WO2014071234A1 - Composés peptidiques gb1 et leurs procédés de préparation et leur utilisation - Google Patents

Composés peptidiques gb1 et leurs procédés de préparation et leur utilisation Download PDF

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WO2014071234A1
WO2014071234A1 PCT/US2013/068137 US2013068137W WO2014071234A1 WO 2014071234 A1 WO2014071234 A1 WO 2014071234A1 US 2013068137 W US2013068137 W US 2013068137W WO 2014071234 A1 WO2014071234 A1 WO 2014071234A1
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peptidic
compound
terminal
subject
protein
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PCT/US2013/068137
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Sachdev S. Sidhu
Maruti Uppalapati
Dana Ault-Riche
Stephen B. H. Kent
Kalyaneswar MANDAL
Dong Jun Lee
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The Governing Council Of The University Of Toronto
Reflexion Pharmaceuticals, Inc.
The University Of Chicago
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Publication of WO2014071234A1 publication Critical patent/WO2014071234A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • 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
    • C07K14/4722—G-proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • Protein ligands can form large binding surfaces with multiple contacts to a target molecule that leads to binding events with high specificity and affinity.
  • antibodies are a class of protein that has yielded specific and tight binding ligands for various target proteins.
  • vascular endothelial growth factor has a key role in the regulation of normal and abnormal angiogenesis and in the development and differentiation of the vascular system (Ferrara et al. Endocr. Rev. 18:4 25 (1997)). Furthermore, VEGF has been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders (Ferrara et al.). Therefore, compounds that inhibit VEGF action are of interest for the treatment of, e.g., solid tumors and various intraocular neovascular disorders.
  • GB 1 peptidic compounds that specifically bind to a target molecule are provided. Both L- peptidic and D-peptidic compounds are included.
  • the subject compounds include a GB 1 motif and one or more extension moieties connected to the N-terminal or C-terminal of the GBl motif via optional linkers.
  • the extension moieties may be peptidic, and as such may include a protein domain, or a peptidic sequence of up to 50 residues.
  • GB l multimers that include two or more GBl peptidic compounds connected to each other, e.g., via a branching moiety. Methods for making and using the compounds are provided, including methods for in vivo diagnosis or imaging of a disease or condition associated with angiogenesis where the compound is
  • Figure 1 shows HPLC-MS chromatograms of three synthetic peptide segments for use in native chemical ligation of a GB l compound: A) D-Met'-Glu ⁇ -aCOSCHzCHzSOsH after DNP removal and prep HPLC purification; B) crude D-Thz 37 -Ala 51 -aCOSCH 2 CH 2 COArg 6 ; C) crude D- Cys 52 -Trp(CHO) 61 -Lys 79 -aCOOH.
  • NCL native chemical ligation
  • Figure 4 shows: A) CD spectrum of D-RFX035; B) Tm spectra of D-RFX035; and C) SPR data of D-RFX035 against L-VEGF-A.
  • Figure 5 illustrates the results of inhibition of VEGF-A driven cell proliferation in a HUVEC assay by exemplary GB 1 peptidic compounds and multimer.
  • Figure 6 illustrates the results of inhibition of VEGFR2 phosphorylation in cells by exemplary GB 1 peptidic compounds and multimer.
  • Figure 7 illustrates sequences of exemplary GB 1 peptidic motifs and compounds that bind specifically to VEGF.
  • Figure 8 illustrates mutation of a D-VEGF binding GB 1 L-peptidic compound to include charged residues such as Arg at several positions located at surface positions not involved in direct contact with the target: A) representation of a VEGF binding surface of the GBl compound
  • peptidic refers to a moiety that is composed of amino acid residues.
  • the term “peptidic” includes compounds in which the conventional backbone has been replaced with non-naturally occurring or synthetic backbones, and peptides in which one or more naturally occurring amino acids have been replaced with one or more non-naturally occurring or synthetic amino acids, or a D-amino acid version thereof.
  • a peptidic moiety may further include one or more non-peptidic moieties, such as a specific binding moiety (e.g., a biotin moiety) and/or a linker.
  • any of the depictions of sequences found herein may represent a L- amino acid or a D-amino acid version of the sequence.
  • the distinction between capital and small letter codes for L- and D-amino acid residues, respectively, is not utilized.
  • capital letter codes for amino acids are used to represent sequences of compounds and amino acid residues, and are meant to encompass both L- and D-versions of the same.
  • polypeptide As used herein, the terms “polypeptide” and “protein” are used interchangeably.
  • polypeptide also includes post translational modified polypeptides or proteins.
  • polypeptide includes polypeptides in which the conventional backbone has been replaced with non- naturally occurring or synthetic backbones, and peptides in which one or more of the conventional amino acids have been replaced with one or more non-naturally occurring or synthetic amino acids.
  • polypeptides may be of any length, e.g., 2 or more amino acids, 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids, 60 or more amino acids, 100 or more amino acids, 300 or more amino acids, 500 or more or 1000 or more amino acids.
  • naturally occurring amino acid and “non-naturally occurring amino acid” may be used to refer to both L- and D-versions of these amino acids.
  • a D- peptidic compound may be described as including naturally occurring amino acids, e.g., D- enantiomers of L-amino acids such as A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.
  • the term "scaffold” or “scaffold domain” refers to a peptidic framework from which a compound arose, and against which the compound is able to be compared.
  • variant amino acids When a compound arises from amino acid mutations at various positions within a scaffold, the amino acids at those positions are referred to as “variant amino acids.” Such variant amino acids may confer on the resulting peptidic compounds different functions, such as specific binding to a target protein.
  • mutation refers to a deletion, insertion, or substitution of an amino acid(s) residue or nucleotide(s) residue relative to a reference sequence, such as a scaffold sequence.
  • domain refers to a continuous or discontinuous sequence of amino acid residues.
  • region refers to a continuous sequence of amino acid residues.
  • GB l scaffold domain and "GBl scaffold” refer to a scaffold that has a structural motif similar to the B l domain of Protein G (GB l), where the structural motif is characterized by a motif including a four stranded ⁇ -sheet packed against a helix (also referred to as a 4 ⁇ -1 ⁇ motif). The arrangement of four ⁇ -strands and one oc-helix may form a hairpin-helix-hairpin motif.
  • An exemplary GB l scaffold domain is depicted in Figure 1 of US2012/0178682, the disclosure of which is herein incorporated by reference.
  • GB 1 scaffold domains include members of the family of IgG binding B domains, e.g., Protein L B l domain.
  • Exemplary GBl scaffold domain sequences include those described by SEQ ID NOs:314-348.
  • a GB l scaffold domain may be a native sequence of a member of the B domain protein family, a B domain sequence with preexisting amino acid sequence modifications (such as additions, deletions and/or substitutions), or a fragment or analogue thereof.
  • a GBl scaffold domain may be L-peptidic, D-peptidic or a combination thereof. In some cases, a "GB l scaffold domain" may also be referred to as a "parent amino acid sequence.”
  • GB l peptidic compound refers to a compound composed of peptidic residues that has a parent GB 1 scaffold domain.
  • parent amino acid sequence and "parent polypeptide” refer to a polypeptide comprising an amino acid sequence from which a variant GB 1 peptidic compound arose and against which the variant GBl peptidic compound is being compared. In some cases, the parent polypeptide lacks one or more of the modifications disclosed herein and differs in function compared to a variant GB 1 peptidic compound as disclosed herein.
  • the parent polypeptide may comprise a native GB 1 sequence or GB 1 scaffold sequence with pre-existing amino acid sequence modifications (such as additions, deletions and/or substitutions).
  • variable region refers to a continuous sequence of residues that includes one or more variant amino acids.
  • a variable region may include one or more conserved amino acids at fixed positions.
  • fixed region refers to a continuous sequence of conserved residues that does not include any mutations or variant amino acids, and is conserved across a library of compounds.
  • variable domain refers to a domain that includes all of the variant amino acids of a GB 1 scaffold.
  • the variable domain may include one or more variable regions, and may encompass a continuous or a discontinuous sequence of residues.
  • the variable domain may be part of the GB 1 scaffold domain.
  • non-core mutation refers to an amino acid mutation of a GB1 peptidic compound that is located at a position in the 4 ⁇ -1 ⁇ structure that is not part of the hydrophobic core of the structure. Amino acid residues in the hydrophobic core of a GB 1 peptidic compound are not significantly solvent exposed but rather tend to form intramolecular hydrophobic contacts. Unless explicitly defined otherwise, a hydrophobic core residue or core position, as described herein, of a GB 1 scaffold domain that is described by SEQ ID NO: l is defined by one of positions 2, 4, 6, 19, 25, 29, 33, 38, 42, 51 and 53 of the GB1 scaffold.
  • surface mutation refers to an amino acid mutation in a GB1 scaffold that is located at a position in the 4 ⁇ -1 ⁇ structure that is solvent exposed. Such variant amino acid residues at surface positions of a GB 1 peptidic compound are capable of interacting directly with a target molecule, whether or not such an interaction occurs.
  • boundary mutation refers to an amino acid mutation in a GB1 scaffold that is located at a position in the 4 ⁇ -1 ⁇ structure that is at the boundary between the hydrophobic core and the solvent exposed surface.
  • variant amino acid residues at boundary positions of a GB 1 peptidic compound may be in part contacting hydrophobic core residues and/or in part solvent exposed and capable of some interaction with a target molecule, whether or not such an interaction occurs.
  • One criteria for describing core, surface and boundary residues of a GB1 peptidic structure is described by Mayo et al. Nature Structural Biology, 5(6), 1998, 470-475. Such methods and criteria can be modified for use with the GB 1 scaffold domain.
  • linking sequence refers to a continuous sequence of amino acid residues, or analogs thereof, that connect two peptidic motifs.
  • a linking sequence is the loop connecting ⁇ -strands in a ⁇ -hairpin motif.
  • stable refers to a compound that is able to maintain a folded state under physiological conditions at a certain temperature, such that it retains at least one of its normal functional activities, for example binding to a target protein.
  • the stability of the compound can be determined using standard methods. For example, the "thermostability" of a compound can be determined by measuring the thermal melt (“Tm”) temperature. The Tm is the temperature in degrees Celsius at which half of the compound becomes unfolded. In some instances, the higher the Tm, the more stable the compound.
  • linker As used herein, the terms “linker”, “linkage”, “linking group” and “crosslink” are used interchangeably and refer to a linking moiety that covalently connects two groups and has a backbone of 100 atoms or less (such as 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less) in length.
  • a linking moiety may be a covalent bond that connects two groups or a chain of between 1 and 100 atoms in length, for example of about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40 or 50 carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom.
  • one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom.
  • the bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone.
  • the linker may include one or more substituent groups, for example an alkyl, aryl or alkenyl group.
  • a linker may include, without limitations, oligo(ethylene glycol), ethers, thioethers, disulfide, amides, carbonates, carbamates, tertiary amines, alkyls, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like.
  • the linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone.
  • a linker may be cleavable or non-cleavable.
  • a linker may be peptidic, e.g., a linking sequence of residues.
  • the compounds may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids and polypeptides.
  • the present invention is meant to include all such possible isomers, as well as, their racemic, diastereomeric, and optically pure forms.
  • the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.
  • a target protein refers to all members of the target family, and fragments and enantiomers thereof, and protein mimics thereof.
  • the target proteins of interest that are described herein are intended to include all members of the target family, and fragments and enantiomers thereof, and protein mimics thereof, unless explicitly described otherwise.
  • the target protein may be any protein of interest, such as a therapeutic or diagnostic target, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors and immunoglobulins.
  • target protein is intended to include recombinant and synthetic molecules, which can be prepared using any convenient recombinant expression methods or using any convenient synthetic methods, or purchased commercially, as well as fusion proteins containing a target molecule, as well as synthetic L- or D-proteins.
  • the term "protein mimic” refers to a peptidic compound that mimics a binding property of a protein of interest, e.g., a target protein.
  • the target protein mimic includes an essential part of the original target protein (e.g., an epitope or essential residues thereof) that is necessary for forming a potential binding surface, such that the target protein mimic and the original target protein are each capable of binding specifically to a binding moiety of interest, e.g., an antibody or a D-peptidic compound.
  • the part(s) of the original target protein that is essential for binding is displayed on a scaffold such that potential binding surface of the original target protein is mimicked.
  • a target protein mimic includes residues or fragments of the original target protein that are incorporated into a protein scaffold, where the scaffold mimics a structural motif of the target protein.
  • the protein mimic may present a potential binding surface that mimics that of the original target protein.
  • the native structure of the fragments of the original target protein are retained using methods of conformational constraint. Any convenient methods of conformationally constraining a peptidic compound may be used, such as but not limited to, bioconjugation, dimerization (e.g., via a linker), multimerization, or cyclization.
  • VEGF vascular endothelial growth factor
  • VEGF refers to the protein products encoded by the VEGF gene.
  • the term VEGF includes all members of the VEGF family, such as, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and fragments and enantiomers thereof.
  • the term VEGF is intended to include recombinant and synthetic VEGF molecules, which can be prepared using any convenient recombinant expression methods or using any convenient synthetic methods, or purchased commercially (e.g. R & D Systems, Catalog No.
  • VEGF is involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature) and can also be involved in the growth of lymphatic vessels in a process known as lymphangiogenesis.
  • vasculogenesis the de novo formation of the embryonic circulatory system
  • angiogenesis the growth of blood vessels from pre-existing vasculature
  • lymphangiogenesis the growth of lymphatic vessels in a process known as lymphangiogenesis.
  • Members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation.
  • the VEGF receptors have an extracellular portion containing 7 immunoglobulin-like domains, a single transmembrane spanning region and an intracellular portion containing a split tyrosine-kinase domain.
  • VEGF-A binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1).
  • VEGFR-2 appears to mediate several of the cellular responses to VEGF.
  • VEGF, its biological activities, and its receptors are well studied and are described in Matsumoto et al. (VEGF receptor signal transduction Sci STKE. 2001 :RE21 and Marti et al (Angiogenesis in ischemic disease. Thromb Haemost. 1999 Suppl 1:44-52).
  • VEGFs vascular endothelial growth factor receptors
  • NCBI's Genbank database Amino acid sequences of exemplary VEGFs that may be employed herein are found in the NCBI's Genbank database and a full description of VEGF proteins and their roles in various diseases and conditions is found in NCBI's Online Mendelian Inheritance in Man database.
  • GB 1 peptidic compounds that specifically bind to target proteins are provided.
  • the subject compounds are D-peptidic and specifically bind a L-target protein.
  • the subject compounds are L-peptidic and specifically bind a L-target protein.
  • the subject GB1 peptidic compounds have a GB 1 motif.
  • a number of GB 1 peptidic compounds are provided.
  • the subject compounds include one or more extension moieties connected to the N-terminal or C-terminal of the GB 1 motif via optional linkers.
  • the extension moieties may be peptidic, and as such may include a protein domain or a peptidic sequence of up to 30 residues.
  • GB1 multimers that include two or more GB 1 peptidic compounds connected to each other, e.g., via a branching moiety.
  • extension moieties e.g., N-terminal and/or C-terminal extension moieties provide for GB 1 peptidic compounds having enhanced and/or modified properties and functions including but not limited to solubility, binding affinity for target, non-specific binding, stability, affinity for a complementary binding moiety or secondary target (such that the GB 1 peptidic compound may be referred to as having bi-specific binding properties), in vivo half -life, improved protease resistance, decreased elimination in vivo, a detectable property such as fluorescence, or immobilization to a support.
  • the GB 1 motif is of the same structural motif as the Bl domain of Protein G (GB 1), where the structural motif of GB 1 is characterized by a motif that includes an arrangement of four ⁇ -strands and one oc-helix around a hydrophobic core (also referred to as a 4 ⁇ -1 ⁇ motif).
  • the GB 1 peptidic compounds of the invention include mutations at non-core positions, e.g., variant amino acids at positions within a GB 1 scaffold domain that are not part of the hydrophobic core of the structure.
  • a sample is contacted with a peptidic GB 1 compound that specifically binds with high affinity to a target protein.
  • a method including administering to a subject a GB 1 peptidic compound of the invention that specifically binds with high affinity to a target protein, is also provided.
  • the subject methods and compounds find use in a variety of applications, including research and therapeutic applications.
  • aspects of the invention include GB 1 peptidic compounds that specifically bind with high affinity to a target protein.
  • the GB 1 peptidic compounds are characterized by having a GB1 motif, e.g., as described herein.
  • the target protein is a L-protein and the compound is a D-peptidic compound.
  • the target protein is a synthetic D-protein and the compound is a L-peptidic compound.
  • the target protein is a L-protein and the compound is a L-peptidic compound.
  • the GB1 peptidic compounds may further include one or more extension moieties, e.g., a N- terminal extension moiety or a C-terminal extension moiety.
  • extension moieties e.g., a N- terminal extension moiety or a C-terminal extension moiety.
  • extension moiety and “extension” are used interchangeably and refer to a moiety that is attached to one or both of the terminal residues of the GB 1 motif of a GB 1 peptidic compound, thereby extending the length of the resulting compound.
  • the extension moiety is covalently attached via the alpha-amino group of the N-terminal residue, or is covalently attached to the alpha-carboxyl acid group of the C-terminal residue.
  • an extension moiety may be attached to the GBl motif via a sidechain residue of the N-terminal or C-terminal residue.
  • the GBl peptidic compound includes a GB 1 motif and at least one of a N-terminal extension and a C-terminal extension.
  • a N-terminal extension is present.
  • a C-terminal extension is present.
  • both a N-terminal and C-terminal extension is present.
  • extension moieties may be utilized as N-terminal and/or C-terminal extensions in the subject GBl peptidic compounds.
  • the extension moieties may be peptidic or not peptidic, naturally occurring or synthetic. A variety of extension moieties find use in the subject modified compounds.
  • Moieties of interest suitable for adapting for use in N-terminal and/or C-terminal extensions include, but are not limited to, any convenient moiety suitable for attachment to the amino terminal or the carboxylic acid terminal of a peptide or protein, a protein domain, a polypeptide, a peptide tag, a specific binding moiety, a polymeric moiety such as a polyethylene glycol (PEG), a carbohydrate, a dextran or a polyacrylate, a linker, and a chemoselective functional group, a moiety that imparts desirable drug-like properties, a label, a support, a half-life extending moiety.
  • PEG polyethylene glycol
  • N-terminal and/or C-terminal extension moieties may confer on the resulting GBl peptidic compounds enhanced and/or modified properties and functions, e.g., as described above.
  • the N-terminal and/or C-terminal extension moieties may be referred to as "enhancing extensions" and/or "modifying extensions.”
  • the extension moiety may include a polypeptide or a protein domain. In some cases, the inclusion of such an extension moiety at the N-terminal or C-terminal of the GB 1 motif may confer on the GB 1 motif enhanced stability, solubility, protein folding, and/or binding affinity, or reduced aggregation. Any convenient polypeptide or protein domain may be utilized as an extension moiety. Any convenient protein domain may be utilized in the extension moieties, such as protein domains that find use in fusion proteins. Any convenient polypeptides may be utilized in the extension moieties. The polypeptide may be a sequence of 50 residues or less, such as 40 residues or less, 30 residues or less, 25 residues or less, or 20 residues or less.
  • the polypeptide includes a majority of hydrophilic residues. In certain cases, the polypeptide includes a majority of charged residues (e.g., basic or acidic residues) under physiological conditions. In certain instances, the polypeptide includes a poly(His) (e.g., 6 to 10-His), poly(Arg), poly(Lys) sequence, or mixtures thereof.
  • Polypeptides and protein domains of interest include, but are not limited to: gD tags, c-Myc epitopes, FLAG tags, His tags, fluorescence proteins (e.g., GFP), beta-galactosidase protein, GST, albumins, immunoglobulins, Fc domains, or similar antibody-like fragments, leucine zipper motifs, a coiled coil domain, a hydrophobic region, a hydrophilic region, a polypeptide comprising a free thiol which forms an intermolecular disulfide bond between two or more multimerization domains, a "protuberance -into- cavity" domain, beta-lactoglobulin, or fragments thereof.
  • the GB l compound includes a GB l motif and an extension moiety that includes a polypeptide or protein domain, e.g., as described herein.
  • the GBl motif is D-peptidic and the extension moiety includes a L-peptidic polypeptide or protein domain, where the L-peptidic polypeptide or protein domain may be prepared by any convenient means, e.g., recombinant or synthetic methods.
  • the GB l motif is D-peptidic and the extension moiety includes a D-peptidic polypeptide or protein domain.
  • a D-peptidic GB 1 motif may be attached to an extension moiety that includes a polypeptide or protein domain using any convenient method, e.g., synthetic methods, bioconjugation methods, or recombinant methods.
  • the GB l compound may be termed a fusion of a GB 1 motif and an extension moiety that includes a polypeptide or protein domain. It is understood that although fusion proteins are typically prepared by recombinant means, D-peptidic versions of the sequences are also meant to be included in the subject compounds, e.g., where the GB l motif may be D-peptidic and the extension moiety may be D-peptidic or L- peptidic.
  • half-life extending moiety refers to a pharmaceutically acceptable moiety, domain, or "vehicle” covalently linked or conjugated to the subject compound, that prevents or mitigates in vivo proteolytic degradation or other activity-diminishing chemical modification of the subject compound, increases half-life or other pharmacokinetic properties (e.g., rate of absorption), reduces toxicity, improves solubility, increases biological activity and/or target selectivity of the subject compound with respect to a target of interest, increases manufacturability, and/or reduces
  • the extension moiety is a half-life extending moiety (e.g., a polypeptide that binds serum proteins, such as an immunoglobulin (e.g., IgG) or a serum albumin (e.g., human serum albumin (HSA)).
  • a half-life extending moiety e.g., a polypeptide that binds serum proteins, such as an immunoglobulin (e.g., IgG) or a serum albumin (e.g., human serum albumin (HSA)).
  • a serum albumin e.g., human serum albumin (HSA)
  • Polyethylene glycol is an example of a useful half-life extending moiety.
  • Exemplary half-life extending moieties include a polyalkylene glycol moiety (e.g., PEG), a serum albumin or a fragment thereof, a transferrin receptor or a transferrin-binding portion thereof, and a moiety comprising a binding site for a polypeptide that enhances half -life in vivo, a copolymer of ethylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-l,3-dioxolane, a poly-l,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid (e.g., polylysine), a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene
  • polyoxyethylated polyol a polyvinyl alcohol, a linear or branched glycosylated chain, a polysialic acid, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group, an
  • immunoglobulin Fc domain see, e.g., U.S. Pat. No. 6,660,843
  • an albumin e.g., human serum albumin; see, e.g., U.S. Pat. No. 6,926,898 and US 2005/0054051 ; U.S. Pat. No. 6,887,470
  • TTR transthyretin
  • TSG thyroxine-binding globulin
  • the half-life extending moiety is a fatty acid.
  • Any convenient fatty acids may be used in the subject modified compounds. See e.g., Chae et al., "The fatty acid conjugated exendin-4 analogs for type 2 antidiabetic therapeutics", J. Control Release. 2010 May 21 ; 144(1): 10-6.
  • the extension moiety includes a multimerizing group.
  • a multimerizing group is any convenient group that is capable of forming a multimer (e.g., a dimer, a trimer, or a dendrimer), e.g., either by mediating binding between two or more GB1 compounds (e.g., directly or indirectly via a multivalent binding moiety), or by connecting two or more compounds via a covalent linkage.
  • the multimerizing group is a chemoselective reactive functional group that is capable of conjugating to a compatible function group on a second GB 1 peptidic compound.
  • the multimerizing group is a specific binding moiety (e.g., biotin or a peptide tag) that is capable of specifically binding to a multivalent binding moiety (e.g., a streptavidin or an antibody).
  • a multivalent binding moiety e.g., a streptavidin or an antibody.
  • the multimerizing group is a specific binding moiety that is capable of forming a homodimer or a heterodimer directly with a second specific binding moiety of a second GB 1 compound.
  • the GB1 compound may be part of a multimer of GB1 compounds.
  • the GB 1 compound may be a monomer that is capable of being multimerized either directly with one or more other GB1 compounds, or indirectly via binding to a multivalent binding moiety.
  • the extension moiety includes a specific binding moiety.
  • the specific binding moiety is a moiety that is capable of specifically binding to a second moiety that is complementary to it.
  • the specific binding moiety binds to the complementary second moiety with an affinity of at least 10 "7 M (e.g., as measured by a K D of lOOnM or less, such as 30nM or less, lOnM or less, 3nM or less, InM or less, 300pM or less, or 100 pM or even less).
  • Complementary binding moiety pairs of specific binding moieties include, but are not limited to, a ligand and a receptor, an antibody and an antigen, complementary polynucleotides, complementary protein homo- or heterodimers, an aptamer and a small molecule, a polyhistidine tag and nickel, and a chemoselective reactive group (e.g., a thiol) and an electrophilic group (e.g., with which the reactive thiol group can undergo a Michael addition).
  • the specific binding pairs may include analogs, derivatives and fragments of the original specific binding member.
  • an antibody directed to a protein antigen may also recognize peptide fragments, chemically synthesized, labeled protein, derivatized protein, etc.
  • Protein domains of interest that find use as specific binding moieties in the subject GB 1 multimers include, but are not limited to, Fc domains, or similar antibody-like fragments, leucine zipper motifs, a coiled coil domain, a hydrophobic region, a hydrophilic region, a polypeptide comprising a free thiol which forms an intermolecular disulfide bond between two or more multimerization domains, or a "protuberance-into-cavity" domain (see e.g., WO 94/10308; U.S. Pat. No. 5,731,168, Lovejoy et al. (1993), Science 259: 1288-1293; Harbury et al. (1993), Science 262: 1401-05; Harbury et al. (1994), Nature 371 :80-83; Hakansson et al. (1999), Structure 7: 255-64.
  • the specific binding moiety is an affinity tag such as a biotin moiety.
  • biotin moieties include biotin, desthiobiotin, oxybiotin, 2'-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc.
  • the biotin moiety is capable of specifically binding with high affinity to a chromatography support that contains immobilized avidin, neutravidin or streptavidin.
  • Biotin moieties bind to streptavidin with an affinity of at least 10 "8 M.
  • a monomeric avidin support may be used to specifically bind a biotin-containing compound with moderate affinity thereby allowing bound compounds to be later eluted competitively from the support (e.g., with a 2mM biotin solution) after non-biotinylated polypeptides have been washed away.
  • the biotin moiety is capable of binding to an avidin, neutravidin or streptavidin in solution to form a multimeric GB 1 compound , e.g., a dimeric, or tetrameric complex of GB1 compounds with the avidin, neutravidin or streptavidin.
  • a biotin moiety may also include a linker, e.g.,— LC -biotin,— LC-LC- Biotin,— SLC -Biotin or— PEG n -Biotin where n is 3-12 (commercially available from Pierce
  • the extension moiety includes a label.
  • labels include labels that permit both the direct and indirect measurement of the presence of the GB 1 peptidic compound.
  • labels that permit direct measurement of the compound include radiolabels, fluorophores, dyes, beads, nanoparticles (e.g., quantum dots), chemiluminescers, colloidal particles, paramagnetic labels and the like.
  • Radiolabels may include radioisotopes, such as 35 S, 14 C, 125 1, 3 H, 64 Cu and 131 I.
  • the subject compounds can be labeled with the radioisotope using any convenient techniques, such as those described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed.
  • labels which permit indirect measurement of the presence of the modified compound include enzymes where a substrate may provide for a colored or fluorescent product.
  • the compound may include a covalently bound enzyme capable of providing a detectable product signal after addition of suitable substrate.
  • the compound may include a first member of specific binding pair which specifically binds with a second member of the specific binding pair that is conjugated to the enzyme, e.g. the compound may be covalently bound to biotin and the enzyme conjugate to streptavidin.
  • suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such enzyme conjugates may be readily produced by any convenient techniques.
  • the label is a fluorophore.
  • fluorophore refers to a molecule that, when excited with light having a selected wavelength, emits light of a different wavelength, which may emit light immediately or with a delay after excitation.
  • Fluorophores include, without limitation, fluorescein dyes, e.g., 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6- FAM), 2',4',1,4,-tetrachlorofluorescein (TET), 2',4', 5',7',1,4-hexachlorofluorescein (HEX), and 2 T- dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE); cyanine dyes, e.g. Cy3, CY5, Cy5.5,
  • QUASARTM dyes etc. dansyl derivatives
  • fluorophore includes excimers and exciplexes of such dyes.
  • the compound includes a detectable label, such as a radiolabel.
  • the radiolabel suitable for use in PET, SPECT and/or MR imaging.
  • the radiolabel is a PET imaging label.
  • the compound is radiolabeled with 18F, 64Cu, 68Ga, l l lln, 99mTc or 86Y.
  • the label may be attached to the compound at any convenient position and via any convenient chemistry.
  • Methods and materials of interest include, but are not limited to those described by USP 8,545,809; Meares et al., 1984, Acc Chem Res 17:202-209; Scheinberg et al., 1982, Science 215:1511- 13; Miller et al., 2008, Angew Chem Int Ed 47:8998-9033; Shirrmacher et al., 2007, Bioconj Chem 18:2085-89; Hohne et al., 2008, Bioconj Chem 19: 1871-79; Ting et al., 2008, Fluorine Chem 129:349- 58, the labeling method of Poethko et al.
  • the label is connected to the compound via an optional linker. In certain embodiments, the label is connected to the N-terminal of the compound. In certain embodiments, the label is connected to the C-terminal of the compound. In certain embodiments, the label is connected to a non-terminal residue of the compound, e.g., via a side chain moiety. In certain embodiments, the label is connected to the N-terminal peptidic extension moiety of the compound via an optional linker. In some cases, the N-terminal peptidic extension moiety is modified to include a reactive functional group which is capable of reacting with a compatible functional group of a radiolabel containing moiety.
  • any convenient reactive functional groups, chemistries and radiolabel containing moieties may be utilized to attach a radiolabel to the compound, including but not limited to, click chemistry, an azide, an alkyne, a cyclooctyne, copper-free click chemistry, a nitrone, a chelating group (e.g., selected from DOTA, TETA, NOTA, NODA, (tert-Butyl) 2 NODA, NETA, C-NETA, L-NETA, S- NETA, NODA-MPAA, and NOD A-MPAEM) , a propargyl-glycine residue, etc.
  • a chelating group e.g., selected from DOTA, TETA, NOTA, NODA, (tert-Butyl) 2 NODA, NETA, C-NETA, L-NETA, S- NETA, NODA-MPAA, and NOD A-MPAEM
  • the compound includes an extension moiety that is useful as a pharmaceutical (e.g., a protein, nucleic acid, organic small molecule, etc.).
  • a pharmaceutical e.g., a protein, nucleic acid, organic small molecule, etc.
  • exemplary pharmaceutical proteins include, e.g., cytokines, antibodies, chemokines, growth factors, interleukins, cell-surface proteins, extracellular domains, cell surface receptors, cytotoxins, etc.
  • Exemplary small molecule pharmaceuticals include small molecule toxins or therapeutic agents.
  • the compound is a chimeric molecule that comprises a fusion of a GB 1 motif with a protein or polypeptide extension moiety, such as a pharmaceutical protein, a protein domain or a polypeptide (e.g., a tag polypeptide).
  • a protein or polypeptide extension moiety such as a pharmaceutical protein, a protein domain or a polypeptide (e.g., a tag polypeptide).
  • the compound includes a GB1 motif with an extension moiety that is a tag polypeptide and which provides an epitope to which an anti-tag antibody can selectively bind.
  • the polypeptide is an epitope tag placed at the amino- or carboxyl-terminus of the GB 1 motif, but could also be placed in any convenient region that does not significantly disrupt binding to the target, e.g. a loop region.
  • the presence of such epitope-tagged forms of a GB 1 peptidic compound can be detected using an antibody against the tag polypeptide.
  • provision of the epitope tag allows the GB 1 peptidic compound to be readily isolated or purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • the chimeric molecule may include a fusion of a subject GB 1 peptidic compound with an extension moiety that includes an immunoglobulin or a particular region of an immunoglobulin.
  • the chimeric molecule may include a cell penetrating peptide (e.g., tat). The cell penetrating peptide may facilitate cellular uptake of a chimeric molecule.
  • tag polypeptides and their respective antibodies may be used.
  • examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.
  • tag polypeptides include the Flag- peptide [Hopp et al., BioTechnology 6: 1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science 255: 192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem. 266: 15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. U.S.A. 87:6393-6397 (1990)].
  • the extension moieties may be attached to the subject compound via any convenient method.
  • an extension moiety is attached via covalent conjugation to a terminal amino acid residue, e.g., at the amino terminal or at the carboxylic acid terminal.
  • the extension moiety may be attached to the GBl peptidic domain via a single bond or a suitable linker, e.g., a PEG linker, a peptidic linker including one or more amino acids, or a saturated hydrocarbon linker.
  • a suitable linker e.g., a PEG linker, a peptidic linker including one or more amino acids, or a saturated hydrocarbon linker.
  • Any convenient reagents and methods may be used to include an extension moiety in a subject GB l peptidic domain, for example, conjugation methods as described in G. T.
  • X is a GB l motif that specifically binds to a target molecule
  • L is an optional linking group
  • Y is an extension moiety, where L is attached to X at any convenient location (e.g., the N- terminal, C-terminal or the sidechain of a surface residue not involved in binding to the target).
  • the extension moiety may be peptidic. It is understood that a peptidic extension moiety may further include one or more non-peptidic groups including, but not limited to, a biotin moiety and/or a linker.
  • the GB l peptidic compound includes at least one of a N-terminal peptidic extension moiety and a C-terminal peptidic extension moiety. Any convenient peptidic extension moiety may be utilized as a N-terminal or C-terminal extension.
  • the at least one of a N-terminal peptidic extension and a C-terminal peptidic extension comprises a protein domain. Any convenient protein domains may be adapted and utilized as extension moieties in the subject GB l peptidic compounds.
  • the subject compounds may be modified in a way to form chimeric molecules comprising a GB 1 motif fused to a heterologous protein domain, polypeptide or amino acid sequence.
  • Protein domains of interest include, but are not limited to, any convenient serum protein, serum albumin (e.g., human serum albumin; see, e.g., U.S. Pat. No. 6,926,898 and US 2005/0054051; U.S. Pat. No. 6,887,470), a transferrin receptor or a transferrin-binding portion thereof, immunoglobulin (e.g., IgG), an immunoglobulin Fc domain (see, e.g., U.S. Pat. No. 6,660,843), a transthyretin (TTR; see, e.g., US 2003/0195154; 2003/0191056), a thyroxine-binding globulin (TBG), or a fragment thereof.
  • serum albumin e.g., human serum album
  • the GB l peptidic compound further comprises a linker between the at least one N-terminal peptidic extension and C-terminal peptidic extension and the GB 1 motif.
  • the N-terminal and C-terminal peptidic extensions independently include a sequence of 1 to 30 residues, such as, 2 to 30 residues, 2 to 25 residues, 2 to 22 residues, 2 to 20 residues, 4 to 20 residues, 6 to 20 residues, 8 to 20 residues, 10 to 20 residues.
  • the peptidic extension moiety includes 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, or 20 or more residues.
  • the peptidic extension moiety includes 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 8 or less, 10 or less, 12 or less, 14 or less, 16 or less, 18 or less, or 20 or less residues.
  • the GB l peptidic compound includes at least one of the N-terminal and C- terminal peptidic extensions including a sequence described by the formula:
  • A-T where A is an extension of 2 to 30 residues in length (e.g., 2 to 24 residues in length), and T is an optional linker attached to the N-terminal of the GB1 motif.
  • T is a peptidic linker of 1 to 10 residues in length, such as, 1 to 8 residues, 1 to 6 residues, 2 to 6 residues, or 2 to 4 residues in length.
  • A may be referred to as a peptidic extension or an enhancing extension.
  • A is a hydrophilic sequence of residues, e.g., a sequence of residues where 50% or more (e.g., 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more) of the A residues are independently selected from His, Lys, Arg, Asp, Ser and Glu.
  • A is basic, e.g., includes a basic sequence of residues where 50% or more (e.g., 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more) of the A residues are independently selected from His, Lys and Arg.
  • A includes a sequence of residues where 50% or more (e.g., 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more) of the A residues are histidine residues. In certain instances, A includes a sequence of residues where 50% or more (e.g., 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more) of the A residues are arginine residues. In certain cases, A includes a His tag.
  • the linker T is peptidic and includes 1 to 10 residues, where 50% or more (e.g., 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more) of the T residues are independently selected from Gly, ⁇ -Ala and Ser.
  • the GB1 peptidic compound includes a N-terminal peptidic extension described by the formula A N -T N -, where A N is an sequence comprising 6 to 16 residues, and T N is an optional linker to the N-terminal (e.g., a peptidic linker comprising 1 to 6 residues).
  • the GB 1 peptidic compound includes a C-terminal peptidic extension described by the formula -T c - A c , where A c is an enhancing sequence comprising 1 to 6 residues, and T c is an optional linker to the C-terminal (e.g., a peptidic linker comprising 1 to 6 residues).
  • the GB1 peptidic compound further includes a multimerizing group Z attached to the at least one N-terminal peptidic extension and C-terminal peptidic extension.
  • the N-terminal peptidic extension is described by the formula: Z-A N -T N -, where Z is a multimerizing group and A N and T N are as described above.
  • the N-terminal peptidic extension is described by the formula: -T c -A c -Z, where where Z is a multimerizing group and A c and T c are as described above.
  • a multimerizing group is any convenient group that is capable of forming a multimer (e.g., a dimer, a trimer, or a dendrimer), e.g., by mediating binding between two or more compounds (e.g., directly or indirectly via a multivalent binding moiety), or by connecting two or more compounds via a covalent linkage.
  • the multimerizing group Z is a chemoselective reactive functional group that conjugates to a compatible function group on a second GB 1 peptidic compound.
  • the multimerizing group is a specific binding moiety (e.g., biotin or a peptide tag) that specifically binds to a multivalent binding moiety (e.g., a streptavidin or an antibody).
  • a multivalent binding moiety e.g., a streptavidin or an antibody.
  • the GB 1 compound includes a multimerizing group and is a monomer that has not yet been multimerized.
  • Chemoselective reactive functional groups for inclusion in the subject GB 1 peptidic compounds include, but are not limited to: an azido group, an alkynyl group, a phosphine group, a cysteine residue, a C-terminal thioester, aryl azides, maleimides, carbodiimides, N- hydroxysuccinimide (NHS)-esters, hydrazides, PFP-esters, hydroxymethyl phosphines, psoralens, imidoesters, pyridyl disulfides, isocyanates, aminooxy-, aldehyde, keto, chloroacetyl, bromoacetyl, and vinyl sulfones.
  • the GB 1 peptidic compound includes at least one of a N-terminal extension and a C-terminal extension where:
  • the N-terminal peptidic extension includes a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence set forth in one of the following sequences: MKIEEHHHHHHHHHHGGS (SEQ ID NO:76), MKIEEHHHHHHHHHH (SEQ ID NO:77), HHHHHHGGS (SEQ ID NO:78), (His) n -GGS
  • the C-terminal peptidic extension includes a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence set forth in one of the following sequences: GGSDK (SEQ ID NO:85), or _ (G m S 0 ) p - X l X 2 .. .X q , where each m and o are each independently 0, 1 , 2, 3 or 4, p is an integer of 1 to 6, and q is 10 or less, and X 1 to X q are each independently amino acid residues.
  • the GB 1 peptidic compound includes at least one of a N-terminal extension and a C-terminal extension where:
  • the N-terminal peptidic extension includes a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence set forth in one of the following sequences: MKIEEHHHHHHHHHHGGS (SEQ ID NO:86); and
  • the C-terminal peptidic extension includes a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence set forth in the following sequence: GGSDK (SEQ ID NO: ).
  • the GB 1 peptidic compound includes a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence set forth in the following sequence:
  • the GB 1 peptidic compound includes a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence set forth in the following sequence:
  • the extension moiety is not an L-peptidic sequence of residues.
  • the GB 1 compound includes at least one of a N-terminal and C-terminal extension that is D-peptidic.
  • the GB 1 compound includes a N-terminal and/or a C-terminal extension that is not an N-terminal leader sequence or a C-terminal linker sequence used for intracellular expression.
  • the GB 1 compound includes a N-terminal extension that does not include a His-tag, such as a lOxHis tag, e.g., a sequence of residues including
  • the GB 1 compound includes a N-terminal extension that does not include a FLAG tag, e.g., a sequence of residues including SMGDYKDDDDKGGS (SEQID NO: 91). In some instances, the GB 1 compound includes a C-terminal extension that does not include a His tag or a FLAG tag. In some instances, the GB1 compound includes a C-terminal extension that does not include a sequence of residues such as, GGSDK (SEQ ID NO: 92), DK, or GGSDKHHHHHHHHHH (SEQ ID NO: 93). In certain instances, the GB 1 compound does not include a C-terminal extension.
  • any convenient GB 1 peptidic compound e.g., as described herein may be multimerized, to result in a multimer (e.g., a dimer, a trimer, a tetramer, dendrimer, etc) of GB 1 peptidic compounds.
  • the multimer includes two or more GBl peptidic compounds, where each GB 1 peptidic compound includes a GB 1 motif.
  • the two or more GBl peptidic compounds each independently include at least one of a N-terminal peptidic extension and a C-terminal peptidic extension.
  • the at least two GBl peptidic compounds each independently include a first specific binding moiety (e.g., a biotin or a peptide tag) and the at least two GB l peptidic compounds are bound to each other via a multivalent binding moiety (e.g., a streptavidin, an avidin or an antibody) that specifically binds the at least two first specific binding moieties.
  • a first specific binding moiety e.g., a biotin or a peptide tag
  • a multivalent binding moiety e.g., a streptavidin, an avidin or an antibody
  • the at least two GB l peptidic compounds each include a specific binding moiety that is a biotin moiety.
  • the at least two GB l peptidic compounds are covalent linked to each other via the at least one of a N-terminal peptidic extension and a C-terminal peptidic extension.
  • the GB l multimer is described by the formula:
  • Y is a multivalent linker; n is an integer greater than one; and GB l is a GBl peptidic compound comprising a GB 1 motif.
  • the multimer is described by one of the following formulas:
  • each GBl motif is attached to at least one of A N and A c via the optional linker.
  • any convenient multivalent linker may be utilized in the subject multimers.
  • multivalent is meant that the linker includes two or more terminal groups suitable for attachment to a GB 1 compound, e.g., as described herein.
  • the multivalent linker is divalent or trivalent.
  • the multivalent linker Y is a dendrimer scaffold.
  • Any convenient dendrimer scaffold may be adapted for use in the subject GBl multimers.
  • the dendrimer scaffold is a branched molecule that includes at least one branching point and two or more terminals suitable for connecting to the N- terminal or C-terminal of a GB l motif via optional linkers.
  • the dendrimer scaffold may be selected to provide a desired spatial arrangement of two or more GBl motifs. In some cases, the spatial arrangement of the two or more GB 1 motifs is selected to provide for a desired binding affinity and avidity for the target protein.
  • GB 1 peptidic compounds including GB 1 motifs of interest are described by Sidhu et al. in copending U.S. application 13/294,097, the disclosure of which is entirely incorporated herein by reference.
  • GB l motif and "4 ⁇ -1 ⁇ motif ' are used interchangeably and refer to that part of the GB 1 peptidic compounds corresponding to a GB 1 scaffold domain of the same structural motif as the Bl domain of Protein G (GB l), where the structural motif of GB l is characterized by a motif that includes an arrangement of four ⁇ -strands and one ⁇ -helix (also referred to as a 4 ⁇ -1 ⁇ motif) around a hydrophobic core.
  • the GB l motif of the subject GBl peptidic compounds include mutations at various non-core positions of the underlying GB 1 scaffold domain, e.g., variant amino acids at non-core positions within a GBl scaffold domain.
  • GBl scaffold domains of interest include, but are not limited to, those domains and sequences described in copending U.S. application 13/294,097, Gronenborn et al , FEBS Letters 398 (1996), 312-316; Kotz et al , Eur. J. Biochem. 271, 1623-1629 (2004); Malakaukas et al , Nature Structural Biology, 5(6), 1998, p.470-475; Minor Jr.
  • the GB1 scaffold domain is described by the following sequence: (T/S)Y(K/R)L(Z1)(Z1)(N/K)G(K/NA ⁇ /A)T(L/F)(K/S)GET(T/A/S)T(K/E)(A/T)(V/I)D(A/T/V)(A/E)(T /V)AE(K/Q)(A/E r/V)F(K/R)(Q/D)YA(N T)(A/D/E/K)N(G/N)(Z3)(D/T)G(E/V)W(A/T/S)YD(D/A/Y /T)ATKT(Z1)T(Z1)TE (SEQ ID NO:94), where each Zl is independently a hydrophobic residue.
  • the GB1 scaffold domain is described by the following sequence:
  • GB 1 scaffold domain includes the following sequence:
  • the GB1 scaffold domain includes the following sequence:
  • the GB1 scaffold domain includes the following sequence:
  • any one of the GB 1 scaffold domains described above may further include one or more additional residues at the N-terminal and/or C-terminal of the sequences shown.
  • the GB1 scaffold domains described above include one or more additional N-terminal residues.
  • the GB 1 scaffold domain includes a sequence of 54 or more residues, such as 55 or more, 56 or more, 57 or more, 58 or more, 59 or more or 60 residues or more.
  • the GB l scaffold domain includes 55, 56 or 57 residues.
  • GB 1 peptidic compounds include mutations at various non-core positions of the GB 1 scaffold domain.
  • a mutation in a scaffold domain may include a deletion, insertion, or substitution of an amino acid residue at any convenient position to produce a sequence that is distinct from the reference scaffold domain sequence.
  • the positions to be mutated are selected to minimize structural perturbations of the GB 1 scaffold domain and to ensure that the subject GB l peptidic compounds can maintain a folded state under physiological conditions.
  • Another aspect of the subject compounds is the selection of amino acid positions to be mutated such that the amino acids can form a potential binding surface in the GB 1 scaffold domain, whether or not the residues actually contact a target protein.
  • One way of determining whether an amino acid position is part of a potential binding surface involves examining the three dimensional structure of the GB 1 scaffold domain, using a computer program such as the UCSF Chimera program. Other ways include crystallographic and genetic mutational analysis. Any convenient method may be used to determine whether an amino acid position is part of a potential binding surface.
  • the mutations may be found at positions in the GB 1 scaffold domain where the residue is at least in part solvent exposed.
  • solvent exposed positions include the following residues (shown in bold):
  • TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: l).
  • the mutations of the parent GB l domain may be concentrated at one of several different potential binding surfaces of the scaffold domain.
  • Several distinct arrangements of mutations of the GB 1 scaffold domain at non-core positions of the hairpin-helix-hairpin scaffold domain are provided.
  • the majority of the mutations are at non-core positions of the parent GB l domain (e.g., solvent exposed or boundary positions) however in some cases one or more mutations (e.g., 1 or 2 mutations) may be located at hydrophobic core positions.
  • mutations at hydrophobic core positions may be tolerated without significantly disrupting the GB l scaffold structure.
  • core mutations are selected in a loop region.
  • mutations at boundary positions may also be tolerated without significantly disrupting the GB l scaffold structure.
  • solvent exposed residues located at a surface of the molecule not involved in target binding may be mutated to impart a desired property to the resulting GB l compounds, such as solubility, stability, bispecific binding to a second target of interest, pi, chemoselective functional group, a certain structural property, etc.
  • the sequence of the GBl scaffold domain is optimized for stability.
  • mutations at hydrophobic core and/or boundary positions are included to optimize the stability of a GB 1 peptidic structure.
  • optimization of the stability is performed when the GB l structure is disrupted by mutations at a target protein-binding surface. For example, one or more mutations in the variable domain of a GB 1 peptidic compound that provide for specific binding to a target protein may lead to disruption of the hydrophobic core structure of the compound.
  • one or more further mutations may be introduced, or optimization of the underlying scaffold sequence may be performed, to optimize stability and minimize any disruptions to the structure and produce a stable GB l peptidic compound that specifically binds to the target protein.
  • the one or more stabilizing mutations may include mutations to hydrophobic core and/or boundary residues, and may be located in a fixed domain or a variable domain of the structure.
  • the positions of the mutations in the parent GB 1 domain may be described herein either by reference to a structural motif or region, or by reference to a position number in the primary sequence of the domain. Any convenient alternate GB 1 peptidic domain sequence may be substituted for the underlying GBl scaffold sequence of the subject GBl compounds, and the positions of the mutations that define the subject compound may be transferred from one scaffold to another.
  • a first GBl scaffold domain sequence may be aligned with a second GB l scaffold domain sequence that is one or more amino acids longer or shorter.
  • the second GB 1 scaffold domain may have one or more additional amino acids at the N-terminal or C-terminal relative to the first GB 1 scaffold, or may have one or more additional amino acids in one of the loop regions of the structure.
  • a numbering scheme such as is described herein for insertion mutations may be used to relate two GB l scaffold domain sequences.
  • a subject GB l peptidic compound may include a hairpin-helix-hairpin domain described by formula (I):
  • PI and P2 are independently beta-hairpin domains and ocl is a helix domain and PI, ocl and P2 are connected independently by linking sequences of between 1 and 10 residues in length.
  • PI is ⁇ — ⁇ 2 and P2 is ⁇ 3— ⁇ 4 such that the compound is described by formula (II): ⁇ — ⁇ 2— ⁇ — ⁇ 3— ⁇ 4
  • each linking sequence is independently of 3, 4, 5, 6, 7 or 8 residues in length. In certain embodiments, each linking sequence is independently of 3, 4, 5, 6, 7 or 8 residues in length, such as 4 or 5 residues in length.
  • the linking sequences may form a loop or a turn structure.
  • the two antiparallel ⁇ -strands of a hairpin motif may be connected via a loop.
  • Mutations in a linking sequence that includes insertion or deletion of one or more amino acid residues may be tolerated without significantly disrupting the GB1 scaffold domain structure.
  • the subject compound in formulas (I) and (II), includes mutations in one or more linking sequences. In certain embodiments, 80% or more, 90% or more, 95% or more, or even 100% of the mutations are at positions within the regions of the linking sequences.
  • At least one of the linking sequences is one or more (e.g., such as 2 or more) residues longer in length than the corresponding linking sequence of the GB1 scaffold domain. In certain embodiments, in formulas (I) and (II), at least one of the linking sequences is one or more residues shorter in length than the corresponding linking sequence of the GB 1 scaffold domain.
  • one or more positions in the scaffold may be selected as positions at which to include insertion mutations, e.g., mutations that include the insertion of 1 or 2 additional amino acid residues in addition to the amino acid residue being substituted.
  • the insertion mutations are selected for inclusion in one or more loop regions, or at the N-terminal or C-terminal of the scaffold.
  • the positions of the variant amino acids that are inserted may be referred to using a letter designation with respect to the numbered position of the mutation, e.g., an insertion mutation of 2 amino acids at position 38 may be referred to as positions 38a and 38b.
  • the subject compound includes a mutation at position 38 that includes insertion of 0, 1 or 2 variant amino acids. In certain embodiments, the subject compound includes a mutation at position 19 that includes insertion of 0, 1 or 2 variant amino acids. In certain embodiments,
  • the subject compound includes a mutation at position 1 that include insertion of 2 variant amino acids, and at positions 19 and 47 that each include insertion of 0, 1 or 2 variant amino acids. In certain embodiments, the subject compound includes mutations at positions 9 and 38 that each include insertion of 0, 1 or 2 variant amino acids, and at position 55 that includes insertion of 1 variant amino acid. In certain embodiments, the subject compound includes a mutation at position 9 that includes insertion of 0, 1 or 2 variant amino acids, and at position 55 that includes insertion of 1 variant amino acid. In certain embodiments, the subject compound includes a mutation at position 1 that includes insertion of 1 variant amino acid, and at position 47 that includes insertion of 0, 1 or 2 variant amino acids.
  • the resulting GB 1 compound variants may be aligned with the parent GB l scaffold in different ways.
  • an insertion mutation including 2 additional variant amino acids at position 38 of the GBl scaffold may lead to GB l compound variants where the loop regions between the ocl and ⁇ 3 regions can be aligned with the GB 1 scaffold domain in two or more distinct ways.
  • the resulting GBl compounds may encompass various distinct loop sequences and/or structures that align differently with the parent GB 1 scaffold domain.
  • the various distinct loop sequences are produced when the insertion mutation is in a variable loop region (e.g. where most of the loop region is being mutated).
  • the subject compound includes 4 or more, such as, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more mutations at different positions of the hairpin-helix-hairpin domain.
  • the mutations may involve the deletion, insertion, or substitution of the amino acid residue at the position being mutated.
  • the mutations may include substitution with any naturally or non-naturally occurring amino acid, or an analog thereof.
  • the subject compound includes 3 or more different non-core mutations, such as, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more , 10 or more, 11 or more, or 12 or more different non-core mutations in a region outside of the ⁇ 1- ⁇ 2 region.
  • the subject compound includes 3 or more different non-core mutations, such as, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more or 11 or more different non-core mutations in the ocl region.
  • the subject compound includes 3 or more different non-core mutations, such as 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more different non- core mutations in the ⁇ 3- ⁇ 4 region.
  • the subject compound includes at least 5 or more different non-core mutations, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more different non-core mutations in the ⁇ 1- ⁇ 3 region.
  • the subject compound includes ten or more different mutations, where the ten or more different mutations are located at positions selected from the group consisting of positions 21-24, 26, 27, 30, 31, 34, 35, 37-41.
  • one or more mutations e.g., 1 or 2 mutations
  • one or more mutations are at boundary positions.
  • ten or more mutations are at solvent exposed positions.
  • the subject compound includes ten or more different mutations, where the ten or more different mutations are located at positions selected from the group consisting of positions 18-24, 26-28, 30-32, 34 and 35.
  • one or more mutations e.g., 1 mutation
  • ten or more mutations are at solvent exposed positions.
  • the subject compound includes ten or more different mutations, where the ten or more different mutations are located at positions selected from the group consisting of positions 1, 18-24 and 45-49.
  • one or more mutations e.g., 1 mutation
  • ten or more mutations are at solvent exposed positions.
  • the subject compound includes ten or more different mutations, where the ten or more different mutations are located at positions selected from the group consisting of positions 7-12, 36-41, 54 and 55.
  • one or more mutations e.g., 1 mutation
  • one or more mutations e.g., 2, 3 or 4 or more mutations
  • five or more mutations e.g., 6, 7, 8, 9 or more mutations
  • the subject compound includes ten or more different mutations, where the ten or more different mutations are located at positions selected from the group consisting of positions 3, 5, 7-14, 16, 52, 54 and 55.
  • one or more mutations e.g., 2, 3 or more mutations
  • nine or more mutations e.g., 10, 11, 12 or more mutations
  • the subject compound includes ten or more different mutations, where the ten or more different mutations are located at positions selected from the group consisting of positions 1, 3, 5, 7, 41, 43, 45-50 52 and 54.
  • one or more mutations e.g., 2 or more mutations
  • nine or more mutations e.g., 10, 11, 12 or more mutations
  • the subject compound includes five or more different mutations in the ocl region.
  • five or more different mutations are located at positions selected from the group consisting of positions 22-24, 26, 27, 30, 31, 34 and 35.
  • the subject compound includes ten or more different mutations in the ocl region. In certain embodiments, the ten or more different mutations are located at positions selected from the group consisting of positions 22-24, 26, 27, 28, 30, 31, 32, 34 and 35.
  • the subject compound includes three or more different mutations in the ⁇ 3- ⁇ 4 region. In certain embodiments, the three or more different mutations are located at positions selected from the group consisting of positions 41, 54 and 55. In certain embodiments, the three or more different mutations are located at positions selected from the group consisting of positions 52, 54 and 55.
  • the subject compound includes five or more different mutations in the ⁇ 3- ⁇ 4 region. In certain embodiments, the five or more different mutations are located at positions selected from the group consisting of positions 45-49.
  • the subject compound includes nine or more different mutations in the ⁇ 3- ⁇ 4 region. In certain embodiments, the nine or more different mutations are located at positions selected from the group consisting of positions 41, 43, 45-50 52 and 54.
  • the subject compound includes two or more different mutations in the region between the ocl and ⁇ 3 regions, e.g., mutations in the linking sequence between ocl and ⁇ 3.
  • the two or more different mutations are located at positions selected from the group consisting of positions 37-40.
  • the subject compound includes three or more, four or more, five or more, six or more, or ten or more different mutations in the ⁇ 1- ⁇ 2 region.
  • the ten or more different mutations in the ⁇ 1- ⁇ 2 region are located at positions selected from the group consisting of positions 3, 5, 7-14 and 16.
  • the GBl peptidic compound includes an intramolecular crosslink.
  • the intramolecular crosslink is a disulfide bond between two Cys residues of the GBl compound. Any convenient ligation chemistries, linking moieties and functional groups may be used to produce an intramolecular crosslink.
  • the subject compound is described by a formula independently selected from the group consisting of:
  • F1-V1-F2 (III); F3-V2-F4 (IV);
  • V9-F11-V10 V9-F11-V10 (VII).
  • variable regions of any one formula include a combined total of three or more different non-core mutations in a region outside of the ⁇ 1- ⁇ 2 region.
  • the subject compound is described by formula (III), where:
  • Fl comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence
  • TYKLILNGKTLKGETTTEA SEQ ID NO:2;
  • F2 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to an amino acid sequence TYDDATKTFTVTE (SEQ ID NO: 3); and
  • VI comprises a sequence that comprises 10 or more mutations (e.g., 11, 12, 13, 14 or 15 or more mutations) compared to the parent amino acid sequence VDAATAEKVFKQYANDNGVDGEW (SEQ ID NO:4).
  • VI comprises a sequence of the following formula: VXXXXAXXVFXXYAXXNXXXXXW (SEQ ID NO: 5), where each X is a variant amino acid.
  • Fl comprises the sequence
  • TYKLILNGKTLKGETTTEA (SEQ ID NO:2)
  • F2 comprises the sequence TYDDATKTFTVTE (SEQ ID NO:3)
  • VI comprises a sequence of the following formula:
  • VXXXXAXXVFXXYAXXNXXXXXW (SEQ ID NO: 6) where each X is independently selected from the group consisting of A, D, F, S, V and Y.
  • the mutation at position 19 in VI includes insertion of 0, 1 or 2 variant amino acids.
  • the subject compound is described by formula (IV), where:
  • F3 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence TYKLILNGKTLKGETT
  • F4 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to an amino acid sequence
  • V2 comprises a sequence that comprises 10 or more mutations (e.g., 11, 12, 13, 14 or 15 or more mutations) compared to the parent amino acid sequence TEAVDAATAEKVFKQYANDN (SEQ ID NO:9).
  • V2 comprises a sequence of the formula:
  • F3 comprises the sequence TYKLILNGKTLKGETT (SEQ ID NO:7)
  • F4 comprises the sequence GVDGEWTYDDATKTFTVTE (SEQ ID NO:8)
  • V2 comprises a sequence of the formula: TXXXXXXAXXXFXXXAXXN (SEQ ID NO: 11) where each X is independently selected from the group consisting of A, D, F, S, V and Y.
  • the mutation at position 3 of V2 includes insertion of 0, 1 or 2 variant amino acids.
  • the subject compound is described by formula (V), where:
  • F5 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence KLILNGKTLKGETT (SEQ ID NO: 12);
  • F6 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to an amino acid sequence
  • EKVFKQYANDNGVDGEWT (SEQ ID NO: 13);
  • F7 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to an amino acid sequence FTVTE (SEQ ID NO: 14);
  • V3 comprises a sequence that comprises one or more mutations (e.g., 2 or more mutations) compared to the parent amino acid sequence TY;
  • V4 comprises a sequence that comprises 3 or more mutations (e.g., 4, 5, 6 or 7 or more mutations) compared to the parent amino acid sequence TEAVDAATA (SEQ ID NO: 15); and
  • V5 comprises a sequence that comprises 3 or more mutations (e.g., 4 or 5 or more mutations) compared to the parent amino acid sequence YDDATKT (SEQ ID NO: 16).
  • V3 comprises a sequence of the formula XY
  • V4 comprises a sequence of the formula TXXXXXXA (SEQ ID NO: 17)
  • V5 comprises a sequence of the formula YXXXXT (SEQ ID NO: 18) where each X is a variant amino acid.
  • F5 comprises the sequence KLILNGKTLKGETT (SEQ ID NO: 12)
  • F6 comprises the sequence EKVFKQYANDNGVDGEWT (SEQ ID NO: 13)
  • F7 comprises the sequence FTVTE (SEQ ID NO: 14)
  • V3 comprises a sequence of the formula XY
  • V4 comprises a sequence of the formula TXXXXXXA (SEQ ID NO: 19)
  • V5 comprises a sequence of the formula YXXXXT (SEQ ID NO:20) where each X is independently selected from the group consisting of A, D, F, S, V and Y.
  • the mutation at position 1 of V3 includes insertion of + 2 variant amino acids
  • the subject compound is described by formula (VI), where:
  • F8 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence TYKLI (SEQ ID NO:21);
  • F9 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence
  • F10 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence TYDDATKTFT (SEQ ID NO:23);
  • V6 comprises a sequence that comprises 3 or more mutations (e.g., 4, 5 or 6 or more mutations) compared to the parent amino acid sequence LNGKTLKG (SEQ ID NO:24);
  • V7 comprises a sequence that comprises 3 or more mutations (e.g., 4, 5 or 6 or more mutations) compared to the parent amino acid sequence DNGVDGEW (SEQ ID NO:25);
  • V8 comprises a sequence that comprises one or more mutations (e.g., 2 or more mutations) compared to the parent amino acid sequence VTE.
  • V6 comprises a sequence of the formula
  • V7 comprises a sequence of the formula DXXXXXW (SEQ ID NO: 27)
  • V8 comprises a sequence of the formula VXX where each X is a variant amino acid.
  • F8 comprises the sequence TYKLI (SEQ ID NO:21), F9 comprises the sequence ETTTEAVDAATAEKVFKQYAN (SEQ ID NO:22), F10 comprises the sequence TYDDATKTFT (SEQ ID NO:23), V6 comprises a sequence of the formula LXXXXXG (SEQ ID NO:28), V7 comprises a sequence of the formula DXXXXXW (SEQ ID NO:29), and V8 comprises a sequence of the formula VXX where each X is independently selected from the group consisting of A, D, F, S, V and Y.
  • the mutations at position 4 of V6 and V7 each include insertion of 0, 1 or 2 variant amino acids, and the mutation at position 3 of V8 includes insertion of 1 variant amino acid.
  • the subject compound is described by formula (VII), where:
  • Fl l comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to an amino acid sequence
  • V9 comprises a sequence that comprises 6 or more mutations (e.g., 7, 8, 9, 10 or 11 or more mutations) compared to the parent amino acid sequence TYKLILNGKTLKGETTT (SEQ ID NO:31); and
  • V10 comprises a sequence that comprises 2 or more mutations (e.g., 3 or more mutations) compared to the parent amino acid sequence FTVTE (SEQ ID NO:32).
  • V9 comprises a sequence of the formula
  • V10 comprises a sequence of the formula FXVXX (SEQ ID NO:34), where each X is a variant amino acid.
  • Fl l comprises the sequence
  • V9 comprises a sequence of the formula TYXLXLXXXXXXXTXT (SEQ ID NO:35), and V10 comprises a sequence of the formula FXVXX (SEQ ID NO:36), where each X is independently selected from the group consisting of A, D, F, S, V and Y.
  • the mutation at position 9 of V9 includes insertion of 0, 1 or 2 variant amino acids
  • the mutation at position 5 of V10 includes insertion of 1 variant amino acid.
  • the subject compound is described by formula (VIII), where:
  • F12 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence
  • VI 1 comprises a sequence that comprises 3 or more mutations (e.g., 4 or more mutations) compared to the parent amino acid sequence TYKLILNG (SEQ ID NO: 38);
  • V12 comprises a sequence that comprises 5 or more mutations (e.g., 6, 7, 8, 9 or 10 or more mutations) compared to the parent amino acid sequence GEWTYDDATKTFTVTE (SEQ ID NO:39).
  • VI 1 comprises a sequence of the formula
  • V12 comprises a sequence of the formula
  • F12 comprises the sequence
  • VI 1 comprises a sequence of the formula XYXLXLXG (SEQ ID NO:42), and V12 comprises a sequence of the formula
  • GXWXYXXXXXXFXVXE (SEQ ID NO:43), where each X is independently selected from the group consisting of A, D, F, S, V and Y.
  • the mutation at position 8 of V12 includes insertion of 0, 1 or 2 variant amino acids
  • the mutation at position 1 of VI 1 includes insertion of 2 variant amino acids.
  • the GB l peptidic compound includes a peptidic sequence of between 30 and 90 residues, such as between 30 and 80 residues, between 40 and 70 residues, between 45 and 60 residues, between 54 and 60 residues, or between 55 and 58 residues. In certain instances, the GBl peptidic compound includes a peptidic sequence of between 55 and 90 residues, such as between 60 and 80 residues, between 70 and 80 residues. In certain embodiments, the GBl peptidic compound includes a peptidic sequence of 55 or more residues, such as 60 or more residues, 65 or more residues, 70 or more residues, 75 or more residues, 80 or more residues, 90 or more residues, or even 100 or more residues.
  • the GBl peptidic compound includes a GB l motif having a peptidic sequence of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 residues.
  • the GBl motif is a sequence of 54, 55, 56 or 57 residues, such as 55 or 56 residues.
  • the subject GB l compounds may be further mutated to incorporate residues at surface positions of the GB 1 scaffold domain not involved in contacting the target protein.
  • the residues may be selected to confer on the resulting GB 1 compound an enhancing property, such as solubility, stability, pi, aggregation, reduced non-specific binding, specific binding to a second target protein.
  • the positions of the mutations may selected so as to minimize any disruption to the structure of the GBl motif or specific binding to the target protein, e.g., by selecting positions on opposite sides of the scaffold from the binding surface.
  • the GB l motif includes two or more, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more surface mutations at positions that are not part of the binding surface to the target protein.
  • the two or more surface mutations may be located at positions in the GB l motif including, but not limited to, position 1, 3, 5, 7, 9, 10, 12, 14, 16, 52, 54, as depicted in Figure 8.
  • surface mutations include mutating the residue to a hydrophilic residue. In some cases, surface mutations include mutating the residue to a charged residue. In some cases, surface mutations include mutating the residue to a basic residue. In some cases, surface mutations include mutating the residue to an acidic residue. Such surface mutations may include residues independently selected from Arg, Lys and His. Such surface mutations may include residues independently selected from Glu and Asp. Such surface mutations may include Ser and Gly residues.
  • the GB l motif includes 2 or more arginine or lysine surface mutations, such as 3 or more, 4 or more, 5 or more, or even 6 or more arginine or lysine surface mutations.
  • the GBl motif includes a sequence having 60% or more (e.g., 70, 80, 90% or more) amino acid sequence identity to the amino acid sequence set forth in one of the sequences of RFX031 to RFX034 depicted in Figure 7.
  • the subject compound includes a GB l scaffold domain and a variable domain.
  • the variable domain may be a part of the GB 1 scaffold domain and may be either a continuous or a discontinuous sequence of residues.
  • a variable domain that is defined by a discontinuous sequence of residues may include contiguous variant amino acids at positions that are arranged close in space relative to each other in the structure of the compound.
  • the variable domain may form a potential binding interface of the subject compound.
  • the variable domain may define a binding surface area of a suitable size for forming protein-protein interactions of high affinity (e.g., 300nM or less, such as 100 nM or less, 30nM or less, lOnM or less, 3nM or less, or 1 nM or less) and specificity.
  • the variable domain may include a surface area of between 600 and 1800 A 2 , such as between 800 and 1600 A 2 , between 1000 and 1400 A 2 , between 1100 and 1300 A 2 , or about 1200 A 2 .
  • Any GBl scaffold as defined herein may be selected as a scaffold for a subject compound.
  • the positions of the mutations in the GB 1 scaffold domain may be selected as described herein.
  • the nature of the mutation at each variant amino acid position may be selected, e.g., substitution with any naturally occurring or non naturally occurring amino acid and may confer on the compound variant a desirable property (e.g., increased solubility, stability or specific binding to a target molecule).
  • Certain variant amino acid positions may be selected as positions where mutations can include the insertion or deletion of amino acids, e.g., the insertion of 1 or 2 amino acids where the variant amino acid position occurs in a loop or turn region of the scaffold.
  • the mutations can include the insertion or amino acids at one or more positions selected from positions 1, 9, 19, 38, 47 and 55.
  • the individual sequences of the subject GB 1 motifs can be determined.
  • To the GB 1 motifs may be added a N-terminal extension moiety and/or a C-terminal extension moiety, e.g., as described above.
  • a subject compound may include two distinct groups of mutations that define two distinct potential binding surfaces of the GB 1 scaffold domain.
  • the two distinct groups of mutations may be described by any two of formulas (III) to (VIII), as defined above, e.g., the subject compound includes two distinct arrangements of variable regions that define two distinct variable domains in the same compound.
  • the two distinct groups of mutations include a first group of mutations defined by one of formulas (III)-(V) and a second group of mutations defined by one of formulas (VI)-(VIII).
  • the subject compound may be bifunctional in the sense that the compound has specific binding properties for two target molecules.
  • the compound may include two potential binding surfaces for the same target molecule, where the overall binding affinity of the compound may be modulated by an avidity effect.
  • the compound may include a first potential binding surface for a first target molecule and a second binding surface that recruits a second target molecule.
  • GB 1 peptidic compounds that have been optimized for binding to a target molecule by affinity maturation, e.g., second generation GB 1 peptidic compounds based on a parent GB 1 peptidic compound that binds to a certain target molecule, where the second generation GB 1 peptidic compounds are optimized for binding affinity and specificity.
  • the subject peptidic compounds specifically bind to a target protein with high affinity, e.g., as determined by an SPR binding assay or an ELISA assay.
  • the subject compounds may exhibit an affinity for a target protein of 1 uM or less, such as 300nM or less, ⁇ or less, 30nM or less, ⁇ or less, 5 nM or less, 2 nM or less, 1 nM or less, 600pM or less, 300pM or less, or even less.
  • the subject peptidic compounds may exhibit a specificity for a target protein, e.g., as determined by comparing the affinity of the compound for the target protein with that for a reference protein (e.g., an albumin protein), that is 5: 1 or more 10: 1 or more, such as 30: 1 or more, 100: 1 or more, 300: 1 or more, 1000: 1 or more, or even more.
  • a reference protein e.g., an albumin protein
  • the GB 1 peptidic compounds may be optimized for any desirable property, such as protein folding, protease stability, thermostability, compatibility with a pharmaceutical formulation, etc. Any convenient methods may be used to select second generation GB 1 peptidic compounds, e.g., structure-activity relationship (SAR) analysis, affinity maturation methods, or phage display methods.
  • SAR structure-activity relationship
  • GB 1 peptidic compounds that have high thermal stability. In some cases, the compounds have a melting temperature of 50°C or more, such as 60°C or more, 70°C or more, 80°C or more, or even 90°C or more). Also provided are GB 1 peptidic compounds that have high protease stability.
  • the GB1 peptidic compounds are resistant to proteases and have long serum and/or saliva half -lives.
  • the D-GB1 peptidic compounds have 10% or greater, such as 20% or greater, 30% or greater, 40% or greater, 50% or greater, 100% or greater, 200% or greater stability to a protease compared to a L-peptidic compound, in a protease stability assay such as that described by Tugyi et al. (2005), "Partial D-amino acid substitution: Improved enzymatic stability and preserved Ab recognition of a MUC2 epitope peptide", PNAS, 102, 413-418; and Fischer, P M. (2003).
  • half-life refers to the time required for a measured parameter, such the potency, activity and effective concentration of a compound to fall to half of its original level, such as half of its original potency, activity, or effective concentration at time zero.
  • the parameter such as potency, activity, or effective concentration of a polypeptide molecule is generally measured over time.
  • half-life can be measured in vitro or in vivo.
  • the GB1 peptidic compound has a half -life of 1 hour or longer, such as 2 hours or longer, 6 hours or longer, 12 hours or longer, 1 day or longer, 2 days or longer, 7 days or longer, or even longer.
  • Stability in human blood may be measured by any convenient method, e.g., by incubating the compound in human EDTA blood or serum for a designated time, quenching a sample of the mixture and analyzing the sample for the amount and/or activity of the compound, e.g., by HPLC-MS, by an activity assay, e.g., as described herein.
  • GB 1 peptidic compounds that have low immunogenicity, e.g., are non- immunogenic.
  • the D-peptidic compounds have low immunogenicity compared to an L-peptidic compound.
  • the D-peptidic compounds are 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 70% or less, or 90% or less immunogenic compared to an L-peptidic compound, in an immunogenicity assay such as that described by Dintzis et al., "A Comparison of the Immunogenicity of a Pair of Enantiomeric Proteins" Proteins: Structure, Function, and Genetics 16:306-308 (1993).
  • the affinity maturation of a subject compound may include holding a fraction of the variant amino acid positions as fixed positions while the remaining variant amino acid positions are varied to select optimal amino acids at each position.
  • a parent GB 1 peptidic compound that specifically binds to a certain target molecule may be selected as a scaffold for an affinity maturation compound.
  • a number of affinity maturation compounds are prepared that include mutations at limited subsets of the variant amino acid positions of the parent (e.g., mutations at 4 of 15 variable positions), while the rest of the variant positions are held as fixed positions.
  • the positions of the mutations may be tiled through the scaffold sequence to produce a series of compounds such that mutations at every variant position is represented and a diverse range of amino acids are substituted at every position (e.g., all 20 naturally occurring amino acids). Mutations that include deletion or insertion of one or more amino acids may also be included at variant positions of the affinity maturation compounds.
  • An affinity maturation compound may be prepared and screened using any convenient method, e.g., phage display library screening, to identify second generation compounds having an improved property, e.g., increased binding affinity for a target molecule, protein folding, protease stability, thermostability, compatibility with a pharmaceutical formulation, etc.
  • the affinity maturation of a subject compound may include holding most or all of the variant amino acid positions in the variable regions of the parent GB 1 compound as fixed positions, and introducing contiguous mutations at positions adjacent to these variable regions. Such mutations may be introduced at positions in the parent GB 1 compound that were previously considered fixed positions in the original GB 1 scaffold domain. Such mutations may be used to optimize the GB 1 compound variants for any desirable property, such as protein folding, protease stability, thermostability, compatibility with a pharmaceutical formulation, etc.
  • the subject compound specifically binds to a VEGF protein, such as VEGF-A, VEGF-B, VEGF-C, VEGF-D or VEGF-E, where the VEGF protein may be a synthetic D- protein or a L-protein.
  • a VEGF protein such as VEGF-A, VEGF-B, VEGF-C, VEGF-D or VEGF-E, where the VEGF protein may be a synthetic D- protein or a L-protein.
  • the VEGF protein is a D-protein and the subject compound is L- peptidic. In certain embodiments, the VEGF protein is a L-protein and the subject compound is D- peptidic. In some embodiments, the VEGF protein is VEGF-A. In certain embodiments, the subject compound is described by formula (II) as defined above, and includes a GB 1 scaffold domain and a variable domain, where the variable domain encompasses at least part of al. In certain embodiments, the variable domain encompasses positions 21 to 41 of the scaffold domain.
  • the subject VEGF-binding compound is described by formula (III) as defined above, where the subject VEGF-binding compound includes a GB 1 scaffold domain and a variable domain.
  • the GB 1 scaffold domain includes a first sequence having 80% or more (e.g., 90% or more) amino acid sequence identity to an amino acid sequence set forth in SEQ ID NO:2 of US2012/0178682, and a second sequence having 80% or more (e.g., 90% or more) amino acid sequence identity to an amino acid sequence set forth in SEQ ID NO:3.
  • variable domain comprises a sequence having 80% or more (e.g., 90% or more) amino acid sequence identity to an amino acid sequence set forth in one of SEQ ID NOs: 44-75 of US2012/0178682. In certain embodiments, the variable domain comprises a sequence having 80% or more (e.g., 90% or more) amino acid sequence identity to an amino acid sequence set forth at positions 20-42 in one of SEQ ID NOs:76-162 and 529-809 of US2012/0178682.
  • the mutation at position 38 in VI includes insertion of 0, 1 or 2 variant amino acids.
  • the subject VEGF-binding compound comprises a sequence having 80% or more amino acid sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 98% or more amino acid sequence identity) to an amino acid sequence set forth in one of SEQ ID NOs:76-162 and 529-809 of US2012/0178682.
  • the subject VEGF-binding compound comprises a sequence having 80% or more amino acid sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 98% or more amino acid sequence identity) to that of clone E01 (SEQ ID NO:79 of US2012/0178682).
  • the VEGF-binding compound further includes one or more point mutations, selected from V22L, Y23F, D24G, D27E, V31A, A34S, S35R, S35E, S35G, S38K, S38G, D39F, F40I and D41V.
  • the subject VEGF-binding compound is described by formula (III) (F1-V1-F2), where Fl comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequences
  • TYKLILNGKTLKGETTTEA SEQ ID NO:2.
  • the subject VEGF-binding compound is described by formula (III) (F1-V1-F2), where Fl comprises a sequence TYKX 1 ILNGKTX 2 KX 3 EX 4 TX 5 EA (SEQ ID NO:99), where X 1 is selected from M and L, X 2 is selected from R, Q, M, L, C, S, Q, F, W, V, A, K, X 3 is selected from F, R, D, S, W, D, G, A, E and C, X 4 is selected from T, I, V, L, M, A, H, Y, C and F, and X 5 is selected from T, I, A, L, V, M and V.
  • Fl comprises a sequence TYKX 1 ILNGKTX 2 KX 3 EX 4 TX 5 EA (SEQ ID NO:99)
  • X 1 is selected from M and L
  • X 2 is selected from R, Q, M, L, C, S, Q, F, W, V, A
  • X 2 is L.
  • X 3 is S or G.
  • X 4 is C or F.
  • X 5 is T or L.
  • Fl comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence:
  • Fl comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence: TYKLILNGKTLKGEFTLEA (SEQ ID NO: 101).
  • the subject VEGF-binding compound is described by formula (III) (F1-V1-F2), where F2 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence:
  • the subject VEGF-binding compound is described by formula (III) (F1-V1-F2), where F2 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence: (T/R/I/V)YDDATKTFTVTE (SEQ ID NO: 102).
  • the subject VEGF-binding compound is described by formula (III) (F1-V1-F2), where VI comprises a sequence of the following formula:
  • VDXXXAFXVLXXCAXXXFXXIXXW (SEQ ID NO: 103), where each X is a variant amino acid.
  • the Cys residue at position 32 in VI forms an intramolecular crosslink.
  • the variant amino acid at position 22 in VI is selected from L, F, I, M, R, V and W; the variant amino acid at position 23 in VI is selected from F, I, N and Y; the variant amino acid at position 24 in VI is selected from D, A, E, H, N, Q, T and V; the variant amino acid at position 27 in VI is selected from D, A, E, G, H and Q; the variant amino acid at position 30 in VI is selected from F, Y, L, S and W; the variant amino acid at position 31 in VI is selected from V, A, D, E, G, I, L, M, P, S and T; the variant amino acid at position 34 in VI is selected from A, D, E, I, S, T,
  • the variant amino acid at position 35 in VI is selected from A, D, F, N, R, S, T and Y
  • the variant amino acid at position 36 in VI is selected from N, E, H, I, K, Q, R, S, T, V and Y
  • the variant amino acid at position 38 in VI is selected from S, A, D, E, F, G, L, N, P, Q, R, T and Y
  • the variant amino acid at position 39 in VI is selected from F, D, E, H, I, L, N, V and Y
  • the variant amino acid at position 41 in VI is selected from D, E, A, N, Q, S and V
  • the variant amino acid at position 41a in VI is selected from D, E, A, N and S.
  • the variant amino acid at position 22 in VI is selected from L, F, I, M, R, V and W; the variant amino acid at position 23 in VI is selected from F and Y; the variant amino acid at position 24 in VI is selected from D and E; the variant amino acid at position 27 in VI is selected from D and E; the variant amino acid at position 30 in VI is selected from F and Y (e.g., F); the variant amino acid at position 31 in VI is selected from V, A, D, E, G, I, L, M, P, S and T; the variant amino acid at position 34 in VI is selected from A, E, S and T (e.g., A); the variant amino acid at position 35 in VI is selected from A, N, R, S and Y (e.g., S); the variant amino acid at position 36 in VI is selected from N, H, K, Q, S, V and Y (e.g., N); the variant amino acid at position 38 in VI is selected from S, A, D, E, F
  • VI is selected from D, E, A, N, Q, S and V; and the variant amino acid at position 41a in VI is selected from D, E, A, N and S.
  • the subject VEGF-binding compound is described by formula (III) (F1-V1-F2), where:
  • Fl comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence:
  • TYKMILNGKTLKSECTTEA (SEQ ID NO: 104);
  • F2 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence: TYDDATKTFTVTE (SEQ ID NO: 3); and
  • VI comprises a sequence of the following formula:
  • VI comprises a sequence of the following formula:
  • the subject VEGF-binding compound comprises a sequence having 80% or more amino acid sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 98% or more amino acid sequence identity) to the amino acid sequence set forth in SEQ ID NO: 107 (RFX- 002 sequence)
  • the subject VEGF-binding compound is described by formula (III) (F1-V1-F2), where VI comprises a sequence of the following formula:
  • the variant amino acid at position 22 in VI is selected from L, F, M, Q and V; the variant amino acid at position 23 in VI is selected from F, H and Y; the variant amino acid at position 24 in VI is selected from D, E and H; the variant amino acid at position 27 in VI is selected from D, E and G; the variant amino acid at position 30 in VI is selected from F and Y; the variant amino acid at position 31 in VI is selected from V, A, D, E, G and S; the variant amino acid at position 34 in VI is selected from A, G, S and T; the variant amino acid at position 35 in VI is selected from S, A, N and L; the variant amino acid at position 36 in VI is selected from N, A, E, H, I, K, Q, R, S, T and Y; the variant amino acid at position 38 in VI is selected from
  • the variant amino acid at position 22 in VI is selected from L and V; the variant amino acid at position 23 in VI is F; the variant amino acid at position 24 in VI is selected from D and E; the variant amino acid at position 27 in VI is D; the variant amino acid at position 30 in VI is F; the variant amino acid at position 31 in VI is selected from V, A, D, E and G the variant amino acid at position 34 in VI is selected from A, S and T; the variant amino acid at position 35 in VI is selected from S, A and N; the variant amino acid at position 36 in VI is selected from N, H, I, K, T and Y; the variant amino acid at position 38 in VI is selected from S, A, L, P and Y; the variant amino acid at position 39 in VI is selected from F, D, I, L, Q, S, T,
  • the variant amino acid at position 40 in VI is F; and the variant amino acid at position 41 in
  • the subject VEGF-binding compound is described by formula (III) (F1-V1-F2), where:
  • Fl comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence:
  • TYKLILNGKTLKGEFTLEA SEQ ID NO: 109
  • F2 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence: TYDDATKTFTVTE (SEQ ID NO: 3); and
  • VI comprises a sequence of the following formula:
  • VI comprises a sequence of the following formula: VDLF(D/E)AFDVFF(V/G)YA(A/S/T)(S/A)(N/H)FS(F/D/V/Y)FDDW (SEQ ID NO: 111).
  • the subject VEGF-binding compound comprises a sequence having 80% or more amino acid sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 98% or more amino acid sequence identity) to the amino acid sequence set forth in SEQ ID NO: 376 of US2012/0178682 (RFX-025 sequence)
  • the subject VEGF-binding compound comprises a sequence having 80% or more amino acid sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 98% or more amino acid sequence identity) to an amino acid sequence set forth in one of SEQ ID NOs:76-162 and 529-809 of Sidhu et al. Application No. 13/294,097 (US Publication No. 2012/0178682) (see Figures 9-20 of US2012/0178682, the disclosure of which is herein incorporated by reference).
  • the VEGF protein is a D-protein and the subject compound is L-peptidic.
  • the VEGF protein is a L-protein and the subject compound is D-peptidic.
  • the VEGF protein is a L-protein and the subject compound is L- peptidic. In certain embodiments, the VEGF protein is a D-protein and the subject compound is D- peptidic. In some embodiments, the VEGF protein is VEGF-A. In certain embodiments, in formula (II), the variable domain of the subject VEGF-binding compound encompasses at least part of ⁇ and at least part of ⁇ 4. In certain embodiments, the variable domain encompasses positions 3 to 16 and 52 to 55 of the GB1 scaffold domain.
  • the subject VEGF-binding compound is described by formula (VII) (V9-F11-V10) as defined above, where:
  • Fll comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to an amino acid sequence set forth in SEQ ID NO:30;
  • V9 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to an amino acid sequence set forth in one of SEQ ID NOs: 163-167 of US2012/0178682, the disclosure of which is herein incorporated by reference, or to an amino acid sequence set forth at positions 1-17 of SEQ ID NOs:179-228 and 810- 864 of US2012/0178682, the disclosure of which is herein incorporated by reference; and
  • V10 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to an amino acid sequence set forth in one of SEQ ID NOs: 168-172 of US2012/0178682, the disclosure of which is herein incorporated by reference or to an amino acid sequence set forth at positions 51-55 of SEQ ID NOs:179-228 and 810- 864 of US2012/0178682, the disclosure of which is herein incorporated by reference.
  • the subject VEGF-binding compound comprises a sequence having 80% or more amino acid sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 98% or more amino acid sequence identity) to an amino acid sequence set forth in one of SEQ ID NOs: 173- 228 and 810-864 of US2012/0178682, the disclosure of which is herein incorporated by reference ( Figures 21-24 of US2012/0178682).
  • the VEGF protein is a L-protein and the subject compound is L- peptidic.
  • the VEGF protein is VEGF-A.
  • the subject compound is described by formula (II) as defined above, and includes a GB 1 scaffold domain and a variable domain, where the variable domain encompasses at least part of cel . In certain embodiments, the variable domain encompasses positions 21 to 41 of the GB 1 scaffold domain.
  • the subject VEGF-binding compound is described by formula (III) as defined above, where:
  • Fl comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to the amino acid sequence
  • TYKLILNGKTLKGETTTEA SEQ ID NO:2;
  • F2 comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to an amino acid sequence TYDDATKTFTVTE (SEQ ID NO: 3); and
  • VI comprises a sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) amino acid sequence identity to an amino acid sequence set forth in SEQ ID NO :229 (VDFYYAFS VFFS YASSNSSD YVS W) .
  • the subject VEGF-binding compound comprises a sequence having 80% or more amino acid sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 98% or more amino acid sequence identity) to an amino acid sequence set forth in one of SEQ ID NO: 112 (T YKLILNGKTLKGETTTEA VDFYYAFS VFFS YASSNSSD YVS WTYDD ATKTFTVTE) .
  • the VEGF protein is a L-protein and the subject compound is L-peptidic.
  • the GB1 motif may be expanded to include one or more, such as two or more additional residues at the N-terminal. Such one or more additional residues may be considered part of the GB 1 structural motif. Any convenient residues may be included at the N-terminal of the GB1 scaffold domain. In certain embodiments, the one or more additional residues is an Asp or a Glu amino acid residue.
  • polynucleotides that encode GB 1 peptidic compounds as described above.
  • the polynucleotide encodes a GB1 peptidic compound that includes at least 3 mutations at non-core positions in a region outside of the ⁇ 1- ⁇ 2 region.
  • the polynucleotide encodes a GB1 peptidic compound that includes between 30 and 80 residues, such as between 40 and 70, between 45 and 60 residues, or between 50 and 56 residues. In certain embodiments, the polynucleotide encodes a GB1 peptidic compound that includes a peptidic sequence of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 residues. In certain embodiments, the peptidic sequence is of 54, 55, 56 or 57 residues, such as 55 or 56 residues.
  • the polynucleotide is a replicable expression vector that includes a nucleic acid sequence encoding a GB 1 peptidic compound that may be expressed in a protein expression system. In certain embodiments, the polynucleotide is a replicable expression vector that includes a nucleic acid sequence encoding a gene fusion, where the gene fusion encodes a fusion protein including the GB 1 peptidic compound fused to all or a portion of a viral coat protein.
  • the subject polynucleotides are capable of being expressed and displayed in a cell-based or cell-free display system. Any convenient display methods may be used to display GB1 peptidic compounds encoded by the subject polynucleotides, such as cell-based display techniques and cell-free display techniques.
  • cell-based display techniques include phage display, bacterial display, yeast display and mammalian cell display.
  • cell-free display techniques include niRNA display and ribosome display.
  • a subject polynucleotide is expressed in a cell to produce a GB1 peptidic compound that specifically binds to a target within the cell, thereby modulating the activity of the target.
  • the modulation of the activity of the target within the cell imparts one or more desirable traits on the cell, such as, tolerance to an environmental stimulus, pathogen or disease; increased size, or modified growth rate.
  • the cell is a cell in a plant.
  • the target is a transcription factor, a pathogen or a disease related target protein.
  • Methods of making the subject compounds include, but are not limited to, protein expression methods, solid phase peptide synthesis methods, solution phase synthesis methods and native chemical ligation methods.
  • the subject compounds are prepared in a protein expression system.
  • the nucleic acids encoding a subject compound may be introduced directly into a host cell, and the cell incubated under conditions sufficient to induce expression of the encoded compound.
  • Any convenient cell for expression may be used as a host cell.
  • yeast, insect, plant, etc. cells.
  • the subject compounds are prepared using stepwise solid phase peptide synthesis methods, e.g., such as the stepwise addition of amino acids in a solid-phase Merrifield-type synthesis. Such methods may be used to prepare compounds of high purity that are free from undesirable side products.
  • D-amino acids or protected D- amino acids are utilized rather than the L-amino acids.
  • Any convenient protecting group strategies may be used such as, but are not limited to, Fmoc solid-phase peptide synthesis and Boc solid-phase peptide synthesis strategies.
  • Boc solid-phase peptide synthesis a Boc-amino protecting group is used at the amino terminal and benzyl or benzyl-based or other convenient protecting groups may be used for the protection of sidechain functional groups.
  • Fmoc solid-phase peptide synthesis a Fmoc-amino protecting group is used at the amino terminal and tert-butyl or benzyl-based or other convenient protecting groups may be used for protection of sidechain functional groups.
  • the subject compounds are prepared using solution phase chemical synthesis methods, such as but not limited to, the hybrid method.
  • the hybrid method includes the solid phase synthesis of protected peptide segments, that may be synthesized and purified on a large scale. The protected segments may then be covalently condensed in solution using any convenient solution phase method, before final deprotection.
  • the hybrid method was used in the manufacture of the peptide drug Fuzeon.
  • the subject compounds are prepared by the assembly of polypeptide building blocks using native chemical ligation methods.
  • two or more polypeptide segments are first synthesized, that contain termini adapted for native chemical ligation or for kinetically controlled ligation.
  • stepwise chemical synthesis and cleavage from their respective solid phase resins, and after purification e.g. by reverse phase HPLC two of the two or more polypeptides are mixed and reacted to join the adapted termini and form a larger, linear polypeptide that includes the two polypeptides linked by a native amide bond.
  • Further native chemical ligation and/or kinetically controlled ligation reactions can then be performed to add further polypeptide segments in a convergent synthetic strategy.
  • the method includes: synthesizing 2 or more segments of the GB 1 peptidic compound, where a first segment comprises a C-terminal thioester, or precursor thereof, and a second segment includes a N-terminal cysteine residue, or protected version thereof; and contacting the two or more segments under conditions by which the C-terminal thioester of the first segment ligates to the N-terminal cysteine of the second segment.
  • the method further includes desulfurizing the N-terminal cysteine residue of the second segment to produce an alanine residue at that position of the GB 1 compound.
  • the 2 or more segments are 3 segments.
  • a first segment includes residues 1-18 of the GB 1 motif
  • a second segment includes residues 19-33 of the GB1 motif
  • a third segment includes residues 34-56 of the GB1 motif.
  • a subject compound may be optionally purified or used without further purification. Purification may be performed using any convenient method, for example, by chromatography (e.g., RP-HPLC, ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other convenient technique for the purification of proteins.
  • chromatography e.g., RP-HPLC, ion exchange, affinity, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other convenient technique for the purification of proteins.
  • the peptidic compound that is produced exists in a random coil or an unfolded state.
  • the peptidic compound may then be folded using any convenient method, such that the compound folds from the random coil or unfolded state into a characteristic three-dimensional structure.
  • folding the compound includes dissolving the compound in an aqueous buffer under conditions that mimic physiological conditions (e.g., conditions of pH, ionic strength, temperature, and the like) and allowing the compound to fold into a characteristic three-dimensional structure in solution over a period of time (e.g., 2 days).
  • the progress of folding of the compound may be followed using any convenient methods, such as HPLC, circular dichroism, etc.
  • the above-described compounds may be employed in a variety of methods.
  • One such method includes contacting a subject compound with a target of the compound under conditions suitable for binding of the target to produce a complex.
  • a method of blocking binding of a ligand to its receptor is provided.
  • the method includes administering a GB 1 peptidic compound to a subject, where the compound binds to either the receptor or the ligand in said subject and blocks binding thereof.
  • a subject compound may inhibit at least one activity of its target in the range of 10% to 100%, e.g., by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.
  • a subject compound may inhibit its target with an IC 50 of 1 x 10 "5 M or less (e.g., 1 x 10 "6 M or less, 1 x 10 "7 M or less, 1 x 10 "8 M or less, 1 x 10 "9 M or less, 1 x 10 "10 M or less, or 1 x 10 "11 M or less).
  • a subject compound may inhibit its target with an IC 20 of 1 x 10 "6 M or less (e.g., 500nM or less, 200nM or less, ⁇ or less, 30nM or less, lOnM or less, 3nM or less, orl nM or less).
  • a subject compound may inhibit its target with an ICi 0 of 1 x 10 "6 M or less (e.g., 500nM or less, 200nM or less, ⁇ or less, 30nM or less, lOnM or less, 3nM or less, orl nM or less).
  • a subject compound may have an ED 50 of less than 1 ⁇ g/mouse (e.g., 1 ng/mouse to about 1 ⁇ g/mouse).
  • the protocols that may be employed in these methods are numerous, and include but are not limited to cell-free assays, e.g., binding assays; cellular assays in which a cellular phenotype is measured, e.g., gene expression assays; and in vivo assays that involve a particular animal (which, in certain embodiments may be an animal model for a condition related to the target).
  • the assay may be a vascularization assay.
  • the target protein is VEGF and the compound inhibits VEGF dependent cellular proliferation.
  • the target protein is VEGF and the compound inhibits VEGFR2 phosphorylation.
  • the subject method is an in vitro method that includes contacting a sample with a subject compound that specifically binds with high affinity to a target molecule.
  • the sample is suspected of containing the target molecule and the subject method further comprises evaluating whether the compound specifically binds to the target molecule.
  • the target molecule is a naturally occurring L-protein and the compound is D- peptidic.
  • the subject compound is a modified compound that includes a label, e.g., a fluorescent label
  • the subject method further includes detecting the label, if present, in the sample, e.g., using optical detection.
  • the compound is modified with a support, such that any sample that does not bind to the compound may be removed (e.g., by washing).
  • the specifically bound target protein if present, may then be detected using any convenient means, such as, using the binding of a labeled target specific probe or using a fluorescent protein reactive reagent.
  • the sample is known to contain the target protein.
  • the target protein is a synthetic D-protein and the compound is L- peptidic.
  • the subject method includes administering to a subject a compound that specifically binds with high affinity to a target molecule.
  • the subject is human.
  • the compound is administered as a pharmaceutical preparation.
  • the compound is D-peptidic.
  • the compound is a modified compound that includes a detectable label, and the method further includes detecting the label in the subject.
  • the selection of the label depends on the means of detection. Any convenient labeling and detection systems may be used in the subject methods, see e.g., Baker, "The whole picture," Nature, 463, 2010, p977-980.
  • the compound includes a fluorescent label suitable for optical detection.
  • the compound includes a radiolabel for detection using positron emission tomography (PET) or single photon emission computed tomography (SPECT).
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • the compound includes a paramagnetic label suitable for tomographic detection.
  • the subject compound may be labeled, as described above, although in some methods, the compound is unlabelled and a secondary labeling agent is used for imaging.
  • the subject methods include diagnosis of a disease condition in a subject by comparing the number, size, and/or intensity of labeled loci, to corresponding baseline values.
  • the base line values can represent the mean levels in a population of undiseased subjects, or previous levels determined in the same subject.
  • the subject compounds may bind to receptors associated with angiogenesis. These compounds may thus be useful for treatment, in vivo diagnosis and imaging of diseases and conditions associated with angiogenesis.
  • the term "diseases and conditions associated with angiogenesis” includes those diseases and conditions referred to below. Reference is also made in this regard to WO 98/47541.
  • Diseases and conditions associated with angiogenesis include different forms of cancer and metastasis, for example, breast, skin, colorectal, pancreatic, prostate, lung or ovarian cancer.
  • Other diseases and conditions associated with angiogenesis are inflammation (for example, chronic inflammation), atherosclerosis, rheumatoid arthritis and gingivitis.
  • angiogenesis diseases and conditions associated with angiogenesis are arteriovenous alformations, astrocytomas, choriocarcinomas, glioblastomas, gliomas, hemangiomas (childhood, capillary), hepatomas, hyperplastic endometrium, ischemic myocardium, endometriosis, Kaposi sarcoma, macular degeneration, melanoma, neuroblastomas, occluding peripheral artery disease, osteoarthritis, psoriasis, retinopathy (diabetic, proliferative), scleroderma, seminomas and ulcerative colitis.
  • a subject compound or a salt thereof finds use in medicine, particularly in the in vivo diagnosis or imaging, for example by PET, of a disease or condition associated with angiogenesis.
  • radiolabelled compounds may be administered to subjects for PET imaging in amounts sufficient to yield the desired signal.
  • the radionuclide dosage is of 0.01 to 100 mCi, such as 0.1 to 50 mCi, or 1 to 20 mCi, which is sufficient per 70 kg bodyweight.
  • the radiolabelled compounds may therefore be formulated for administration using any convenient physiologically acceptable carriers or excipients.
  • the compounds optionally with the addition of pharmaceutically acceptable excipients, may be suspended or dissolved in an aqueous medium, with the resulting solution or suspension then being sterilized.
  • a radiolabelled compound or a salt thereof as described herein for the manufacture of a radiopharmaceutical for use in a method of in vivo imaging, e.g., PET imaging, such as imaging of a disease or condition associated with angiogenesis; involving administration of the radiopharmaceutical to a human or animal body and generation of an image of at least part of said body.
  • the method is a method for in vivo diagnosis or imaging of a disease or condition associated with angiogenesis involving administering a radiopharmaceutical to said body, e.g. into the vascular system and generating an image of at least a part of said body to which said radiopharmaceutical has distributed using PET, wherein said radiopharmaceutical comprises a radiolabelled compound or a salt thereof.
  • the method is a method of monitoring the effect of treatment of a human or animal body with a drug to combat a condition associated with cancer, e.g., angiogenesis, e.g. a cytotoxic agent, said method comprising administering to said body a radiolabelled compound or a salt thereof and detecting the uptake of the compound by cell receptors, such as endothelial cell receptors, e.g., alpha. v.beta.3 receptors, the administration and detection optionally being effected repeatedly, e.g. before, during and after treatment with said drug.
  • a condition associated with cancer e.g., angiogenesis, e.g. a cytotoxic agent
  • cell receptors such as endothelial cell receptors, e.g., alpha. v.beta.3 receptors
  • the method is a method for in vivo diagnosis or imaging of a disease or condition associated with angiogenesis comprising administering to a subject a GB1 peptidic compound and imaging at least a part of the subject.
  • the imaging comprises PET imaging and the administering comprises administering the compound to the vascular system of the subject.
  • the method further comprising detecting uptake of the compound by cell receptors.
  • the target is VEGF and the subject is human.
  • the method includes comprising administering avastin to the subject, wherein the disease or condition is a condition associated with cancer.
  • the subject methods may be diagnostic methods for detecting the expression of a target protein in specific cells, tissues, or serum, in vitro or in vivo.
  • the subject method is a method for in vivo imaging of a target protein in a subject.
  • the methods may include administering the compound to a subject presenting with symptoms of a disease condition related to a target protein.
  • the subject is asymptomatic.
  • the subject methods may further include monitoring disease progression and/or response to treatment in subjects who have been previously diagnosed with the disease.
  • the subject methods include administering a compound, such as a VEGF-binding compound, and then detecting the compound after it has bound to its target protein, e.g., VEGF.
  • a compound such as a VEGF-binding compound
  • the same compound can serve as both a therapeutic and a diagnostic compound.
  • the subject compounds can be used in subject methods for binding to one or more target molecules (e.g., a target protein, nucleic acid, organic small molecule, etc.).
  • target molecules e.g., a target protein, nucleic acid, organic small molecule, etc.
  • the subject compounds are selected to bind to a disease-specific target protein.
  • Disease-specific proteins are proteins that are expressed exclusively, or at a significantly higher level, in one or several diseased cells or tissues compared to other non-diseased cells or tissues in an animal.
  • diseases include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus
  • lymphocytotoxins erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic,
  • Exemplary disease or conditions include, e.g., MS, SLE, ITP, IDDM, MG, CLL, CD, RA, Factor VIII Hemophilia, transplantation, arteriosclerosis, Sjogren's Syndrome, Kawasaki Disease, anti- phospholipid Ab, AHA, ulcerative colitis, multiple myeloma, Glomerulonephritis, seasonal allergies, and IgA Nephropathy.
  • the subject compounds find use in the treatment of disease or conditions including: age-related macular degeneration (AMD), diabetic macular edema (DME), graft survival of transplanted corneas, osteoarthritis pain, chronic lower back pain, cancer-related pain, angiogenesis and metastasis in a variety of cancers, lymphangioleiomyomatosis (LAM), and ideopathic pulmonary fibrosis (IPF).
  • AMD age-related macular degeneration
  • DME diabetic macular edema
  • graft survival of transplanted corneas osteoarthritis pain, chronic lower back pain
  • cancer-related pain angiogenesis and metastasis in a variety of cancers
  • LAM lymphangioleiomyomatosis
  • IPF ideopathic pulmonary fibrosis
  • the target molecules may be any type of protein-binding or antigenic molecule, such as proteins, nucleic acids, carbohydrates or small molecules.
  • the target molecule is a therapeutic target molecule or a diagnostic target molecule.
  • the target molecule is a hormone, a growth factor, a receptor, an enzyme, a cytokine, an osteoinductive factor, a colony stimulating factor or an immunoglobulin.
  • the target molecule may be one or more of the following: growth hormone, bovine growth hormone, insulin like growth factors, human growth hormone including n- methionyl human growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, amylin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), leutinizing hormone (LH), hemapoietic growth factor, Her-2, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factors, mullerian inhibiting substance, mouse gonadotropin-associated polypeptide, inhibin, activin, vascular endothelial growth factors, integrin, nerve growth factors such as NGF-beta, insulin-like growth factor-I and II, erythropoietin, osteoinductive factors, interferons, colony stimulating factors, interleukins (e.g., an IL-4 or an IL-8 protein), bone morphogenetic proteins, L
  • the target molecule may be a therapeutic target protein for which structural information is known, such as, but not limited to: Raf kinase (a target for the treatment of melanoma), Rho kinase (a target in the prevention of pathogenesis of cardiovascular disease), nuclear factor kappaB (NF-.kappa.B, a target for the treatment of multiple myeloma), vascular endothelial growth factor (VEGF) receptor kinase (a target for action of anti-angiogenetic drugs), Janus kinase 3 (JAK-3, a target for the treatment of rheumatoid arthritis), cyclin dependent kinase (CDK) 2 (CDK2, a target for prevention of stroke), FMS-like tyrosine kinase (FLT) 3 (FLT-3; a target for the treatment of acute myelogenous leukemia (AML)), epidermal growth factor receptor (EGFR) kinase (a
  • p21-activated kinase a target for the treatment of breast cancer
  • mitogen- activated protein kinase MAPK
  • MAPK mitogen- activated protein kinase
  • JNK c-Jun NH.sub.2- terminal kinase
  • AMPK AMP-activated kinase
  • lck kinase a target for immuno-suppression
  • phosphodiesterase PDE4 a target in treatment of inflammatory diseases such as rheumatoid arthritis and asthma
  • Abl kinase a target in treatment of chronic myeloid leukemia (CML)
  • CML chronic myeloid leukemia
  • phosphodiesterase PDE5 (a target in treatment of erectile dysfunction), a disintegrin and
  • ADAM33 human immunodeficiency virus (HIV)-l protease and HIV integrase (targets for the treatment of HIV infection), respiratory syncytial virus (RSV) integrase (a target for the treatment of infection with RSV), X-linked inhibitor of apoptosis (XIAP, a target for the treatment of neurodegenerative disease and ischemic injury), thrombin (a therapeutic target in the treatment and prevention of thromboembolic disorders), tissue type plasminogen activator (a target in prevention of neuronal death after injury of central nervous system), matrix metalloproteinases (targets of anti -cancer agents preventing angiogenesis), beta secretase (a target for the treatment of Alzheimer's disease), src kinase (a target for the treatment of cancer), fyn kinase, lyn kinase, zeta-chain associated protein 70 (ZAP-
  • the target molecule is a target protein that is selected from the group consisting of a VEGF protein, a RANKL protein, a NGF protein, a TNF-alpha protein, a SH2 domain containing protein, a SH3 domain containing protein, an IgE protein, a BLyS protein (Oren et al., "Structural basis of BLyS receptor recognition", Nature Structural Biology 9, 288 - 292, 2002), a PCSK9 protein (Ni et al., "A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake", J.
  • a target protein that is selected from the group consisting of a VEGF protein, a RANKL protein, a NGF protein, a TNF-alpha protein, a SH2 domain containing protein, a SH3 domain containing protein, an Ig
  • the target protein is a VEGF protein.
  • the target protein is a SH2 domain containing protein (e.g., a 3BP2 protein), a SH3 domain containing protein (e.g., a ABL or a Src protein), or a cytokine (e.g., an IL-13 protein, a TNF-alpha protein, a BAFF protein, or a RANKL protein).
  • a SH2 domain containing protein e.g., a 3BP2 protein
  • a SH3 domain containing protein e.g., a ABL or a Src protein
  • a cytokine e.g., an IL-13 protein, a TNF-alpha protein, a BAFF protein, or a RANKL protein.
  • the target protein is selected from VEGF-D, PDGF, Robo4, Influenza hemagglutinin, R-spondin, MD-2 (myeloid differentiation protein 2) , TNF-alpha, RANKL, IL-1 alpha, IL-6 , IL-12, IL-13, IL-17 , IL-23, CTLA-4, PCSK9, BlyS, NGF, Ang2, CXCL5, CD5, CD40, IGF-1R, Sclerostin, EGFR, HER2, amyloid beta and Navl.7.
  • the subject compounds are selected to bind to a tissue-specific target protein.
  • Tissue-specific proteins are proteins that are expressed exclusively, or at a significantly higher level, in one or several particular tissue(s) compared to other tissues in an animal.
  • Exemplary tissues where target proteins may be specifically expressed include, e.g., liver, pancreas, adrenal gland, thyroid, salivary gland, pituitary gland, brain, spinal cord, lung, heart, breast, skeletal muscle, bone marrow, thymus, spleen, lymph node, colorectal, stomach, ovarian, small intestine, uterus, placenta, prostate, testis, colon, colon, gastric, bladder, trachea, kidney, or adipose tissue.
  • a subject compound that binds to the target protein is linked to the pharmaceutical protein or small molecule such that the resulting complex or fusion is targeted to the specific tissue or disease-related cell(s) where the target protein is expressed.
  • Subject compounds for use in such complexes or fusions can be initially selected for binding to the target protein and may be subsequently selected by negative selection against other cells or tissue (e.g., to avoid targeting bone marrow or other tissues that set the lower limit of drug toxicity) where it is desired that binding be reduced or eliminated in other non-target cells or tissues.
  • tissue-specific or disease-specific proteins can be found in, e.g., Tables I and II of U.S. Patent Publication No 2002/0107215.
  • the target protein is a VEGF protein.
  • the VEGF-binding compounds of the invention are therapeutically useful for treating any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition, or reduction of a VEGF protein.
  • the VEGF protein is VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, or a fragment thereof.
  • Neoplasms and related conditions that are amenable to treatment include breast carcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervical carcinomas, endometrial carcinoma, endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma, glioblastoma, Schwannoma
  • Non-neoplastic conditions that are amenable to treatment include rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, nephrotic syndrome, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion.
  • rheumatoid arthritis rheumatoid arthritis
  • psoriasis atherosclerosis
  • diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, thyroid hyperplasia
  • AMD Age-related macular degeneration
  • AMD Age-related macular degeneration
  • the exudative form of AMD is characterized by choroidal neovascularization and retinal pigment epithelial cell detachment. Because choroidal neovascularization is associated with a dramatic worsening in prognosis, the subject VEGF-binding compounds find use in reducing the severity of AMD.
  • Lymphangioleiomyomatosis is a rare, progressive, frequently fatal cystic lung disease that affects women almost exclusively. It occurs in up to 40% of women with the tuberous sclerosis complex, a tumor-suppressor syndrome associated with seizures, cognitive impairment, and hamartomas in multiple organs, and can also occur in a nonheritable sporadic form that involves only the lung, lymphatics, and kidney.
  • VEGF is a major angiogenic growth factor produced by malignant cells.
  • VEGF-D a ligand for the lymphatic growth-factor receptor VEGFR-3/FU-4, induces formation of lymphatics and promotes the spread of tumor cells to lymph nodes.
  • VEGF- D levels of VEGF- D, but not VEGF-A or VEGF-C, are elevated in patients with sporadic lymphangioleiomyomatosis as compared with healthy controls.
  • Serum VEGF-D may be a clinically useful diagnostic test that can distinguish sporadic lymphangioleiomyomatosis from other cystic and chylous lung diseases, potentially decreasing the need for lung biopsy. See e.g., Young et al. "Diagnostic Potential of Serum VEGF-D for Lymphangioleiomyomatosis," N. Engl. J. Med. 358, 2, 2008, 199-200.
  • LAM cells proliferate along lymphatic channels in the lung and in
  • LAM cells are divided into fascicles or bundles by channels lined by lymphatic endothelial cells.
  • LAM cells produce VEGF-C, and the degree to which it is produced by LAM cells correlated, in some cases, with the degree of lymphangiogenesis observed.
  • the lymphatic channels recruited by LAM cells tend to divide the cells into clusters that are then shed from the lesion. This mechanism may account for the ability of LAM cells to metastasize to distant sites and facilitate the progressive invasion of the lung parenchyma by LAM cells. See e.g., Juvet et al., "Molecular Pathogenesis of Lymphangioleiomyomatosis," Am. J. Respir. Cell Mol. Biol., Vol 36. pp 398-408, 2007.
  • modulation of VEGF-C and VEGF-D is of interest in the treatment of LAM.
  • a subject compound may be contacted with a cell in the presence of VEGF, and a VEGF response phenotype of the cell monitored.
  • Exemplary VEGF assays include assays using isolated protein in a cell free systems, in vitro using cultured cells or in vivo assays.
  • Exemplary VEGF assays include, but are not limited to a receptor tyrosine kinase inhibition assay (see, e.g., Cancer Research June 15, 2006; 66:6025-6032), an in vitro HUVEC proliferation assay (FASEB Journal 2006; 20: 2027-2035; Wells et al., Biochemistry 1998, 37, 17754-17764), an in vivo solid tumor disease assay (USPN 6,811,779) and an in vivo angiogenesis assay (FASEB Journal 2006; 20: 2027-2035). The descriptions of these assays are hereby incorporated by reference.
  • the subject method is a method of modulating angiogenesis in a subject, the method comprising administering to the subject an effective amount of a subject compound that specifically binds with high affinity to a VEGF protein.
  • the method further comprises diagnosing the presence of a disease condition in the subject.
  • the disease condition is a condition that may be treated by enhancing angiogenesis.
  • the disease condition is a condition that may be treated by decreasing angiogenesis.
  • the subject method is a method of inhibiting angiogenesis, the target protein is a VEGF protein and the compound is a VEGF antagonist.
  • the subject method is a method of treating a subject suffering from a cellular proliferative disease condition, the method including administering to the subject an effective amount of a subject compound that specifically binds with high affinity to a VEGF protein so that the subject is treated for the cellular proliferative disease condition.
  • the subject method is a method of inhibiting tumor growth in a subject, the method comprising administering to a subject an effective amount of a subject compound that specifically binds with high affinity to the VEGF protein.
  • the tumor is a solid tumor. In certain embodiments, the tumor is a non-solid tumor.
  • the subject VEGF-binding compounds may be used as affinity purification agents.
  • the compounds are immobilized on a solid phase such a Sephadex resin or filter paper, using any convenient methods.
  • the subject VEGF-binding compound is contacted with a sample containing the VEGF protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the VEGF protein, which is bound to the immobilized compound. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the VEGF protein from the immobilized compound.
  • a suitable solvent such as glycine buffer, pH 5.0
  • the subject VEGF-binding compounds may also be useful in diagnostic assays for VEGF protein, e.g., detecting its expression in specific cells, tissues, or serum. Such diagnostic methods may be useful in cancer diagnosis.
  • the subject compound may be modified as described above.
  • the target protein is a SH2 domain and SH3 domain containing protein.
  • the subject compounds of the invention are therapeutically useful for treating any disease or condition which is improved, ameliorated, inhibited or prevented by modulation of the activity of a SH2 domain or SH3 domain containing protein.
  • 3BP2 (or SH3BP2 for Abl-SH3 Binding Protein-2) is an adapter protein composed of an amino-terminal pleckstrin homology (PH) domain, a central proline -rich (PR) region, and a carboxyl- terminal SH2 domain that was originally identified as a c-Abl SH3 binding protein in 1993. Functional studies have implicated a role for 3BP2 in immunoreceptor signaling through its interaction with a number of signaling molecules, including Src and Syk families
  • the Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine -binding module present in many signaling molecules.
  • the SH2 domain In cytoplasmic tyrosine kinases, the SH2 domain is located N- terminally to the catalytic kinase domain (SHI) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity.
  • SHI catalytic kinase domain
  • structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members.
  • biochemical characterization showed that the presence of the SH2 domain is frequently required for catalytic activity, suggesting a crucial function stabilizing the active state of many nonreceptor tyrosine kinases.
  • the structure of the SH2-kinase domain of Fes revealed that the SH2 domain stabilizes the active kinase conformation by direct interactions with the regulatory helix aC. Stabilizing interactions between the SH2
  • the subject compounds find use in treating a SH2 domain-related disorder.
  • the SH2 domain-related disorder is selected from lung cancer, X- linked agammaglobulinemia (XLA), therapy-resistant Epstein-Barr virus (EBV)-positive B cell proliferation.
  • XLA X- linked agammaglobulinemia
  • EBV Epstein-Barr virus
  • the subject method is a method of treating an SH2 domain-mediated disorder that includes administering a therapeutically effective amount of a subject compound that specifically binds to the SH2 domain.
  • c-Src and Bcr-Abl are two non-receptor or cytoplasmic tyrosine kinases (TKs) that play important roles in the development of solid and hematological malignancies. Indeed, Src is overexpressed or hyperactivated in a variety of solid tumors, while Bcr-Abl is the causative agent of chronic myeloid leukemia (CML), where Src is also involved.
  • CML chronic myeloid leukemia
  • Src is a non-receptor or cytoplasmic tyrosine kinase (TK), belonging to a family with nine currently identified members. Src affects cell proliferation, differentiation, migration, invasion, apoptosis and angiogenesis, by interaction with a diverse array of molecules, including growth factor receptors, cell— cell adhesion receptors, integrins and steroid hormone receptors. Given the role of Src in all these functions, its inhibition would be of interest for the treatment of patients with Src- dependent cancers.
  • TK cytoplasmic tyrosine kinase
  • the subject compounds find use in treating a Src-mediated disorder.
  • the Src-mediated disorder is CML or a lymphoma, e.g., diffuse large B-cell lymphoma (DLBCL).
  • the Src-mediated disorder is selected from breast, prostate, lung, pancreatic, and head and neck, CNS (including glioblastoma), ovarian cancers, mesothelioma, sarcomas (including chondrosarcoma), neuroblastoma, and unspecified adult and childhood solid tumors.
  • the subject method is a method of treating a Src-mediated disorder that includes administering a therapeutically effective amount of a subject compound that specifically binds to Src.
  • the subject method is a method of treating a Src- or Abl-mediated disorder that includes administering in combination therapeutically effective amounts of a subject compound that specifically binds to a Src protein and a subject compound that specifically binds to a Abl protein.
  • the subject method is a method of treating a disease condition mediated by a SH3 domain containing protein. In some embodiments, the subject method is a method of treating an Abl-mediated disorder that includes administering a therapeutically effective amount of a subject compound that specifically binds to Src. In some embodiments, the Abl-mediated disorder is CML.
  • TNF-OC tumor necrosis factor-oc
  • TNF-alpha tumor necrosis factor-oc
  • TNF-OC is a pleiotropic cytokine that is primarily produced by activated macrophages and lymphocytes; but is also expressed in endothelial cells and other cell types.
  • TNF-alpha is a major mediator of inflammatory, immunological, and
  • TNF-alpha exerts its biological effects through interaction with high-affinity cell surface receptors.
  • Two distinct membrane TNF-alpha receptors that have been cloned and characterized include a 55 kDa species, designated p55 TNF-R and a 75 kDa species designated p75 TNF-R
  • TNF receptors exhibit 28% similarity at the amino acid level. This is confined to the extracellular domain and consists of four repeating cysteine-rich motifs, each of approximately 40 amino acids. Each motif contains four to six cysteines in conserved positions.
  • TNF-a assays include in vitro assays using cell free systems or using cultured cells or in vivo assays.
  • TNF-a assays include in vitro human whole blood assay and cell mediated cytotoxicity assay (USPN 6,090,382 ), in vitro tumor human killing assay (see, e.g., published U.S. patent application 20040185047), in vivo tumor regression assay (USP Application 20040002589). Additional TNF-a assays are described in a variety of publications, including 20040151722, 20050037008,
  • the ability of the subject compounds to modulate the binding of a TNF-alpha protein to a TNF receptor protein such as p55 and p75 may be determined by any convenient method. Suitable assays include, but are not limited to, e.g., quantitative comparisons comparing kinetic and equilibrium binding constants.
  • the kinetic association rate (Kon) and dissociation rate (Kofi), and the equilibrium binding constants (Kd) may be determined using surface plasmon resonance on a BIAcore instrument following the standard procedure in the literature [Pearce et al., Biochemistry 38:81-89 (1999)].
  • the subject methods are methods of treating a TNF-alpha mediated disease comprising the administration of the TNF-alpha binding molecule of the present invention to a subject (e.g., a human).
  • the administration is under conditions such that the symptoms of the TNF-alpha mediated disease are reduced or eliminated.
  • the TNF-alpha mediated disease is selected from sepsis, an autoimmune disease, rheumatoid arthritis, allergy, multiple sclerosis, autoimmune uveitis, nephrotic syndrome, an infectious disease, a malignancy, transplant rejection, graft-versus-host disease, systemic lupus erythematosus, thyroidosis, scleroderma, diabetes mellitus, Graves' disease, a pulmonary disorder, a bone disorder, an intestinal disorder, a cardiac disorder, cachexia, circulatory collapse, shock resulting from acute or chronic bacterial infections, acute and chronic parasitic and/or infectious diseases, chronic inflammatory pathologies, vascular inflammatory pathologies, sarcoidosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, disseminated intravascular coagulation, atherosclerosis, Kawasaki's pathology, neurodegenerative diseases, demyelinating diseases, multiple sclerosis, acute transverse myelitis,
  • abetalipoprotemia ataxia, telangiectasia, mitochondrial multi-system disorder, disorders of the motor unit, neurogenic muscular atrophies, anterior horn cell degeneration, amyotrophic lateral sclerosis, infantile spinal muscular atrophy, juvenile spinal muscular atrophy, Alzheimer's disease, Down's Syndrome, Diffuse Lewy body disease, senile dementia of Lewy body type, Wernicke-Korsakoff syndrome, chronic alcoholism, Creutzfeldt-Jakob disease, subacute sclerosing panencephalitis, Hallerrorden-Spatz disease, dementia pugilistica, malignant pathologies involving TNF-.alpha.- secreting tumors or other malignancies involving TNF, such as, leukemias (acute, chronic myelocytic, chronic lymphocytic and/or myelodyspastic syndrome), lymphomas (e.g., Hodgkin's, non-Hodgkin's and Burkitt's lymph
  • the TNF-. alpha, mediated disease is selected from the group consisting of juvenile and adult rheumatoid arthritis, Crohn's disease, psoriasis, ulcerative colitis, psoriatic arhritis, ankylosing spondylitis, and other spondyloarthropathies, Wegener's granulomatosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, oncology, and graft-versus-host disease.
  • Nerve growth factor was the first neurotrophin to be identified, and its role in the development and survival of both peripheral and central neurons has been well characterized.
  • NGF nerve growth factor
  • NGF upregulates expression of neuropeptides in sensory neurons (Lindsay and Harmer, Nature 337:362-364 (1989)) and its activity is mediated through two different membrane-bound receptors.
  • TrkA tyrosine kinase receptor mediates high affinity binding and the p75 receptor, which is structurally related to other members of the tumor necrosis factor receptor family, mediates low affinity binding (Chao et al., Science 232:518- 521 (1988)).
  • NGF In addition to its effects in the nervous system, NGF has been increasingly implicated in processes outside of the nervous system.
  • NGF is produced by a number of cell types including mast cells (Leon et al., Proc. Natl. Acad. Sci. U.S.A. 91 :3739-3743 (1994)), B-lymphocytes (Torcia et al., Cell 85:345-356 (1996),
  • keratinocytes Di Marco et al., J. Biol. Chem. 268:22838-22846)
  • smooth muscle cells Ueyama et al., J. Hypertens. 11: 1061-1065 (1993)
  • NGF neurotrophic factor
  • NGF plays a role in regulating innervation in the skin and also is known to upregulate neuropeptides, suggesting that increased NGF levels may be responsible for the upregulation of neuropeptides and the increased cutaneous innervation seen with psoriasis, normal cell death, leading to psoriasis (Pincelli et al., J. Derm. Sci. 22:71-79 (2000)).
  • NGF has been shown to affect mast cell degranulation (Bruni et al., FEBS Lett. 138: 190-193 (1982)) and substance P release (Donnerer et al., Neurosci. 49:693-698 (1992)), implicating it in the pathogenesis of arthritis.
  • NGF neurotrophic factor
  • NGF has been shown to regulate the development of increased airway hyperactive response, a hallmark of bronchial asthma (Braun et al., Eur. J. Immunol. 28:3240-3251 (1998)). Indeed, in one study, treatment of allergen-sensitized mice with anti-NGF antibody prevented the development of airway hyperresponsiveness following local allergen challenge (Braun et al., Int. Arch. Allergy Immunol. 118: 163-165 (1999)).
  • the subject compounds find use in modulating the activity of NGF.
  • the subject compounds can be identified by incubating the compound with NGF and monitoring binding and modulation of a biological activity of NGF.
  • the binding assay may be performed with purified NGF polypeptide(s), or with cells naturally expressing, or transfected to express, NGF polypeptide(s).
  • the binding assay is a competitive binding assay, where the ability of a subject compound to compete with a known anti-NGF antibody for NGF binding is evaluated.
  • the assay may be performed in various formats, including the ELISA format.
  • the ability of a subject compound to modulate a biological activity of NGF can, for example, be carried out by monitoring the ability of the subject compound to inhibit NGF mediated survival in the embryonic rat dorsal root ganglia survival bioassay as described in Hongo et al. (Hybridoma 19:215-227 (2000)).
  • a kinase-induced receptor activation (KIRA) assay may be used to measure the effect of the subject compounds on the NGF-dependent TrkA autophosphorylation in transfected cells in response to stimulation with a ligand, such as hNGF (Sadick et al., Exp. Cell Res. 234:354-361 (1997)).
  • the subject compounds finds use in inhibiting the binding of hNGF to human TrkA (hTrkA) in vivo.
  • the subject compounds find use in treating a NGF-related disorder.
  • the inflammatory condition may be selected from the group consisting of asthma, arthritis, multiple sclerosis, lupus erythematosus and psoriasis.
  • the NGF binding compounds may be used to prevent the onset of the active disease state, to treat symptoms that are currently being experienced and to treat the underlying disease itself.
  • the subject compounds find use in treating other diseases that may be associated with increased levels of NGF including, for example, lupus erythematosus, shingles, postherpetic neuralgia, hyperalgesia, and chronic pain.
  • diseases including, for example, lupus erythematosus, shingles, postherpetic neuralgia, hyperalgesia, and chronic pain.
  • IgE immunoglobulin E
  • IgE exists in a membrane bound form and in a secreted form. These distinct forms appear to be splice variants. Previous approaches to achieve therapeutic effect by down regulating IgE targeting primarily the secreted form (e.g., XOLAIR.RTM. omalizumab), so as prevent or disarm further "arming" of the immune system.
  • secreted form e.g., XOLAIR.RTM. omalizumab
  • Targeting the ⁇ segment of IgE with anti-IgE antibodies can result in inducing apoptosis of the B-cell.
  • the progeny of activated B-cells can result in plasma cells that make and secrete the secreted form of IgE, the depletion of the IgE-producing B-cell through apoptosis offers one therapeutic approach to the treatment of allergy.
  • the subject compounds find use in treating a IgE-mediated disorder.
  • the IgE-mediated disorder is selected from allergic rhinitis, asthma (e.g., allergic asthma and non-allergic asthma), atopic dermatitis, allergic gastroenteropathy, hypersensitivity (e.g., anaphylaxis, urticaria, food allergies etc.), allergic bronchopulmonary aspergillosis, parasitic diseases, interstitial cystitis, hyper-IgE syndrome, ataxia-telangiectasia, Wiskott-Aldrich syndrome, athymic lymphoplasia, IgE myeloma and graft-versus-host reaction.
  • the IgE-mediated disorder is food allergy, anaphylaxis, contact dermatitis or allergic purpura.
  • the subject method is a method of treating an IgE-mediated disorder including administering a therapeutically effective amount of a subject compound that specifically binds to the IgE and induces apoptosis in IgE-expressing B-cells.
  • the subject compound specifically binds to the ⁇ segment of IgE.
  • RANKL receptor activator of NF-kB ligand, also referred to as OPGL, TRANCE or ODF
  • OPGL receptor activator of NF-kB ligand
  • TRANCE TRANCE
  • TNF tumour necrosis factor
  • the cytokine RANKL (receptor activator of NF-kB ligand) triggers migration of human epithelial cancer cells and melanoma cells that express the receptor RANK.
  • RANK is
  • Osteoporosis is a systemic skeletal disorder characterized by low bone mass
  • RANKL is a pivotal regulator of osteoclast activity is a target for treatment of osteoporosis, e.g., denosumab, a highly specific anti-RANKL antibody, rapidly and substantially reduces bone resorption.
  • the subject compounds find use in treating a RANKL-mediated disorder.
  • the subject method is a method of treating an RANKL-mediated disorder that includes administering a therapeutically effective amount of a subject compound that specifically binds to a RANKL protein.
  • the compounds of the invention find use in a variety of applications.
  • Applications of interest include, but are not limited to: therapeutic applications, research applications, and screening applications. Each of these different applications are now reviewed in greater details below.
  • the subject compounds find use in a variety of therapeutic applications.
  • Therapeutic applications of interest include those applications in which the activity of the target is the cause or a compounding factor in disease progression.
  • the subject compounds find use in the treatment of a variety of different conditions in which the modulation of target activity in the host is desired.
  • the subject compounds are useful for treating a disorder relating to its target.
  • the disease conditions include, but are not limited to: cancer, inhibition of angiogenesis and metastasis, osteoarthritis pain, chronic lower back pain, cancer-related pain, age-related macular degeneration (AMD), diabetic macular edema (DME), LAM:
  • lymphangioleiomyomatosis IPF: Ideopathic pulmonary fibrosis and graft survival of transplanted corneas.
  • the invention provides a method of treating a subject for a VEGF-related condition.
  • the method generally involves administering a subject compound to a subject having a VEGF-related disorder in an amount effective to treat at least one symptom of the VEGF-related disorder.
  • VEGF-related conditions are generally characterized by excessive vascular endothelial cell proliferation, vascular permeability, edema or inflammation such as brain edema associated with injury, stroke or tumor; edema associated with inflammatory disorders such as psoriasis or arthritis, including rheumatoid arthritis; asthma; generalized edema associated with burns; ascites and pleural effusion associated with tumors, inflammation or trauma; chronic airway inflammation; capillary leak syndrome; sepsis; kidney disease associated with increased leakage of protein; and eye disorders such as age related macular degeneration and diabetic retinopathy.
  • Such conditions include breast, lung, colorectal and renal cancer.
  • the subject compounds may be administered in combination with one or more additional compounds or therapies, including a second target-binding molecule, a chemotherapeutic agent, surgery, catheter devices, and radiation.
  • Combination therapy includes administration of a single pharmaceutical dosage formulation which contains the subject compound and one or more additional agents; as well as administration of the subject compound and one or more additional agent(s) in its own separate pharmaceutical dosage formulation.
  • a subject compound and a cytotoxic agent, a chemotherapeutic agent or a growth inhibitory agent can be administered to the patient together in a single dosage composition such as a combined formulation, or each agent can be administered in a separate dosage formulation.
  • the subject compound and one or more additional agents can be administered concurrently, or at separately staggered times, e.g., sequentially.
  • the subject compounds and methods find use in a variety of research applications.
  • the subject compounds and methods may be used to analyze the roles of target proteins in modulating various biological processes, including but not limited to, angiogenesis, inflammation, cellular growth, metabolism, regulation of transcription and regulation of phosphorylation.
  • Other target protein binding molecules such as antibodies have been similarly useful in similar areas of biological research. See e.g., Sidhu and Fellhouse, "Synthetic therapeutic antibodies," Nature Chemical Biology, 2006, 2(12), 682-688.
  • Such methods can be readily modified for use in a variety of research applications of the subject compounds and methods.
  • the subject compounds and methods find use in a variety of diagnostic applications, including but not limited to, the development of clinical diagnostics, e.g., in vitro diagnostics or in vivo tumor imaging agents. Such applications are useful in diagnosing or confirming diagnosis of a disease condition, or susceptibility thereto. The methods are also useful for monitoring disease progression and/or response to treatment in patients who have been previously diagnosed with the disease.
  • clinical diagnostics e.g., in vitro diagnostics or in vivo tumor imaging agents.
  • Such applications are useful in diagnosing or confirming diagnosis of a disease condition, or susceptibility thereto.
  • the methods are also useful for monitoring disease progression and/or response to treatment in patients who have been previously diagnosed with the disease.
  • Diagnostic applications of interest include diagnosis of disease conditions, such as those conditions described above, including but not limited to: cancer, inhibition of angiogenesis and metastasis, osteoarthritis pain, chronic lower back pain, cancer-related pain, age-related macular degeneration (AMD), diabetic macular edema (DME), LAM: lymphangioleiomyomatosis, IPF:
  • the disease condition is a target-mediated condition where the target is selected from the group consisting of a VEGF protein, a RANKL protein, a NGF protein, a TNF-alpha protein, a SH2 domain containing protein, a SH3 domain containing protein, a Src protein, an IL-4 protein, an IL-8 protein, an IgE protein and fragments thereof.
  • the same compound can serve as both a treatment and diagnostic reagent.
  • target protein binding molecules such as aptamers and antibodies
  • aptamers and antibodies have also found use in the development of clinical diagnostics.
  • Such methods can be readily modified for use in a variety of diagnostics applications of the subject compounds and methods, see for example, Jayasena , "Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics," Clinical Chemistry. 1999;45: 1628-1650.
  • compositions that include a compound (either alone or in the presence of one or more additional active agents) present in a pharmaceutically acceptable vehicle.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
  • vehicle refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is formulated for administration to a mammal.
  • Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
  • the compounds and compositions of the invention and pharmaceutically acceptable vehicles, excipients, or diluents may be sterile.
  • an aqueous medium is employed as a vehicle when the compound of the invention is administered intravenously, such as water, saline solutions, and aqueous dextrose and glycerol solutions.
  • compositions can take the form of capsules, tablets, pills, pellets, lozenges, powders, granules, syrups, elixirs, solutions, suspensions, emulsions, suppositories, or sustained- release formulations thereof, or any other form suitable for administration to a mammal.
  • the pharmaceutical compositions are formulated for administration in accordance with routine procedures as a pharmaceutical composition adapted for oral or intravenous administration to humans. Examples of suitable pharmaceutical vehicles and methods for formulation thereof are described in Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro ed., Mack Publishing Co.
  • Administration of compounds of the invention may be systemic or local. In certain embodiments administration to a mammal will result in systemic release of a compound of the invention (for example, into the bloodstream).
  • Methods of administration may include enteral routes, such as oral, buccal, sublingual, and rectal; topical administration, such as transdermal and intradermal; and parenteral administration.
  • Suitable parenteral routes include injection via a hypodermic needle or catheter, for example, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intraarterial, intraventricular, intrathecal, and intracameral injection and non-injection routes, such as intravaginal rectal, or nasal administration.
  • the compounds and compositions of the invention are administered orally.
  • This may be achieved, for example, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • the compounds can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • formulations suitable for oral administration can include (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, or saline; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
  • Lozenge forms can include the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles including the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are described herein.
  • the subject formulations of the present invention can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluorome thane, propane, nitrogen, and the like. They may also be formulated as pharmaceuticals for non-pressured preparations such as for use in a nebulizer or an atomizer.
  • formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried
  • sterile liquid excipient for example, water
  • injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Formulations suitable for topical administration may be presented as creams, gels, pastes, or foams, containing, in addition to the active ingredient, such carriers as are appropriate.
  • the topical formulation contains one or more components selected from a structuring agent, a thickener or gelling agent, and an emollient or lubricant.
  • Frequently employed structuring agents include long chain alcohols, such as stearyl alcohol, and glyceryl ethers or esters and oligo(ethylene oxide) ethers or esters thereof.
  • Thickeners and gelling agents include, for example, polymers of acrylic or methacrylic acid and esters thereof, polyacrylamides, and naturally occurring thickeners such as agar, carrageenan, gelatin, and guar gum.
  • emollients include triglyceride esters, fatty acid esters and amides, waxes such as beeswax, spermaceti, or carnauba wax, phospholipids such as lecithin, and sterols and fatty acid esters thereof.
  • the topical formulations may further include other components, e.g., astringents, fragrances, pigments, skin penetration enhancing agents, sunscreens (e.g., sunblocking agents), etc.
  • a compound of the invention may also be formulated for oral administration.
  • suitable excipients include pharmaceutical grades of carriers such as mannitol, lactose, glucose, sucrose, starch, cellulose, gelatin, magnesium stearate, sodium saccharine, and/or magnesium carbonate.
  • the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in solid or liquid form suitable for hydration in an aqueous carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, preferably water or normal saline.
  • composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers.
  • auxiliary substances such as wetting agents, emulsifying agents, or buffers.
  • a compound of the invention may also be incorporated into existing nutraceutical formulations, such as are available conventionally, which may also include an herbal extract.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors.
  • unit dosage forms for injection or intravenous administration may include the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • Dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Desired dosages for a given compound are readily determinable by a variety of means.
  • the dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to effect a prophylactic or therapeutic response in the animal over a reasonable time frame, e.g., as described in greater detail below. Dosage will depend on a variety of factors including the strength of the particular compound employed, the condition of the animal, and the body weight of the animal, as well as the severity of the illness and the stage of the disease. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound.
  • the compounds may be administered in the form of a free base, their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
  • a pharmaceutical composition includes a subject compound that specifically binds with high affinity to a target protein, and a pharmaceutically acceptable vehicle.
  • the target protein is a VEGF protein and the subject compound is a VEGF antagonist.
  • kits that include compounds of the invention.
  • Kits of the invention may include one or more dosages of the compound, and optionally one or more dosages of one or more additional active agents.
  • the formulations may be provided in a unit dosage format.
  • an informational package insert describing the use of the subject formulations in the methods of the invention, e.g., instructions for using the subject unit doses to treat cellular proliferative disease conditions.
  • These instructions may be present in the subject systems and kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc.
  • Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded.
  • Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
  • a kit in some embodiments, includes a first dosage of a subject pharmaceutical composition and a second dosage of a subject pharmaceutical composition. In certain embodiments, the kit further includes a second angiogenesis modulatory agent.
  • GB 1 peptidic compounds may be synthesized utilizing solid phase peptide synthesis and/or native chemical ligation (NCL) methods. The following synthetic strategy and protocols can be adapted as needed in preparing the subject GB 1 peptidic compound. Sequences of exemplary GB1 motifs and compounds prepared using the methods described herein are depicted in Figure 7.
  • C37 and C52 are desulfurized to A37 and A52 residues.
  • All D-peptide segments were synthesized using D-amino acids and glycine via solid phase peptide synthesis (SPPS) using Boc chemistry in situ neutralization protocols. Segments 2 and 3 were cleaved using anhydrous HF, triturated with cold ether, dissolved in 1: 1 MeCN:H 2 0 + 0.1% TFA solution then lyophilized for use in native chemical ligation without further purification.
  • Segment 1 was cleaved using anhydrous HF, triturated with cold ether, then dissolved in 6 M GnHCl/0.2 M Na 2 HP0 4 containing 2- mercaptoethanesulfonate (MESNa, 300 mM) at pH 6.8 in order to remove the side chain DNP groups of histidine residues.
  • the deprotection reaction was complete in 30 min, and the product was purified using CI 8 reverse-phase semiprep to afford the D-peptide -thioester segment Met ⁇ Glu 36 - aCOSCH 2 CH 2 S0 3 H. It was notable that there were significant deletions of one and two histidine residues that were not separated from the desired His 10 peptide under the purification conditions used.
  • the LCMS profiles of the chemically synthesized D-peptide segments are shown in Figure 1.
  • Figure 1 A) D-Met 1 -Glu 36 -aCOSCH 2 CH 2 S0 3 H after DNP removal and prep HPLC purification; B) crude D-Thz 37 -Ala 51 - aCOSCH 2 CH 2 COArg 6 ; C) crude D-Cys 52 -Trp(CHO) 61 -Lys 79 - aCOOH.
  • the D-peptides Thz37-Ala51-aCOSCH 2 CH 2 COArg 6 (0.9 mM) and D-Cys 52 -Trp(CHO) 61 - Lys 79 -aCOOH (1 mM) were dissolved in 6 M GnHCl/0.2 M Na 2 HP0 4 containing 4- mercaptophenylacetic acid (MPAA, 50 mM) and tris(2-carboxyethyl) phosphine.HCl (TCEP.HC1, 20 mM) at pH 7.0 and allowed to react at room temperature.
  • MPAA 4- mercaptophenylacetic acid
  • TCEP.HC1 tris(2-carboxyethyl) phosphine.HCl
  • the reaction mixture was adjusted to pH 4.5 and kept at 50 °C for 10 h which resulted in adequate conversion of Thz to Cys (Figure 2C).
  • the isoelectric point (pi) of the ligated product was calculated to be 3.6, therefore the reaction mixture was diluted to a final GnHCl concentration of 0.01 M and the pH was adjusted to 3.6.
  • the ligation product precipitated and was readily recovered by centrifugation. The supernatant was decanted and the precipitate resuspended in 0.1 M Na 2 HP0 4 buffer (pH 3.6), then re-centrifuged after which the supernatant was decanted to yield pure product as the precipitate (Figure 2D).
  • the precipitated D-peptide Cys 37 -Lys 79 -aCOOH (3 mM) was dissolved in 6 M GnHCl/0.2 M Na 2 HP0 4 containing MPAA (50 mM) and TCEP (20 mM) at pH 6.8. Then, the D-peptide Met 1 -Glu 36 -aCOSCH 2 CH 2 S0 3 H (4.5 mM) was added and the pH was readjusted to 6.8. After 2 h, formation of more than 50 % of the desired D-polypeptide Met 1 -
  • Segment 1 synthesized with a aCOSCH 2 CH 2 CO-Arg 4 Ala tag but replaced with MESNa during DNP removal of His(DNP).
  • ⁇ PI 4.07, +ve/-ve 4/10; GRAVY: -0.061 ⁇
  • D-RFX027 was chemically synthesized using native chemical ligation as well as stepwise SPPS. The synthetic strategy by native chemical ligation from two unprotected peptide segments was performed using D-Thr 1 -Val 31 -aCOSR and D-Cys 3 J 2 -Glu 5 3 6 °-aC0 2 H segments. However, folding of the product gave low recovery and purity of the folded protein molecule. D-(Arg) 4 -RFX027 was prepared using similar methods. Folding of the D-(Arg) 4 -RFX027 gave similar recovery and purity as D- RFX027. D-RFX028, D-RFX029 and D-RFX030 were prepared using similar methods, and showed improved folding and handling properties, recovery and purity.
  • Ahx (6-aminohexanoic acid) may be used as a spacer in between the peptide and the biotin.
  • L-peptidic GB1 compounds may be prepared using protein expression methods. The following protocols may be adapted as needed in preparing the subject GB1 peptidic compounds. Proteins may be expressed in BL21(DE3) cells with induction at 24°C for 12h under control of ptac promoter. Proteins with arginine codons were induced at 0.1 mM IPTG while the other proteins were induced at 0.5 mM IPTG.
  • RFX.T1.4.83.29.50.37 + extensions RFX033 variant: MKKNIAFLLASMFVFSIATNAYA/MKIEEHHHHHHHHHHGGSTYKMRLHGRTLTSECSTEAR DWFHAFDVLFGCAASNFPLIEDWTYDDATKTFTVTEGGSDK (SEQ ID NO: 146), where ' / ' indicates cleavage site after export to periplasm.
  • the GB1 compounds were expressed in BL21(DE3) cells with induction at 24°C for 12 hr with 0.5 mM IPTG.
  • proteins with signal sequences get exported to periplasm (variants 1, 3, 5, 7, 8, 9) while other proteins remain in cytoplasm. Some of the sequences did not express and some expressed at lower levels.
  • the following protocols may be adapted as necessary to measure properties and activities of the subject GB 1 peptidic compounds.
  • the binding affinity of GB 1 peptidic compounds for a target may be measured using any convenient SPR binding assay (e.g., an octet-SPR or standard SPR assay).
  • SPR binding assay e.g., an octet-SPR or standard SPR assay.
  • exemplary protocol below is described for VEGF, the protocol can be adapted as needed to measure binding of any convenient compound to any target.
  • Binding affinities are measured using the Biacore SPR system. SPR analysis is performed on a ProteOn XPR36 Protein Interaction Array System (BioRad). Chemically synthesized L-VEGF, D- VEGF and VEGF165 (Peprotech) are immobilized in 50 mM sodium acetate (pH 5.5) to a non-dilute ED AC/ sulfo-NHS activated GLC surface on separate channels using a flow rate of 30 ml/min for 5 minutes in the vertical direction. Immobilization levels are monitored to ensure immobilization of approximately 500 response units of each protein. The domains were then stabilized with PBS for 30 seconds and 0.85% H 3 P0 4 for 18 seconds each at 100 ml/min.
  • GB1 peptidic compounds are diluted in PBS plus 0.05% Tween 20 at a starting concentration of 200 nM.
  • the binders are further diluted with PBST 2-fold in series to produce 5 concentrations of variants.
  • a PBST blank is also included.
  • Compound injection parameters are: 100 ml/min, 60 seconds contact time, and 600 seconds dissociation time, in the horizontal direction.
  • VEGF proteins are regenerated with an injection of 0.85% H 3 P0 4 at a flow rate of 100 mL/min followed by a PBST wash of 30 seconds at 100 mL/min flow rate.
  • the expressed GB 1 peptidic compounds prepared above showed specific binding to D-VEGF as measured by SPR assay.
  • the thermal melting experiment of the synthetic D-protein RFX035 was recorded on an AVIV -202 instrument. An aqueous solution of the synthetic D-protein was transferred to a CD cuvette of path length 0.1 cm. The thermal melting was recorded at 222 nm over the range 10 °C-98 °C with increments of 1.5 °C with 90 sec equilibration time and averaging of 5 sees.
  • the D-protein RFX035 had a Tm value of 52.5 °C ( Figure 4B).
  • L-VEGF-A was bound in separate flow cells of a GLM sensor chip in a ProteOnTM Protein Interaction Array System (Bio-Rad) using an amine coupling kit. Sensorgrams were generated by injecting several concentrations of the D-protein RFX035 over the chip. The interactions were analyzed by local and global kinetic analysis using nonlinear regression fits to a 1 :1 Langmuir binding isotherm. Under the conditions used for this experiment the observed global dissociation constant value, K D , was 3.3 nM (averaged over the four highest concentrations) (Figure 4C).
  • RFX-001D- and L-VEGF were bound in separate flow cells of a GLM sensor chip in a ProteOnTM XPR36 Protein Interaction Array System (Bio-Rad) with 4000 RU of L-VEGF or 3500 RU of D- VEGF using an amine coupling kit. Sensorgrams were generated by injecting several concentrations of protein ligand over the chip. The interactions were analyzed by local and global kinetic analysis using nonlinear regression fits to a 1 : 1 Langmuir binding isotherm. Under the conditions used for this experiment the mean of the global KD values for L-RFX001 and Z)-RFX001 gives an observed K D of 70 ⁇ 17 nM.
  • HUVEC Proliferation Assay protocol Human umbilical vein endothelial cells (HUVEC) are plated in a 96 well plate coated with 2% gelatin at a density of 5000 cells/well.
  • HUVECs are initially cultured in starvation media including DMEM, 10% FBS, penicillin (100 units/ml), streptomycin (100 ⁇ g/ml), and glutamine (4mM) for 18-20 hours.
  • Experimental compounds of interest are diluted in serum-free DMEM and tested in triplicate at 1, 10, 20, 50, 100, and 200 nM.
  • Biotinylated compounds are preincubated with streptavidin at a molar ratio of 4: 1 for 30 minutes.
  • Avastin is used as a positive control and tested at 0.01, 0.1, 1, and 10 nM.
  • the experimental compounds and Avastin are preincubated in DMEM/10% FBS with VEGF (10 ng/ml; R&D Systems, Minneapolis, MN) for 30 minutes prior to them replacing the starvation media. The compounds are replaced after 72 hours. After 6 days in treatment media, cell viability is measured by WST-1 assay (Roche) per the manufacturer's instructions. The absorbance was measured using a microplate reader at 450 nm and a reference wavelength of 600 nm was subtracted.
  • Figure 5 shows the results of inhibition of VEGF-A driven cell proliferation in a HUVEC assay.
  • Bevacizumab is used as a positive control.
  • HUVECs are cultured in EGM-2 MV media (Lonza) and plated at a density of 5 x 10 5 cells per well in a 6-well plate and incubated for 24 hours.
  • Cells are starved in EBM-2 (Lonza) + 0.1% BSA (EBM-2/BSA) for 16 hours.
  • VEGF (10 ng/ml) resuspended in EBM-2/BSA is incubated with VEGF inhibitors at indicated concentrations for 30 min at 37 ° C.
  • Biotinylated compounds are pre -incubated with streptavidin at a molar ratio of 4: 1 in 10 ⁇ PBS for 10 min at room temperature.
  • Starvation medium is aspirated and cells receive prepared media containing VEGF +/- inhibitors or EBM-2/BSA only (control). Cells are incubated for 5 min at 37 ° C, immediately placed on ice, and washed once with ice-cold PBS. Cells from each well are scraped into 30 ⁇ RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails (Roche). Lysates are transferred to tubes and incubated on ice for 30 min with occassional vortexing. Lysates are cleared by centrifugation (16000xg, 10 min, 4 ° C) and supplemented with reducing Laemmli sample buffer.
  • Membranes are washed three times with TBST and incubated with peroxidase-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch diluted 1 : 10000 in TBST/milk for lh at room temperature. Blots are washed three times with TBST, and incubated with ECL substrate (Perkin Elmer) for 1 min. Films are exposed to blots for 1-3 min and developed.
  • Figure 6 illustrates the results of inhibition of VEGFR2 phosphorylation in cells.
  • Bevacizumab is used as a positive control.
  • Mutated GB 1 compounds may be prepared and screened for a desirable property by adapting methods, such as those described by Fellouse & Sidhu, "Making antibodies in bacteria. Making and using antibodies” Howard & Kaser, Eds., CRC Press, Boca Raton, FL, 2007.
  • a D-VEGF binding GB 1 L-peptidic compound was mutated to include charged residues such as Arg or Lys at several positions selected as suitable for mutation because they are located at surface positions not involved in direct contact with the target ( Figure 8).
  • the library of compounds (theoretical diversity 1.3 x 10 8 , covered in a library of 1 xlO 10 mutants) was prepared and screened for binding to target protein. Selected clones were identified and synthesized as D-peptidic versions of the compounds, as described above.
  • Codons used to introduce mutations (residue mutations): ASG (T/R); ARG (K/R); MKT (I/R/L/S); MRT (N/R/H/S); MST (T/R/P/S); ASA (K/R); RRA (E/R/K/G); SKG (V/R/L/G); SRT (D/R/H/G); RSA (A/T/R/G); AGS (S/R); CKG (L/R).

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Abstract

L'invention concerne des composés peptidiques GB1 qui se lient spécifiquement à une molécule cible. Tant des composés L-peptides que D-peptidiques sont inclus. Dans certains modes de réalisation, les composés selon l'invention incluent un motif GB1 et un ou plusieurs fragments de prolongement relié(s) à l'extrémité N ou C du motif GB1 via des lieurs éventuels. Les fragments de prolongement peuvent être peptidiques et en tant que tels peuvent inclure un domaine protéinique ou une séquence peptidique de jusqu'à 50 résidus. Des multimères de GB1 qui incluent deux composés peptidiques GB1 ou plus reliés via un fragment de ramification sont également décrits. L'invention concerne également des procédés de préparation et d'utilisation des composés, incluant des procédés pour le diagnostic ou l'imagerie in vivo d'une maladie ou d'un état associé à l'angiogenèse, où le composé est radiomarqué. Ces composés et procédés sont utilisés dans diverses applications dans lesquelles une liaison spécifique à des molécules cibles, par exemple des protéines cibles, est souhaitée.
PCT/US2013/068137 2012-11-02 2013-11-01 Composés peptidiques gb1 et leurs procédés de préparation et leur utilisation WO2014071234A1 (fr)

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WO2018180205A1 (fr) * 2017-03-31 2018-10-04 株式会社カネカ Matrice de dissociation par affinité à utiliser pour la purification d'immunoglobulines
JPWO2018180205A1 (ja) * 2017-03-31 2020-02-06 株式会社カネカ 免疫グロブリン精製用アフィニティー分離マトリックス
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WO2020198074A1 (fr) * 2019-03-22 2020-10-01 Reflexion Pharmaceuticals, Inc. Composés d-peptidiques pour facteur de croissance endothélial vasculaire (vegf)
WO2020198075A3 (fr) * 2019-03-22 2020-12-10 Reflexion Pharmaceuticals, Inc. Composés d-peptidiques multivalents pour protéines cibles
CN114144433A (zh) * 2019-03-22 2022-03-04 反射制药有限公司 用于目标蛋白的多价d-肽化合物
CN114174334A (zh) * 2019-03-22 2022-03-11 反射制药有限公司 针对vegf的d-肽化合物
CN114989298A (zh) * 2019-03-22 2022-09-02 反射制药有限公司 针对vegf的d-肽化合物

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