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WO2024191776A2 - Liants protéiques à haute affinité conçus de novo se liant à sites cibles de protéine convexe sur tgfbrii, ctla -4 et pd-l1 - Google Patents

Liants protéiques à haute affinité conçus de novo se liant à sites cibles de protéine convexe sur tgfbrii, ctla -4 et pd-l1 Download PDF

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Publication number
WO2024191776A2
WO2024191776A2 PCT/US2024/019019 US2024019019W WO2024191776A2 WO 2024191776 A2 WO2024191776 A2 WO 2024191776A2 US 2024019019 W US2024019019 W US 2024019019W WO 2024191776 A2 WO2024191776 A2 WO 2024191776A2
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polypeptide
cancer
amino acid
acid sequence
seq
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WO2024191776A8 (fr
WO2024191776A3 (fr
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David Baker
Wei Yang
Derrick Hicks
Brian COVENTRY
Inna GORESHNIK
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University of Washington
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University of Washington
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Publication of WO2024191776A8 publication Critical patent/WO2024191776A8/fr
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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Naturally occurring high affinity' protein-protein interfaces generally exhibit considerable shape complementarity, which enables concerted interatomic interactions and solvation free energy reduction needed to overcome the entropic cost of macromolecular association.
  • Design of proteins that bind to convex protein target sites are difficult due to the requirement for overall shape matching. Methods for designing proteins which bind to convex target sites could considerably expand the power and scope of de novo binder design.
  • the disclosure provides polypeptides comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-9.
  • the polypeptides comprise the amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, wherein the polypeptide binds to CTLA-4.
  • the polypeptides comprise the amino acid sequence selected from the group consisting of SEQ ID NO:4-6, wherein the polypeptide binds to PD-Ll .
  • the polypeptides comprise the amino acid sequence selected from the group consisting of SEQ ID NO:7-9, wherein the polypeptide binds to TGFbRII.
  • the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from SEQ ID NO: 10-16, not including any insertions.
  • the disclosure provides fusion proteins comprising the polypeptide of any embodiment herein and one or more functional domains at the N-terminus and/or at the C-terminus of the polypeptide.
  • the disclosure also provides nucleic acids encoding the polypeptide or fusion protein of any embodiment or combination of embodiments herein, expression vectors comprising tlie nucleic acid operatively linked to a promoter, host cells comprising the polypeptide, fusion protein, nucleic acid, or expression vector of any embodiment, and pharmaceutical compositions, comprising the polypeptide, the nucleic acid, the expression vector, and/or the host cell of embodiment; and a pharmaceu tically acceptable carrier.
  • the disclosure provides methods for treating cancer or tissue fibrosis, comprising administering to a subject in need thereof an amount of the polypeptide, fission protein, nucleic acid, expression vector, host cell, and/or pharmaceutical composition effective to treat the cancer or tissue fibrosis.
  • Figure 1 Design of 5HCS scaffolds to target convex interfaces.
  • A Distribution of protein-protein interface curvatures from the PDB and designed protein binders.
  • Previously designed protein binders for these, the designed binders are partner 1 and the targets, partner 2
  • Examples of native protein complexes v: PDB ID, 5XXB; vi: TGFpIIl/TGFpRll complex, PDB ID. 1KTZ, vii: CD86/CTLA-4 complex, PDB ID, 1185, viii, PD-1 /PD-Ll, PDB ID, 3bik.
  • the TGFpRII and CTLA-4 functional interfaces showed high convexity, which we used as case studies to design concave binders.
  • the 5HCS scaffolds described in the examples can target convex binding sites.
  • the distribution of convexity of the 5HCS scaffolds shows that the 5HCS scaffolds are diverse enough to cover most of the naturally existing convex interfaces, (B) Design models of complexes highlighted in panel a.
  • i,ii,ii are PDCsFR, 1GF1R, H3 in complex with corresponding de novo minibinders; iv, 5HCS binder in complex with TGFpRII; v, PDB ID: 5XXB; vi, TGFpiII/TGFpRII complex, PDB ID: 1KTZ.
  • C Design workflow.
  • Column 1 5FICS concave scaffolds with a wide range of curvatures were designed with three helices forming the concave surfaces (Cbeta labeled as spheres ) and two helices butressing at the back side.
  • Column 2 Docking of 5HCS scaffolds to target binding sites.
  • Column 3 Following docking, the interface sequencing is optimized for high affinity binding.
  • FIG. 1 Concave 5HCS binder to TGFpRII.
  • A Design model of 5HCS_TGFBR2_1 (cartoon) binding to TGFpRII (PDB ID: 1KTZ).
  • 5HCS_TGFBR2_I is shown by Shannon entropy from the site saturation mutagenesis results at each position from low entropy, conserved, to high entropy, not conserved.
  • B Circular dichroism spectra from 25 °C to 95 °C for 5HCS TGFBR2 1.
  • C Biolayer interferometry characterization of 5HCS_TGFBR2_1.
  • Biotinylated TGFpRII were loaded to Streptavidin (SA) tips and incubated with 2.7 nM, 0.9 nM and 0.3 nM of 5HCS TGFBR2 1 to measure the binding affinity'. The binding responses are shown in solid lines and fited curves shown in dotted lines.
  • FIG. 3 Designed 5HCS CTLA-4 binder.
  • A Model of 5HCS CTLA4J (cartoon) binding to CTLA-4 (PDB ID: 1185 ) shown by Shannon entropy’ from site saturation mutagenesis results.
  • B Circular dichroism spectra from 25 °C to 95 °C tor 5HCS__CTLA4 __1.
  • C Biolayer interferometry characterization of 5HCS__ CTLA4__1. Biotinylated CTLA-4 was loaded to Streptavidin (SA) tips and these were incubated with 2.7 nM, 0.9 nM and 0.3 nM of 5HCS CTLA4 1 to measure the binding affinity.
  • D Log enrichments for the 5HCS CTLA4 1 SSM library selected with 10 nM CTLA-4 at representative positions. The annotated ammo acid in each column indicates the residue from the parent sequence.
  • FIG. 4 Designed 5HCS binder to PD-Ll .
  • A Model of 5HCS PDL1 1 (cartoon) binding to PD-Ll (PDB ID: 3BIK), with 5HCS PDL1 1 shown by Shannon entropy from site saturation mutagenesis results.
  • B Circular dichroism spectra from 25 °C to 95 °C for 5HCS PDL1 1.
  • C Biolayer interferometry characterization of 5HCS PDL1 1. Biotinylated PD-Ll was loaded to Streptavidin (SA) tips and these were incubated with 8 nM, 2.7 nM and 0.9 nM of 5HCS PDL1 1 to measure the binding affinity .
  • SA Streptavidin
  • FIG. 1 TGF0RII binding protein binding site and SSM analysis.
  • A Complex structure of the TGF0RH and the TGF0-3.
  • B 5HCS_TGFBR2J binds to the TGF0-3 binding site on the TGF0RII.
  • C Heat map representing the log enrichments for the 5HCS_TGFBR2_1 SSM libraiy selected with 1.6 nM TGF0RII.
  • FIG. 7 Examples of Immunoglobulin domain head-to-head interactions.
  • FIG. 1 CTLA-4 binding protein binding site and SSM analysis.
  • A Complex structure of the CTLA-4 and the CD86.
  • B 5HCS_CTLA4_1 binds to the CD86 binding site on the CTLA-4.
  • C Heat map representing the log enrichments for the 5HCS CTLA4 1 SSM library selected with 10 nM CTLA-4.
  • Figure 9 PD-L1 binding protein binding site and SSM analysis.
  • A Complex structure of the PD-L1 and the PD-1.
  • B 5HCS PDL1 1 binds to the PD-1 binding site on the PD-L1.
  • C Heat map representing the log enrichments for the 5HCS PDL1 1 SSM library' selected with 6 nM PD-L1.
  • amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Tip; W), tyrosine (Tyr; Y), and valine (Vai; V).
  • Any N-terminal methionine residue in any polypeptide of the disclosure may be present or may be deleted.
  • 1, 2, 3, 4, or 5 residues may be deleted from the N-terminus and/or the C-terroinus of the polypeptide while retaining activity.
  • the disclosure provides polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-9.
  • the polypeptides of the disclosure are high affinity binders to convex protein target sites on transforming growth factor beta receptor type 2 (TGFbRII), cytotoxic T-lymphocyte associated protein 4 (CTLA- 4), or programmed death-ligand 1 (PD-L1).
  • TGFbRII transforming growth factor beta receptor type 2
  • CTLA- 4 cytotoxic T-lymphocyte associated protein 4
  • PD-L1 programmed death-ligand 1
  • Hie ammo acid sequence of SEQ ID NO: 1-9 are shown in Tables 1 -3.
  • Table 1 provides sequences and permissible substitutions for CTLA4 mini-binders (SEQ ID NO: 1-3), Table 2 provides sequences and permissible substitutions for PD-L1 minibinders (SEQ ID NO:4 ⁇ 6), and Table 3 provides sequences and permissible substitutions for TGFBR2 minibinders (SEQ ID NO:7-9).
  • each row in the table represents one residue in the polypeptide and the different columns show permissible substitutions from the “native” amino acid residue at that position (i.e., “native” meaning a residue present in a specific design shown in Table 4 below), based on site saturated mutagenesis studies as described in the examples.
  • the first column in Tables 1-3 provides the residue number of the reference polypeptide
  • the second column lists the amino acid sequence of an exemplary design listed in Table 4
  • the third column indicates whether the residue is present in a loop of the polypeptide
  • the fourth column indicates whether the residue is present at an interface between the polypeptide and its target (i.e., TGFbRII, CTLA-4, or PD-L1).
  • Columns 5-7 provide all residues that can be present at defined positions (the starting residue from column 2, and permissible substitutions), as determined by site saturation mutagenesis studies.
  • residue 1 can be P, Q, T, S, M, E, A, L, N, W, D, V, H, F, Y, C, K, I, or G.
  • residue 1 can be N, W, M, D, V, E, A, T, L, P, Y, S, H, K, I, G, or Q.
  • residue 1 is N.
  • Table 4 provides exemplary binder designs, which are described in detail in the examples. Table 4. Specific Designs
  • the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, wherein the polypeptide binds to CTLA-4.
  • the polypeptides may be used, for example, in cancer immunotherapy.
  • Exemplary tumor types that can be treated with the CTLA-4 binders of the disclosure include, but are not limited to, melanoma, lung cancer (such as non-small cell lung cancer), bladder cancer, head and neck cancer, renal cell carcinoma, ovarian cancer, and colorectal cancer.
  • the polypeptide comprises the amino acid sequence of SEQ ID NO:3.
  • the polypeptide comprises an amino acid sequence at least 50%, 55%. 60%, 65%, 70%. 75%, 80%, 85%.
  • polypeptide is identical relative to tire reference polypeptide at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, or all interface residues. Interface residues are shown in column 4 of Table 1, and are at positions 8, 11-16, 18-20, SO- 51, 54-55, 58, 89-90, 92-94, 96-98, and 100.
  • the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO:4-6, wherein the polypeptide binds to PD-L1.
  • the polypeptides may be used, for example, in cancer immunotherapy.
  • Exemplary tumor types that can be treated with the PD-L1 binders of the disclosure include, but are not limited to, non-small cell lung cancer, melanoma, bladder cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, renal cell carcinoma, gastric or gastroesophageal junction adenocarcinoma, cervical cancer, and breast cancer (including but not limited to triple-negative breast cancer).
  • the polypeptide comprises the amino acid sequence of SEQ ID NO:6.
  • the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence selected from the group consisting of SEQ ID NO: 15 or 16, Exemplary substitutions are as disclosed in Table 2 above.
  • the amino acid sequence of SEQ ID NO: 15- 16 are provided in Table 4, and are discussed in detail in the examples.
  • the polypeptide is identical relative to the reference polypeptide at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, or all interface residues.
  • Interface residues are shown in column 4 of Table 2, and are at positions 2, 6, 9-10, 13, 45, 48-49. 52-53, 56, 59, 86-88, 91-92, 95-96. 99, and 102.
  • the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO:7-9, wherein the polypeptide binds to TGFbRII.
  • the polypeptides may be used, for example, in in treating cancer and tissue fibrosis, by interfering with the TGF-p pathway.
  • Exemplary tumor types that can be treated with the TGFbRII binders of the disclosure include, but are not limited to, pancreatic cancer, breast cancer, colorectal cancer, lung cancer, prostate cancer, liver cancer, gastric cancer, ovarian cancer, melanoma, and glioblastoma.
  • Exemplary uses for treating tissue fibrosis include, but are not limited to, idiopathic pulmonary’ fibrosi s (IPF), cirrhosis of the liver, renal fibrosis, cardiac fibrosis, scleroderma, keloids, and hypertrophic scarring.
  • the polypeptide comprises the amino acid sequence of SEQ ID NO:9.
  • the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14.
  • Exemplary’ substitutions are as disclosed in Table 3 above.
  • the amino acid sequence of SEQ ID NO: 14 is provided in Table 4, and is discussed in detail in the examples.
  • the polypeptide is identical relative to the reference polypeptide at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, or all interface residues.
  • Interface residues are shown in column 4 of Table 3, and are at positions 3, 6, 10, 42-43, 45-46, 48-50, 52-53, 56-57, 85-86, 88-89, 92-93, 95-96, and 100-101.
  • the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from SEQ ID NO: 10-16, not including any insertions.
  • Tables 1-3 show in column 3 the position of loop regions in the polypeptides of the disclosure.
  • the loop regions can accommodate amino acid insertions.
  • Tirus in another embodiment the polypeptides comprise an insertion in one or more loop regions of the polypeptide.
  • ammo acids or amino acid domains (such as a functional domain) may be inserted in the loop region.
  • the polypeptides of the disclosure may include any such insertion, and in these embodiments the polypeptide would still comprise the reference amino acid sequence, with an interruption at tire site of insertion.
  • substitutions relative to the reference sequence are conservative amino acid substitutions.
  • conservative amino acid substitutions involve replacing a residue by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, 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 known.
  • Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp.
  • residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: 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,
  • the position of residues in the polypeptides of the disclosure are “relative to” the position of residues in the reference sequence; this does not necessarily mean that the residue number in the polypeptide of the disclosure will be identical to the residue number in the reference sequence.
  • the polypeptides of the disclosure may be fused to other functional domains (such as the fusion proteins or polypeptides with insertions described below), including N-terminal domains, or comprise insertions, such that the residue numbers in the polypeptides relative to the reference polypeptide sequence may differ.
  • the disclosure provides fusion proteins, comprising: (a) the polypeptide of any embodiment or combination of embodiments herein; and
  • any functional domain may be inserted (as an insertion at a loop region, and/or at one or both termini of tire fusion protein).
  • the functional domain may comprise, for example, a targeting domain, a detectable domain, a. scaffold domain, an oligomerization domain, a secretion signal, an Fc domain, or a further therapeutic peptide domain.
  • the functional domain comprises an oligomerization domain, As described in the examples, fusing the binders to an oligomerization domain, optionally via a linker sequence (including but not limited to a flexible linker such as GS-rich linker), can improve avidity of binding to the target.
  • oligomerization domain comprises the amino acid sequence of SEQ ID NO: 17.
  • SEQ ID NO: 12 amino acid sequence of SEQ ID NO: 17.
  • the polypeptide or fusion protein binds its target with nanomolar or picomolar affinit y, as determined using biolayer interferometry and a. protocol as defined in the examples and figure legends.
  • the disclosure provides nucleic acids encoding the polypeptide or fusion protein of any embodiment or combination of embodiments of the disclosure .
  • the nucleic acid sequence may comprise single stranded or double stranded RNA or DNA in genomic or cDNA form, or DNA -RNA hybrids, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • Such nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purificati on of the encoded peptide or chimeric molecular construct, 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 polypeptide or fusion protein of the disclosure.
  • the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence, such as a promoter.
  • a suitable control sequence such as a promoter.
  • “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 disclosure 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 “operably 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, 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).
  • 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 disclosure provides host cells that comprise the polypeptide, fusion protein nucleic acid or expression vector (i.e.: episomal or chromosomally integrated) 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 disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate coprecipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.
  • compositions comprising:
  • the carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • the saline comprises isotonic phosphate-buffered saline.
  • neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefore.
  • a composition comprising an immunomodulatory fusion protein is prepared for storage by mixing the selected composition having the desired degree of puri ty with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising an immunomodulatory fusion protein is formulated as a lyophilizate using appropriate excipients such as sucrose.
  • compositions may be used, for example, in the methods disclosed herein.
  • the compositions may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) atonicity 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 composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose.
  • the composition includes a preservative e.g.
  • the composition includes a bulking agent, like glycine.
  • the composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof.
  • the 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 composition additionally includes a stabilizer, e.g., a molecule which substantially prevents or reduces chemical and/or physical instability of the nanostructure, in lyophilized or liquid form.
  • Exemplar ⁇ ' stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.
  • the polypeptide, fusion protein, nucleic acid, expression vector, and/or host cell maybe the sole active agent in the composition, or the composition may further comprise one or more other agents suitable for an intended use.
  • such other agents may include angiogenesis inhibitors (including but not limited to) axitinib, bevacizumab, cabozantinib, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, ramucirumab, regorafenib, sorafenib, sunitinib, thalidomide, vandetanib, and ziv-aflibercept), immune checkpoint inhibitors (including, but not limited to, pembrolizumab, nivolumab, and cemiplimab as anti-PD-1 antibodies, ipilimumab as an anti-CTLA-4 antibody, and atezolizumab, avelumab, and durvalumab
  • the disclosure provides methods for treating cancer, comprising administering to a subject in need thereof an amount of the polypeptide, fusion protein, nucleic acid, expression vector, host cell, and/or pharmaceutical composition of any preceding claim effective to treat the cancer.
  • exemplary tumor types that can be treated with the CTLA-4 binders of the disclosure include, but are not limited to, melanoma, lung cancer (such as non-small cell lung cancer), bladder cancer, head and neck cancer, renal cell carcinoma, ovarian cancer, and colorectal cancer.
  • the Exemplary' tumor types that can be treated with the PD-L1 binders of the disclosure include, but are not limited to, non-small cell lung cancer, melanoma, bladder cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, renal cell carcinoma, gastric or gastroesophageal junction adenocarcinoma, cervical cancer, and breast cancer (including but not limited to triple-negative breast cancer).
  • tire polypeptides may be used, for example, in in treating cancer and tissue fibrosis, by interfering with the TGF ⁇ p pathway.
  • Exemplaiy tumor types that can be treated with the TGFbRII binders of the disclosure include, but are not limited to, pancreatic cancer, breast cancer, colorectal cancer, lung cancer, prostate cancer, liver cancer, gastric cancer, ovarian cancer, melanoma, and glioblastoma.
  • Exemplary uses for treating tissue fibrosis include, but are not limited to, idiopathic pulmonary fibrosis (IPF), cirrhosis of the liver, renal fibrosis, cardiac fibrosis, scleroderma, keloids, and hypertrophic scarring.
  • IPF idiopathic pulmonary fibrosis
  • cirrhosis of the liver renal fibrosis
  • cardiac fibrosis cardiac fibrosis
  • scleroderma scleroderma
  • keloids hypertrophic scarring
  • treat or “treating” means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder! s) being treated; (d) limiting or preventing recurrence of the disorder! s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).
  • "treat” or “treating” means accomplishing one or more of the following: (a) reducing the size or volume of tumors and/or metastases in the subject; (b) limiting any increase in the size or volume of tumors and/or metastases in the subject; (c) increasing survival; (d) reducing the severity of symptoms associated with cancer; (e) limiting or preventing development of symptoms associated with cancer; and (f) inhibiting worsening of symptoms associated with cancer.
  • Hie subject may be any subject that has a relevant disorder.
  • the subject is a mammal, including but not limited to humans, dogs, cats, horses, cattle, etc.
  • an “‘effective” amount refers to an amount of the polypeptide, fusion protein, nucleic acid, expression vector, and/or host cell that is effective for treating the disorder.
  • the polypeptides, fusion proteins nucleic acids, expression vectors, and/or host cells are typically formulated as a pharmaceutical composition, such as those disclosed above, and can be administered via any suitable route, including but not limited to orally, by inhalation spray, ocularly, intravenously, subcutaneously, intraperitoneally, and intravesicularly in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • any suitable dosage range may be used as determined by attending medical personnel. Dosage regimens can be adjusted to provide the optimum desired response.
  • a suitable dosage range for the polypeptides or fusion proteins 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 recommended dose could be lower than 0.1 mcg/kg, especially if administered locally (such as by mtra-tumoral injection). In other embodiments, the recommended dose could be based on weight/m 2 (i.e.
  • polypeptides, fusion proteins, nucleic acids, expression vectors, and/or host cells 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.
  • polypeptides, fusion proteins, nucleic acids, expression vectors, and/or host cells made be administered as the sole therapeutic agent, or may be administered together with (i.e.: combined or separately) one or more other therapeutic agents, including but not limited to tumor resection, chemotherapy, radiation therapy, and immunotherapy (such as checkpoint inhibitors).
  • agents that may be administered in the methods of the disclosure include angiogenesis inhibitors (including but not limited to) axitinib, bevacizumab, cabozantinib, everolimus, lenalidomide, lenvatmib mesylate, pazopanib, ramucirumab, regorafenib, sorafenib, sunitinib, thalidomide, vandetanib, and ziv-aflibercept), immune checkpoint inhibitors (including, but not limited to, pembrolizumab, nivolumab, and cerniplimab as anti-PD-1 antibodies, ipilimumab as an anti- CTLA-4 antibody, and atezolizumab, avelumab, and durvalumab as anti-PD-Ll antibodies), and other cancer growth inhibitors including but not limited to tyrosine kinase inhibitors (including but not limited to)
  • PI3K inhibitors including but not limited to copanlisib, alpelisib, idelal i sib, duvelisib and ombralisib
  • histone deacetylase inhibitors including but not limited to vorinostat, romidepsin, panobinostat, and belinostat
  • Hedgehog pathway blockers including but not limited to vismodegib, sonidegib, and glasdegib.
  • varying curvature protein surfaces vary considerably in shape, and to enable close complementary matching of a wide range of targets, a set of proteins with varying curvature and surface topography would be ideal.
  • high stability the higher the stability of the base scaffold, tire more room for customizing the binding interface for high affinity binding, and the more robust the resulting binders.
  • the overall length of the binder scaffolds should be minimal (80-120 aa). With these selection criteria, we set out to construct a set of scaffolds.
  • 5HCS_TGFBR2_1 The highest affinity binder, 5HCS_TGFBR2_1, was found using biolayer interferometry to have an affinity less than 1nM for TGF ⁇ RII (Fig.2c, Fig.8a).
  • the sequence identity between 5HCS_TGFBR2_0 and 5HCS_TGFBR2_1 is 88.12% (Fig.7a).
  • the circular dichroism spectra indicates a helical structure with peaks at 208 nm and 222 nm, consistent with the design model (Fig.2a,b), and was only slightly changed by heating to 95 °C, indicating high stability (Fig.2b).
  • the high resolution (1.24 ⁇ ) X-ray crystal structure is very close to the computational design model (Fig.2f, g; root mean square deviation (rmsd) over C ⁇ atoms of 0.55 ⁇ over the full complex), showing 5HCS_TGFBR2_1 binds to the TGF- ⁇ 3 binding site on TGF ⁇ RII utilizing the concave surface as designed.
  • SSM site saturation mutagenesis
  • H1 N10 hydrogen bonds with TGF ⁇ RII D142 (Fig 2g, top panel); in H3, S46 and S49 hydrogen bond to the backbone atoms of strand S72 - S75 (Fig 2g, middle panel); and in H5, N93 hydrogen bonds to the backbone atoms of I76 (Fig 2g, lower panel).
  • HEK293 cells with luciferase reporter for the TGF ⁇ SMAD2/3 signaling pathway were stimulated using 10 pM TGF- ⁇ 3 and varying concentrations of 5HCS_TGFBR2_1. Dose-dependent inhibition of the TGF ⁇ SMAD2/3 signaling was observed with an IC 50 of 30.6 nM (Fig.2d).
  • Design and structural validation of CTLA-4 binders An important class of convex targets are the portions of the extracellular domains of transmembrane receptors which interact with their biological partners. These frequently consist of immunoglobulin fold domains, which our large-scale shape analyses indicate are generally quite convex.
  • Immunoglobulin domain recognition plays important roles in immune receptor functions; in particular the cancer immunotherapy target Cytotoxic--T- -lymphocyte--antigen--4 (CTLA-4) have extracellular Ig fold domains that are the targets of therapeutic antibodies. Because of the therapeutic importance of the target, and receptor extracellular Ig domains more generally, we next sought to evaluate the generality of our approach by designing 5HCS based binders to CTLA-4.
  • CTLA-4 plays an important role in peripheral tolerance and the prevention of autoimmune disease by inhibition of T cell activation.
  • Antibody CTLA-4 targeting checkpoint inhibitors have been used for melanoma and non-small cell lung cancer (NSCLC) therapy.
  • 5HCS_CTLA4_1 has a sequence similarity of 82.86% compared to 5HCS_CTLA4_0 (Fig.7b).5HCS_CTLA4_1 had an off rate too slow and a binding affinity for CTLA-4 too tight ( ⁇ 100 pM) to be measurable by biolayer interferometry (Fig.3c, Fig. S5b).
  • the unbound crystal structure of 5HCS_CTLA4_2 aligns with the structure of binder in 5HCS_CTLA4_1 bound structure well with a rmsd. of 0.416 ⁇ .
  • 5HCS_CTLA4_1 binds to the CD86 binding site on CTLA-4 using a concave binding surface formed by H1, H3 and H5 covering both the CTLA-4 beta-turn (L98 to Y104) and hydrophobic pocket which interacts with CD86.
  • H1 interacts with the hydrophobic beta-turn (L128 to Y136) through hydrophobic interactions between Y18 and M135 and aromatic interactions between H19 and Y136 (Fig.3g, top panel). Substitution of this residue with H or Y improves binding affinity (Fig 3E).
  • PD-L1 binders Programmed death-ligand 1 (PD-L1), is upregulated on many tumors, and interacts with PD-1 on T-cells to downregulate T-cell activation.
  • Therapeutic antibodies against PDL1 have shown considerable promise for checkpoint inhibition in cancer immunotherapy.
  • binders using the methods described above to target the binding site of PD-1 on PD-L1 (PDB ID:3BIK ) and block the interaction between the two proteins (Fig.1a).
  • Two PD-L1 binders were obtained from a set of 96 designs.
  • H1 and PD-L1 include aromatic packing of Y9 and Y123 on PD-L1 and electrostatic interactions between D10 and E13 with K124 and R125 on PD-L1 (Fig.4h).
  • H3 binds to the hydrophobic pocket formed by Y56, M115, A121 and Y123 (Fig.4h).
  • Residues Y9, E13, K56 and Q99 spanning the three helices satisfy the hydrogen bonding requirements of both the side chains and backbone of the PD-L1 edge beta strand (A121-R125) buried at the interface.
  • the refined structure has excellent geometry and reveals the expected helical assembly with five antiparallel helices (Fig.4g).
  • the crystal structure of 5HCS_PDL1_1 superimposes on the computational design model with a rmsd of 0.75 A over 105 aligned Ca atoms (Fig. 4g; the substitutions which increase affinity relative to 5HCS PDL1 0 do not alter the backbone structure).
  • the shape and electrostatic potential of the designed target binding interfaces are nearly identical between the crystal structure and the computational design model.
  • the 5HCS Hl, H3 and H5 interface helices interact with hydrophobic pockets and patches on tire target surface in ways not possible with 50-65 residue miniprotein scaffolds in which the secondary’ structure elements at the interface are necessarily all very close together.
  • the binders designed to TGFpRII and PD-L1 the dense and extended networks of hydrogen bonding residues that the 5FICS designs are able to satisfy the hydrogen bonding requirements of exposed target beta-strand backbone polar atoms, which enables binding modes which span both sides of the beta sheet; this is almost impossible to achieve with smaller miniproteins.
  • Tire 5HCS binders can interact with beta-stands either parallelly using helix H3 with Hl and H5 flanking the sheet (5HCS TGBR2 1) or perpendicularly with sides chains from all HI, H3 and H5 (5HCS PDL1 1).
  • the backbones were designed by taking a library' of loops and helices drawn from previous successfill mini-proteins and assembling them into helix-tum- helix-tum modules of 30-50 amino acids. The modules were then repeated 3 times to give a repeat protein. Ail possibilities of N- and C- terminal truncation were assessed and the most concave compact structure under 120 amino acids was chosen .
  • the backbones were diversified using the Rosetta 1M HybrizeMover using the backbones themselves as templates.
  • Protein complex structure extraction Pairs of interacting chains were extracted from high quality crystals from PDB. The pairs of protein complex structures were filtered by interfacial profiles, including the length of each partner's and delta solvent accessible surface area (dSASA). Then we clustered them 40% sequence identity on both chains, and selected representatives favoring higher resolution and shorter proteins.
  • dSASA delta solvent accessible surface area
  • the 5HCS libraries were docked to the target-binding site using the previously reported method 3 . Docked poses of the 5HCS library were filtered by binding orientation. Only designs with interfacial residues as the concave surfaces were kept. Interface sequence design was performed using previously reported protocol. Tire designs were later filtered by ddG (less than -40), contact molecular surface (larger than 400). Finally, 4600 designs from 5HCS passed the filters and were tested experimentally.
  • the 5HCS libraries were docked to the target binding site using the previously reported method 3 . Docked poses of the 5HCS library were filtered by binding orientation. Only designs with interfacial residues as the concave surfaces were kept. Interface sequence design was performed using ProteinMPNMTM with target sequences fixed as native sequences as previously reported. The designs were later filtered by ddG (less than - 40), contact molecular surface (larger than 400) and pAE (less than 10) from AlphaFold2 1M initial guess. Finally, 96 designs from 5HCS libraries passed the filters and were tested experimentally.
  • Saccharomyces cerevisiae EBY 100 strain cultures were grown in C-Trp-Ura medium supplemented with 2% (w/v) glucose.
  • yeast cells were centrifuged at 4,000g for 1 min and resuspended in SGCAA medium supplemented with 0.2% (w/v) glucose at the cell density of 1 x 10 7 cells per ml and induced at 30 °C for 16 -24 h.
  • Cells were washed with PBSF (PBS with 1% (w/v) BSA) and labeled with biotinylated targets using two labeling methods: with-avidity and without-avidity labeling.
  • the cells were incubated with biotinylated target, together with anti-c-Myc fluorescein isothiocyanate (FITC, Miltenyi Biotec) and streptavidin-phycoeiythrin (SAFE, ThermoFisher).
  • FITC anti-c-Myc fluorescein isothiocyanate
  • SAFE streptavidin-phycoeiythrin
  • the concentration of SAPE in the with-avidity method was used at one- quarter of the concentration of the biotinylated targets.
  • the cells were first incubated with biotinylated targets, washed and secondarily labeled with SAPE and FITC.
  • Synthetic genes were optimized for E. colt expression and purchased from IDT (Integrated DN A Technologies) as plasmids in pET29b vector with a TEV -cleavable hexahistidine affinity tag. Plasmids were transformed into BL21* (DE3) E. coli competent cells (Invitrogen). Single colonies from agar plate with 100 ing/L kanamycin were inoculated in 50 mL of Studier autoinduction media 45, and the expression continued at 37 °C for over 24 hours.
  • the cells were harvested by centrifugation at 4000 g for 10 min, and resuspended in a 35 mL lysis buffer of 300 mM NaCI, 25 mM Tris pH 8.0 and 1 mM PMSF. After lysis by sonication and centrifugation at 14000 g for 45 min, the supernatant was purified by Ni ?i immobilized metal affinity chromatography (IMAC) with Ni-NTA Superflow 1M resins (Qiagen).
  • IMAC immobilized metal affinity chromatography
  • Resins with bound cell lysate were washed with 10 mL (bed volume 1 mL) of washing buffer (300 mM NaCI, 25 mM Tris pH 8.0, 60 mM imidazole) and eluted with 5 mL of elution buffer (300 mM NaCI, 25 mM Tris pH 8.0, 300 mM imidazole). Both soluble fractions and full cell culture were checked by SDS-PAGE. Soluble designs were further purified by size exclusion chromatography (SEC). Concentrated samples were run in 150 mM NaCI, 25 mM Tris pH 8.0 on a SuperdexTM 75 Increase 10/300 gel filtration column (Cytiva). SEC-purified designs were concentrated by 10K concentrators (Ami con) and quantified by UV absorbance at 280 nm.
  • washing buffer 300 mM NaCI, 25 mM Tris pH 8.0, 60 mM imidazole
  • elution buffer 300 mM
  • Binding assays were performed on an OctetRED96 1M BLI system (ForteBio) using streptavidin-coated biosensors. Biosensors were equilibrated for at least 10 min in Octet 1M buffer (10 mM Hepes pH 7.4, 150 mM NaCI, 3 mM EDTA, 0.05% Surfactant P20) supplemented with 1 mg/mL bovine serum albumin (SigmaAldrich). For each experiment, the biotinylated target protein was immobilized onto the biosensors by dipping the biosensors into a solution with 50 nM target protein for 200 to 500 s, followed by dipping in fresh Octet TM buffer to establish a baseline for 200 s.
  • Wavelength scans were measured from 260 to 190 nm at 25 and 95 °C and again at 25 °C after fast refolding (about 5 min). Temperature melts monitored the dichroism signal at 222 nm in steps of 2 °C min–1 with 30 s of equilibration time. Wavelength scans and temperature melts were performed using 0.3 mg ml –1 protein in PBS buffer (20 mM NaPO 4 , 150 mM NaCl, pH 7.4) with a 1 mm path-length cuvette. Cell assays TGF- ⁇ luciferase reporter assay.
  • the TGF- ⁇ inhibition assays utilizing HEK-293 cells stably transfected with the CAGA 12 TGF- ⁇ reporter 23 were performed as previously described 24 .
  • Cells were maintained in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
  • FBS fetal bovine serum
  • Cells were plated at 3x10 4 cells per well in a treated 96-well plate. After 24 hours, the media was removed and replaced with fresh DMEM containing 0.1% bovine serum albumin (BSA) and a two-fold concentration series of 5HCS_TGF ⁇ R2_1. After 30 minutes, cells were stimulated with 10 pM TGF- ⁇ 3.
  • BSA bovine serum albumin
  • CTLA-4 blockade cell assay The CTLA-4 Blockade Bioassay (Promega) was used as described in the product literature to compare bioacitivity of our novel high affinity CTLA-4 binders with Ipilimumab. Briefly, 25 uL of CTLA-4 effector cells prediluted into complete RPMI media supplemented with 10% FBS were added to wells of a 96-well flat- bottomed white luminescence plate (Costar).
  • PD-L1 aAPC/CHO-K1 cells were thawed in a 37 ⁇ water bath until just thawed and transferred to pre-warmed media (90% Ham’s F12 / 10% FBS).
  • Cells were mixed and immediately seeded to the inner 60 wells of a 96 well flat bottom white cell culture plates at 100 ul volume.100 ul of media was also added to the outside wells to prevent evaporation. Cells were incubated for 16 hours in a 37 ⁇ , 5% CO ⁇ incubator. At the end of the incubation period, 95 ul of media was removed from each of the wells. Immediately after 40 ul of appropriate antibody or binder dilutions were added to individual wells.
  • PD-1 effector cells were thawed in similar fashion as for PD-L1 aAPC/CHO-K1 cells and transferred to pre-warmed assay buffer (99% RPMI 1640 / 1% FBS).40 ul of PD-1 effector cells were added to the inner 60 wells of the assay plate.80 ul of assay buffer was added to outside wells to prevent evaporation. The assay plate was incubated for 6 hours in a 37 ⁇ , 5% CO ⁇ incubator. At the end of incubation plates were removed from the incubator and equilibrated to ambient temperature (22 ⁇ 25 ⁇ ).80 ul of Bio-Glo TM reagent was added to each well and incubated for 10 mins.
  • Luminescence was measured using the BioTek TM Synergy Neo2 TM multi-mode reader. EC50 values were calculated using the four parameters logistic regression by python scripts. Specificity Determination Cell surface receptor knockouts A431 cells had PD-L1 knocked out via CRISPR RNP transfection. RNP complexes were formed by incubating 4 ul of 80 uM guide RNA (IDT guides: Hs.Cas9.CD274.1.AA, Hs.Cas9.CD274.1.AB) with 4 ul of 80 uM tracrRNA (IDT cat.1072533) at 37°C for 30 minutes.
  • IDTT guides Hs.Cas9.CD274.1.AA, Hs.Cas9.CD274.1.AB
  • the 5HCS_TGFBR2_1 used for crystallization was prepared as described above, followed by digestion for 12 h at 25°C with TEV protease (1:25 mass ratio) in 25 mM Tris, 100 mM Tris, pH 8.0, 1 mM DTT, 1 mM EDTA.
  • Thermo UltiMateTM UHPLC coupled to Broker Compact QqTOF ESI quadrupole TOF mass spectrometer Thermo UltiMateTM UHPLC coupled to Broker Compact QqTOF ESI quadrupole TOF mass spectrometer.
  • the TbRII:5HCS_TGFBR2_l complex was isolated by size exclusion chromatography using a HiLoad SuperdexTM 75 26/60 column (GE Healthcare, Piscataway, NJ) in 25mM HEPES pH 7.5, 100 mM NaCl at a 1: 1.1 ratio, with 5HCS TGFBR2 1 being in slight excess.
  • the complex peak fractions were pooled and concentrated to 33 mg/mL for crystal screening.
  • Cells were harvested by centrifugation at 14,000 x g and suspended in buffer containing 20 mM HEPES (pH 7.5), 500 mM NaCl, 20 mM imidazole, 0.1% IGEPAL, 20% sucrose, 1 mM P-mercaptoethanol (BME). Cells were disrupted by sonication and debris was removed by centrifugation at 45,000 x g. The supernatants were applied to a chromatography column packed with 10 ml His60 SuperFlowTM resin (Clontech Laboratories) that had been equilibrated with buffer A (50 mM HEPES pH 7.5, 30 mM imidazole, 500 mM NaCl, and 1 mM BME).
  • buffer A 50 mM HEPES pH 7.5, 30 mM imidazole, 500 mM NaCl, and 1 mM BME).
  • the columns were washed with buffer A and the Hise-binder proteins were eluted with buffer B (20 mM HEPES, pH 7.5, 350 mM NaCl, 400 mM imidazole, and 1 mM BME), The His* tags were removed by overnight digestion at 4 °C with the TEV protease at a 1500: 1 ratio of binder: TEV. Tire tag-free binders were then separated from Hise-tags by SuperdexTM 200 gel filtration equilibrated with a buffer containing 20 mM HEPES pH 7.5, 350 mM NaCl .
  • the CTLA-4 and PD-L1 binders migrated through gel filtration as discrete peaks with estimated molecular weights of 14 kDa and 12 kDa, respectively, indicating that they are monomers in solution.
  • the purity of the binders was judged by SDS-PAGE and Coomassie blue staining.
  • the peak fractions from the gel filtration step were pooled and concentrated to 20-25 mg/ml in a buffer containing 20 mM HEPES (pH 7.5) and 150 mM NaCl.
  • 5HCS_CTLA4_l:CTLA-4 complex were purified using size exclusion chromatography (SuperdexTM S200) equilibrated with a buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl. The peak fractions were pooled and concentrated to 7.5 mg/ml The preparations were flash frozen in liquid nitrogen and stored at -80 °C for long-term storage.
  • Crystals of the TpRII:5HCS__TGFBR2_l complex were formed using hanging drop vapor diffusion in 24-well plates with 300 pL of well solution and siliconized glass cover slips. Crystals formed in 1- 2 days at 25 °C with drops prepared by mixing 0,4 pL 25 mg/mL protein complex and 0.4 pL of 20% (w/v) PEG-MME 5K, 0.4 M (NH02 SO-i, 0.1 M Tris pH 7.4, and 16 - 32 % glycerol. The crystals were mounted in nylon loops without additional cryoprotectants and with excess well solution wicked off.
  • the diffraction data for the TpRII:5HCS_TGFBR2_l complex was collected at the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory.
  • Phasing was performed with Phaser 34 , initially with the 1.1 A TpRII X-ray structure (PDB 1M9Z), followed by the predicted 5HCS_TGFBR2_1 structure. Several cycles of refinement using RefinacS 3 ’" 42 and model building using COOT 43 were performed to determine the final structure.
  • CTLA-4-binder complex crystals were collected on a Dectris EIGER X 9M detector, with a wavelength of 0.98 A, on the 17-ID-2 (FMX) beamline at the Brookhaven National Laboratory. Tire datasets were indexed, integrated, and scaled using fastDP, XDS 23 and aimless 44 , respectively.
  • the PD-L1 crystals belong to tetragonal space group and diffracted to 1.88 A.
  • Hie CTLA-4-binder complex crystal belongs to C2 space group and diffracted to 2.72 A.
  • TGF- ⁇ transforming growth factor ⁇

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Abstract

L'invention concerne des polypeptides ayant l'acide aminé de l'un des SEQ ID NO : 1-9 qui se lient à des sites cibles de protéine convexe sur le récepteur bêta de facteur de croissance transformant de type 2 (TGFbRII), la protéine 4 associée aux lymphocytes T cytotoxiques (CTLA -4), ou le ligand de mort programmée 1 (PD-L1), ainsi que des procédés de leur utilisation dans le traitement du cancer ou de la fibrose tissulaire.
PCT/US2024/019019 2023-03-10 2024-03-08 Liants protéiques à haute affinité conçus de novo se liant à sites cibles de protéine convexe sur tgfbrii, ctla -4 et pd-l1 Pending WO2024191776A2 (fr)

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