 ± 500 mm' and 680 ± 410 mm5, respectively, at day 1 1 when the first mice were euthanized due to excessive tumor burden. Due to the therapeutic effects of aCD 19 coupled to an endosomoiytic polymer, both scaffold 3LHP(S) and BINDI treatment groups had reduced tumor sizes, though volumes were significantly smaller (unpaired t test, P = 0.003) in the BINDI ( 140 ± 60 mm") than 3LHP(S) (330 ± 140 mm3) treatment group (Figure 13D-E), Lifespan was extended in the BINDI-treated mice compared to the scaffold treatment (log-rank test, P = 0.006), with median survival of 15 days for PBS treatment, 16 days for cliemo-only, and 21 days for 3LHP(S) treatment, extending to 24 days following BINDI treatment (Figure I3F), In addition to validating BHRF'l as a therapeutic target in EBV-positive B lymphoma, our data represent the first demonstration that a de novo computationally-designed protein can treat cancer in a preclinical model.
BCL2 family proteins share similar sequences (> 50% similarity between any two family members) and similar structures (~ 3 A RMSD). It therefore seemed likely that the BINDI protein, having high complementarity with the binding pocket of BHRFl, could serve as an excellent scaffold for engineering new specificities to other BCL2 proteins. Since earlier variants of BIND! prior to exhaustive optimization bound Mcl- 1 with high affinity, we began by repurposing the BINDI protein as a Mcl-1 binder. First, BINDI (PDB 40YD chain D) was 'docked' into the hydrophobic binding cavity of existing crystaliographic models of Mcl-1. in these models, Mcl-1 is bound to nonspecific BH3 peptides from Bim (PDBID
2PQK), Bax (PDBID 3PK1 ), or the Mcl-1 specific peptide MB 7 (PDBID 3KZ0). The bound peptide was used to align the BFI3-equivalent residues of BINDI. The docked complex was then designed (Figure 15A-B). Residues of BINDI within 8 A of the interface were computationally mutated to minimize the bound proteins' energy, keeping critical residues shared with Bim-BH3 fixed. Since design calculations use repeated random sampling, the process is done numerous times to give different possible sequences. Genes encoding six Mcll -targeted computationally designed proteins, M-CDPOl to M-CDP06, were synthesized (Tables 5 and 6) and five expressed in E. coli. The affinities of the five proteins for BCL2 family members were tested by biolayer interferometry (BIT), with the specific Mcl-1 - binding peptide MB1 tested as a positive control. Ail five proteins had tight affinity for Mcl- 1 due to slow off rates, and two appeared to be highly specific (Figure 16). This is despite only interactions with Mcf-1 being designed; specificity was achieved without explicitly designing against interactions with other BCL2 proteins.
When exposed to chemical denaturants and measuring the loss of helical structure by CD, two partially-specific binders (M-CDP02 and M-CDP05) unfolded over broad denaturant concentration ranges, suggestive of poorly packed or 'molten' cores (Figure 15C). Binders specific for just Mcl- 1 have narrow, cooperative unfolding transitions. A well - packed structure therefore appears to be necessary for specificity. We chose highly specific M-CDP04 (subsequently called MINDI for Mci- 1 -inhibiting design acting intracellular ly) for accurate determination of binding affinities in BLI experiments (Figure 15D-E). MINDI bound Mcll with 150 ± 60 pM affinity, with over ten thousand-fold weaker affinity for other BCL2 family members.
We sought to evolve a partially-specific Mcl-1 binder (M-CDP02) to specifically associate with single BCL2 proteins. However, this approach enriched for mutations that damaged regions of structure (data not shown). Since our aim is to engineer specific binders that are compact and well-folded, we abandoned directed evolution at this point and instead explicitly designed proteins to bind each BCL2 family member.
The structure of BINDI (PDB 40YD chain D) was docked into the BH3 binding cavity in the structures of Bax-BH3 -bound Bcl-2 (PDB 2XA0), small molecule inhibitors bound to Bcl-2 (PDBs 4AQ3, 4IEH and 4LVT), Bim-BH3 -bound Bcl-XL (PDB 1 PQ1 ; structure of mouse Bcl-XL, which is 97 % identical to the hitman sequence), modified Bim peptides bound to Bcl-XL (PDBs 2YQ6 and 2YQ7), Bax-bound Bcl-XL (PDB 3PL7), a Puma-derived αβ peptide bound to Bcl-XL (PDB 4BPK), Bim-bound Bcl-B (PDB 4B4S), and Bak-bound Bfl-1 (PDB 3ΠΗ). Critical interaction residues from the peptide ligand were grafted to the BINDI scaffold, or alternatively, residues of the BINDI BH3~fike motif were kept fixed (Tables 5 and 6). Then, surrounding residues at the edges of the interface were computationally designed. The designed proteins were filtered for favorable binding energies, shape complementarity with the Bcl-2 homoiog's BH3 binding cavity, and minimal buried unsatisfied polar atoms. Codon-optimized genes were synthesized and the proteins were expressed and purified from E. coli.
Figure imgf000059_0001
Figure imgf000060_0001
CDP1 K L54, 157,
1 G58
1 0-
ABA 158, A59, 1,62, 165,
CDPO Bim-BH3»Bcl-B - - + S G66, D67, N70
1
F-
158, A59, L62, 165,
CDPO 3ΠΗ Bak-BH3*Bfl-l - - +
G66, D67, N70
1
Table 6. Sequences of computationally designed proteins (CDPs) prior to experimental optimization and evolved combinatorial mutants (ECM) selected for BL1 screening.
> M-CDP01
ADPKKVLDKAK13QAE RVRELKQELEELY KAR LDL'T'QEERRKLEEEAIAALLRAIGDiYN AIQQAL EADKL AGLVNSQQLDEL RRLEELK EAS XARDYGLEFFEKLDY (SEQ ID NO: 28)
> M-CDP02
ADPK.KVLDKAKDQAENRVRELKQELEELYKEARKLDLTQEERRKLEESYIAAMLRAIGDTFN AIMQAKNEADKLKKAGLVNSQQLDELRRRLEELRKEASL AEDYGREFQEKLEY (SEQ ID NO: 29)
> M-CDP03
ADPKKVI,DKAKDQAENRVRELKQDLERLYX.EARKLDLTQEMRRKLQEKAAAAMIRA[GDI NNArYQALQEADKLKKAGIA^NSQQLDELKRRLEELQKEASR-KAQAYGEEFMLKLEY (SEQ ID NO: 30)
> M-CDP04
ADP KVLDKi KDQAENRVREL QVLEELYKEARKLDLTQEMRK LIERYA^^AilR/VIGDr N AIYQAKQEAEKLKKAGLVNSQQLDELLRRLDELQKEASR A2SIEYGREFELKLEY (SEQ ID NO: 2)
> M-CDP05
ADP KVLDKAKDQAENRVRELKQ LEELY EARKLDLTQEERHRLE'T' ALSALLAAiGDiL DA1MQALQEAAKLKKAGLV¾SQQLDELKRRLEELRKEASRKARDYGREFWLKLDY (SEQ ID NO: 32)
> M-CDP06
ADPKKVLDKAKDQAENRVRELKQKLEELYKEARKLDLTQEEREKLKTKYLSAMLAAIGDTL DAIMQALNEAQKLKKAGLVNSQQLDEL RRLEELRKEASRKARDYGREFELKLDY (SEQ ID NO: 33)
> 2-CDPOl
ADPKKVLDKAKDQAENVVRKLKQELEELYKEARKLDLTQDMREKTKLRAEAAELQAIGDTF QAILQAKMEAKKLYDAGLVNSQQLDELKRRLEELAKEAEDRAAKLGKEFLQKLEYG (SEQ ID NO: 34)
> 2-CDP02
ADPKKVLDKAKDRAENAVRELKQKLEELYKEARKLDLTQDMRNKLIMKAIAAELRAIGDIF QAILEAKAEAKKIXDAGLVNSQQFDELKRRLEELEEEAAERARKLGDEFRQKLEYG (SEQ ID NO: 35)
> 2-CDP03
ADPKX\XDKAKJ3QAENR\7RELKQKLEELYKEARKLDLTQEMRRELKERALAARLQAVGDI FYAILQAKSEADKXKi AGLVNSQQLDELKRRLEELAEEAQR ARDYGIEFALKLEY (SEQ ID NO: 36)
> 2-CDP04
ADPK V DKA JDQAENRVRELKQKLEELYKEARKLDLTQEMREKXQEQALAAWLNAAGDI
1EAI SRALQEADKLKKAGLVNSQQLDELKRRLEEL AEEA ARKAEKYGEEFKKKLE Y (SEQ ID NO: 37) > 2-CDP05
A-DPKKVLDKAKDQAENRVRELKQKLEELYKEARKLDLTQEMRAEL ARFAAATLAAAGDTI NAISEALAEAD L KAGLVNSQQLDELKRRLEELAQEAERKAEEYGQEFLLKLEY (SEQ ID NO: 38)
> 2-CDP06
ADPKK\^DKAKDEAENRVRFXKQKLEELYKEARKLDLTQEMRQELVDKARAASLQASGDTF YAILRALAEAEKLKKAGLVNSQQLDELKRMEELAEEARRKAEKLGDEFRLKLEY (SEQ ID NO: 39)
> 2-CDP07
ADPKKVLDKAKDDAENRVRELKQKLEELYKEARKLDLTQEERDELKLKAIAASLQASGDIY
NAILRALEEARKLKKAGLVNSQQLDELKRRLEELAEEAQRKANKLGDEFRLKLEY (SEQ ID NO: 40)
> X-CDPOl
ADPKX\T.DKAKJ3QAENRVRELKQKLEELYKEARKLDLTQEMRRELQARYIAAMLAAAGDI MEAIQQAKNEADKLKKAGLVNSQQLDELKRRLEELAKEAAR J EDYGREFQLKLEY (SEQ ID NO: 41)
> X-CDP02
ADPKKVLDKAKI3QAENRVRELKQKLEELYKEAR LDL1'QEMRKELVARYIAAMLAAAGD1 VQAIQDAKNEAD L KAGLVNSQQLDELKRRLEELAKEAAR ATDYGREFQL LEY (SEQ ID NO: 42)
> X-CDP03
ADPK.KVLDKAKDQAENRVRELKQKLEELY EARKLDLTQEMRRELRNRATAAILQATGDLL NATQQAKDEADKLKKAGLVNSQQLDELKRRLEELQNEAAEKAADYGEEFWLKLEY (SEQ ID NO: 43)
> X-CDP04
ADPKKVLDKAiQ^QAENRVRELKQKLEELY EARKLDLTQEDRKRLLLQYlAAMLAAIGDLE NATRW KREADKLKKAGLVNSQQLDELKRRLEELAKE AAEKAADYGEEFNLKLEY (SEQ ID NO: 44)
> X-CDP05
ADPKKVXDK^ KDQA£NRVRELKQKLEELYKEARK1.DLTQEMRRQLRDQY1AAMLAAIGDL LNAIMQAKREADKLKKAGLWSQQLDELKRRLEELEEEAAQKA DYGQEFLLKLEY(SEQ ID NO: 45)
> X-CDP06
.\i)P \'i.i K \ ΠΙ·ξ \l N \ n ΚΚΓ ΙΛ Κ HARK 1.: 1. Ί Ql > N UN \A\i.\ Λ'νΐΙΛ AT D!I HAIQEAKEEAKKLKKAGLA'NSQQLDELKRRLDELDEEAAQRAEKLGKEFNL FEY (SEQ ID NO: 46)
> X-CDP07
ADP KVLDKA DRAENVA'R L KELEELYKEARKLDLTQEMRDRIRLAAIAARIAAFGDIFH AIMEALEEAR L KAGLVNSQQLDELKRRLEELDEEAAQRAEKLGKEFELKLEY(SEQ ID NO: 47)
> X-CDP08
ADPKKVLDKAKDRAENRVTKLKKELE LYKEARKLDLTQEQRDRIINAAIAAMIAAFGDIFH AIMEAKEEAR LKKAGLVNSQQLDELKRRLDELDEEAAQRAEKLGKEFRLKFEY (SEQ ID NO: 48)
> X-CDP09
ADPKK\XDKAKDQAENRVRELKQKLEELYKEARKLDLTQEMR KLIQKALSALLKAIGDIL DAIARAKAEADKLKKAGIA^SQQLDELKRRLEELLKEAARKALDYGREF rXKLEY (SEQ ID NO: 49)
> X-CDP10
ADPKX\XDKAKJ3QAENR\7RELKQKLEELYKEARKLDLTQEMRRELRERYIAAMLAjAGDL ^^AITQA READKLKKAGLVNSQQLDELKRRLEELLEEAAR AEDYGEEFRLKLEY (SEQ ID NO: 50)
>X-CDP11
ADPKKVLDKAKI3QAENRVRELKQKLEELY EAR LDL1'QEMRRELRDRYIAAMLAAIGDLF NAIQWAKQEADKLKKAGLVNSQQLDELKRRLEELAEEAARKAEDYGEEFKLKLEY (SEQ ID NO: 51)
> 10-CD.P01
A.DPKKVLDKAKDQAENRVRELKQELERL YKEARKLDLTQEMRRKLEWRYIAAMLKAIGDIL NAIAQAENEADKLKKAGLVTSJSQQLDELRRRLEELAKEAARKAHDYGREFQLKLEY (SEQ ID NO: 52)
> F-CDPOl
ADPKK^DKAKDQAE R.YRELKQKLEELYKEARKLDLTQEMRKKLQYAAIGAMLAAIGDT LNAIMQAKQEADKLKKAGLV SQQLDELKRRLEELKEEALRKAHDYGSEFYLKLEY (SEQ ID NO: 53)
> X-ECMOl
Figure imgf000063_0001
HAIKU ALREARKLKKAGL VN SQQLDELKRRLEELDEEAEQRAEKLGKEFELKLEYG (SEQ ID NO: 54)
> X-ECM02
A Di'K K S S ! ) .\K ! )RAHN\ V R K I .K K i [ HKL Y .KAR K U H Qi Ni DRi R R Γ.Μ ΛΛΚ ί QAl lGi M F
HAIKEAKREARKLKKAGLYNSQQLDELKRRLEELDEEAEQRAEKLGKEFELKLEYG (SEQ ID NO: 55)
> X-ECM03
ADPKKVLDKAKDRAENVrVRKLKKELEELYKEARKLDLT'QEMRDRiRRAAIAARFAAHGDIF HAIKEAKEEARKLKKAGLVNSQQLDELKRRLRELDEEAEQRAEKlXiKEFRLKLEYG (SEQ ID NO: 56)
> X-ECM04(XINDI)
ADPKKVLDKAKDRAENWRKLKKELEELYKEARKLDLTQEMRDRIRRTAlAARFQAHGDiF HATKHAKEEARKLKKAGLVNSQQLDELKRRLRELDEEAEQRAEKLGKEFRLKLEYG (SEQ ID NO: 4)
> iO-ECMOl
ADPKKILDKA.KDQVENRVRELKQELERLYKEARKLDLTQEMRRKLHVR.YIAAMLKAIAAIL NATAQAENEADKLKKAGLVNSQQLDELRRRLEELTEEAAQKAHDYGREFQLKLEYG (SEQ ID NO: 58)
> 10-ECM02
ADPKKILDK^^KDQ^NRVRELKQELERLYKEARKLDLTQEMRRKLHVRYIAAMLKAIASIL NAIAQAENEADKLKKAGLWSQQLDELRRRLEELTEEAAQKAIIDYGREFQLKLEYG (SEQ ID NO: 59)
> 10-ECM03
ADPKKILDKAKDQVENRVRELKQELERLYKEARKLDLTQEMRRKLHVRYIAAMLKAIADIL NA1AQAENEADKLKKAGL VN SQQLDELRRRLEELTEEAARKAHDYGREFQLKLE Y G (SEQ ID NO: 60)
> 10-ECM04
ADPKKiLDKAKDQVENRVRELKQELERLYKEARKLDLTQEMRRKLHWRYIAAMLKAiADiL NAIAQAENEADKLKKAGLVTSJSQQLDELRRRLEELTEEAARKAHDYGREFQLKLEYG (SEQ ID NO: 61)
> F-ECMOl
ADPKKVLDKAKDQAENRVRELKQKLEELYKEARKLDLTQEMRKKLETAALGAVLAAHGDT LNAIMQAKEEADKLKKAGLVNSQQLDELKRRLEELKEEALRKASDYGKEFHLKRQYG (SEQ ID NO: 62)
> F-ECM02
ADPKK\^DKAKDQAENRVRELKQKLEELYKEARKLDLTQEMRKKLETAAIXJAVI.AAHGDT LNAIMQAKEEADKLKKAGLVNSQQLDELKRRLEELKEEALRKASDYGKEFHLKRRYG (SEQ ID NO: 63)
> F-ECM03
ADPKX\^DKAKJ3QAENR\7RELKQKLEELYKEARKLDLTQEMRKKLE\rAAIXxAVLAAHGDI LNAIMQAKEEADKLKK^^GLVNSQQLDELKRRLEELKEEALRKASDYGKEFHLKRQYG (SEQ ID NO: 64)
> F-ECM04
ADPKKVLDKAKDQAENRVRELKQKLEELYKEARKLDLTQEMRKKLEVAAL iAV^LAAHGDl LNA.iMQAKEEADKLKKAGLVNSQQLDELKRRLEELKEEA.LRKASDYGKEFHLKRRYG (SEQ ID NO: 65)
> F-ECM05
ADPKKVLDKAKDQAENRVRELKQKLEELYKEARKLDLTQEMRKKLQIAALGAMLAAIGDIL NAIMQAKEEADKLKKAGLVNSQQLDEL RRLEELKEEALRKASDYGKEFHL RQYG (SEQ ID NO: 66)
> F-ECM06
ADPKK\^DKAKDQAENRVRELKQKLEELYKEARKLDLTQEMR KLQIAALGAMLAAIGDIL NAIMQAKEEADKLKKAGLVNSQQLDELKRRLEELKEEALRKA.SDYGSEFHLKREYG (SEQ ID NO: 3 1)
> F-ECM07
ADPKX\^DKAKJ3QAENRVRELKQKLEELYKEARKLDLTQEMRKKLQIAALGAMLAAIGDIL NAIMQAKEEADKLKKAGLVNSQQLDEL RRLEELKEEALR J SDYGSEFHLKLEYG (SEQ ID NO: 57)
Initial screening by BLI indicated designed proteins generally bound their intended targets with nanomolar affinity and moderate specificity, but lacked the exceptional specificity of MINDI for Mcl-i or BINDI for BHRF 1. The designed proteins were therefore now improved by directed evolution. Selecting individual designs with promising partial specificity for each target BCL2 protein, the genes were diversified at every codon position to encode all possible single amino acid substitutions, and the libraries were transformed into yeast as Aga2p fusions for surface display. Each library was selected by one round of FACS for high affinity binding to the intended target (biotinylated for detection with streptavidin- phycoerythr n), with the other five human BCL2 proteins (unlabeled) added to the binding reaction as competitors to favor specific interactions. The pre- and post-sort populations were deep sequenced and enrichment ratios for all single amino acid substitutions calculated. From these sequence-fitness landscapes, mutations were chosen that were highly enriched during selection (Table 7). in the cases of the designed ikl-Xi .. Bcl-B, and Bfl-l binders, these enriching mutations were then combined in a combinatorial library that was selected by five (Bel-XL binder) or three (Bfl-l and Bcl-B binders) rounds of FACS to find variants with significantly improved affinity and/or specificity, each round under more stringent conditions including lower concentrations of target Bcl-2 paralogue and/or higher concentrations of competitors (Tables 7 and 8). Another round of directed evolution was required to further improve specificity of the Bfl-l and Bcl-B binders. In these cases, the most specific evolved combinatorial mutants (10-ECMOl and F-ECM04) were diversified by error prone PCR, expressed on the yeast cell surface and selected as previously (Tables 7 and 8). In the case of the designed Bcl-2 binder, the computationally designed protein 2-CDP06 bound Bcl-2 with high affinity prior to in vitro evolution. Therefore, 20 point mutants indicating improved affinity and specificity in the sequence-fitness landscape were screened by BLI in lieu of further evolution. Point mutants that improved affinity for Bcl-2 while diminishing affinity for other paralogues were combined. Ultimateiy protein variants were found that bind each BCL2 paralogue with high affinity and specificity (Figure 17 and Table 9).
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000066_0002
10-ECMO 1 error-prone
1 0,5 40
PCR
10-ECMO 1 error-prone 2 0,2 40
PCR
10-ECMO 1 error-prone 3 0.2 0
PCR
10-ECMO 1 error-prone
4 0.1 40
PCR
10-ECMO 1 error-prone 5 0,1 40
PCR
F-CDPO l SSM 1 4 4
F-CDPOl SSM 4
F-CDPOI combinatorial 1 4 8
F-CDPO l combinatorial 9 2 8
F-CDPO 1 combinatorial 3 z 16
F-CDPO 3 combinatorial 4 2 36
F-ECM04 error-prone
1 0,75 40
PCR
F-ECM04 error-prone
2 0,5 40
PCR
F-ECM04 error-prone 3 0,5 40
PCR
F-ECM04 error-prone
4 0.5 40
PCR
F-ECM04 error-prone
5 0.5 40
PCR
Figure imgf000067_0001
The final variants that specifically bind Bci-2, Bcl-XL, Bcl-B/BCL2L10, and Bfl- I with high affinity are named 2-INDI, XINDI, 10-INDI and FINDI, respectively. Based on BLI measurements at multiple analyte concentrations (Figure 17), 2-INDI binds Bcl-2 with KD 0.839 ± 0.005 nM and > 2,000-fold weaker affinity for the next tightest binding BCL2 family protein; XINDI binds Bel-XL with Κυ 5.59 ± 0.03 nM and > 660-fold weaker affinity for other BCL2 proteins; 10-INDl binds Bcl-B with 24.7 ± 0.1 nM affinity, and 1000-fold specificity; and FINDI binds Bfl-1 with KD 0.91 ± 0.01 nM and > 350-fold specificity (Table 10). These affinities and specificities are similar or better than other engineered peptides or small molecule ligands of BCL2 family proteins. When exposed to the chemical denaturant guanidinium hydrochloride, all the optimized inhibitors had sharp unfolding transitions as measured by loss of CD absorbance for helical structure (Figure 18). For 2-INDI, XINDI and FINDI, the protem stabilities were slightly to moderately decreased from the original computational designs. However, unfolding was still a cooperative reaction over narrow guanidinium concentrations, suggestive of a well-packed protein core.
Figure imgf000068_0001
Using the experimental sequence-fitness landscapes described above, we could determine the allowed sequence variability for the designed proteins (Figure 19, Tables 1 1- 20). While our saturation mutagenesis data are for the original computational designs, they nonetheless likely capture the capacity of the fina l optimized v ariants to tolerate mutations. As described for BINDI earlier, sequence conservation varies across the protein sequence, and while some positions are reasonably conserved for high affinity and specific interaction with a BCL2 family member, other positions are not. The BINDI scaffold is able to tolerate many mutations while preserving function. The different BCL2 inhibitors differ from each other by as many as 39 mutations, yet when any of the sequences is queried against GenBank for homologues by BLAST (E -value threshold 0.1), the proteins are found to be related only to each other, without homologous natural proteins. We have therefore designed an unnatural protein scaffold that can be easily repurposed for binding any BCL2 family member. Any modified v ersion of BIND! or its derivatives will similarly belong to our designed protein family but lack homology to any natural protein, and should therefore be covered by the claims in this patent.
An alignment of the optimized binders demonstrates that some amino acids differ in j ust one or a couple of the proteins, while other residu es diverge among most of the binders and are likely strong determinants of specificity (Figure 20 A). When mapped to the structure of BHRF 1 -bound BIND!, residues that differ in just a couple of the binders tend to be localized to the extreme edges of the interface where there is minimal direct contact, with a few positions in the very center of the interface that are conserved for binding across the BCL2 family (Figure 20B). By comparison, the primary specificity-determining residues are localized around the interface core at sites of direct contact (Figure 20B). Our interface can therefore be divided into three regions from the center outwards: (i) a conserved core for binding all BCL2 family members, (si) a region that principally determines specific interactions, and (iii) an extreme periphery that can offer an occasional specificity contact.
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000082_0002
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000084_0002
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000086_0002
Figure imgf000087_0001
Table 18
Allowable residues for 10-INDI based on experimental saturation mutagenesis data (enrichment ratios of -1 or greater after two rounds of sorting). (SEQ ID NO: 10)
Figure imgf000088_0001
Figure imgf000088_0002
Figure imgf000089_0001
Table 19
Allowable residues for FINDI based on experimental saturation mutagenesis data (enrichment ratios of -1 or greater after two rounds of sorting). (SEQ ID NO: 1 1)
Figure imgf000090_0001
Figure imgf000090_0002
Figure imgf000091_0001
Table 20
Allowable residues for MINDl SEQ ID NO: 12)
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
EXPERIMENTAL PROCEDURES
Computational methods: General information
ROSETTA® software can be downloaded from the Rosetta Commons web site, wherein online documentation and RosettaScripts® syntax can be found.
Computational methods: Side chain grafting on a fixed backbone
A suitable helical region of the scaffold protein was aligned to the Bim-BH3 motif of PDB 2WH6 (Bim-BH3»BHRF1 ) using PyMOL (Schrodinger, LLC). The structural alignment was visually inspected for minimal backbone clashes between the scaffold protein and BHRF1 (side chain clashes may be fixed later by sequence design of the scaffold and by retainer repacking on the target). Based on the structural alignment, scaffold residues were mutated in PyMol to the corresponding Bim-BH3 residue within the interface core; this 'grafted' important Bim interaction residues to the scaffold surface by mutation. A new FDB file containing the partially mutated scaffold bound to BHRFl was saved and used as the input for ROSETTA- based design.
Design with ROSETTA. An example command line to launch ROSETTA (Leaver-Fay et al., 2011) and example recipe/protocol file (Fleishman et ai., 201 l a) was developed. The design run was launched ten times. The consensus sequence was chosen for experimental validation after minor manual modification (e.g. a less-represented amino acid amongst the set of ten designs may be substituted for the consensus residue based on user preference).
Filtering. Proteins that passed the interface design filters (buried SASA > 800 A2, calculated AAG < -15 REU, unsatisfied buried polar atoms < 20) were further filtered based on properties of the unbo und designed protein. The lowest scoring 10-20 designs for monomer energy, unsatisfied buried polar atoms, and RosettaHoles score were selected for manual inspection. Designs were human modified to increase packing within the hydrophobic core and increase surface hydrophilicity, using the ROSETTA graphical user interface Foldlt (Cooper et al., 2010). Those designs considered most promising by the human eye were then selected for experimental validation.
For the 'direct- from-computer' designs tested in a high-throughput yeast display library (Figure 1C), 5,000 structures were initially assembled using the FFL procedure. The lowest scoring 1 ,000 were designed at the interface, with 423 designs passing the minimum threshold for interface binding energy. From these 423, the 74 designs with the lowest number of buried unsatisfied hydrogen bonding atoms in the unbound monomer were chosen for experimental testing.
Other computational methods
Predicted binding probabilities for BbpD04 point mutants were calculated using the method of ( Whitehead et al., 2012), with mutations ranked according to specificity
improvements based on the electrostatics term in the score function.
Plasmids, gene synthesis and mutagenesis
Genes encoding Bcl-2 proteins were synthesized (Genscript) and cloned with C-terminal avi-6his tags (GLNDIFEAQ 1EWHEGSHHHHHH (SEQ ID NO: 75)) into plasmid pET29b (Ndel-Xhol sites; Novagen): human Bcl-2 a.a. 1-207 (Accession No. NP_ 000624.2), Bcl-w a.a. 1-182 (AAB09055.1), Bfl-1 a.a. 1-153 (C4S mutation; NP 004040), Bci-B a.a. 1 1 -175
(NS> 065129.1), Mcl-1 a.a. 172-327 (Q07820.3), Bcl-XL a.a. 1 -205 (CAA80661), and EBV BHRF1 a.a. 1-161 (YP 401646). For later BLi analysis, Bcl-B and Bfl-1 were genetically fused to C-terminai maltose-binding-protein with an avi-6his tag for improved solution properties. Codon usage was optimized for E. coli expression. Human Bim-BH3 (a.a. 141-166, Accession No. 043521) was cloned into pETCON (Ndel-Xhol sites). The genes for individually-tested designed proteins were assembled from oligos (Hoover and Lubkowski, 2002) and cloned into pET29b (Ndel-Xhol sites) with C-terminal 6his tags for purification from E. coli, or cloned into PETCON (Ndel-Xhol sites: (Fleishman et al., 2011)) for yeast surface expression. Alternative tags were added using PGR methods. Point mutations were made by overlapping PGR (Procko et al., 2013), Error-prone PGR with an average error rate of 1.3 amino acid substitutions per clone used GeneMorph II Random Mutagenesis (Agilent Technologies).
Protein purification
E, coli BL21 * (DE3) (Invitrogen) transformed with the relevant plasmid were grown at 37 °C in terrific broth with 50 .ug/ml kanamycin to ODeoo 0.5-0.8, transferred to 21 °C and expression induced overnight with 0.1 mM IPTG. Centrifuged cells were resuspended in lysis buffer (20 mM Tris-Cl pH 8.0, 20 mM imidazole, 300 mM NaCl, 0.5 mM PMSF) supplemented with 0.2 mg/ml lysozyme and 0.06 mg/ml DNase I, and sonicated. Cleared lysate was incubated with NiNTA-agarose at 4 °C for 5 h and collected in a chromatography column. The resin was washed with 500 CV lysis buffer and protein was eluted with 6 CV elution buffer (20 mM Tris- Cl pH 8.0, 250 mM imidazole, 300 mM NaCl, 0.5 mM PMSF, 0.05% β-mercaptoethanol).
Proteins were concentrated using a centrifugal ultrafiltration device (Sartorius) and separated from remaining contaminants by SEC using a Sephacryl-100 16/600 column (GE Healthcare) with running buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM DTT). Fractions containing pure protein were pooled, concentrated to 5-20 mg ml based on calculated extinction coefficients for absorbance at 280 nm, and aliquots snap frozen in liquid N2 for storage at -80 °C. For animal studies, endotoxin was removed with a high-capacity endotoxin removal spin column (Pierce) and reducing agent was removed with a PD-10 desalting column (GE Healthcare).
Enzymatic ligand biotinylation
Purified avi-6his-tagged ligands (20 μΜ) in reaction buffer (250 mM potassium glutamate, 20 mM Tris-Cl [pH 7.5], 50 mM bicine [pH 8.3], 10 mM ATP, 10 mM MgOAc, 100 μΜ d-biotin) were enzymaticaily biotinylated with 150 U/μΙ BirA (Avidity) at room temperature overnight, followed by purification with NiNTA-agarose and SEC. Biotinylated iigands were stored at 4 °C in 150 mM NaCi, 20 mM Tris-Cl (pH 7.5), 1 mM DTT, 0.02% sodium azide. Yeast surface display
Transformed yeast were cultured, induced and binding of surface displayed protein to biotinylated Iigands was assessed by flow cytometry as reported (Chao et ai., 2006; Procko et ai., 2013), All yeast displayed protei s had C-terminal myc epitope tags for detection with F1TC- conjugated anti-myc (Immunology Consultants Laboratory). Binding of biotinylated protein to the yeast surface is detected with phycoerythrin-conjugated streptavidin (Invitrogen).
Deep sequencing analysis
Yeast ceils were sorted on a BD Influx cytometer operated by Spigot (BD Biosciences) and recovered in SDCAA media at 30 °C overnight. Yeast were lysed with 125 U/ml Zymolase at 37 °C for 5 h, and DNA was harvested (Zymoprep kit from Zymo Research). Genomic DNA. was digested with 2 U./μΙ Exonuclease I and 0.25 U/μΙ Lambda exonuclease (New England Biolabs) for 90 min at 30 °C, and plasmid. DNA purified with a QIAquick kit (Qiagen). DNA. was deep sequenced with a MiSeq sequencer (Illumina) and sequences were analyzed with adapted scripts from Enrich (Fowler et ai., 20 1).
For the library of designs in Figure 1C, genes were synthesized (Gen9) with barcodes downstream of the stop codon for easy identification during deep sequencing (Table 3). After yeast cell transformation, expression, sorting and plasmid DNA purification, the genes were PCR amplified using primers that annealed to external regions within the plasmid, followed by a second round of PCR to add flanking sequences for annealing to the Illumina fl ow cell oligonucleotides and a 6-bp sample identification sequence. PCR rounds were 12 cycles each with high-fidelity Phusion polymerase (New England Biolabs). Barcodes were read on a MiSeq sequencer using a 50-cycle reagent kit (Illumina). 257,812 sequences passing the chastity filter were read in the naive population (ranging from 260 to 17,192 reads per gene, with a median of 2,492). The sorted populations had 1 17,720 to 232,195 reads.
For the single-site saturation mutagenesis library (Figure 6), the BbpD04.3 gene was amplified as two overlapping fragments to provide complete sequencing coverage, and additional flanking DNA for annealing to the Illumina flow cell was added by PCR as described above. Gel-purified DNA was sequenced on a MiSeq sequencer using a 300-cycle paired-end reads reagent kit (lilumina). 3,058,244 sequences passing the chastity filter were read for the naive population. Each single amino acid substitution had 10 to 10,856 reads, with a median of 451 reads per mutant, and only mutation E109F was not represented. Parental protein sequences accounted for -25% of reads. 2,930,499 and 2,548,997 sequences passing the chastity filter were read for the affinity and affinity-specificity sorted populations, respectively.
Analytical size exclusion chromatography
Proteins (20 nmol each) were injected in a 200 μΐ loop in line with a Superdex-75 10/300 column (GE Healthcare) and separated with running buffer (20 niM Tris-Cl pH 7.5, 150 niM NaCl, 1 mM DTT) at room temperature.
Proteolysis susceptibility assay
Substrates (0.5 mg ml) were incubated at 37°C with protease (0.01 mg/ml) in 50 mM Tris-HCl (pH 8.0), 10 mM CaCl2. Reactions were terminated with benzamidine (12.5 mM final), PMSF (1.25 mM final) and 4x load dye. Samples were run on 18% SDS-polyacrylamide gels, stained with Coomassie dye, and the decrease in full-length protein quantified using Image! software (National Institute of Mental Health).
Circular dichroism
CD spectra were recorded with a Model 420 spectrometer (AVIV Biomedical). Unless stated othenvise, proteins were at 10 μΜ in PBS and data were collected at 25°C.
Bio-layer interferometry
Data were collected on an Octet RED96 (Forte Bio) and processed using the instrument's integrated software. Enzymatically-biotinylated Bcl-2 proteins (25 nM) in binding buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% non-fat dry milk) were immobilized for 360 s at 30 °C to streptavidin biosensors. Biosensors were dipped in solutions containing the analyte of interest to measure association, and transferred back to empty binding buffer for monitoring dissociation. Kinetic constants were determined from the mathematical fit of a 1 : 1 binding model.
Cytochrome c release
Ceils H0: were equilibrated in 5 ml of homogenization buffer (0.25 M sucrose, 1 mM EGTA, 10 mM HEPES/NaOH, 0.5% BSA, pH 7.4, Roche Complete protease inhibitors) for 5 min. Samples were kept on ice or at 4 °C until assayed. Cells were homogenized under N2 pressure (400 psi) in a steel disruption vessel (Parr Instrument Company) for 10 min, then centrifuged (750 g) for 10 min to remove intact cells. Supernatant was centrifuged again (12,000 g) for 12 min to collect mitochondria. The pellet was resuspended in 300 μΐ wash buffer (0.25 M sucrose, 1 niM EDTA, 10 raM Tris/HCi pH 7.4). Proteins at the indicated concentrations were incubated with mitochondria (25 g mitochondrial protein based on BCA assay, Sigma) in 50 id final volume of experimental buffer (125 mM KG, 10 mM Tris-MOPS pH 7.4, 5 mM glutamate, 2.5 mM malate, 1 mM K-PQ4, 10 μΜ EGTA-Tris pH 7.4) for 30 min at room temperature.
Reaction solutions were centrifuged (18,000 g) for 10 min at 4 °C and cytochrome c release was quantified using a Cytochrome c ELISA kit (Life Technologies). Complete cytochrome c release was quantified by treatment with 0.5% Triton-XlOO.
Cell viability assay
A 25,000 Da diblock copolymer (PoBOO) composed of 95% polyethylene glycol methacrylate (300 Da) for stabilit and 5% pyridyl disulfide methacryate for conjugation in the first block, and 60% diethylaminoethyl methacrylate and 40% butyl methacrylate in the second block, was synthesized by reversible addition-fragmentation chain transfer. Development, and characterization of the diblock copolymer will be published in a separate article. After purification, PoBOO was dissolved in ethanol at 100 mg/'ml then diluted into PBS at 1 mg/ml and spin filtered to remove ethanol. Proteins with exposed terminal cysteines were incubated with PoBOO at a molar ratio of 2: 1 (proteinrpolymer) overnight. Protein-polymer conjugation was quantified by measuring pyridyl disulfide release and the absorbance of 2-mercaptopyridine at 343 nm with 8,080 M^cm"1 as the extinction coefficient. For cell viability studies, protein and protein-polymer conjugates were incubated with Ramos or Ramos-AW cells in a 96 well round bottom plate with 50,000 cells per well in 100 μ.1 media. Cells were cultured in RPM1 1640 containing L-glutamine and 25 mM HEPES supplemented with 1 % penicillin-streptomycin (GIBCO) and 10% fetal bovin serum (Invitrogen) at 37 °C and 5% C02. After 24 h, cell viability was measured using a CeilT iter 96 Aqueous One Solution Cell Proliferation Assay, MTS (Pro mega).
Tissue culture
Ramos, Ramos-AW, Daudi, Raji, DOHH2, JVM-2, and JVM- 13 were grown in RPMI 1640 containing L-glutamine and 25 mM HEPES supplemented with 1% penicillin-streptomycin (GIBCO) and 10% fetal bovine serum (FBS, nvitrogen). Jeko-1 were grown in similar RPM 1640 media supplemented with 20% FBS. Granta-519 and K562 were grown in Iscove's DMEM supplemented with 10% FBS. All cell lines were maintained in log growth phase at 37 °C and 5% C02.
Xenograft mouse model
To prepare mAb-polymer-protein conjugates, a 44,000 Da diblock copolymer (Pol950) composed of 80% polyethylene glycol methacrylate (950 Da), 10% pyridyl disulfide
methacrylate, and 10 % biotin-hydroxy ethyl methacrylate for mAb-streptavidin conjugation in the first block, and 60% diethylaminoethyl methacrylate and 40% butyl methacrylate in the second block, was synthesized by reversible addition- fragmentation chain transfer. Development and characterization of the Pol950 diblock copolymer will be published in a separate article, Pol950 was dissolved in ethanol at 100 mg/mL, then diluted in PBS at 10 mg/ml and spin filtered to remove ethanol. Proteins were incubated with Pol950 at an equimolar ratio overnight and conjugation was quantified by A343 absorbance. ctCD19 was conjugated to protein-poylmer through the streptavidin linkage at a molar ratio of 90: 1 (polymer:mAb).
BALB/c nu/nu mice (6 to 8 weeks old) were used from Harian Sprague-Dawley and housed under protocols approved by the FHCRC Institutional Animal Care and Use Committee. Mice were placed on biotin-free diet (Purina Feed) for the duration of study. To form tumor- xenografts, Ramos-AW cells were resuspended in PBS (5 x 10' cells/mL) and injected in the right, flank with 10' cells/mouse. Tumors were allowed to grow for 6 days to a volume of 50 rmrf . Mice with similar sized tumors were sorted randomly into treatment groups (n = 8 to 10). On days 6, 9, and 12, mice were injected intraperitoneally with cyclophosphamide (35 mg/kg) and bortezomib (0.5 mg/kg). After 30 rnin, mice were injected via tail vein with conjugates at a dose of 15 mg/kg (aCD19), 300 mg/kg (Pol950) and 105 mg/kg (BINDI or 3LHP). Body weight was monitored for toxicity and tumor sizes were measured while blinded to treatment groups. Measurements were performed in the x, y, and z plane using calipers three times a week. Mice were euthanized when tumors reached a volume of 1250 mm3. Tumor volumes and deaths were recorded into Prism (GraphPad Software, Inc.) for statistical analysis and a log-rank (Mantel- Cox) test was performed to determine if survival curves and trends were statistically different (P < 0.0001). Significance in tumor volumes was verified by an unpaired t test with Welch's correction. By breaking free of the conformational constraints imposed by repurposing pre-existing scaffolds and instead building a new protein with structure tailored for the target surface, a remarkably tight and specific binder of the EBV apoptosis regulator BHRFl was designed. The elevated toxicity of the engineered BIND I protein towards EBV-positive cancer lines supports the hypothesis that BHRFl is necessary for survival in at least some EBV-associated cancers. BINDI should provide a useful tool for characterizing primary isolates of EBV-associated cancers in which the molecular mechanisms of cell transformation remain poorly understood, including EBV-positive BL, Hodgkin's lymphoma, and nasopharyngeal and gastric carcinomas (Young and Murray, 2003).
BINDI has a structure and amino acid sequence found after computationally filtering thousands of potential designed conformations for optimum interactions with BHRFl , The ability to custom-tailor the backbone conformation to the challenge at hand helped achieve very high affinity and specificity.
BIND! is an artificial polypeptide sequence that folds to a designed structure, with no identifiable homologues in nature. We demonstrate how sequence variants of BINDI (see Figures 2, 4, 6 and 14) can also bind BHRFl with high affinity and specificity. Redesigning BINDI to bind other BCL2 family proteins yielded a set of related sequences (MINDI, 2-INDI, XTNDI, 10-INDI and FINDI), with any two differing by as many as 39 mutations (34% of the protein). Each of these redesigned BINDI variants were related to each other but not to any naturally occurring proteins. Saturation mutagenesis of all these designed proteins consistently revealed that significant sequence diversity is tolerated (Figure 19 and Table 5 5 -20). We have therefore designed a new farnily of proteins that share a common structure and architecture. We have shown that many sequence homologues can maintain our artificially designed structure and functional inhibition of BCL2 family proteins..
Finally, we demonstrate that BI DI can slow progression of EB V-positive B lymphoma and prolong survival in a human xenograft mouse model. More doses, higher dosage, alternative targeting antibodies, and copolymer optimization may all increase therapeutic efficacy.
Intracellular delivery of BINDI, either of encoding nucleic acid or of the polypeptide, is expected to have therapeutic effects in Epstein-Barr related diseases generally. Quantitative analysis of mRNA expression has shown that different cancer lines overexpress different BCL2 family members. The designed proteins described herein can specifically inhibit BCL2 family members at the protein level, thereby demonstrating which BC.L2 proteins are functionally important for preventing apoptosis in different cancers. This will lead to better tumor characterization and future diagnostics, in addition targeted therapies as described for BINDI delivery to EBV- positive cancer.
Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "above," and "below" and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.
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Claims

We claim:
1. A polypeptide comprising an amino acid sequence having at least 50% amino acid sequence identity over its length relative to the amino acid sequence of SEQ ID NO.: 1, wherein the polypeptide selectively binds to a protein selected from the group consisting of Epstein Barr protein BHFR1, and B cell lymphoma family proteins selected from the group consisting of myeloid cell leukemia 1 (Mcl-1), B-cell lymphoma 2 (Bcl-2), Bcl-2-like protein 1 (BCL2L1/Bcl-XL), Bcl-2-like protein 10 (BCL2L10/Bcl-B), and Bcl-2-like protein Al (Al/Bfl- 1)·
2. The polypeptide of claim 1, comprising an amino acid sequence having at least 60% identity over its length relative to the amino acid sequence of SEQ ID NO,: 1.
3. The polypeptide of claim 1, comprising an amino acid sequence having at least 66% identity over its length relative to the amino acid sequence of SEQ ID NO,: 1.
4. The polypeptide of any one of claims 1-3, wherein the polypeptide comprises an amino acid sequence having at, least 50% amino acid sequence identity over its length relative to the amino acid sequence selected from the group consisting of SEQ ID NOS:2~6,
5. The polypeptide of claim 4, wherein the polypeptide comprises an amino acid sequence having at least 60% amino acid sequence identity over its length relative to the amino acid sequence selected from the group.
6. The polypeptide of claim 4, wherein the polypeptide comprises an amino acid sequence having at least 66% amino acid sequence identit over its length relative to the amino acid sequence selected from the group.
7. The polypeptide of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 7, and wherein the polypeptide binds to BHFR1 .
8. The polypeptide of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 8, and wherein the polypeptide binds to Bcl-2.
9. The polypeptide of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO:9, and wherein the polypeptide hinds to hinds to Bcl-2- like protein 1.
10. The polypeptide of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 10, wherein the polypeptide binds to Bcl-2-iike protein 10.
11. The polypeptide of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 11 , and wherein the polypeptide binds to Bcl-2-like protein Al (Al/Bfl-1).
12. The polypeptide of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 12, and wherein the polypeptide binds to Bei-2-like protein Mcl- 1.
13. The polypeptide of any one of claims 1-12, wherein the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NQ8: 1-6,
14. The polypeptide of any one of claims 1-3, wherein the polypeptide comprises an amino acid sequence having at least 50% identity over its length relative to the amino acid sequence of SEQ ID NO: 13.
15. The polypeptide of claim 14, comprising an amino acid sequence having at least 60% identity over its length relative to the amino acid sequence of SEQ ID NO.: 13.
16. The polypeptide of claim 14, comprising an amino acid sequence having at least 66% identity over its length relative to the amino acid sequence of SEQ ID NO.: 13.
17. The polypeptide of any one of claims 14-16, comprising at least one conservative substitution corresponding to residues 3, 13, 21, 28, 31 , 33, 46, 48, 49, 61, 62, 65, 79, 84, 103, and 104 of the amino acid sequence of SEQ ID NO: 13.
18. The polypeptide of claim 17, comprising the substitutions K3.1 E, E48R, and E65 . 59. The polypeptide of claim 1 8, further comprising the substitutions 125 L, Q79L, L84Q, and H104 .
20. The polypeptide of claim 19, further comprising the substitution C103A.
21. The polypeptide of claim 20, further comprising substitutions W3P, I13Q, F28L, M33R, M46E, W49Y, and F61 Y.
22. The polypeptide of claim 21 , further comprising the substitution N62S.
23. The polypeptide of any one of claims 1-22, further comprising a cell-penetrating peptide.
24. The polypeptide of claim 23, wherein the cell-penetrating peptide is selected from the group consisting of SEQ ID NOS : 14-26.
25. A pharmaceutical composition, comprising the polypeptide of any one of claims 1-24 and a. pharmaceutically acceptable carrier.
26. The pharmaceutical composition of claim 25 further comprising an antibody.
27. The pharmaceutical composition of claim 25 or 26, wherein the carrier comprises a polymer.
28. The pharmaceutical composition of claim 27, wherein the polymer comprises a hydrophiiic block and an eiidosomolytic block,
29. The pharmaceutical composition of claim 28, wherein the hydrophiiic block comprises polyethylene glycol methacrylate, and wherein the endosomolytic block comprises a diethylaminoethyl metha cry late-butyl methacrylate copolymer.
30. The pharmaceutical composition of any one of claims 27-29, wherein the polymer is a. stimuli-responsive polymer that responds to one or more stimuli selected from the group consisting of pH, temperature, UV-visible light, photo- irradiation, exposure to an electric field, ionic strength, and the concentration of certain chemicals by exhibiting a property change.
31 . A recombinant nucleic acid encoding the polypeptide of any one of claims 1 -24.
32. A recombinant expression vector comprising the nucleic acid of claim 31 operatively linked to a promoter.
33. A recombinant host cell comprising the recombinant expression vector of claim 32.
34. A method of treating an Epstein-Barr v ir u s - related disease comprising administering to a subject in need thereof a therapeutically effective amount of one or more of th e polypeptides of any one of claims 1-7, 13-20, or 23-24, or salts thereof, pharmaceutical compositions thereof, a recombinant nucleic acid encoding the one or more polypeptides, a recombinant expression vector comprising the recombinant nucleic acids, and/or a recombinant host cells comprising the recombinant expression vector, to treat and/or limit Epstein-Barr vims related diseases wherein the polypeptide or encoded polypeptide selectively inhibits BHRFL
35. The method of claim 34, wherein the Epstein-Barr v i r u s - related disease is selected from the group comprising o f infectious mononucleosis, Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma, nasopharyngeal carcinoma, multiple sclerosis, Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy.
36. The method of claim 34 wherein the Epstein-Barr v i r u s - related disease is Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma, or nasopharyngeal carcinoma.
37. A method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of one or more of th e polypeptides of any one of claims 1-6, 8- 12, or 23-24, or salts thereof, a pharmaceutical composition thereof, a recombinant nucleic acid encoding the one or more polypeptides, a recombinant expression vector comprising the recombinant nucleic acid, and/or a recombinant host cell comprising the recombinant expression vector, to treat cancer, wherein the polypeptide or encoded polypeptide selectively inhibits one or more of Mel- 5 , Bcl-2, BCL2L1/Bc!-XL, BCL2L10/Bc!~B, and A l/Bfl-5.
38. The method of claim 36, wherein the method further comprises administering
chemotherapy to the subject.
39. A me thod for determining the Bcl-2 phenotype of a tumor, comprising contacting tumor cells, tumor cell l sates or tumor cellular components with one or more polypeptides selected from the group consisting of SEQ ID NOS: 2-6 and 8-12, under conditions suitable to promote apoptosis signaling in cells of the tumor that express a bcl-2 homologue targeted by the one or more polypeptides; and
determining bcl-2 dependency of the tumor based on the polypeptide that causes apoptosis or apoptotic signaling in the cells of the tumor.
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